Covalent fluorescent probes for cannabinoid receptor 2

Abstract

The present invention provides: a covalent fluorescent probe for cannabinoid receptor 2 ("CB2") having the general formula (I) wherein X, n, p and R1 to R4 are as described herein; comprising the compound; processes for preparing the compounds; and methods of using the compounds. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a method for treating cannabinoid receptor 2 (CB 2 The probes are organic compounds that are affinity-driven covalent probes of R. These probes are realized through a novel, convergent synthetic blueprint derived from a central reactive motif. Background Art

[0002] Dysregulation of the endocannabinoid system, specifically CB 2 Dysregulation of signaling pathways involving CB R has been implicated in a range of diseases, including tissue damage, neurodegenerative conditions, and inflammation. 2 The importance of R is obvious, but no compound has yet entered the clinic. This feature is mainly attributed to the 2 The biology of CB is poorly understood, especially its expression and localization in health and disease. Fluorescent imaging probes have recently emerged as high-resolution tools to study CB in living cells and in vivo. 2 R localization, expression level and distribution (RC 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 to CB 2 The agonist CB1 binds to the orthosteric site of the R and triggers the relevant signal transduction pathway with its strong agonist ligand, which eventually leads to desensitization through receptor internalization. 2 In the case of R probes (S. Singh, et al., ACS Med. Chem. Lett. 2019, 10, 209-214), the probes also regulate cellular homeostasis because they continuously occupy the CB 2 R and thus directly alter cellular tone by preventing binding of endogenous ligands. Therefore, there are currently no available probes to study CB 2 R without simultaneously altering receptor signaling pathways, localization, expression, trafficking and / or distribution. Summary of the invention

[0003] The present invention solves the above limitations. The present invention reports CB carrying a cleavable motif 2R affinity driven probe, the motif allows the reporter gene unit to be covalently transferred to study the target protein while releasing the targeting ligand (T.Tamura, I.Hamachi, J.Am.Chem.Soc.2019,141,2782-2799). Such probes can be applied to flow cytometry fluorescence activated cell sorting (FACS) experiments or cell studies using confocal live cell imaging, for example, to quantify the transport of low-abundance targets. In addition, such probes provide the potential to generate equilibrium and kinetic binding data in a high-throughput manner without the need to use, for example, time-resolved fluorescence resonance energy transfer (TR-FRET) to process radioactive substances. In addition, these probes are particularly suitable for constructing FRET biosensors on the surface and inside of living 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 gene transfer, such probes are very suitable for targeting low-expression targets in healthy brains, such as CB 2 R. These probes can also support the translation of preclinical pharmacology animal data to the clinic.

[0004] In a first aspect, the present invention provides a compound of formula (I)

[0005]

[0006] or a pharmaceutically acceptable salt thereof, wherein X, n, p and R 1 To R 4 As defined herein.

[0007] In another aspect, the present invention provides methods for preparing fluorescent and bioorthogonal probes for proteins of interest, including compounds of formula (I) as described herein.

[0008] In another aspect, the present invention provides a method capable of orthogonally forming amides in the presence of labile, highly electrophilic functional groups.

[0009] In another aspect, the present invention provides certain synthetic intermediates useful in the methods of the present invention.

[0010] In another aspect, the present invention provides compounds of formula (I) as described herein as cannabinoid receptor 2 (CB 2 R) or the use of the probe for secondary bioorthogonal conjugation. DETAILED DESCRIPTION

[0011] definition

[0012] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process disclosed thereby may be combined in any combination, except for combinations in which at least some of these features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel step or any novel combination of steps of any method or process disclosed thereby.

[0013] The term "alkyl" refers to a monovalent or polyvalent (e.g., monovalent or divalent) straight or branched chain saturated hydrocarbon group containing 1 to 12 carbon atoms. In some preferred embodiments, the alkyl group contains 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5 or 6 carbon atoms ("C 1 -C 6 -alkyl"). In other embodiments, the alkyl group contains 1 to 3 carbon atoms, such as 1, 2, or 3 carbon atoms. Some non-limiting examples of alkyl include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl, and 2,2-dimethylpropyl. A particularly preferred but non-limiting example of an alkyl is methyl.

[0014] As used herein, the term "cycloalkyl" refers to a ring of 3 to 10 carbon atoms ("C 3-10 In some preferred embodiments, the cycloalkyl group is a saturated monocyclic hydrocarbon group having 3 to 8 ring carbon atoms. Preferably, the cycloalkyl group is a saturated monocyclic hydrocarbon group having 3 to 6 ring carbon atoms, in particular having 3 to 5 ring carbon atoms, for example having 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.

[0015] The term "halogen" or "halo" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Preferably, the term "halogen" or "halo" refers to fluorine (F), chlorine (Cl) or bromine (Br). Particularly preferred but non-limiting examples of "halogen" or "halo" are fluorine (F) and chlorine (Cl).

[0016] The term "cyano" refers to a -CN (nitrile) group.

[0017] The term "azide" refers to -N 3 Group.

[0018] The term "pharmaceutical salt" refers to those salts that retain the biological effect and properties of free alkali or free acid, which are not undesirable in biology or other aspects. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. (particularly hydrochloric acid) and 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, methylsulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc. In addition, these salts can be prepared by adding inorganic bases or organic bases to free acids. 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.). Specific pharmaceutically acceptable salts of the compounds of formula (I) are hydrochlorides and trifluoroacetates.

[0019] The compounds of formula (I) may contain several asymmetric centers and can exist in the form of optically pure enantiomers, mixtures of enantiomers (eg racemates), optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

[0020] According to the Cahn-Ingold-Prelog Convention, an asymmetric carbon atom can be in either the "R" or "S" configuration.

[0021] The abbreviation "CB2" refers to cannabinoid receptor 2.

[0022] Compounds of the present invention

[0023] In the first aspect (A1), the present invention provides a compound of formula (I)

[0024]

[0025] or a pharmaceutically acceptable salt thereof, wherein:

[0026] (i)R 1 for and

[0027] X is selected from O, S and SO; or

[0028] (ii) R 1 for

[0029] and X is selected from O, S, SO and NHCO;

[0030] R 2 and R 3 Selected from hydrogen and C 1 -C 4 -alkyl; or

[0031] R 2 and R 3 Together with the carbon atom to which they are attached, they form C 3 -C 5 - cycloalkyl;

[0032] R 4 is selected from hydrogen, halogen, cyano and azide;

[0033] R 5 Selected from:

[0034]

[0035] R 6 Selected from:

[0036]

[0037] R 7 Selected from:

[0038]

[0039]

[0040]

[0041] R a , R b and R c are each independently selected from hydrogen and halogen;

[0042] n is selected from 1, 2, 3, 4, 5 and 6;

[0043] p is selected from 1, 2, 3, 4, 5, 6 and 7;

[0044] q is selected from 1, 2, 3, 4 and 5;

[0045] r is selected from 1 and 2;

[0046] s is selected from 1, 2, 3 and 4;

[0047] t is selected from 0, 1, 2, 3, 4 and 5;

[0048] u is selected from 0, 1, 2, 3, 4 and 5;

[0049] W is carbonyl or absent;

[0050] Y is selected from Se, S and O.

[0051] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0052] (i)R 1 for and

[0053] X is selected from O, S and SO; or

[0054] (ii) R 1 for and

[0055] X is selected from O, S, SO and NHCO;

[0056] R 2 and R 3 Selected from hydrogen and C 1 -C 4 -alkyl; or

[0057] R 2 and R 3 Together with the carbon atom to which they are attached, they form C 3 -C 5 - cycloalkyl;

[0058] R 4 is selected from hydrogen, halogen, cyano and azide;

[0059] R 5 Selected from:

[0060]

[0061] R 6 Selected from:

[0062]

[0063] R 7 Selected from:

[0064]

[0065] R 8 Selected from:

[0066]

[0067]

[0068] R a , R b and R c are each independently selected from hydrogen and halogen;

[0069] n is selected from 1, 2, 3, 4, 5 and 6;

[0070] p is selected from 1, 2, 3, 4, 5, 6 and 7;

[0071] q is selected from 1, 2, 3, 4 and 5;

[0072] r is selected from 1 and 2;

[0073] s is selected from 1, 2, 3 and 4;

[0074] u is selected from 0, 1, 2, 3, 4 and 5;

[0075] v is selected from 1, 2, 3 and 4;

[0076] W is carbonyl or absent;

[0077] Y is selected from Se, S and O.

[0078] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0079] (i)R 1 for and

[0080] X is selected from O, S and SO; or

[0081] (ii) R 1 for and

[0082] X is NHCO;

[0083] R 5 Selected from:

[0084]

[0085] R 6 Selected from:

[0086]

[0087] R 7 Selected from:

[0088]

[0089] R 8 Selected from:

[0090]

[0091] p is selected from 3, 4 and 5;

[0092] q is selected from 2, 3 and 5;

[0093] r is selected from 1 and 2;

[0094] s is selected from 1 and 4;

[0095] u is selected from 0, 4 and 5; and

[0096] W is carbonyl or absent;

[0097] Y is selected from Se and O.

[0098] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0099] (i)R 1 for and

[0100] X is O; or

[0101] (ii) R 1 for

[0102] and

[0103] X is NHCO;

[0104] R 5 Selected from:

[0105]

[0106] R 6 Selected from:

[0107]

[0108] R 7 Selected from:

[0109]

[0110] R 8 Selected from:

[0111]

[0112] p is 3;

[0113] q is selected from 2, 3 and 5;

[0114] r is 1;

[0115] s is 1; and

[0116] u is 5.

[0117] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0118] R1 for

[0119] R 5 for

[0120] R 6 for

[0121] R 7 for

[0122] R 8 for

[0123] X is NHCO;

[0124] p is 3;

[0125] q is 2 or 3;

[0126] r is 1; and

[0127] u is 5.

[0128] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0129] R 1 for and

[0130] X is selected from O, S and SO.

[0131] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0132] R 1 for and

[0133] X is selected from O, S and SO.

[0134] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0135] R 1 for and

[0136] X is NHCO;

[0137] R 5 , R 6 and q are as defined herein.

[0138] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0139] R 1 for and

[0140] X is NHCO;

[0141] R 8 and r are as defined herein.

[0142] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0143] R 1 for and

[0144] X is NHCO;

[0145] R 5 , R 6 , R 8 , q and r are as defined herein.

[0146] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0147] R 5 Selected from:

[0148] in

[0149] R 7 , W and u are as defined herein.

[0150] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0151] R 5 for in

[0152] R 7 and u are as defined herein.

[0153] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0154] R 6 yes

[0155] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0156] R 6 yes

[0157] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0158] R 7 Selected from:

[0159]

[0160] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0161] R 7 yes

[0162] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0163] R 7 yes

[0164] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0165] R 8 Selected from:

[0166] in

[0167] s is as defined herein.

[0168] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0169] R 8 yes

[0170] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0171] R 8 for in

[0172] s is as defined herein.

[0173] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0174] R 8 yes

[0175] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0176] p is selected from 1, 3, 4 and 5.

[0177] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0178] p is 1.

[0179] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0180] p is 3.

[0181] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0182] p is 4.

[0183] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0184] p is 5.

[0185] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0186] q is selected from 2, 3 and 5.

[0187] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0188] q is 2 or 3.

[0189] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0190] r is selected from 1 and 2.

[0191] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0192] r is 1.

[0193] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0194] s is selected from 1 and 4.

[0195] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0196] u is selected from 0, 4 and 5.

[0197] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0198] u is 5.

[0199] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0200] Y is selected from Se and O.

[0201] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0202] s is 1.

[0203] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0204] u is 5.

[0205] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0206] R 2 and R 3 Selected from hydrogen and C 1 -C 4 -alkyl;

[0207] R 4 is hydrogen or azide; and

[0208] n is 6.

[0209] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0210] R 2 and R 3 Both are C 1 -C 4 -alkyl;

[0211] R 4 is azide; and

[0212] n is 6.

[0213] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0214] R 2 and R 3 Both are methyl;

[0215] R 4 is azide; and

[0216] n is 6.

[0217] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0218] R 2 and R 3 Selected from hydrogen and C 1 -C 4 -alkyl.

[0219] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0220] R 2 and R 3 Both are C 1 -C 4 -alkyl.

[0221] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0222] R 2 and R 3 Both are hydrogen.

[0223] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0224] R 2 and R 3 Both are methyl groups.

[0225] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0226] R 4 is hydrogen or azide.

[0227] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0228] R 4 For hydrogen.

[0229] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0230] R 4 Azide.

[0231] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0232] n is 6.

[0233] In one embodiment, the present invention provides a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein:

[0234] (i)R 1 for and

[0235] X is selected from O, S and SO; or

[0236] (ii) R 1 for and

[0237] X is NHCO;

[0238] R 2 and R 3 Selected from hydrogen and C 1 -C 4 -alkyl;

[0239] R 4 is hydrogen or azide;

[0240] R 5 Selected from:

[0241]

[0242] R 6 Selected from:

[0243]

[0244] R 7 Selected from:

[0245]

[0246] R 8 Selected from:

[0247]

[0248] n is 6;

[0249] p is selected from 1, 3, 4 and 5;

[0250] q is selected from 2, 3 and 5;

[0251] r is selected from 1 and 2;

[0252] s is selected from 1 and 4;

[0253] u is selected from 0, 4 and 5;

[0254] W is carbonyl or absent;

[0255] Y is selected from Se and O.

[0256] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0257] (i)R 1 for and

[0258] X is O; or

[0259] (ii) R 1 for and

[0260] X is NHCO;

[0261] R 2 and R 3 Both are C 1 -C 4 -alkyl;

[0262] R 4 is azide;

[0263] R 5 Selected from:

[0264]

[0265] R 6 Selected from:

[0266]

[0267] R 7 Selected from:

[0268]

[0269] R 8 Selected from:

[0270]

[0271] n is 6;

[0272] p is 3;

[0273] q is selected from 2, 3 and 5;

[0274] r is 1;

[0275] s is 1; and

[0276] u is 5.

[0277] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0278] R 1 for

[0279] R 2 and R 3 Both are methyl;

[0280] R 4 is azide;

[0281] R 5 for

[0282] R 6 for

[0283] R 7 for

[0284] R 8 for

[0285] X is NHCO;

[0286] n is 6;

[0287] p is 3;

[0288] q is 2 or 3;

[0289] r is 1; and

[0290] u is 5.

[0291] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein the compound of formula (I) is selected from:

[0292] 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;

[0293] 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;

[0294] 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;

[0295] 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;

[0296] 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;

[0297] 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;

[0298] 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-nitrobenz[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide;

[0299] 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-nitrobenz[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide;

[0300] 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;

[0301] 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]silanol-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;

[0302] 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)heptylamide;

[0303] 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;

[0304] 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;

[0305] 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]silanol-10,1'-isobenzofuran]-6'-carboxamide;

[0306] 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-ynyl)sulfamoyl)benzamide;

[0307] 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;

[0308] 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;

[0309] 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;

[0310] 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]silanol-10,1′-isobenzofuran]-6′-yl)oxy)heptylamide;

[0311] 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;

[0312] 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-azaeicosyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide;

[0313] 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.

[0314] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein the compound of formula (I) is selected from:

[0315] 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;

[0316] 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;

[0317] 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;

[0318] 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;

[0319] 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;

[0320] 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;

[0321] 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-nitrobenz[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide;

[0322] 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-nitrobenz[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide;

[0323] 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;

[0324] 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]silanol-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;

[0325] 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)heptylamide;

[0326] 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;

[0327] 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;

[0328] 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]silanol-10,1'-isobenzofuran]-6'-carboxamide;

[0329] 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-ynyl)sulfamoyl)benzamide;

[0330] 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;

[0331] 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;

[0332] 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

[0333] 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]silanol-10,1′-isobenzofuran]-6′-yl)oxy)heptylamide.

[0334] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein the compound of formula (I) is selected from:

[0335] 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;

[0336] 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;

[0337] 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-nitrobenz[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide;

[0338] 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]silanol-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;

[0339] 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)heptylamide;

[0340] 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;

[0341] 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]silanol-10,1'-isobenzofuran]-6'-carboxamide;

[0342] 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-ynyl)sulfamoyl)benzamide;

[0343] 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

[0344] 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]silanol-10,1′-isobenzofuran]-6′-yl)oxy)heptylamide.

