Thiocycloheptane derivatives and uses thereof

Abstract

The present invention relates to novel thiocycloheptyne derivatives of general formula (I), and specifically to thioheterocyclic alkyne sulfonimide derivatives and their synthesis. The present invention also relates to the use of novel thiocycloheptyne derivatives in coupling reactions with linkers and drugs. The present invention also relates to the use of novel thiocycloheptyne in bioorthogonal (copper-free) click reactions. The present invention also relates to the use of novel thiocycloheptyne derivatives in the production of advanced multifunctional drug delivery systems (drug-loaded) nanoparticles. #imgabs0#

Description

Field of the Invention

[0001] The present invention relates to novel thiocycloheptyne derivatives, in particular thioheterocyclic alkynyl sulfonimide derivatives and their synthesis. The present invention also relates to the use of novel thiocycloheptyne derivatives in various coupling reactions, such as coupling reactions involving linkers, drugs and drug delivery systems, proteins, imaging agents, dyes, chromophores, ligands, etc. The present invention also relates to the use of novel thiocycloheptyne in copper-free click reactions. The present invention also relates to the use of novel thiocycloheptyne derivatives in the production of various systems, such as targeted and / or labeled delivery systems, such as nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates, drug polymer conjugates, etc. Background of the Invention

[0003] Bioorthogonal chemistry is used for the study of biomolecules and physiological processes. Bioorthogonal chemistry can be used when other more conventional research tools or medical treatments are unavailable or insufficient. Typically, the protocol begins by labeling target biomolecules in cells or living organisms with bioorthogonal functional groups. Probe molecules with complementary functions are provided to the system, and the bioorthogonal chemical reaction delivers the probe specifically to the target of interest. Kinetic optimization and yield optimization (e.g., by making purification easier) are considered key factors for the further development of this useful method. In the past, strain-promoted cycloaddition reactions of azides and cyclooctynes ​​have been found to be well-tolerated bioorthogonal reactions. Strain-promoted cycloaddition reactions of azides and cyclooctynes, also known as Cu-free (or copper-free) click chemistry, were inspired by the classic work of Krebs and Kimling in the 1970s. They observed that cyclooctynes ​​react easily with the same substrate at room temperature, compared to unactivated straight-chain alkynes that undergo 1,3-dipolar cycloadditions with azides only at elevated temperatures. The increased reactivity of cyclooctyne is attributed to the ring strain generated by the bond angle deformation of the alkyne.

[0004] Almeida et al. have described a group of cyclooctynes ​​that have been developed and used in bioorthogonal coupling reactions over the years. Almeida studied the effect of intracyclic sulfur on the activity of cyclooctynes ​​and synthesized many thiocycloalkynes. It was found that 3,3,6,6-tetramethylthioheptyne (TMTH) was more reactive in cycloaddition reactions than previous cyclooctyne compounds. King et al. (Chem Commun 2102, 48, 9308) described the reasonable stability of TMTH only when it was derivatized with benzyl bromide at the sulfur atom. It was also found that other derivatives of TMTH were difficult to synthesize, and sulfur was difficult to further derivatize. Dommerholt et al. (NATURE COMMUNICATIONS 2014, 5, 5378, Top Curr Chem (Z) 2016, 374: 16) showed that TMTH had poor stability and could not be isolated into a pure form. Li et al. (Molecules, 2016, 21, 1393) showed that TMTH is the fastest SPAAC reported to date, but cannot be equipped with labels which hinders its application in bioorthogonal reactions.

[0005] To exploit the advantages of TMTH in copper-free click reactions, more compounds for copper-free click reactions that are novel, readily available, and reactive and at least as reactive in 1,3-dipolar cycloadditions with azides, nitrones, and other 1,3 dipoles are needed. Summary of the invention

[0006] The present inventors have discovered a novel class of sulfur-containing cycloalkynes, in particular thiocycloheptynes. The cycloalkynes of the present invention form a novel class of compounds which can be used for a variety of purposes, one of which is that they are reactive in copper-free click chemistry.