[0345] 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 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 ((E)-2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate.

[0346] 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.

[0347] 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.

[0348] 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-(3,7-Di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silanol-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.

[0349] 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)heptylamide.

[0350] 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 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.

[0351] 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 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-dimethyl (bicyclo[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]silanol-10,1'-isobenzofuran]-6'-carboxamide.

[0352] 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-ynyl)sulfamoyl)benzamide.

[0353] 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.

[0354] 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]silanol-10,1'-isobenzofuran]-6'-yl)oxy)heptylamide.

[0355] In a particular embodiment, the invention provides a pharmaceutically acceptable salt of a compound according to formula (I) as described herein. In another particular embodiment, the invention provides a compound according to formula (I) as described herein as a free base.

[0356] Preparation process

[0357] The preparation of the compound of formula (I) of the present invention can be carried out according to sequential or concurrent synthetic routes. The synthesis of the present invention is shown in the following general scheme. The skills required for performing the reaction and purifying the resulting product are known to those skilled in the art. Unless otherwise indicated, the substituents and indices used in the following method descriptions have the meanings provided herein.

[0358] If one of the starting materials, intermediates or compounds of formula (I) contains one or more functional groups that are unstable or reactive under the reaction conditions of one or more reaction steps, appropriate protecting groups may be introduced before applying key steps of methods well known in the art (such as TW Greene and PGM Wutts in "Protective Groups in Organic Chemistry" 5th edition, 2014, John Wiley & Sons, NY). Such protecting groups may be removed at a later stage of the synthesis using standard methods described in the literature.

[0359] If the starting material or intermediate contains a stereogenic center, the compound of formula (I) can be obtained in the form of a mixture of diastereomers or enantiomers, which can be separated by methods well known in the art (e.g., chiral HPLC, chiral SFC or chiral crystallization). Racemic compounds can be separated into corresponding enantiomers, for example, by diastereomeric salts, which are crystallized with optically pure acids, or by specific chromatography separations of enantiomers using chiral adsorbents or chiral eluents. Starting materials and intermediates containing stereogenic centers can also be separated to provide starting materials and intermediates enriched in diastereomers / enantiomers. Using such diastereomer / enantiomer-enriched starting materials and intermediates in the synthesis of compounds of formula (I) will generally produce diastereomers / enantiomer-enriched compounds of the corresponding formula (I).

[0360] Those skilled in the art will recognize that in the synthesis of compounds of formula (I) (if not desired), an "orthogonal protecting group strategy" will be applied, which allows multiple protecting groups to be cleaved each time without affecting other protecting groups in the molecule. The principle of orthogonal protection is well known in the art and has also been reported in the literature (e.g., Barany and RB Merrifield, J. Am. Chem. Soc. 1977, 99, 7363; H. Waldmann et al., Angew. Chem. Int. Ed. Engl. 1996, 35, 2056).

[0361] Those skilled in the art will recognize that the order of reactions may vary depending on the reactivity and nature of the intermediates.

[0362] In more detail, the compound of formula (I) can be manufactured by the method given below, by the method given in the examples or by similar methods. The appropriate reaction conditions for each reaction step are known to those skilled in the art. Similarly, the reaction conditions affecting the reaction reported in the relevant literature are described, for example: Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2nd Edition, Richard C. Larock. John Wiley & Sons, New York, NY. 1999. It is convenient to react in the presence or absence of a solvent. There are no particular restrictions on the nature of the solvent used, as long as it has no adverse effect on the reaction or the reagents involved and can dissolve the reagents at least to a certain extent. The described reaction can occur over a wide temperature range, and the precise reaction temperature is not critical to the present invention. The above reaction can be conveniently performed in a temperature range of -78°C to reflux. The time required for the reaction can also vary over a wide range, depending on many factors, particularly the reaction temperature and the nature of the reagents. However, it usually takes 0.5 hours to several days to obtain the intermediates and compounds. The reaction sequence is not limited to the one shown in the schemes, but, depending on the starting materials and their respective reactivity, the order of the reaction steps can be freely varied.

[0363] If starting materials or intermediates were not commercially available or their synthesis was not reported in the literature, they could be prepared by analogy to existing preparations of close analogs or as outlined in the experimental section.

[0364] The following abbreviations are used in this text:

[0365] AcOH = acetic acid, ACN = acetonitrile, Boc = tert-butyloxycarbonyl, CAS RN = Chemical Abstracts Registry Number, CH 2 Cl 2 = dichloromethane, COMU = (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylamino-morpholino-carbonium hexafluorophosphate, [Cu(MeCN) 4 ]PF 6 = 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-Pr 2NEt = N,N-diisopropylethylamine, EDCI = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, EDCI·HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, ESI = electrospray ionization, Et 3 N = triethylamine, Et 2 O = diethyl ether, EtOAc = ethyl acetate, EtOH = ethanol, h = hour, GABA = γ-aminobutyric acid, H 2 O = 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, K 2 CO 3 = Potassium carbonate, MeOH = methanol, MgSO 4 = magnesium sulfate, min = minute, mL = milliliter, MS = mass spectrometry, NaH = sodium hydride, NaHCO 3 =Sodium bicarbonate, NaOH =Sodium hydroxide, Na 2 SO 4 =Sodium sulfate, NEt 3 = triethylamine (TEA), NH 4 Cl = ammonium chloride, O t Bu = tert-butyloxy, PG = protecting group, PyAOP = (7-azabenzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate, PyBOP = (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate, R = any group, rt = room temperature, SiO 2 = silica, T3P = propylphosphonic anhydride, TBTU = O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, TFA = trifluoroacetic acid, THF = tetrahydrofuran, TLC = thin layer chromatography.

[0366] Where R 4 , R 7 , p, r, s, t, W Compounds of formula I as described herein can be synthesized analogously to the literature and / or as depicted, for example, in Scheme 1.

[0367]

[0368] Solution 1.

[0369] Accordingly, the ω-amino ester linker of type A is reacted with 6-hydroxynicotinic acid to give B (step a). This type of amide coupling can be accomplished by using one of the well-known coupling reagents (such as DCC, HATU, EDCI, TBTU, T3P, etc.) and a base (such as i-Pr 2 NEt, Et 3 N or DMAP, in a suitable solvent (such as DMF, DMA, CH 2 Cl 2 or dioxane), preferably between 0°C and room temperature. Subsequently, B is reacted with an activated sulfonate (such as but not limited to a sulfonyl halide) in the presence of a base (preferably K0 t Bu, but can also be i-Pr 2 NEt, TEA, DMAP) in a suitable solvent (such as THF or CH 2 Cl 2 or dioxane) to give C (step b). One skilled in the art will recognize that the order of addition of reagents and the choice of base and solvent may be important in such reactions due to the reactivity and stability of the two tautomers of the deprotonated 2-pyridone anion leading to the formation of N- or O-sulfonylpyridone structural isomers. Type C compounds can be directly linked to ligands for covalent transfer of common cargoes such as alkynes to further functionalize by secondary conjugation (such as with azide) in the presence of a metal catalyst. Alternatively, type C N-pyridone motifs can be optionally reacted with a metal catalyst such as Cu I )-catalyzed [3+2]-azide-alkyne cycloaddition coupling to afford the functionalized D-type N-pyridone scaffold (step c). Preferentially, a protic non-nucleophilic solvent mixture (CH 2 Cl 2 , AcOH), which allows the reaction to proceed in higher yields at ambient temperature, but is not necessary because, for example, MeOH, CH 2 Cl 2 It also works at 40°C. The functional group is preferably a fluorescent label, but can also be any cargo for secondary reactions in vitro or in vivo, preferably bioorthogonal such as strained alkenes, strained alkynes, tetrazenes, alkynes, azides, diaziridines, aldehydes, boronic acids. The D-type compound is then reacted in a range of solvents (such as CH 2 Cl 2 , Et 2The ligand is deprotected by an acid (such as HCl or TFA) in a suitable solvent (such as DMF, DMA, CH 4 ) and the acid is coupled to the desired protein ligand of interest in one step without further purification to produce E (step d). The ligand carries an amino functional group for coupling by one of the well-known coupling reagents (DCC, EDCI, TBTU, T3P), preferably HATU, PyAOP or an activating reagent that usually produces HOAt esters from carboxylic acids. The reaction is carried out in a suitable solvent (such as DMF, DMA, CH 4 ). 2 Cl 2 or dioxane) and acid / base buffer systems (such as i-Pr 2 NEt, TFA), preferably at a temperature between 0°C and room temperature.

[0370] The key part of the present invention is the discovery that N-sulfonyl pyridone C can actually be used only on the O-pyridone isomer using KO in THF t Not formed.

[0371] Another key part of the present invention is the discovery and use of an acid / base buffer system, such as TFA / i-Pr 2 NEt. Preferentially, with TFA / i-Pr 2 NEt buffer system is used together with the reaction (step d) of HATU mediation.HOAt active ester is conducive to the rapid formation of amide bond, but free HOAt, if not buffered, can react and mediate degradation with electrophilic N-sulfonyl pyridone.Selecting suitable acid / base buffer system can prevent this key side reaction, and allows to carry out the formation of final amide bond in the presence of high electrophilic motif.Those skilled in the art will recognize that, for wherein after key formation, activator has enough acidity and nucleophilicity with other coupling reagents of cracking electrophilic reagent, such as TBTU, PyAOP, PyBOP, COMU, similar reactivity will be expected.

[0372] One 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 either functionalization component.

[0373] Where R 4 , R 5 , p, q Compounds of formula I as described herein can be synthesized analogously to the literature and / or as depicted, for example, in Scheme 2.

[0374]

[0375] Solution 2.

[0376] Accordingly, the A-type ω-amino ester linker is reacted with an activated 4-sulfobenzoic acid (such as in the form of an acyl group, a sulfonyl halide), followed by the addition of an amine B to obtain a compound of formula C (step a). Subsequently, the N-alkylsulfonamide C is reacted with a functionalized acid to produce an N-acyl-N-alkylsulfonamide D (step b). This type of amide coupling can be accomplished by using one of the well-known coupling reagents (such as DCC, HATU, EDCI, TBTU, T3P, etc.) and a base (such as i-Pr 2 NEt, Et 3 N or DMAP, in a suitable solvent (such as DMF, DMA, CH 2 Cl 2 or dioxane), preferably between 0°C and room temperature. The D-type compound is then reacted with a series of solvents (such as CH 2 Cl 2 , Et 2 The ligand is deprotected by an acid (such as HCl or TFA) in a suitable solvent (such as DMF, DMA, CH 4 ) and the acid is coupled to the desired protein ligand of interest in one step without further purification to produce E (step c). The ligand carries an amino functional group for coupling by one of the well-known coupling reagents (DCC, EDCI, TBTU, T3P), preferably HATU, PyAOP or an activating reagent that usually produces HOAt esters from carboxylic acids. The reaction is carried out in a suitable solvent (such as DMF, DMA, CH 4 ). 2 Cl 2 or dioxane) and acid / base buffer systems (such as i-Pr 2 NEt, TFA), preferably at a temperature between 0°C and room temperature.

[0377] Where R 4 , R 5 , p, q Compounds of formula I as described herein can be synthesized analogously to the literature and / or as depicted, for example, in Scheme 3.

[0378]

[0379] Solution 3.

[0380] Accordingly, the ω-amino ester linker of type A is reacted with 4-sulfamoylbenzoic acid to produce B (step a). This type of amide coupling can be accomplished by using one of the well-known coupling reagents (such as DCC, HATU, EDCI, TBTU, T3P, etc.) and a base (such as i-Pr 2 NEt, Et 3 N or DMAP, in a suitable solvent (such as DMF, DMA, CH 2 Cl 2or dioxane), preferably between 0°C and room temperature, preferably catalyzed by HOBt. Subsequently, the sulfonamide of B is reacted with a carboxylic acid and N-acylated with one of the well-known coupling reagents (such as DCC, HATU, EDCI, TBTU, T3P, etc.) and a base (such as i-Pr 2 NEt, Et 3 N), in a suitable solvent (such as DMF, DMA, CH 2 Cl 2 N-acylation is carried out in 4% to 5% ethyl acetate or dioxane, preferably catalyzed by DMAP, preferably between 0°C and room temperature to produce C1 and C2 (step b).

[0381] Following the approach of Example 10, N-acylsulfonamide C1 is alkylated with iodoacetonitrile in the presence of a suitable base to produce type D compound (step c). The tert-butyl ester of type D compound is then reacted in a range of solvents (such as CH 2 Cl 2 , Et 2 The ligand is deprotected by an acid (such as HCl or TFA) in a suitable solvent (such as DMF, DMA, CH 4 ) and the acid is coupled to the desired protein ligand of interest in one step without further purification to produce E (step d). The ligand carries an amino functional group for coupling by one of the well-known coupling reagents (DCC, EDCI, TBTU, T3P), preferably HATU, PyAOP or an activating reagent that usually produces HOAt esters from carboxylic acids. The reaction is carried out in a suitable solvent (such as DMF, DMA, CH 4 ). 2 Cl 2 or dioxane) and acid / base buffer systems (such as i-Pr 2 NEt, TFA), preferably at a temperature between 0°C and room temperature.

[0382] Following the approach of Example 15, C2 is deprotected with an acid (such as HCl or TFA) and reacted with an activated carboxylic acid derivative (such as an N-hydroxysuccinimide ester or anhydride) to produce an N-acylsulfonamide F (step e). Compounds of type F are then coupled to the desired protein ligand of interest. The ligand carries a complementary functional group, such as an amine or alcohol, to be coupled via one of the well-known coupling reagents (DCC, HATU, EDCI, TBTU, T3P, etc.) and a base (such as i-Pr 2 NEt, Et 3 N or DMAP, in a suitable solvent (such as DMF, DMA, CH 2 Cl 2 or dioxane), preferably between 0°C and room temperature. Finally, alkylation of the intermediate N-acylsulfonamide with the desired alkyl halide provides probe G (step f).

[0383] Synthesis of Examples 11 to 14, in step g, B is acylated with 4-pentynoic acid under conditions similar to step b to give H. In step h, the N-acylsulfonamide in H is alkylated under conditions similar to step c to produce the activated N-acyl-N-alkylsulfonamide platform reagent I.

[0384] Following the approach of Example 14, in a series of solvents (such as CH 2 Cl 2 , Et 2 I is deprotected by acid (such as HCl or TFA) in 4% paraformaldehyde (D, dioxane) and the acid is coupled to the desired protein ligand of interest in one step without further purification (step i).

[0385] In the syntheses of Examples 11 to 13, the alkyne of I is functionalized with an azide-bearing cargo (such as a fluorophore) in a manner similar to Scheme 1, step c, to produce K (step j). K is then deprotected and coupled with the desired ligand in analogy to the conditions of Scheme 1, step d, to afford the final compound type L (step k).

[0386] A key part of the present invention is the discovery and use of an acid / base buffer system, such as TFA / i-Pr 2 NEt. Preferentially, with TFA / i-Pr 2 The NEt buffer system is used together with the reaction mediated by HATU. HOAt active esters are conducive to the rapid formation of amide bonds, but free HOAt, if not buffered, can react and mediate degradation with electrophilic N-acyl-N-alkyl sulfonamide. Selecting a suitable acid / base buffer system can prevent this key side reaction, and allows the formation of final amide bonds in the presence of high electrophilic motifs. Those skilled in the art will recognize that for other coupling reagents wherein the activator has enough acidity and nucleophilicity to crack electrophilic reagents after the key is formed, such as TBTU, PyAOP, PyBOP, COMU, similar reactivity will be expected.

[0387] Those skilled in the art will recognize that the formation of amide bonds depends on the acidity differences of nucleophilic catalyst activators (e.g., HOAt), carboxylic acids, and acid / base (e.g., TFA / i-Pr2NEt) buffer systems. If different activators are used together with matching buffer systems, it is expected that the same reaction results will be produced. In the above case, the acidity of HOAt (pKa≈3.3) is between carboxylic acids (pKa≈5) and TFA (pKa≈0.5). Therefore, during the reaction, any released nucleophilic HOAt anion is immediately protonated by TFA, and therefore does not participate in the nucleophilic degradation of the electrophilic motif. The resulting non-nucleophilic TFA anion will not cleave the electrophilic motif. Using the above exemplary logic, those skilled in the art can construct a system that matches its preferred coupling reagents and buffer systems.

[0388] One 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 either functionalizing component.

[0389] Where R 1 , R 4 , p, X Compounds of formula I as described herein can be synthesized analogously to the literature and / or as depicted, for example, in Scheme 4.

[0390]

[0391] Solution 4.

[0392] Accordingly, an ester of type A, wherein X may be N, O, S, Se, P, is reacted with a fluorophore carrying a leaving group (L) to give a compound of type B (step a). The ester of B is then cleaved with an acid using standard conditions to reveal a carboxylic acid coupled to the desired targeting ligand, thereby producing a compound of type C similar to the methods previously described herein (step b). Alternatively, B may be oxidized to produce a compound of type D (step c). This may be achieved by an oxidizing agent such as hydrogen peroxide, meta-chloroperbenzoic acid, dimethyldioxirane or potassium persulfate. In a similar manner to C, a compound of type E is obtained after deprotection and formation of an amide bond with the ligand of interest (step d).

[0393] In one aspect, the present invention provides a method for preparing a fluorescent probe P1 or P2 for a protein of interest,

[0394]

[0395] where LG is the ligand for the protein of interest, and W, R 6 , R 7 , p, q, r and s are as defined herein, the method comprising:

[0396] (a) Make alkyne C or I

[0397]

[0398] Where R 6 , p, q and r are as defined herein, reacting with the azide-substituted fluorophore M

[0399]

[0400] Among them, W, R 7 , s and t are as defined herein;

[0401] To form compound D or K

[0402]

[0403] Among them, W, R 6 , R 7 , p, q, r and s are as defined herein;

[0404] After that

[0405] (b) removing the t-Bu protecting group from compound D or K using an acid such as HCl or TFA,

[0406] To obtain compound D1 or K1

[0407]

[0408] Among them, W, R 6 , R 7 , p, q, r and s are as defined herein;

[0409] After that

[0410] (c) reacting compound D1 or K1 with an amino-substituted ligand N for the protein of interest,

[0411]

[0412] where LG is the ligand for the protein of interest,

[0413] The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system;

[0414] To obtain the fluorescent probe P1 or P2 for the protein of interest.

[0415] In the above-described process according to the invention, the reaction order may be reversed so that steps (b) and (c) are carried out before step (a).

[0416] Therefore, in another aspect, the present invention provides a method for preparing a fluorescent probe P1 or P2 for a protein of interest,

[0417]

[0418] where LG is the ligand for the protein of interest, and W, R 6 , R 7 , p, q, r and s are as defined herein, the method comprising:

[0419] (a) removing the t-Bu protecting group from compound C or I using an acid such as HCl or TFA,

[0420]

[0421] Where R 6 , p, q and r are as defined herein,

[0422] To obtain compound Ci or Ii

[0423]

[0424] Where R 6 , p, q and r are as defined herein,

[0425] After that

[0426] (b) reacting compound Ci or Ii with an amino-substituted ligand N for the protein of interest,

[0427]

[0428] where LG is the ligand for the protein of interest,

[0429] The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system;

[0430] To obtain compound Cii or Iii

[0431]

[0432] where LG is the ligand for the protein of interest and R 6 , p, q, r and r are as defined herein;

[0433] After that

[0434] (c) reacting the compound Cii or Iii with an azide-substituted fluorophore M

[0435]

[0436] Among them, W, R 7 , s and t are as defined herein;

[0437] To obtain the fluorescent probe P1 or P2 for the protein of interest.

[0438] In another aspect, the present invention provides a method for preparing a fluorescent probe P1 or P2 for a protein of interest,

[0439]

[0440] where LG is the ligand for the protein of interest, and W, R 6 , R 7 , p, q, r and s are as defined herein, the method comprising:

[0441] (a) Compound D1 or K1

[0442]

[0443] Among them, W, R 6 , R 7 , p, q, r and s are as defined herein;

[0444] Reaction with an amino-substituted ligand N for the protein of interest,

[0445]

[0446] where LG is the ligand for the protein of interest,

[0447] The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system;

[0448] To obtain the fluorescent probe P1 or P2 for the protein of interest.

[0449] In another aspect, the present invention provides a method for preparing a fluorescent probe P1 or P2 for a protein of interest,

[0450]

[0451] where LG is the ligand for the protein of interest, and W, R 6 , R 7 , p, q, r and s are as defined herein, the method comprising:

[0452] (a) Compound Cii or Iii

[0453]

[0454] where LG is the ligand for the protein of interest and R 6 , p, q, r and r are as defined herein;

[0455] Reaction with azide-substituted fluorophore M

[0456]

[0457] Among them, W, R 7 , s and t are as defined herein;

[0458] To obtain the fluorescent probe P1 or P2 for the protein of interest.

[0459] In one aspect, the present invention provides a method for preparing compound D or K,

[0460]

[0461] Among them, W, R 6 , R 7 , p, q, r and s are as defined herein, the method comprising:

[0462] (a) Make alkyne C or I

[0463]

[0464] Where R 6 , p, q and r are as defined herein, reacting with the azide-substituted fluorophore M

[0465]

[0466] Among them, W, R 7 , s and t are as defined herein;

[0467] To form the compound D or K.

[0468] In another aspect, the present invention provides a method for preparing compound Cii or Iii,

[0469]

[0470] where LG is the ligand for the protein of interest and R 6 , p, q and r are as defined herein, the method comprising:

[0471] (a) making the compound Ci or Ii

[0472]

[0473] Where R 6 , p, q and r are as defined herein,

[0474] Reaction with an amino-substituted ligand N for the protein of interest,

[0475]

[0476] where LG is the ligand for the protein of interest,

[0477] The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system;

[0478] To obtain the compound Cii or Iii.

[0479] In another aspect, the present invention provides a method for preparing an amide of formula 1

[0480]

[0481] Where R x and R y Both are chemical moieties attached to the parent amide functional group through a carbon atom; and wherein R x Contains N-sulfonylpyridone (SP) motif i, or N-acyl-N-alkylsulfonamide (NASA) motif ii

[0482]

[0483] Where R 6 As defined herein;

[0484] The method comprises:

[0485] Make carboxylic acid 2

[0486]

[0487] Reaction with amine 3

[0488]

[0489] The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system;

[0490] To obtain the amide 1.

[0491] In one embodiment, the coupling reagent used in the amide coupling according to the present invention is selected from the group consisting of DCC, PyBOP, COMU, EDCI, TBTU and T3P.

[0492] In a preferred embodiment, the coupling reagent used in the amide coupling according to the present invention produces HOAt esters from carboxylic acid in the presence of a suitable catalyst. Examples of coupling reagents that produce HOAt esters from 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 such coupling reactions are well known in the art. Non-limiting examples include EDCI and HOAt, and DIC and HOAt.

[0493] In a particularly preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is selected from HATU and PyAOP.

[0494] In another particularly preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is HATU.

[0495] In one embodiment, 1 to 5 equivalents, such as 1, 2, 3, 4 or 5 equivalents of coupling reagent relative to the corresponding carboxylic acid are used in the amide coupling according to the present invention.

[0496] In a preferred embodiment, 1 to 2 equivalents of coupling reagent relative to the corresponding carboxylic acid are used in the amide coupling according to the present invention.

[0497] In a particularly preferred embodiment, 1.1 equivalents of coupling reagent relative to the corresponding carboxylic acid are used in the amide coupling according to the invention.

[0498] In another particularly preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is PyAOP.

[0499] In one embodiment, 1 to 2 equivalents of coupling reagent relative to the corresponding carboxylic acid are used in the amide coupling according to the present invention.

[0500] In a preferred embodiment, 1 to 1.5 equivalents of the coupling reagent relative to the corresponding carboxylic acid are used in the amide coupling according to the present invention.

[0501] In a particularly preferred embodiment, 1.1 equivalents of coupling reagent relative to the corresponding carboxylic acid are used in the amide coupling according to the invention.

[0502] In one embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt.

[0503] In one embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt, in which TFA and i-Pr 2 The ratio of NEt is 1 / 3–11 / 13, such as 1 / 3, 3 / 5, 5 / 7, 7 / 9, 9 / 11 or 11 / 13.

[0504] In a preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt, in which TFA and i-Pr 2 The ratio of NEt is 3 / 5–3.5 / 5.5.

[0505] In a particularly preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt, in which TFA and i-Pr 2 The ratio of NEt is 3.3 / 5.3.

[0506] In a preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt, wherein 3 to 3.5 equivalents of TFA and 5 to 5.5 equivalents of i-Pr are used relative to the corresponding carboxyl group. 2 NEt.

[0507] In a preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt, where 3.3 equivalents of TFA and 5.3 equivalents of i-Pr were used relative to the corresponding carboxyl group. 2 NEt.

[0508] In one embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr 2 NEt, wherein 1 to 10 equivalents of TFA are used relative to the coupling reagent.

[0509] 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-Pr 2 NEt.

[0510] In one embodiment, the amide coupling of the present invention is carried out in a medium selected from DMF, DMSO, DMA, CH 2 Cl 2 and dioxane or a mixture thereof.

[0511] In a preferred embodiment, the amides of the present invention are coupled in DMF and CH 2 Cl 2 in a mixture of.

[0512] In a preferred embodiment, the ligand for the protein of interest is a ligand for CB2, in particular the following ligand:

[0513]

[0514] Where R 2 , R 3 and R 4 As defined herein, and the wavy line represents the point of attachment of the ligand to the rest of the compound of formula (I) as described herein.

[0515] In another aspect, the present invention provides a compound selected from:

[0516]

[0517]

[0518] Where R 2 , R 3 , R 4 , R 6 , R 7 , p, q, r, s, t are as described herein, and LG is the ligand for the protein of interest.

[0519] TR-FRET hCB2R binding assay

[0520] Cell culture: cells were cultured at 37°C and 5% CO 2 The cells were maintained in a humidified environment in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and blasticidin (5 μg / mL; Invitrogen) and Zeocin (20 μg / mL; Invitrogen). For inducible expression, SNAP-tagged human CB in pcDNA4 / TO was incubated with PEI. 2 The receptor cDNA was introduced into HEK293TR cells (Invitrogen, which express the Tet repressor to allow inducible expression) by transfection. Stable lines of mixed populations were selected by resistance to blasticidin (TR vector, 5 μg / mL) and Zeocin; (receptor plasmid, 20 μg / mL). For inducible expression of the receptor, cells were plated at t175 cm 2 Flasks were grown to 70% confluence and supplemented with DMEM containing 1 μg / ml tetracycline. After 24 h, cells were labeled with SNAP-Lumi4-Tb (CisBio) and membranes were prepared as described in detail below.

[0521] Terbium labeling of HEK293-TR cells with SNAP-tagged CB2R: From confluent adherent CB 2 The cell culture medium was removed from the t175 cm2 flask of HEK293-TR cells. The cells were washed once in PBS (GIBCO Carlsbad, CA) and then washed with 1x Tag-lite labeling medium (LABMED, CisBio) to remove excess cell culture medium. Then 10 ml of LABMED containing 100 nM SNAP-Lumi4-Tb was added to the flask and incubated at 37°C in 5% CO. 2Incubate for 1 h at 4 °C. Wash the cells once in PBS (GIBCO Carlsbad, CA) to remove excess SNAP-Lumi4-Tb, then detach the cells using 5 mL of GIBCO enzyme-free Hank's base cell dissociation buffer (GIBCO, Carlsbad, CA) and collect them in a vial containing 5 ml of DMEM (Sigma-Aldrich) and 10% fetal bovine serum. The cells were precipitated by centrifugation (5 min at 1500 rpm) and the precipitate was frozen to -80 °C. To prepare the membrane, a homogenization step was performed at 4 °C (to avoid receptor degradation) as described in CK Herenbrink, et al., Nat. Commun., 2016, 7, 10842.

[0522] Fluorescent ligand binding assay: All fluorescent ligand binding experiments were performed in white 384-well Optiplate plates, and the assay binding buffer was Hanks balanced salt solution (HBSS), 5mM HEPES, 0.5% BSA, 0.02% Pluronic F-127 pH 7.4 and 100μM GppNHp. GppNHp was included to remove the G protein-coupled receptor population, which may result in two different binding site populations in the membrane preparation because the Motulsky-Mahan model is only applicable to ligands that compete at a single site. In all cases, nonspecific binding was determined by the presence of 1μM SR144528. Data are presented as mean ± SEM from 3 to 8 experiments representative of the present invention.

[0523] Fluorescent ligand binding kinetics and equilibrium affinity assays: To accurately determine the association rate (kon) and dissociation rate (koff) values, the observed association rate (kob) was calculated using at least five different concentrations of 8-SiR (for 8-SiR and assay instructions, see T. Gazzi et al. Chem. Sci., 2022, 13, 5539-5545). Appropriate concentrations of fluorescent ligand binding were incubated with human CB2R HEK293-TR cell membranes (4 μg per well) in assay binding buffer (final assay volume, 40 μL). The extent of fluorescent ligand binding to the receptor was assessed at multiple time points by HTRF detection to construct an association kinetic curve. The resulting data were globally fitted to the association kinetic model (Equation 1, see the Signal Detection and Data Analysis section below) to derive a single best fit estimate of kon and koff, as described in Data Analysis. Fluorescent ligand binding affinity was determined by performing a saturation analysis at equilibrium by simultaneously fitting the total binding and nonspecific (NSB) binding data (Eq. 2, see Signal Detection and Data Analysis section below).

[0524] Competitive binding: To determine CB2 We used a simple competitive kinetic binding assay to determine the affinity of CB for specific ligands. 2 Both fluorescent ligand and competitor were added to the R preparation. 62.5 nM 8-SiR (a concentration to avoid ligand depletion in the assay volume) was added simultaneously with increasing concentrations of unlabeled compound to 40 μL of assay buffer in a 384-well plate. 2 R cell membranes (4 μg per well) were incubated at room temperature with orbital mixing. The extent of fluorescent ligand binding to the receptor was assessed by HTRF detection at equilibrium. Nonspecific binding was determined as the amount of HTRF signal detected in the presence of SR144528 (1 μM) and subtracted from total binding to calculate the IC 50 Specific binding curve.

[0525] Signal detection and data analysis: Signal detection was performed on a Pherastar FSX (BMG Labtech, Offenburg, Germany). The terbium donor was always excited with eight laser flashes at a wavelength of 337 nm. When the red acceptor fluorescent ligand 8-SiR was used, TR-FRET signals were collected at 665 nm (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 non-regression using Prism 8.0 (GraphPad Software, San Diego, USA).

[0526] The fluorescent ligand association data were fitted to a global fit model using GraphPad Prism 8.0 as follows to simultaneously calculate k using the following equation on and k off ,

[0527] Y=Y max *(1-exp(-1*k ob *X))

[0528] (Equation 1)

[0529] Among them, k ob =[L]*k on +k off

[0530] Among them, k ob is equal to the observed ligand association rate, and k on and k off are the association rate constant and dissociation rate constant of the fluorescent ligand, respectively. In this globally fitted tracer binding model, the tracer concentration [L] is fixed and k on and koff is a shared parameter, and k obs Here, Y is the level of receptor-bound tracer, Y max is the level of tracer binding at equilibrium, X is the time unit (e.g., min), and k obs is the rate of approaching equilibrium (e.g. min -1 ).

[0531]

[0532] Saturation binding data were analyzed by global fit to total binding and NSB by nonlinear regression according to the one-site equation. The fluorescent ligand dissociation constant (K) was calculated using the following equation: d ), where L is the fluorescent ligand concentration:

[0533] (Simultaneously) globally fit the total binding dataset and the NSB dataset, sharing the slope value, K d and B max Both provide a best fit value.