[0007] In a first aspect, the present invention relates to compounds of formula (I)

[0008]

[0009] in:

[0010] n and m are independently 0, 1 or 2, and n+m is 2;

[0011] X is O or NR 9 ,

[0012] Y is NR 10 ,

[0013] R 1 , R 2 , R 3 , R 4 independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl, wherein O, N, P and S are further independently hydrogen, halogen (F, Cl, Br, I), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 The alkyl radicals are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the alkyl radicals, (hetero)aryl radicals, alkyl (hetero)aryl radicals and (hetero)arylalkyl radicals are independently optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkyloxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkyloxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkyloxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the silyl group is represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy and C3-C 12 Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S;

[0014] R5 , R 6 , R 7 , R 8 independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl, wherein O, N, P and S are further independently hydrogen, halogen (F, Cl, Br, I), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 The alkyl radicals are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the alkyl radicals, (hetero)aryl radicals, alkyl (hetero)aryl radicals and (hetero)arylalkyl radicals are independently optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkyloxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkyloxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkyloxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the silyl group is represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy and C3-C 12Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S;

[0015] Wherein, optionally, R 1 and R 7 , R 1 With R 8 , R 2 With R 7 , R 2 With R 8 , R 3 With R 5 , R 3 With R 6 , R 4 With R 5 and / or R 4 With R 6 independently form fused ring systems, such as cycloalkyl-, cyclo(hetero)aryl-, cycloalkyl(hetero)aryl, -cyclo(hetero)arylalkyl systems,

[0016] wherein the alkyl group of the fused ring system is optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups of the fused ring system are independently optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkyloxy, halogen, amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkyloxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkyloxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the silyl group is represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12Alkynyloxy and C3-C 12 Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S;

[0017] R 9 , R 10 are independently selected from hydrogen, halogen (F, Cl, Br, I), R 12 、-CH=C(R 12 2. -C≡CR 12 、-[C(R 12 )2C(R 12 )2O] q -R 12 (where q is 1 to 200), -CN, -N3, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12 、-OS(O)2R 12 、-OS(O)OR 12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12 )C(X)XR 12 and -N(R12 )C(X)N(R 12 )2, wherein X is oxygen or sulfur, and wherein R 12 independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl.

[0018] In a second aspect, the present invention relates to a process for preparing a compound of formula (I), said process comprising the following steps:

[0019] a. converting the dihydrazone (2) into the iminodihydrosulfonylimino group (3);

[0020] b. Isolate the obtained iminodihydrosulfonylimino group (3).

[0021] In a third aspect, the present invention relates to compounds, wherein X and / or Y and / or an atom adjacent to the S atom in the thiocycloheptyne ring of the compound of formula (I) are independently coupled with an optional linking group (L) and a functional group (Q) to produce a compound of formula (III):

[0022] (Formula I)-LQ

[0023] (III)

[0024] Wherein the linking group (L) is absent or selected from a linear or branched C1-C 24 Alkylene, C2-C 24 Alkenylene, C2-C 24 Alkynylidene, C3-C 24 Cycloalkylene, C5-C 24 Cycloalkenylene, C5-C 24 Cycloalkynylene, C7-C 24 Alkyl (hetero) arylene, C7-C 24 (Hetero)aryl alkylene, C5-C 24 (Hetero)arylalkenylene, C9-C 24 (Hetero)arylalkynylene, alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl(hetero)arylene, (hetero)arylalkylene, (hetero)arylalkenylene and (hetero)arylalkynylene, which are optionally independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 Cycloalkynyl, C8-C12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl are substituted with one or more substituents, wherein the silyl group can be represented by the formula (R 11 )3Si- represents, where R 11 As defined above;

[0025] The functional group Q is selected from hydrogen, halogen (F, Cl, Br, I), R 12 、-CH=C(R 12 2. -C≡CR 12 、-[C(R 12 )2C(R 12 )2O] q -R 12 (where q is 1 to 200), -CN, -N3, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12 、-OS(O)2R 12 、-OS(O)OR 12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12)C(X)XR 12 and -N(R 12 )C(X)N(R 12 )2, wherein X is oxygen or sulfur, and wherein R 12 independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl.