[0534] The competition displacement binding data were fit to a sigmoidal (variable slope) curve using a "four-parameter logistic equation":

[0535] Y = bottom + (top - bottom) / (1 + 10 (logIC50-X).希尔系数 )

[0536] (Equation 3)

[0537] The IC obtained from the inhibition curves was calculated using the method of Cheng and Prusoff (C. Yung-Chi, WH Prusoff, Biochem. Pharmacol. 1973, 22, 3099-3108). 50 Convert value to K i value.

[0538] Table 1

[0539]

[0540] Use of the Compounds of the Invention

[0541] The compound of formula (I) is 2 R has high affinity for fluorescent or bioorthogonal probes. Therefore, they can be used to study the localization, expression levels and protein distribution in health and disease states, and in living cells. 2The probes are high-resolution tools for the structure, dynamics and function of R. They can also be applied to flow cytometry fluorescence activated cell sorting (FACS) experiments or cellular trafficking studies using confocal live cell imaging. The probes provide an excellent tool to perform these experiments without disturbing cellular homeostasis because they are functionalized with cleavable motifs that allow the ligand to dissociate after covalent labeling. Due to the covalent nature of their labeling, the probes are particularly well suited for imaging low-abundance target proteins. The probes can be used to construct FRET sensors for membrane-bound and intracellular protein targets and subsequently for real-time studies of ligand-protein interactions by TR-FRET approach.

[0542] In one aspect, the present invention provides a compound of formula (I) as described herein for use as a cannabinoid receptor 2 (CB 2 R) fluorescent probe or bioorthogonal probe.

[0543] In another aspect, the present invention provides compounds of formula (I) as described herein as cannabinoid receptor 2 (CB 2 R) of fluorescent probes or bioorthogonal probes.

[0544] In another aspect, the present invention provides a method for treating cannabinoid receptor 2 (CB 2 R) for imaging, the method comprising allowing the cannabinoid receptor 2 (CB 2 R) is contacted with a compound of formula (I) as described herein.

[0545] Examples

[0546] The present invention will be more fully understood by reference to the following examples. However, the claims should not be interpreted as being limited to the scope of the examples.

[0547] Where the preparation examples are obtained as mixtures of enantiomers, the pure enantiomers can be separated by methods described herein or by methods known to those skilled in the art such as chiral chromatography (eg chiral SFC) or crystallization.

[0548] If not stated otherwise, all reaction examples and intermediates were prepared under argon atmosphere.

[0549] Example 1

[0550] 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

[0551]

[0552] Step a) tert-Butyl 5-(6-hydroxynicotinamido)pentanoate

[0553] 6-Hydroxynicotinic acid d (155 mg, 1.11 mmol, 1.0 equiv, CAS RN 5006-66-6), tert-butyl 5-aminovalerate (193 mg, 1.11 mmol, 1.0 equiv, CAS RN 63984-03-2), HOBt·H 2 O (213 mg, 1.39 mmol, 1.25 equiv), EDCI·HCl (267 mg, 1.39 mmol, 1.25 equiv) and Et 3 A solution of N (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 2 , dry loaded onto SiO 2 On the top, 5% MeOH in CH 2 Cl 2 The title compound (285 mg, 87%) was obtained as a white solid.

[0554] 1 H NMR (500MHz, CD 2 Cl 2 )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, CD 2 Cl2 )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 (pure, νmax / 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] + (Calculated value C 15 H 22 N 2 NaO 4 m / z=317.1472)

[0555] Step b) tert-Butyl 5-(1-((3-(but-3-yn-1-ylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)pentanoate

[0556] 5-(6-Hydroxynicotinamido)pentanoic acid tert-butyl ester (15.0 mg, 51 μmol, 1.0 equiv) was dissolved in anhydrous THF (0.50 mL) at rt and cooled to -78 °C. KO was added dropwise at -78 °C. t Bu (1M in THF, 102 μL, 102 μmol, 2.0 equiv) was added 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 RN 1344252-51-2) dissolved in anhydrous THF (0.30 mL) was added dropwise and the mixture was stirred at -78 °C for 2 h before being allowed to warm to rt. The solvent was removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was purified by flash column chromatography (SiO 2 , dry loaded on SiO 2 1–4% MeOH in CH 2 Cl 2 The obtained product was purified by centrifugation (middle) to afford the title compound (19.5 mg, 72%) as a light yellow waxy solid.

[0557] 1 H NMR (500MHz, CD 3OD)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). 13 CNMR(126MHz,CD 3 OD)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 (pure, νmax / 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] + (Calculated value C 26 H 31 N 3 NaO 7 S m / z=552.1775)

[0558] 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

[0559] 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 RN 2449214-44-0) 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) 4 ]PF 6 (21.1 mg, 56.7 μmol, 5.0 equiv). The mixture was dissolved in dry MeOH (0.11 mL) and CH 2 Cl 2 (0.11 mL) and the reaction mixture was capped and heated to 40 °C and stirred under argon for 16 h. The reaction mixture was directly loaded onto SiO 2 The crude product was analyzed by preparative TLC (SiO 2 , 6–10% MeOH in CH 2 Cl 2 The product was purified by centrifugation with 1% paraformaldehyde (HCl) and triturated with hexanes to afford the title compound as an orange solid (8.2 mg, 88%).

[0560] 1 H NMR (500MHz, CD 3 OD)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),8.1 5(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(dd,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). 13 C NMR (126MHz, CD 3OD)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 (pure, νmax / 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] + (Calculated value C 36 H 42 N 10 NaO 11 S m / z=845.2647)

[0561] 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

[0562] To 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) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at rt for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was then dissolved in dry DMF (50 μL). TFA (0.5 M solution in dry DMF, 30.5 μL, 15.2 μmol, 3.3 equiv) and i-Pr 2NEt (0.5 M solution in dry DMF, 49.0 μL, 24.4 μmol, 5.3 equiv) was added 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 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 dry CH 2 Cl 2 The reaction mixture was then allowed to warm to ambient temperature and stirred for a further 35 min. i-Pr was added at rt. 2 NEt (0.5 M solution in dry DMF, 2.3 μL, 1.1 μmol, 0.25 equiv) was added and the resulting solution was stirred for another 10 min. The residue was dissolved in CH 2 Cl 2 (10 mL) and saturated NH 4 Cl aqueous solution (5 mL), saturated NaHCO 3 The organic layer was washed with aqueous solution (5 mL) and brine (5 mL). 2 SO 4 Dry, filter and concentrate in vacuo. The crude product was purified by flash column chromatography (SiO 2 , 4–5% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to give the product as an orange solid (5.1 mg, 92%).

[0563] 1 H NMR (600MHz, CD 2 Cl 2)δ8.67(d, J = 2.4 Hz, 1H), 8.42(d, J = 8.6 Hz, 1H), 8.37(s, 1H), 8.19(d, J = 8.0 Hz, 1H), 8.09(d, J = 7.8 Hz, 1H), 7.79(dd, J = 9.7, 2.5 Hz, 1H), 7.64–7.56(m, 3H), 7.53(bs, 1H), 6.48(s, 2H), 6.33(d, J = 9.6 Hz, 1H), 6.21(d, J = 5.4 Hz, 1H), 5.80(bs, 1H), 5.59(dt, J = 3.1, 1.5 Hz, 1H), 4.51(t, J = 4.9 Hz, 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 = 7.0 Hz, 2H), 2.95(t, J = 6.4 Hz, 2H), 2.27(t, J = 7.0 Hz, 2H), 2.17–2.11(m, 1H), 2.06(t, J = 5.7 Hz, 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(151 MHz, CD 2 Cl 2 )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(pure, νmax / 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+Na 2 ] 2+ (Calculated value C 59 H 74 N 14 Na 2 O 12 S m / z=624.2558)[α] 25 D = +26.400 ± 0.321 (c = 0.23, CH 2 Cl 2 ).

[0564] Example 2

[0565] 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)methylamino)-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

[0566]

[0567] Step a) tert-Butyl 7-(6-hydroxynicotinamido)heptanoate

[0568] 6-Hydroxynicotinic acid (346 mg, 2.48 mmol, 1.0 equiv), tert-butyl 7-aminoheptanoate (500 mg, 2.48 mmol, 1.0 equiv, CAS RN 105974-64-9), HOBt·H 2 O (476 mg, 3.11 mmol, 1.25 equiv), EDCI·HCl (595 mg, 3.11 mmol, 1.25 equiv) and Et 3 A solution of N (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 2 , dry loaded on SiO 24% (EtOAc:EtOH, 3:1) in hexanes) to afford the title compound (540 mg, 67%) as a white solid.

[0569] 1 H NMR (500MHz, CD 2 Cl 2 )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, CD 2 Cl 2 )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 (pure, νmax / 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] + (Calculated value C 17 H 26 N 2 NaO 4 m / z=345.1785)

[0570] Step b) tert-Butyl 7-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)heptanoate

[0571] 7-(6-Hydroxynicotinamido)heptanoic acid tert-butyl ester (20.5 mg, 63.5 μmol, 1.0 equiv) was dissolved in anhydrous THF (0.50 mL) at rt and cooled to -78 °C. KO was added dropwise at -78 °C. tBu (1M in THF, 127 μL, 127 μmol, 2.0 equiv) was added 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 being allowed to warm to rt. The solvent was removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was purified by flash column chromatography (SiO 2 , dry loaded on SiO 2 4% (EtOAc:EtOH, 3:1) in hexanes) to give the title compound (23.5 mg, 68%) as a white waxy solid.

[0572] 1 H NMR (500MHz, CD 3 OD)δ8.80(dd,J=2.5,0.7Hz,1H),8.55(td,J=1.9,0.5Hz,1H),8.31(ddd,J=8.0,2.0,1.1H z,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). 13 C NMR (126MHz, CD 3 OD)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 (pure, νmax / 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] + (Calculated value C 27 H 33 N 3 NaO 7 S m / z=566.1931)

[0573] 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)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate

[0574] To 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 added [Cu(MeCN) 4 ]PF 6 (13.7 mg, 36.7 μmol, 5.0 equiv). The mixture was dissolved in CH 4 3 degassed with argon. 2 Cl 2 (0.30 mL) and AcOH (10 μL), capped and stirred under argon for 16 h. The reaction mixture was directly loaded onto SiO 2 The crude product was analyzed by preparative TLC (SiO 2 , 5% MeOH in CH 2 Cl 2 The title compound (5.3 mg, 86%) was obtained as an orange solid.

[0575] 1 H NMR (500MHz, CD 3OD)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,2 H),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, CD 3 OD)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 (pure, νmax / 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] + (Calculated value C 37 H 44 N 10 NaO 11 S m / z=859.2804)

[0576] 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)methylamino)-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)methylcarbamoyl To 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) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at rt for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was then dissolved in dry DMF (50 μL). TFA (0.5 M solution in dry DMF, 17.8 μL, 8.8 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 28.5 μL, 14.2 μmol, 5.3 equiv) was added 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 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 dry CH 2 Cl 2 , 29.6 μL, 2.9 μmol, 1.1 equiv). The reaction mixture was then warmed to ambient temperature and stirred for a further 45 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 4% MeOH in CH 2 Cl 2Purification of the product (in Chinese) gave the product as an orange solid (2.6 mg, 79%).

[0577] 1 H NMR (600MHz, CD 2 Cl 2 )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 (151 MHz, CD 2 Cl 2 )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 (pure, νmax / cm- 1):2959,2924,2854,2096,1649,1583,1457,1411,1377,1302,1260,1186,1092,1018. HRMS(ESI):m / z=1217.5570[M+H] + (Calculated value C 60 H 77 N 14 O 12 S m / z=1217.5561)

[0578] Example 3

[0579] 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

[0580]

[0581] 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)methylamino)-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)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide

[0582] To 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) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at rt for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2The residue was then dissolved in dry DMF (50 μL). TFA (0.5 M solution in dry DMF, 21.1 μL, 10.5 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 33.9 μL, 16.9 μmol, 5.3 equiv) was added 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 dry CH 2 Cl 2 The reaction mixture was then warmed to ambient temperature and stirred for a further 45 min. The residue was dissolved in CH 2 Cl 2 (10 mL) and saturated NH 4 Cl aqueous solution (5 mL), saturated NaHCO 3 The organic layer was washed with aqueous solution (5 mL) and brine (5 mL). 2 SO 4 Dry, filter and concentrate in vacuo. 2 , 4% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as an orange solid (2.7 mg, 72%).

[0583] 1 H NMR (500MHz, CD 2 Cl 2)δ8.61(dd, J = 2.5, 0.7 Hz, 1H), 8.46(d, J = 8.7 Hz, 1H), 8.41(t, J = 1.7 Hz, 1H), 8.25(ddd, J = 8.0, 1.9, 1.1 Hz, 1H), 8.12(ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 7.74(dd, J = 9.7, 2.5 Hz, 1H), 7.70(s, 1H), 7.63(td, J = 7.9, 0.5 Hz, 1H), 7.44(s, 1H), 6.71(t, J = 5.7 Hz, 1H), 6.49(s, 2H), 6.35(dd, J = 9.6, 0.7 Hz, 1H), 6.22(d, J = 8.7 Hz, 1H), 5.58(dt, J = 2.9, 1.5 Hz, 1H), 5.53–5.48(m, 1H), 4.65(d, J = 5.7 Hz, 2H), 4.57–4.49(m, 2H), 3.94(t, J = 2.4 Hz, 1H), 3.90(dd, J = 5.4, 4.5 Hz, 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 = 6.6 Hz, 2H), 2.24–1.94(m, 8H), 1.67(d, J = 8.4 Hz, 1H), 1.64–1.05(m, 16H), 1.26(s, 9H), 0.93(s, 3H), 0.85(t, J = 7.7 Hz, 3H). 13 C NMR(126 MHz, CD 2 Cl 2 )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(pure, νmax / 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]+ (Calculated value C 60 H 77 N 11 NaO 12 S m / z=1198.5366)

[0584] Example 4

[0585] 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

[0586]

[0587] 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)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate

[0588] A 50% flask was 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]. 4 PF 6 (15.3 mg, 41.1 μmol, 5.0 equiv) was added to a heart-shaped flask with dry CH 2 Cl 2 (0.3 mL, previously degassed with argon) and 20 μL of AcOH. The flask was then flushed with argon, stoppered with a glass stopper, and the resulting dark orange solution was stirred for 16 h. The reaction mixture was then loaded directly onto a silica plate and analyzed by preparative TLC (SiO 2 , 5% MeOH in CH 2 Cl 2The product was purified by centrifugation (middle) to give the product as an orange solid (7.2 mg, 90%).

[0589] 1 H NMR (500MHz, CD 3 OD)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,1H),8.01(s, 1H),7.86(dd,J=9.6,2.5Hz,1H),7.73(t,J=7.9Hz,1H),6.42(dd,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),3.48–3. 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, 7.0 Hz, 4H), 1.44 (s, 9H), 1.39 (tt, J = 5.2, 2.3 Hz, 4H). 13 C NMR (126MHz, CD 3 OD)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 (pure, νmax / 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] + (Calculated value C 43 H57 N 10 O 14 S m / z=969.3771)

[0590] 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)methylamino)-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)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide

[0591] To 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) was added to dry CH 2 Cl 2 To a solution of 4% paraformaldehyde (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 CH 2 Cl 2 The residue was then dissolved in dry DMF (0.1 mL). TFA (0.5 M solution in dry DMF, 49.0 μL, 24.5 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 78.8 μL, 39.4 μmol, 5.3 equiv) was added and the resulting light 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 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 dry CH 2 Cl 2The reaction mixture was then allowed to warm to ambient temperature and stirred for a further 30 min. Reaction control by LCMS indicated incomplete conversion and more i-Pr 2 NEt (0.5 M solution in dry DMF, 7.4 μL, 3.7 μmol, 0.5 equiv). The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 7% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as an orange solid (5.7 mg, 57%).

[0592] 1 H NMR (500MHz, CD 2 Cl 2)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)。 13 CNMR(126MHz,CD 2 Cl 2)δ=173.55,166.05,165.20,164.06,160.22,159.00,150.05,145.03,140.82,139.01,137.28,137.12,135.4 8,134.74,133.87,133.68,129.67,128.49,124.22,122.82,117.94,115.96,103.29,71.12,71.04,70.91,70 .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 (pure, νmax / 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] + (Calculated value C 66 H 89 N 14 O 15 S m / z=1349.6347)

[0593] Example 5

[0594] 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

[0595]

[0596] 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

[0597] In a glove box, to a flask loaded 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) were added [Cu(MeCN) 4 ]PF 6 (8.9 mg, 23.9 μmol, 5.0 equiv). The solid was dissolved in dry CH degassed with argon. 2 Cl 2 (0.30 mL) and AcOH (10 μL). The flask was then flushed with argon again, stoppered and stirred at rt for 16 h. The reaction mixture was then loaded directly onto the SiO 2 On-plate and by preparative TLC (SiO 2 , 15% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to obtain the product as a red solid (4.6 mg, 82%).

[0598] 1 H NMR (400MHz, CD 3OD, E / Z signal set reported as complete signal) δ = 9.20 (d, J = 1.8 Hz, 1H, Z), 9.06 (d, J = 1.9 Hz, 1H, E), 8.76 (dd, J = 2.5, 0.7 Hz, 1H), 8.57 (dd, J = 8.3, 1.8 Hz, 1H, E), 8.55 (s, 1H), 8.51 (dd, J = 8.3, 1.8 Hz, 1H, Z), 8.42 (d, J = 8.7 Hz, 1H, E), 8.29–8.22 (m, 1H), 8.19 (dt, J = 7.9, 1.3 Hz, 1H), 8.12 (s, 1H, E), 8.09 (d, J = 15.4 Hz, 1H, E), 7.96 (s, 1H), 7.91 (s, 1H, Z), 7.89 (d, J = 8.4 Hz, 1H, Z), 7.85 (dd, J = 9.7, 2.4 Hz, 1H), 7.79 (d, J = 15.3 Hz, 1H, E), 7.71 (t, J = 7.9 Hz, 1H), 7.48 (d, J = 9.1 Hz, 1H, E), 7.36 (d, J = 9.0 Hz, 1H, Z), 7.09 (dd, J = 12.3, 0.9 Hz, 1H, Z), 6.81 (dd, J = 9.0, 2.5 Hz, 1H, E), 6.79 (dd, J = 12.3, 0.9 Hz, 1H, Z), 6.72 (dd, J = 9.0, 2.5 Hz, 1H, Z), 6.52 (d, J = 2.4 Hz, 1H, E), 6.44 (d, J = 2.4 Hz, 1H, Z), 6.40 (dd, J = 9.6, 0.8 Hz, 1H), 4.69 (t, J = 7.8 Hz, 2H), 4.61 (s, 2H), 4.55 (t, J = 4.9 Hz, 2H), 3.86–3.78 (m, 2H), 3.55 (t, J = 7.2 Hz, 2H), 3.52–3.45 (m, 4H), 3.36–3.26 (m, 4H), 2.22 (t, J = 7.3 Hz, 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 = 7.1 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CD 3OD)δ=168.9,161.3,158.3,155.4,154.5,149.2,145.9,143.8,142.8,141.5,135.1,132.3,125.4,116.4,1 14.7,97.6,81.4,70.5,70.1,51.5,46.1,41.1,40.2,36.3,30.8,30.2,29.8,28.4,27.8,26.7,26.1,12.8.

[0599] IR(pure, νmax / 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] + (Calculated value C 57 H 72 N 9 O 14 S 2 m / z=1170.4635)

[0600] 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

[0601] To the mixture of 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) was added to dry CH 2 Cl 2To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the resulting dark red reaction mixture was stirred at rt for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was then dissolved in dry DMF (75 μL). TFA (0.5 M solution in dry DMF, 24.8 μL, 12.4 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 39.9 μL, 19.9 μmol, 5.3 equiv) was added and the resulting light 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 dry CH 2 Cl 2 , 41.4 μL, 4.1 μmol, 1.1 equiv). The reaction mixture was then allowed to warm to ambient temperature and stirred for a further 30 min. Reaction control by LCMS indicated incomplete conversion, and more i-Pr 2 NEt (0.5 M solution in dry DMF, 3.8 μL, 1.9 μmol, 0.5 equiv) was added and the mixture was stirred for another 5 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 10% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as a red solid (4.4 mg, 75%).

[0602] 1 H NMR (600MHz, CD 2 Cl 2,E / Z signal set reported as complete signal) 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, 3.3 Hz, 2H), 4.50–4.44 (m, 2H), 3.94–3.91 (m, 1H), 3.86–3.80 (m, 2H), 3.80–3.74 (m, 2H), 3.72 (s, 6H, Z), 3.71 (s, 6H, E), 3.57–3.51 (m, 2H), 3.48 (q, J = 7.2 Hz, 2H, E), 3.44 (q, J = 7.1 Hz, 2H, Z), 3.34 (dt, J = 12.3, 6.4 Hz, 2H), 3.29 (d, J = 5.6 Hz, 2H), 3.21 (t, J = 7.0 Hz, 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)。. 13 C NMR(151MHz,CD 2 Cl 2)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 ,139.3,139.1,137.2,137.1,136.7,135.6,135.5,135.4,135.3,135.1,134.9,133.2,133.2,131.7,131.3,130.7,130.5,130.3,129.4,128.5,128.3,127.4,127.3,125.0,124.9,124.8,124.1,123.9,122.5,118.1,118.0,117.7,1 16.0,115.9,114.4,114.0,110.8,110.4,109.5,108.7,103.3,97.4,97.3,69.7,69.6,69.6,69.4,66.0,59.1,58.7,56.3,52.1,50.8,50.8,48.0,45.8,45.6,44.9,44.9,44.8,44.8,44.7,41.2,40.5,40.4,40.1,38.5,38.0,37.6,3 7.3,37.1,36.3,36.1,36.1,36.0,32.5,31.7,31.2,30.6,30.3,30.3,30.1,29.9,29.9,29.8,29.8,29.7,29.5,29.4,29.3,28.1,27.7,27.7,27.3,27.2,27.1,26.6,26.4,26.2,26.1,26.0,25.1,25.0,24.9,23.3,14.4,12.8,12.8. IR (pure, νmax / 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+ (Calculated value C 80 H 105 N 13 O15 S 2 m / z=775.8642)

[0603] Example 6

[0604] 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]silanol-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

[0605]

[0606] 3,7-Di(azetidin-1-yl)-N-(2-(2-azidoethoxy)ethyl)-5,5-dimethyl-3′-oxo-3′H,5H-spiro[dibenzo[b,e]silanol-10,1′-isobenzofuran]-6′-carboxamide

[0607] To tert-butyl 3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silanol-10,1'-isobenzofuran]-6'-carboxylate (9.5 mg, 17.2 μmol, 1.0 equiv, prepared as described in Nat. Methods, 2020, 17, 815–821) was added in dry CH 2 Cl 2 To a solution of 4% ethyl acetate (200 μL) was added TFA (100 μL) and the resulting dark blue solution was stirred for 1 h. The solvent was then evaporated in vacuo and the residue was redissolved in CH 2 Cl 2 The crude product was then added with i-Pr 2 NEt (0.5 solution in DMF, 172 μL, 86.4 μmol, 5.0 equiv) was added and the resulting 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 CH 2 Cl 22-(2-Azidoethoxy)ethylamine (2.4 mg, 5.3 μmol, 1.0 equiv, CAS RN 464190-91-8) was added to 4% paraformaldehyde (0.2 mL) and the mixture was allowed to warm to ambient temperature and stirred for a further 30 min. The reaction mixture was then washed with saturated NaHCO 3 Aqueous solution (5 mL) and CH 2 Cl 2 (10 mL) and the phases were separated. The aqueous phase was diluted with CH 2 Cl 2 (2 × 10 mL) and extracted with Na 2 SO 4 Dry and concentrate in vacuo. 2 , 3% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as a blue solid (6.0 mg, 57%).

[0608] 1 H NMR (500MHz, CD 2 Cl 2 )δ=7.96(dd,J=8.0,0.8Hz,1H),7.89(dd,J=8.0,1.4Hz,1H),7.60(dd,J=1.4,0.8 Hz,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=4.8,3.6,1.3 Hz, 4H), 3.37–3.32 (m, 2H), 2.36 (tt, J = 7.8, 6.8Hz, 4H), 0.62 (s, 3H), 0.54 (s, 3H). 13 C NMR (126MHz, CD 2 Cl 2 )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 (pure, νmax / 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] + (Calculated value C 33 H 37 N 6 O 4 Si m / z=609.2640)

[0609] 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]silanol-10,1′-isobenzofuran]-6′-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate

[0610] A precipitate containing 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'H,5H-spiro[dibenzo[b,e]silanol-10,1'-isobenzofuran]-6'-carboxamide (1.5 mg, 2.5 μmol, 1.0 equiv) and Cu[MeCN] was added. 4 PF 6 (4.6 mg, 12.3 μmol, 5.0 equiv) was added to a heart-shaped flask with dry CH 2 Cl 2 (0.3 mL, previously degassed with argon) and 10 μL of AcOH. The flask was then flushed with argon, stoppered with a glass stopper, and the resulting dark blue solution was stirred for 16 h. The reaction mixture was then loaded directly onto a silica plate and analyzed by preparative TLC (SiO 2 , 6% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to obtain the product as a blue solid (1.4 mg, 49%).

[0611] 1 H NMR (500MHz, CD 3OD) 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 C NMR (126 MHz, CD 3 OD) 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, νmax / cm- 1 ) 3310, 2928, 2856, 1756, 1654, 1594, 1545, 1454, 1367, 1306, 1247, 1189, 1151, 1058. HRMS (ESI): m / z = 1152.4678 [M + H] + (calcd for C 60 H 70 N 9 O 11 SSi m / z = 1152.4679)

[0612] 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]silanol-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

[0613] To 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]silanol-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) was added to dry CH 2 Cl 2 To a solution of 50 μL (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 CH 2 Cl 2 The residue was then dissolved in dry DMF (0.1 mL). TFA (0.3 M solution in dry DMF, 38.2 μL, 11.5 μmol μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 43.7 μL, 21.9 μmol, 6.3 equiv) was added and the resulting light 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 (0.05 M solution in dry CH 2 Cl 2The reaction mixture was then allowed to warm to ambient temperature and stirred for a further 30 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 10% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as a blue solid (2.2 mg, 41%).

[0614] 1 H NMR (600 MHz, CDCl 3 )δ=8.66(s,1H),8.50(s,1H),8.37(d,J=8.3Hz,1H),8.12(d,J=7.7Hz,1H),8.01(d ,J=7.9Hz,1H),7.93(d,J=7.9Hz,1H),7.74(s,2H),7.67(s,1H),7.62(t,J=7.9Hz, 1H),6.78(d,J=8.7Hz,2H),6.64(d,J=2.6Hz,2H),6.47(s,2H),6.35(d,J=9.6Hz,1 H),6.24(dd,J=8.7,2.7Hz,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 .9Hz,2H),3.32–3.27(m,1H),3.22(t,J=7.0Hz,1H),2.36(p,J=15.8,7.5Hz,4H),2. 22(t,J=9.8Hz,2H),2.19–2.14(m,1H),2.07–2.04(m,1H),2.01(q,J=6.3Hz,2H),1 .96(s,1H),1.77–1.46(m,9H),1.38–1.16(m,17H),1.16–0.99(m,2H),0.94(s,3H). 13 C NMR (151 MHz, CDCl 3)δ=175.43,158.39,127.92,115.82,113.94,112.50,102.69,55.81,52.29,51.46,47.23,44.42,37.11,35.91,31.93,27.18,25.52,22.69,14.13. IR (pure, νmax / cm- 1 )3358,2923,2853,2096,1742,1658,1634,1595,1537,1467,1411,1377,1259,1187,1092,1016. HRMS(ESI):m / z=1532.7252[M+H] + (Calculated value C 83 H 102 N 13 O 12 SSi m / z=1532.7255)

[0615] Example 7

[0616] 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

[0617]

[0618] Step a) tert-Butyl 9-(6-hydroxynicotinamido)nonanoate

[0619] 6-Hydroxynicotinic acid (91.0 mg, 654 μmol, 1.0 equiv), tert-butyl 9-aminononanoate (150 mg, 654 μmol, 1.0 equiv, CAS RN 134857-22-0), HOBt·H 2 O (125 mg, 818 μmol, 1.25 equiv), EDCI·HCl (157 mg, 818 μmol, 1.25 equiv) and Et 3 A solution of N (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 (SiO 2, dry loaded on SiO 2 4% (0.1% 4% dextrin, 50% (EtOAc:EtOH, 3:1) in hexanes) to afford the title compound (162 mg, 71%) as a white solid.

[0620] 1 H NMR (400MHz, CD 2 Cl 2 )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). 13 C NMR (101 MHz, CD 2 Cl 2 )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 (pure, νmax / 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] + (Calculated value C 19 H 30 N 2 NaO 4 m / z=373.2098)

[0621] Step b) tert-Butyl 9-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)nonanoate

[0622] 9-(6-Hydroxynicotinamido)nonanoic acid tert-butyl ester (20.0 mg, 57.0 μmol, 1.0 equiv) was dissolved in anhydrous THF (0.50 mL) at rt with sonication and cooled to -78 °C. KO was added dropwise at -78 °C. tBu (1M in THF, 114 μL, 114 μmol, 2.0 equiv) was added 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 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 being allowed to warm to rt. The solvent was removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was purified by flash column chromatography (SiO 2 , dry loaded on SiO 2 4% (EtOAc:EtOH, 3:1) in hexanes) to afford the title compound (23 mg, 70%) as a white waxy solid.

[0623] 1 H NMR (400MHz, CD 3 OD)δ8.80(dd,J=2.5,0.7Hz,1H),8.55(td,J=1.9,0.6Hz,1H),8.31(ddd,J=8.0,2.0,1.1H z,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 (101 MHz, CD 3 OD)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 (pure, νmax / 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] + (Calculated value C 29 H 37 N 3 NaO 7 S m / z=594.2244)

[0624] 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)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)nonanoate

[0625] 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) was added [Cu(MeCN) 4 ]PF 6 (22.2 mg, 59.4 μmol, 5.0 equiv). The mixture was dissolved in MeOH (0.12 mL) and CH 2 Cl 2 (0.12 mL) and the reaction mixture was heated to 40 °C and stirred under argon for 16 h. The reaction mixture was directly loaded onto SiO 2 The crude product was analyzed by preparative TLC (SiO 2 , 2–6% MeOH in CH 2 Cl 2 The product was purified by centrifugation (centigrade) and triturated with hexanes to afford the title compound as an orange solid (7.5 mg, 73%).

[0626] 1 H NMR (400MHz, CD 3OD)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=7.9,0.5Hz,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,2H),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). 13 C NMR (101 MHz, CD 3 OD)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 (pure, νmax / 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] + (Calculated value C 39 H 49 N 10 O 11 S m / z=865.3296)

[0627] 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)methylamino)-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)methylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide

[0628] To 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) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at rt for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was then dissolved in dry DMF (50 μL). TFA (0.5 M solution in dry DMF, 22.9 μL, 11.4 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 36.8 μL, 18.3 μmol, 5.3 equiv) was added 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 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 dry CH 2 Cl 2 , 38.2 μL, 3.8 μmol, 1.1 equiv). The reaction mixture was then warmed to ambient temperature and stirred for a further 45 min. The residue was dissolved in CH 2 Cl 2 (10 mL) and saturated NH 4 Cl aqueous solution (5 mL), saturated NaHCO 3 The organic layer was washed with aqueous solution (5 mL) and brine (5 mL).2 SO 4 Dry, filter and concentrate in vacuo. The crude product was purified by flash column chromatography (SiO 2 , 4% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to give the product as an orange solid (3.5 mg, 81%).

[0629] 1 H NMR (500MHz, CD 2 Cl 2 )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),1.63–1.5 0(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 CNMR(126MHz,CD 2 Cl 2)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 (pure, νmax / cm- 1 )3322,2924,2853,2096,1651,1534,1463,1411,1377,1302,1189,1123. HRMS(ESI):m / z=645.2793[M+Na 2 ] 2+ (Calculated value C 62 H 80 N 14 Na 2 O 12 S m / z=645.2793)

[0630] Example 8

[0631] 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-nitrobenz[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide

[0632]

[0633] Step a) tert-Butyl 7-(4-(N-(pyridin-2-ylmethylsulfamoyl)benzamido)heptanoate

[0634] 4-Chlorosulfonylbenzoyl chloride (855 mg, 3.56 mmol, 3.75 equiv, CAS RN 7516-60-1) was dissolved in dry CH 2 Cl 2 The solution in 2% ethanol (30 mL) was cooled to 0°C and Et 3N (1.04 mL, 5.96 mmol, 6.25 equiv), followed by dropwise addition of 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 material was purified by flash column chromatography (SiO 2 , 10% EtOAc in CHCl 3 The sulfonyl chloride was used immediately in the subsequent step.

[0635] tert-Butyl 7-(4-(chlorosulfonyl)benzamido)heptanoate (260 mg, 644 μmol, 1.0 equiv) was dissolved in dry dioxane (1.5 mL). Pyridin-2-ylmethylamine (73 μL, 708 μmol, 1.1 equiv) and i-Pr 2 NEt (170 μL, 966 μmol, 1.5 equiv) was added and the solution was stirred at rt overnight. The mixture was washed with EtOAc (50 mL) and saturated NH 4 The mixture was diluted with aqueous Cl (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) and MgSO 4 Dry, filter and concentrate in vacuo. The crude product was purified by flash column chromatography (SiO 2 , 70% EtOAc in hexanes) to give the title compound as an amorphous solid (232 mg, 76%).

[0636] 1 H NMR (500MHz, CD 3 Cl)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, CD 3Cl)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 (pure, νmax / cm- 1 ):3289,2931,2859,1726,1645,1597,1543,1439,1393,1367,1334,1152,1094. HRMS(ESI):m / z=498.2034[M+Na] + (Calculated value C 24 H 33 N 3 NaO 5 S m / z=498.2033)

[0637] Step b) tert-Butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethylsulfamoyl)benzoylamino)heptanoate

[0638] 6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoic acid (14.5 mg, 44.4 μmol, 1.25 equiv, CAS RN 88235-25-0), EDCI·HCl (14.0 mg, 73.0 μmol, 2.00 equiv), i-Pr 2 NEt (18.6 μL, 106 μmol, 3.00 equiv) and DMAP (5.4 mg, 44.4 μmol, 1.25 equiv) were dissolved in dry DMF (200 μL) and the mixture was stirred at rt 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 in dry DMF (100 μL) and the reaction mixture was stirred at rt overnight. The mixture was heated in CH 2 Cl 2 (20 mL) and saturated NH 4 The layers were separated and the aqueous phase was washed with CH 2 Cl 2 (2 × 20 mL). The combined organic layers were washed with MgSO 4 Dry, filter and concentrate in vacuo. The crude product was purified by flash column chromatography (SiO 2, 70% EtOAc in hexanes) to give the title compound as an orange solid (6.1 mg, 23%).

[0639] 1 H NMR (500MHz, CD 2 Cl 2 )δ8.49–8.44(m,2H),7.93–7.87(m,2H),7.84–7.80(m,2H),7.73(td,J=7.7,1.8Hz,1H), 7.36(d,J=7.8Hz,1H),7.23(ddd,J=7.6,4.9,1.1Hz,1H),6.50(bs,1H),6.29(t,J=5.7Hz ,1H),6.16(d,J=8.7Hz,1H),5.19(s,2H),3.50–3.37(m,4H),2.61(t,J=7.1Hz,2H),2.19 (t,J=7.4Hz,2H),1.75–1.65(m,2H),1.64–1.52(m,6H),1.42(s,9H),1.40–1.26(m,6H). 13 C NMR (126MHz, CD 2 Cl 2 )δ173.54,173.34,166.00,156.01,149.72,144.86,144.50,142.08,140.15,137.36,137.10,129.16,127.56,123.21,122.06,99.07,80.15,51.10,44.04,40.52,35.88,35.75,29.71,29.01,28.41,28.23,26.94,26.35,25.32,23.99. IR (pure, νmax / 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] + (Calculated value C 36 H 45 N 7 NaO 9 S m / z=774.2892)

[0640] 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)methylamino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethylsulfamoyl)benzamide

[0641] Tert-butyl 7-(4-(N-(6-((7-nitrobenz[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 CH 2 Cl 2 (200 μL) and TFA (100 μL) were added to a mixture and the solution was stirred at rt for 2 h. The volatiles were removed in vacuo and the residue was dissolved in dry DMF (0.1 mL) and cooled to 0°C. Subsequently, TFA (0.5 M in DMF, 69 μL, 34.6 μmol, 4.5 equiv), i-Pr 2 A stock solution of NEt (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) was prepared 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 (3.5 mg, 7.6 μmol, 1.0 equiv) was dissolved in dry CH 2 Cl 2 (50 μL) was added and the reaction mixture was warmed to rt and stirred for 45 min. The crude mixture was concentrated in vacuo and analyzed by preparative TLC (SiO 2 , 95% EtOAc in hexanes) and triturated with hexanes to give the title compound as an orange solid (6.2 mg, 71%).

[0642] 1 H NMR 1H NMR (500MHz, CD 2 Cl 2)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, CD 2 Cl 2 )δ173.48,172.89,165.92,158.91,155.65,149.92,140.37,139.09,137.09,129 .05,127.86,123.91,117.92,103.22,56.13,51.93,47.87,44.85,44.64,44.09,4 1.11,40.40,38.36,37.92,36.94,36.02,32.36,30.25,30.12,29.58,29.23,29.14,28.98,28.44,27.98,27.02,26.79,26.46,26.40,25.98,25.02,24.08,21.17. IR (pure, νmax / 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] + (Calculated value C 59 H 77 N 11 NaO 10S m / z=1154.5468)

[0643] Example 9

[0644] 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

[0645]

[0646] 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)methylamino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethylsulfamoyl)benzamide

[0647] Tert-butyl 7-(4-(N-(6-((7-nitrobenz[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 CH 2 Cl 2 (200 μL) and TFA (100 μL) were added to a mixture and the solution was stirred at rt for 2 h. The volatiles were removed in vacuo and the residue was dissolved in dry DMF (0.1 mL) and cooled to 0°C. Subsequently, TFA (0.5 M in DMF, 35.9 μL, 17.9 μmol, 4.5 equiv), i-Pr 2 NEt (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 prepared as stock solutions and the mixture was stirred at 0 °C for 15 min. ((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) was dissolved in dry CH 2 Cl 2(50 μL) was added and the reaction mixture was warmed to rt and stirred for 45 min. Additional i-Pr 2 NEt (0.5 M in DMF, 10 μL, 5.0 μmol, 1.25 equiv) was added and the reaction mixture was stirred at rt for 1.5 h. The crude mixture was concentrated in vacuo and analyzed by preparative TLC (SiO 2 , 95% EtOAc in hexanes) and triturated with hexanes to give the title compound as an orange solid (4.2 mg, 96%).

[0648] 1 H NMR 1H NMR (500MHz, CD 2 Cl 2 )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,4 H),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,3 H), 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, CD 2 Cl 2)δ173.51,172.87,165.96,158.83,156.04,150.03,149.74,142.07,140.14,139.01,137.34 ,137.10,129.12,127.62,123.90,123.19,122.05,117.77,103.17,56.09,51.11,47.81,44.7 8,44.71,44.61,44.04,41.07,40.35,38.34,37.87,36.92,35.88,32.18,30.43,30.10,29.55,29.16,28.94,28.42,27.94,26.75,26.42,26.36,25.95,25.08,24.00,23.07,21.14,14.25. IR (pure, νmax / 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] + (Calculated value C 59 H 78 N 8 NaO 10 S m / z=1113.5454)

[0649] Example 10

[0650] 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

[0651]

[0652] Step a) tert-Butyl 7-(4-sulfamoylbenzamido)heptanoate

[0653] To a solution of 4-sulfamoylbenzoic acid (550 mg, 2.73 mmol, 1.0 equiv, CAS RN 138-41-0) in dry DMF (11.0 mL) were added tert-butyl 7-aminoheptanoate (688 mg, 2.73 mmol, 1.0 equiv, CAS RN 105974-64-9), HOBt·H2 O (628 mg, 4.10 mmol, 1.5 equiv), EDCI·HCl (786 mg, 4.10 mmol, 1.5 equiv) and i-Pr 2 NEt (1.43 mL, 8.20 mmol, 3.0 equiv). The solution was stirred at rt for 16 h. The mixture was washed with EtOAc (100 mL) and saturated NaHCO 3 The mixture was diluted with aqueous solution (50 mL). The layers were separated and the aqueous phase was extracted with EtOAc (2×100 mL). The combined organic phases were washed with brine (30 mL) and MgSO 4 Dry, filter and concentrate in vacuo. The crude product was purified by flash column chromatography (SiO 2 , 4–6% MeOH in CH 2 Cl 2 The obtained product was purified by centrifugation (middle) to give the title compound as a white solid (700 mg, 67%).

[0654] 1 H NMR (400MHz, CD 3 OD)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 (101 MHz, CD 3 OD)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 (pure, νmax / cm- 1 ):3321,2926,2854,1730,1629,1559,1456,1367,1342,1166,1151,1097. HRMS(ESI):m / z=407.1605[M+Na] + (Calculated value C 18 H 28 N 2 NaO 5 S m / z=407.1611)

[0655] Step b) tert-Butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamido)heptanoate

[0656] 6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoic acid (112 mg, 343 μmol, 1.1 equiv, CAS RN 88235-25-0), EDCI·HCl (90.0 mg, 468 μmol, 1.5 equiv), i-Pr 2 NEt (272 μL, 1.56 mmol, 5.0 equiv) and DMAP (11.4 mg, 93.6 μmol, 0.3 equiv) were dissolved in dry DMF (1.2 mL) and the mixture was stirred at rt 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 rt overnight. The mixture was concentrated in vacuo. The crude product was purified by flash column chromatography (1% AcOH, 1-4% MeOH in CH 2 Cl 2 ) to give the title compound as an orange amorphous solid which was dissolved in CHCl 3 Unreacted sulfonamide starting material (62 mg) was also recovered.

[0657] 1 H NMR (400MHz, CD 3 OD)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 (101 MHz, CD 3 OD)δ175.05,173.63,168.34,146.50,145.74,145.43,143.20,140.67,138.49,129.34,128.81,122.86,99.61,81.34,44.48,41.13,36.69,36.30,30.17,29.77,28.34,27.71,27.19,26.07,25.09. IR (pure, νmax / 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] + (Calculated value C 30 H 40 N 6 NaO 9 S m / z=683.2470)

[0658] Step c) tert-Butyl 7-(4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamido)heptanoate

[0659] 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 dry DMF (0.1 mL) was added iodoacetonitrile (8 μL, 106 μmol, 10 equiv) and i-Pr 2 NEt (0.5 M in DMF, 106 μL, 53.0 μmol, 5.0 equiv). The mixture was stirred at rt overnight. The mixture was concentrated in vacuo and the crude product was analyzed by preparative TLC (SiO 2 , 70% EtOAc in hexanes) and triturated with hexanes to give the title compound as an orange solid (7.3 mg, 98%).

[0660] 1 H NMR (500MHz, CD 2 Cl 2 )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,1H),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, CD 2 Cl 2)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 (pure, νmax / 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] + (Calculated value C 32 H 41 N 7 NaO 9 Sm / z=722.2579)

[0661] 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)methylamino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamide

[0662] 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 CH 2 Cl 2 (200 μL) and TFA (100 μL) were added to a mixture and the solution was stirred at rt for 2 h. The volatiles were removed in vacuo and the residue was dissolved in dry DMF (0.1 mL) and cooled to 0°C. Subsequently, TFA (0.5 M in DMF, 46 μL, 23.2 μmol, 4.5 equiv), i-Pr 2A stock solution of NEt (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) was prepared 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 (2.4 mg, 5.1 μmol, 1.0 equiv) was dissolved in dry CH 2 Cl 2 (50 μL) was added and the reaction mixture was warmed to rt and stirred for 45 min. The crude mixture was concentrated in vacuo and analyzed by preparative TLC (SiO 2 , 95% EtOAc in hexanes) and triturated with hexanes to give the title compound as an orange solid (3.2 mg, 58%).

[0663] 1 H NMR (500MHz, CD 2 Cl 2 )δ8.48(d,J=8.7Hz,1H),8.06–7.99(m,4H),6.61(bs,1H),6.56(bt,J=5.7Hz,1H),6.49(s,2H),6.18(d,J=8.7Hz, 1H),5.58(dt,J=2.8,1.5Hz,1H),5.46(bt,J=5.7Hz,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=7.0Hz,2H),2.70(t,J=7.1Hz,2H),2.23–2.12(m,3H),2.10–2.04(m,1H),2.0 4–1.95(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 C NMR (126MHz, CD 2 Cl 2)δ173.03,172.08,165.64,158.84,149.90,141.40,140.67,138.96,137.11,128 .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 (pure, νmax / 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] + (Calculated value C 55 H 74 N 11 O 10 S m / z=1080.5335)

[0664] Example 11

[0665] 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

[0666]

[0667] Step g) tert-Butyl 7-(4-(N-(pent-4-ynyl)sulfamoyl)benzamido)heptanoate

[0668] Pent-4-ynoic acid (60.3 mg, 614 μmol, 1.05 equiv, CAS RN 6089-09-4), EDCI·HCl (168 mg, 878 μmol, 1.5 equiv), i-Pr 2NEt (510 μL, 2.93 mmol, 5.0 equiv) and DMAP (21.6 mg, 176 μmol, 0.30 equiv) were dissolved in dry DMF (1.3 mL) and the mixture was stirred at rt for 15 min. Subsequently, tert-butyl 7-(4-sulfamoylbenzamido)heptanoate (225 mg, 585 μmol, 1.0 equiv) was added in dry DMF (1.0 mL) and the reaction mixture was stirred at rt overnight. The mixture was heated in CH 2 Cl 2 (60 mL) and saturated NH 4 The layers were separated and the aqueous phase was washed with CH 2 Cl 2 (2 × 60 mL). The combined organic layers were washed with MgSO 4 Dry, filter and concentrate in vacuo. The crude product was purified by flash column chromatography (SiO 2 , 0.5% AcOH, 0–4% MeOH in CH 2 Cl 2 The reaction mixture was purified by centrifugation (centrifugation step (200 mL)) to give the title compound as a white solid (190 mg, 70%). Unreacted sulfonamide starting material (52 mg) was also recovered.

[0669] 1 H NMR (400MHz, CD 3 OD)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,2H),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, CD 3 OD)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 (pure, νmax / cm- 1):3254,2928,2856,2510,1712,1635,1570,1480,1464,1434,1415,1351,1172. HRMS(ESI):m / z=487.1862[M+Na] + (Calculated value C 23 H 32 N 2 NaO 6 S m / z=487.1873)

[0670] Step h) tert-Butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynyl)sulfamoyl)benzamido)heptanoate

[0671] To a solution of tert-butyl 7-(4-(N-(pent-4-ynyl)sulfamoyl)benzamido)heptanoate (75.0 mg, 161 μmol, 1.0 equiv) in dry DMF (0.25 ml) was added i-Pr 2 NEt (141 μL, 807 μmol, 5.0 equiv) and iodoacetonitrile (117 μL, 1.61 mmol, 10 equiv). After 10 min, the transparent reaction mixture turned brown. It was further stirred at rt for 16 h. The reaction mixture was then concentrated in vacuo and redissolved in CH 2 Cl 2 The precipitate was neutralized and adsorbed on silica gel. 2 , 30% EtOAc in hexanes) gave the product as a light yellow wax (63.5 mg, 78%).

[0672] 1 H NMR (500MHz, CD 2 Cl 2 )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=7.1Hz,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, CD 2 Cl 2)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 (pure, νmax / cm- 1 )3300,2934,2861,1718,1649,1542,1487,1367,1293,1169,1154,1088,1070,1001. HRMS(ESI):m / z=526.1978[M+Na] + (Calculated value C 25 H 33 N 3 NaO 6 S m / z=526.1982)

[0673] 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

[0674] A 50% slurry was charged with tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynyl)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 RN 2449214-44-0) and Cu[MeCN] 4 PF 6 (21.1 mg, 56.6 μmol, 5.0 equiv) was added to a heart-shaped flask with dry CH 2 Cl 2 (0.3 mL, previously 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 analyzed by preparative TLC (SiO 2 ,8% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to give the product as an orange solid (3.3 mg, 37%).

[0675] 1 H NMR (500MHz, CD 2 Cl 2)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,2H),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 CNMR(126MHz,CD 2 Cl 2 )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 (pure, νmax / 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] + (Calculated value C 35 H 44 N 10 NaO 10 S m / z=819.2855)

[0676] 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)methylamino)-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

[0677] To 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) was added to dry CH 2 Cl 2 To a solution of 4% paraformaldehyde (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 CH 2 Cl 2 The residue was then dissolved in dry DMF (0.1 mL). TFA (0.5 M solution in dry DMF, 27.3 μL, 13.7 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 43.9 μL, 30.0 μmol, 5.3 equiv) was added and the resulting light 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 dry CH 2 Cl 2 The reaction mixture was then allowed to warm to ambient temperature and stirred for a further 30 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 7% MeOH in CH 2 Cl 2 Purification of 4-(2-(4-(2-hydroxy-1-yl)-4-nitropropene)-2-nitropropene ...

[0678] 1 H NMR (500MHz, CD 2 Cl 2)δ=8.46(d,J=8.5Hz,1H),8.04–7.87(m,4H),7.71(s,1H),6.83(s,1H),6.49(s,2H),6.23(d,J=8.7Hz,1H),5.91(s,1H),5.62(s,1H),4.73(b s,2H),4.58(bs,2H),3.96(bs,1H),3.92(bs,2H),3.85(d,J=14.2Hz,2 H),3.79(s,2H),3.72(s,6H),3.70–3.66(m,2H),3.43(d,J=6.4Hz,2H), 3.22(t,J=6.9Hz,2H),3.14(td,J=7.5,4.0Hz,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=4.6Hz,1 H),1.68(d,J=8.4Hz,1H),1.61–1.52(m,9H),1.44–1.36(m,4H),1.31– 1.20(m,4H),1.28(s,3H),1.26(s,6H),1.14–1.07(m,2H),0.94(s,3H). 13 C NMR (126MHz, CD 2 Cl 2 )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(pure,νmax / cm- 1 )3325,2929,2864,2097,1717,1635,1574,1541,1449,1410,1364,1302,1172,1122,1030. HRMS(ESI):m / z=1199.5422[M+Na] + (Calculated value C 58 H 76 N 14 NaO 11 S m / z=1199.5431)

[0679] Example 12

[0680] 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

[0681]

[0682] 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

[0683] In a glove box, [Cu(MeCN)] was added to a heart-shaped flask charged with tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynyl)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). 4 PF 6 ](5.9 mg, 16.0 μmol, 5.0 equiv). The solid was dissolved in dry argon-degassed CH 2 Cl 2 (300 μL) and AcOH (10 μL). The flask was then flushed with argon, stoppered, and the purple solution was stirred at rt for 16 h. The reaction mixture was then loaded directly onto a silica plate and analyzed by preparative TLC (SiO 2 , 15% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to obtain the product as a red solid (3.4 mg, 94%).

[0684] 1 H NMR (500MHz, CD3 OD, E / Z signal set reported as complete signal) δ = 9.17 (dd, J = 1.8, 0.6 Hz, 1H, Z), 9.02 (dd, J = 1.9, 0.5 Hz, 1H, E), 8.58 (dd, J = 8.7, 1.9 Hz, 1H, E), 8.51 (dd, J = 8.3, 1.8 Hz, 1H, Z), 8.44 (d, J = 8.6 Hz, 1H, E), 8.15 (s, 1H, E), 8.11 (d, J = 15.3 Hz, 1H, E), 8.07 (d, J = 8.8 Hz, 2H), 8.02–7.98 (m, 2H), 7.94 (s, 1H, Z), 7.89 (d, J = 8.4 Hz, 1H, Z), 7.81 (d, J = 15.5 Hz, 1H, E), 7.65 (s, 1H), 7.51 (d, J = 9.1 Hz, 1H, E), 7.40 (d, J = 9.1 Hz, 1H, Z), 7.10 (dd, J = 12.3, 0.9 Hz, 1H, Z), 6.83 (dd, J = 9.0, 2.5 Hz, 1H, E), 6.79 (d, J = 12.3 Hz, 1H, Z), 6.75 (dd, J = 9.0, 2.5 Hz, 1H, Z), 6.57 (dd, J = 2.4, 0.6 Hz, 1H, E), 6.47 (d, J = 2.4 Hz, 1H, Z), 4.74–4.63 (m, 2H), 4.48 (dd, J = 5.5, 4.4 Hz, 2H), 3.79 (dd, J = 5.6, 4.5 Hz, 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, 2H), 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 = 7.1 Hz, 6H, E), 1.22–1.18 (m, 6H, Z). 13 CNMR(126MHz,CD 3OD)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(pure, νmax / 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] + (Calculated value C 55 H 71 N 9 NaO 13 S 2 m / z=1152.4505)

[0685] 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)methylamino)-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

[0686] To 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) was added to dry CH 2 Cl 2To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the resulting purple reaction mixture was stirred for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2 The residue was then dissolved in dry DMF (50 μL). TFA (0.5 M solution in dry DMF, 14.6 μL, 7.2 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 23.4 μL, 11.7 μmol, 5.3 equiv) was added and the resulting colorless solution was cooled to 0°C. HATU (0.1 M solution in DMF, 24.3 μL, 2.4 μmol, 1.1 equiv) was then added and the reaction was stirred at 0°C for 15 min. 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 dry CH 2 Cl 2 The reaction mixture was warmed to ambient temperature and stirred for a further 30 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 7% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as a red solid (2.2 mg, 66%).

[0687] HRMS(ESI):m / z=755.8670[M+2H] 2+ (Calculated value C 78 H 105 N 13 O 14 S 2 m / z= 755.8667)

[0689] Example 13

[0690] 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]silanol-10,1'-isobenzofuran]-6'-carboxamide

[0691]

[0692] 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]silanol-10,1′-isobenzofuran]-6′-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamido)heptanoate

[0693] A precipitate containing tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynyl)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]silabenzene-10,1'-isobenzofuran]-6'-carboxamide (2.5 mg, 4.1 μmol, 1.0 equiv) and Cu[MeCN] was added. 4 PF 6 (7.7 mg, 20.5 μmol, 5.0 equiv) was added to a heart-shaped flask with dry CH 2 Cl 2 (0.3 mL, previously 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 analyzed by preparative TLC (SiO 2 , 3% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to obtain the product as a blue solid (3.1 mg, 68%).

[0694] 1 H NMR (500MHz, CD 2Cl 2 )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=10.3Hz,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). 13 C NMR (126MHz, CD 2 Cl 2 )δ=171.90,145.87,140.47,129.06,128.31,122.83(HMBC),122.37(HMBC),86.61,80.50,80.27,69.88,69.51,69.46,50.51,43.32,40.73,40.44,40.31,36.08,35.93,33.93,30.26,29.79,29.21,28.40,27.17,25.50,20.96,17.29(HSQC),14.44(HSQC),9.07,-1.07. IR(pure,νmax / cm- 1 )3344,2926,2855,1755,1722,1654,1594,1546,1480,1365,1291,1235,1159,1091. HRMS(ESI):m / z=1134.4562[M+Na] + (Calculated value C 58 H 69 N 9 NaO 10 SSim / z=1134.4550)

[0695] 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]silanol-10,1′-isobenzofuran]-6′-carboxamide

[0696] To 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]silanol-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) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (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 CH 2 Cl 2 The residue was then dissolved in dry DMF (0.1 mL). TFA (0.5 M solution in dry DMF, 18.4 μL, 9.2 μmol, 3.3 equiv) and i-Pr 2 NEt (0.5 M solution in dry DMF, 35.1 μL, 17.6 μmol, 6.3 equiv) was 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 dry CH 2 Cl 2 Monitoring the reaction by LCMS showed incomplete conversion, and additional i-Pr 2NEt (0.5 M solution in dry 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 a further 30 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH 2 Cl 2 and loaded directly onto silica plates. 2 , 0.5% AcOH, 7% MeOH in CH 2 Cl 2 The product was purified by HPLC (2.1 mg, 38%).

[0697] HRMS(ESI):m / z=1492.7302[M+H] + (Calculated value C 81 H 102 N 13 O 11 SSi m / z=1492.7306)

[0699] Example 14

[0700] 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-ynyl)sulfamoyl)benzamide

[0701]

[0702] 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)methylamino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynyl)sulfamoyl)benzamide

[0703] Tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynyl)sulfamoyl)benzamido)heptanoate (2.4 mg, 4.8 μmol, 1.1 equiv) was dissolved in CH 2 Cl 2(200 μL) and TFA (100 μL) were added to a mixture and the solution was stirred at rt for 1 h. The volatiles were removed in vacuo and the residue was dissolved in dry DMF (50 μL) and cooled to 0°C. Subsequently, TFA (0.5 M in DMF, 29 μL, 14.5 μmol, 3.3 equiv), i-Pr 2 A stock solution of NEt (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) was prepared 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) was dissolved in dry CH 2 Cl 2 (50 μL) was added and the reaction mixture was warmed to rt and stirred for 10 min. The crude mixture was concentrated in vacuo and analyzed by preparative TLC (SiO 2 , 85% EtOAc in hexanes) to give the title compound (2.0 mg, 51%) as a colorless solid.

[0704] 1 H NMR 1H NMR (500MHz, CD 2 Cl 2 )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),4.74(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=7.4,6.8Hz,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=2.7Hz,1 H),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, CD2 Cl 2 )δ173.04(HMBC),170.81(HMBC),165.51,159.01,150.04,140.50,139.17,129.0 7,128.51,124.03,117.98,115.08,103.30,82.37,69.71,56.26,52.06,47.97,4 4.95,44.77,41.23,40.53,38.49,38.04,37.02,36.07,33.93,32.50,30.39,30.26,29.62,29.36,29.28,28.98,28.11,27.16,26.81,26.58,26.06,25.15,21.30. IR (pure, νmax / cm- 1 ):3309,2960,2920,2851,2094,1715,1648,1571,1464. HRMS(ESI):m / z=884.4736[M+H] + (Calculated value C 48 H 66 N 7 O 7 S m / z=884.4739)

[0705] Example 15

[0706] 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

[0707]

[0708] Step b) tert-Butyl 7-(4-(N-(4-((tert-butoxycarbonyl)amino)butyryl)sulfamoyl)benzamido)heptanoate

[0709] To a solution of tert-butyl 7-(4-sulfamoylbenzamido)heptanoate (115 mg, 299 μmol, 1.0 equiv) in dry DMF (1.3 ml) were added Boc-GABA-OH (76 mg, 374 μmol, 1.25 equiv, CAS RN 57294-38-9), EDCI·HCl (86 mg, 449 μmol, 1.5 equiv), i-Pr 2NEt (260 μL, 1.50 mmol, 5.0 equiv), DMAP (11 mg, 90.1 μmol, 0.3 equiv) were added and the solution was stirred at rt overnight. 4 The solution was diluted with aqueous Cl (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) and purified by MgSO 4 Dry, filter and concentrate in vacuo. The crude material was purified by flash column chromatography (SiO 2 , 1% AcOH, 2–5% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to give the product as a white solid (140 mg, 82%).

[0710] 1 H NMR (400MHz, CD 3 OD)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=6.9H z,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 (101 MHz, CD 3 OD)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 (pure, νmax / cm- 1 ):3366,3110,2978,2934,2866,1729,1213,1678,1635,1515,1444,1357. HRMS(ESI):m / z=592.2661[M+Na] + (Calculated value C 27 H 43 N 3 NaO 8 S m / z=592.2663)

[0711] Step e) 7-(4-(N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butyryl)sulfamoyl)benzamido)heptanoic acid

[0712] To tert-butyl 7-(4-(N-(4-((tert-butoxycarbonyl)amino)butyryl)sulfamoyl)benzamido)heptanoate (38.0 mg, 66.7 μmol, 1.0 equiv) was added to CH 2 Cl 2 To a solution of 1% 4-nitropropene (200 μl) was added TFA (200 μl) and the reaction was stirred at rt for 1 h. The volatiles were removed in vacuo and residual TFA was removed by azeotropy with toluene (3×3 mL). The crude material was dissolved in dry DMF (0.2 mL) and i-Pr 2 NEt (58.1 μL, 334 μmol, 5.0 equiv) was added followed by 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 rt 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 washed with MgSO 4 Dry, filter and concentrate in vacuo. The crude material was purified by flash column chromatography (1% AcOH, 5-10% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to give the product as a white solid (31 mg, 85%).

[0713] 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). 13 C NMR (126MHz, MeOD) δ 179.39, 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, νmax / cm- 1):3096,2935,2861,2504,1711,1632,1571,1459,1346,1178. HRMS(ESI):m / z=494.1951[M+H] + (Calculated value C 23 H 32 N 3 O 7 S m / z=494.1955)

[0714] 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

[0715] 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-Pr 2 NEt (3.1 μL, 17 μmol, 4.0 equiv) was added to anhydrous CH 2 Cl 2 ((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 4% paraformaldehyde (25 μL). The reaction mixture was stirred at rt for 1 h and concentrated in vacuo. The crude product was analyzed by preparative TLC (SiO 2 , 1% AcOH, 6% MeOH in CH 2 Cl 2 The intermediate was crudely purified to give a colorless waxy solid, which was directly redissolved in DMF (50 μL). 2-iodoacetonitrile (2.0 μL, 27 μmol, 6.3 equiv) and i-Pr 2 NEt (2.3 μL, 13 μmol, 3.0 equiv) was added and the light yellow mixture was stirred at rt overnight. The volatiles were removed in vacuo and the crude material was analyzed by preparative TLC (SiO 2 , 5–7% MeOH in CH 2 Cl2 The final product was purified by centrifugation (middle) to give the final compound as a white amorphous solid (2.4 mg, 56%).

[0716] 1 H NMR (600MHz, CD 2 Cl 2 )δ8.06–7.99(m,2H),7.99–7.91(m,2H),7.12(t,J=5.2Hz,1H),6.49(s,2H),6.46–6.39(m,1H),5.61–5.55(m,1H),5.48–5.40(m,2 H),4.77(s,2H),3.95(t,J=2.4Hz,1H),3.87–3.74(m,2H),3.72(s,6H),3.43–3.38(m,2H),3.22(t,J=7.0Hz,2H),3.05(q,J=6.6Hz, 2H),2.65(td,J=7.1,0.9Hz,2H),2.21–2.14(m,3H),2.13(d,J=1.3Hz,3H),2.10–2.05(m,1H),2.02(tt,J=6.0,1.8Hz,1H),1.90(d ,J=1.6Hz,1H),1.71–1.48(m,9H),1.45–1.35(m,4H),1.34–1.22(m,6H),1.28(s,3H),1.26(s,6H),1.13–1.07(m,2H),0.95(s,3H). 13 C NMR (151 MHz, CD 2 Cl 2 )δ176.72,173.04,172.43,166.39,159.02,150.04,142.17,140.40,139.21,129.21,128.6 4,128.40,128.02,124.01,118.01,115.45,114.59,103.31,96.58,56.26,52.06,47.98,44 .95,44.77,40.55,38.85,38.49,38.04,37.09,34.09,33.77,30.39,30.22,29.69,29.37,29.28,29.19,28.11,27.16,27.02,26.59,26.16,25.29,25.15,22.86,21.31,14.45,10.95. IR (pure, νmax / cm- 1)3301,3074,2923,2852,2095,1717,1642,1605,1571,1544,1463,1410,1365. HRMS(ESI):m / z=969.5260[M+H] + (Calculated value C 52 H 73 N 8 O 8 S m / z=969.5267)

[0717] Example 16

[0718] 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)heptylamide

[0719]

[0720] Step a) tert-Butyl 7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanoate

[0721] 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 min. Subsequently, 4-fluoro-7-nitrobenzo[c][1,2,5]selenadiazole (32.0 mg, 130 μmol, 1.5 equiv, CAS RN 2351940-09-3) was added, the solution was warmed to rt and stirred overnight. The reaction mixture was stirred overnight by adding saturated NH 4 The mixture was quenched with aqueous Cl (15 mL) and then with CH 2 Cl 2 (3 × 30 mL). The combined organic extracts were washed with MgSO 4 Dry, filter and concentrate in vacuo. The crude oil was purified by flash column chromatography (SiO 2 , 2% MeOH in CH 2 Cl 2 The product was purified by centrifugation (middle) to give the product as a yellow solid (11.5 mg, 31%).

[0722] 1 H NMR (500 MHz, CDCl 3)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). 13 C NMR (126 MHz, CDCl 3 )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 (pure, νmax / cm- 1 ):2932,2859,1727,1610,1532,1511,1463,1406,1392,1320,1249. HRMS(ESI):m / z=452.0698[M+Na] + (Calculated value C 17 H 23 N 3 NaO 5 Se m / z=452.0695)

[0723] 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)heptylamide

[0724] tert-Butyl 7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanoate (2.9 mg, 6.8 μmol, 1.0 equiv) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (200 μL) was added TFA (100 μL) and the resulting dark red reaction mixture was stirred at rt for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CH 2 Cl 2 To the residue dissolved in anhydrous DMF (100 μL) was added anhydrous i-Pr 2 NEt (2.9 μL, 16.5 μmol, 3.0 equiv), HATU (2.6 mg, 6.8 μmol, 1.25 eq.) and the reaction mixture was stirred at rt for 15 min. 2 Cl2 To the mixture in 100 μL of 2% paraformaldehyde (100 μL) was added ((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) and the reaction mixture was stirred at rt for 45 min and then washed with saturated NaHCO 3 The aqueous layer was quenched with CH 2 Cl 2 (3 x 15 mL) and the combined organic extracts were washed with brine (10 mL). The organic layer was washed with Na 2 SO 4 Dry, filter and concentrate in vacuo. The crude product was analyzed by preparative TLC (SiO 2 , 2% MeOH in CH 2 Cl 2 The title product (3.0 mg, 67%) was obtained as a bright yellow solid.

[0725] 1 H NMR (500MHz, CD 2 Cl 2 )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, CD 2 Cl 2)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 (pure, νmax / cm- 1 )3309,2927,2856,2094,1649,1607,1571,1523,1463,1410,1324,1294,1239. HRMS(ESI):m / z=832.3280[M+Na] + (Calculated value C 40 H 55 N 7 NaO 6 Se m / z=832.3275)

[0726] Example 17

[0727] 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

[0728]

[0729] Step a) 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanoic acid

[0730] To 8-thioheptanoic acid (150 mg, 851 μmol, 1.0 equiv, CAS RN 74328-61-3) 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 RN 10199-89-0) followed by pyridine (343 μL, 4.26 mmol, 5.0 equiv). The mixture gradually turned dark and was vigorously stirred at rt 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 purified by MgSO 4Dry, filter and concentrate in vacuo. The crude product was adsorbed onto silica and purified by flash column chromatography (SiO 2 , 30-50% EtOAc, 1% AcOH in heptane) to give the product as a cloudy brown solid (182 mg, 63%).

[0731] 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) δ 174.52, 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, νmax / cm- 1 ):3031,2936,2853,1697,1615,1513,1433,1364,1334,1303. HRMS(ESI):m / z=340.0969[M+H] + (Calculated value C 14 H 18 N 3 O 5 S m / z=340.0962)

[0732] 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

[0733] 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-Pr 2 NEt (3.5 μL, 20 μmol, 3.0 equiv) was added and the reaction mixture was stirred at 0 °C for 10 min, then added to anhydrous CH 2 Cl 2((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 50 μL. The ice bath was removed and the reaction mixture was stirred at rt for 1 h, concentrated in vacuo and analyzed by preparative TLC (SiO 2 , 70% EtOAc in heptane) to give the final product as an orange solid (1.9 mg, 37%).

[0734] 1 H NMR (500MHz, CD 2 Cl 2 )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=5.7H z,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=7.0 Hz,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,2H),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). 13 C NMR (126MHz, CD 2 Cl 2 )δ172.25,158.44,149.47,138.66,131.04,123.35,120.44,117.42,102.71,55.66,51.48,47.40,44.37,44.15,40.65,37.91,37.45,36.66,31.70,29.81,29.68,28.79,28.77,28.70,28.64,27.75,27.51,26.58,26.00,25.64,24.57,20.71. IR (pure, νmax / cm- 1 )3316,2929,2857,2094,1725,1648,1571,1522,1508,1411,1362,1328. HRMS(ESI):m / z=798.3969[M+Na] + (Calculated value C41 H 57 N 7 NaO 6 Sm / z=798.3983)

[0735] Example 18

[0736] 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

[0737]

[0738] Step c) 8-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanoic acid

[0739] At 0 °C, 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanoic acid (35 mg, 108 μmol, 1.0 equiv) was added to dry CH 2 Cl 2 The solution in (0.5 mL) was added dropwise with mCPBA in dry CH 2 Cl 2 (0.15 M, 1.08 mL, 161 μmol, 1.5 equiv). The resulting mixture was allowed to reach rt and stirred for 2 h. The mixture was directly purified by flash column chromatography (30-40% EtOAc, 1% AcOH in heptane) to give the product as a turbid green solid (22.8 mg, 62%).

[0740] 1 H NMR (400MHz, CD 2 Cl 2 )δ8.64(d,J=7.5Hz,1H),8.13(d,J=7.5Hz,1H),3.38(ddd,J=13.5,9.6,6.0Hz,1H),3.12(d dd,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 (101 MHz, CD 2 Cl 2)δ178.69,146.08,143.38,142.97,137.92,129.67,128.25,54.12,33.62,28.65,28.59,28.18,24.43,21.63. IR (pure, νmax / cm- 1 ):3096,2930,2859,1706,1548,1534,1464,1407,1368,1340. HRMS(ESI):m / z=378.0730[M+Na] + (Calculated value C 14 H 17 N 3 NaO 6 S m / z=378.0730)

[0741] 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

[0742] 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-Pr 2 NEt (2.9 μL, 17 μmol, 3.0 equiv) was added and the reaction mixture was stirred at 0 °C for 10 min before being added to anhydrous CH 2 Cl 2 ((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 50 μL. The ice bath was removed and the reaction mixture was stirred at rt for 30 min, concentrated in vacuo and analyzed by preparative TLC (SiO 2 , 80% EtOAc in heptane) to give the final product as a brown solid (1.5 mg, 35%).

[0743] 1 H NMR (500MHz, CD 2 Cl 2)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,1 H),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),1.6 5–1.44(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, CD 2 Cl 2 )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(pure,νmax / cm- 1 )2929,2856,2095,1725,1649,1604,1571,1543,1365,1336,1121. HRMS(ESI):m / z=792.4101[M+H] + (Calculated value C 41 H 58 N 7 O 7 S m / z=792.4113)

[0744] Example 19

[0745] 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]silanol-10,1′-isobenzofuran]-6′-yl)oxy)heptylamide

[0746]

[0747] 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]silanol-10,1′-isobenzofuran]-6′-yl)oxy)heptanoate

[0748] To a solution of tert-butyl 7-hydroxyheptanoate (1.7 mg, 8.3 μmol, 1.1 equiv, CAS RN 86013-78-7) in THF (0.1 mL) was added NaH (60% dispersion in mineral oil, 0.4 mg, 8.3 μmol, 1.1 equiv) at 0° C. to produce a grey slurry. Then, 3,7-di(azetidin-1-yl)-4′,5′,6′,7′-tetrafluoro-5,5-dimethyl-3′H,5H-spiro[dibenzo[b,e]silabenzene-10,1′-isobenzofuran]-3′-one (4.0 mg, 7.6 μmol, 1.0 equiv, prepared as described in Nat. Methods, 2020, 17, 815–821) in THF (0.1 mL) was added and the reaction mixture was stirred at 0° C. for 1 h. The mixture was washed with water (5 mL) and CH 2 Cl 2 (5 mL), the phases were separated and the aqueous phase was concentrated into CH 2 Cl 2 (3×5 mL) extraction. The combined organic layers were purified by Na 2 SO 4 Dry and concentrate in vacuo. 2 , 7% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) afforded the product as a light blue solid (3.9 mg, 5.5 μmol, 72% yield).

[0749] 1 H NMR (500MHz, CD 2 Cl 2)δ=6.74(dd,J=8.6,1.2Hz,2H),6.67(dd,J=2.7,0.5Hz,2H),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),2.4 2–2.34(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, CD 2 Cl 2 )δ=173.43(HMBC),143.53(HMBC),130.48(HMBC),127.86,116.39,112.94,80. 20,76.99,52.92,35.99,30.42,29.30,28.39,25.83,25.56,17.39,1.33,0.60. 19 F NMR (471MHz, CD 2 Cl 2 )δ=-143.02(dd,J=21.9,2.8Hz),-147.22(t,J=19.5Hz),-147.76(d,J=17.6Hz). (Decoupled)IR(pure,νmax / cm- 1 )2930,2854,1768,1728,1595,1484,1405,1365,1306,1265,1164,1103,1005. HRMS(ESI):m / z=707.3123[M+H] + (Calculated value C 39 H 46 F 3 N 2 O 5 Si m / z=707.3123)

[0750] 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]silanol-10,1′-isobenzofuran]-6′-yl)oxy)heptylamide

[0751] To 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]silanol-10,1'-isobenzofuran]-6'-yl)oxy)heptanoate (2.0 mg, 2.8 μmol, 1.0 equiv) was added to dry CH 2 Cl 2 To a solution of 1% ethyl acetate (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 CH 2 CH 2 The crude product was then added with i-Pr 2 NEt (0.5 M solution in DMF, 34.0 μL, 16.9 μmol, 6.0 equiv) was added and the resulting light blue solution was cooled to 0° C. Then, HATU (0.1 M solution in DMF, 34.0 μL, 3.4 μmol, 1.2 equiv) was 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 dry CH 2 Cl 2 The mixture was warmed to ambient temperature and stirred for a further 30 min. The reaction mixture was then saturated with NaHCO 3 (5 mL) aqueous solution and CH 2 Cl 2 The phases were separated and the aqueous phase was diluted with CH 2 Cl 2 (3 × 5 mL) and extracted with Na 2 SO 4 Dry and concentrate in vacuo. 2 , 2% MeOH in CH 2 Cl 2 Purification of the product (in Chinese) gave the product as a bluish solid (0.9 mg, 29%).

[0752] HRMS(ESI):m / z=1087.5695[M+H] + (Calculated value C 62 H 78 F 3 N 6 O 6 Si m / z=1087.5699)

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: (i)R 1 for and X is selected from O, S and SO; or (ii) R 1 for 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 atom to which they are attached, they form a C3-C5-cycloalkyl group; R 4 is selected from hydrogen, halogen, cyano and azide; R 5 Selected from: R 6 Selected from: R 7 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; Y is selected from Se, S and O.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: (i)R 1 for and X is selected from O, S and SO; or (ii) R 1 for 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 atom to which they are attached they form a C3-C5-cycloalkyl group; R 4 is selected from hydrogen, halogen, cyano and azide; R 5 Selected from: R 6 Selected from: R 7 Selected from: R 8 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; Y is selected from Se, S and O.

3. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: (i)R 1 for and X is selected from O, S and SO; or (ii) R 1 for and X is NHCO; R 5 Selected from: R 6 Selected from: R 7 Selected from: R 8 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; Y is selected from Se and O.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 3, wherein: (i)R 1 for and X is O; or (ii) R 1 for and X is NHCO; R 5 Selected from: R 6 Selected from: R 7 Selected from: R 8 Selected from: p is 3; q is selected from 2, 3 and 5; r is 1; s is 1; and u is 5.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 4, wherein: R 1 for R 5 for R 6 for R 7 for R 8 for X is NHCO; p is 3; q is 2 or 3; r is 1; and u is 5.

6. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: R 2 and R 3 is selected from hydrogen and C1-C4-alkyl; R 4 is hydrogen or azide; and n is 6.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 6, wherein: R 2 and R 3 Both are C1-C4-alkyl; R 4 is azide; and n is 6.

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

9. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: (i)R 1 for and X is selected from O, S and SO; or (ii) R 1 for 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 Selected from: R 6 Selected from: R 7 Selected from: R 8 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; Y is selected from Se and O.

10. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 9, wherein: (i)R 1 for and X is O; or (ii) R 1 for and X is NHCO; R 2 and R 3 Both are C1-C4-alkyl; R 4 is azide; R 5 Selected from: R 6 Selected from: R 7 Selected from: R 8 Selected from: n is 6; p is 3; q is selected from 2, 3 and 5; r is 1; s is 1; and u is 5.

11. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 10, wherein: R 1 for R 2 and R 3 Both are methyl; R 4 is azide; R 5 for R 6 for R 7 for R 8 for X is NHCO; n is 6; p is 3; q is 2 or 3; r is 1; and u is 5.

12. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) is selected from: 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-nitrobenz[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-nitrobenz[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]silanol-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)heptylamide; 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]silanol-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-ynyl)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]silanol-10,1'-isobenzofuran]-6'-yl)oxy)heptylamide; 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-azaeicosyl)-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)-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.

13. A method for preparing a compound of formula (I) according to claim 1, comprising: (a) Make alkyne C or I Where R 6 , p, q and r are as defined in claim 1, reacted with the azide-substituted fluorophore M Among them, W, R 7 , s and t as defined in claim 1; To form compound D or K Among them, W, R 6 , R 7 , p, q, r and s as defined in claim 1; After that (b) Removal of the t-Bu protecting group from compound D or K using an acid such as HCl or TFA to give compound D1 or K1 Among them, W, R 6 , R 7 , p, q, r and s as defined in claim 1; After that (c) reacting compound D1 or K1 with amine N1, Where R 2 , R 3 and R 4 As defined in claim 1; The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system; To obtain the compound of formula (I).

14. A compound selected from: Where R 2 , R 3 , R 4 , R 6 , R 7 , p, q, r, s, t as defined in claim 1, and LG is 15. A method for preparing an amide of formula 1 Where R x and R y Both are chemical moieties attached to a parent amide functional group through a carbon atom; and Where R x Contains N-sulfonylpyridone (SP) motif i, or N-acyl-N-alkylsulfonamide (NASA) motif ii Where R 6 As defined in claim 1; The method comprises: Make carboxylic acid 2 Reaction with amine 3 The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system; To obtain the amide 1.

16. A method for preparing a fluorescent probe P1 or P2 for a protein of interest, where LG is the ligand for the protein of interest, and W, R 6 , R 7 , p, q, r and s are as defined herein, the method comprising: (a) Make alkyne C or I Where R 6 , p, q and r are as defined in claim 1, reacted with the azide-substituted fluorophore M Among them, W, R 7 , s and t as defined in claim 1; To form compound D or K Among them, W, R 6 , R 7 , p, q, r and s as defined in claim 1; After that (b) removing the t-Bu protecting group from compound D or K using an acid such as HCl or TFA, To obtain compound D1 or K1 Among them, W, R 6 , R 7 , p, q, r and s as defined in claim 1; After that (c) reacting compound D1 or K1 with an amino-substituted ligand N for the protein of interest, where LG is the ligand for the protein of interest, The reaction is carried out in the presence of a coupling reagent and an acid / base buffer system; To obtain the fluorescent probe P1 or P2 for the protein of interest.

17. A compound of formula (I) according to any one of claims 1 to 12 for use as a fluorescent probe for cannabinoid receptor 2 (CB2R).

18. Use of a compound of formula (I) according to any one of claims 1 to 12 as a fluorescent probe for cannabinoid receptor 2 (CB2R).

19. A method of imaging cannabinoid receptor 2 (CB2R), the method comprising contacting the cannabinoid receptor 2 (CB2R) with a compound of formula (I) according to any one of claims 1 to 12.

20. The invention as hereinbefore described.

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