[0026] In another aspect, the present invention provides a compound comprising a compound of formula (I) according to the present invention coupled at an atom X, Y and / or an atom adjacent to the S atom in the thiocycloheptyne ring, preferably Y, with an optional linking group (L) and a functional group (Q) to produce a compound of formula (III):

[0027] (Formula I)-LQ

[0028] (III)

[0029] wherein L and Q are as defined herein above.

[0030] In another aspect, the present invention relates to a method for coupling a compound of formula (I) or formula (III) with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene, preferably wherein the compound comprising a 1,3-dipole or a 1,3-(hetero)diene is an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, preferably an azide-containing compound. The coupling of the compound of the present invention with an azide generally produces a triazole. The compound comprising a 1,3-dipole or a 1,3-(hetero)diene preferably comprises a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle and / or a carrier.

[0031] In another aspect, the present invention relates to compounds comprising a compound of formula (III) as defined herein, wherein the functional group Q reacts with a target molecule, preferably the target molecule is selected from drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles and carriers.

[0032] In another aspect, the present invention relates to methods for preparing various systems, such as targeted and / or labeled delivery systems, such as nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates, drug-polymer conjugates, etc., wherein a (functionalized) compound of formula (I) or formula (III) is coupled to an azide.

[0033] In another aspect, the present invention relates to the use of a compound of formula (I) or formula (III) in a method for coupling two target molecules, more specifically, the use of a targeted and / or labeled delivery system (such as nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates) to one or more of drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles and carriers using a copper-free click reaction.

[0034] Other aspects include methods for preparing a construct comprising a nanoparticle and an active compound selected from the group consisting of a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, and a carrier, wherein the coupling of the nanoparticle and the active compound comprises coupling of a compound of Formula I or Formula III with an azide-containing compound to form a triazole compound.

[0035] On the other hand, the present invention relates to the use of a compound according to the present invention in a method for coupling two target molecules, wherein optionally, the molecules are independently selected from drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles and carriers. Preferably, one of the target molecules contains a cycloalkyne according to the present invention, and the other target molecule is a compound containing a 1,3-dipole or a 1,3-(hetero)diene, preferably a compound containing an azide, a nitrone-containing compound or a nitrile oxide-containing compound, more preferably an azide-containing compound. The compound containing a 1,3-dipole or a 1,3-(hetero)diene preferably includes a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle and / or a carrier.

[0036] In another aspect, the invention relates to compounds of formula (I) as defined herein, wherein the alkynyl group is coupled to a compound comprising a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably, the compound comprising a 1,3-dipole or a 1,3-(hetero)diene is an azide-containing compound, a nitrone-containing compound or a nitrile-oxide-containing compound, more preferably an azide-containing compound, and the azide-alkyne coupling preferably results in the formation of a triazole compound. The compound comprising a 1,3-dipole or a 1,3-(hetero)diene preferably comprises a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle and / or a carrier.

[0037] In another aspect, the invention relates to a compound comprising a compound of formula (I) as defined herein, coupled with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene, wherein the alkynyl group of the thiocycloheptyne of formula (I) is coupled with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene, wherein preferably, the compound comprising a 1,3-dipole or a 1,3-(hetero)diene is an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, more preferably an azide-containing compound, and the azide-alkyne coupling preferably results in the formation of a triazole compound. The compound comprising a 1,3-dipole or a 1,3-(hetero)diene preferably comprises a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle and / or a carrier.

[0038] In another aspect, the present invention relates to the use of compounds according to the invention in bioorthogonal, optionally copper-free click reactions. Preferably, the compounds according to the invention are coupled with compounds containing 1,3-dipole or 1,3-(hetero)diene, preferably azide-containing compounds, nitrone-containing compounds or nitrile oxide-containing compounds, more preferably azide-containing compounds. The compounds containing 1,3-dipole or 1,3-(hetero)diene preferably include drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles and / or carriers.

[0039] In another aspect, the present invention relates to the use of a compound according to the present invention in a method for coupling nanoparticles to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle and a carrier using a copper-free click reaction. Preferably, the nanoparticle comprises a cycloalkyne compound according to the present invention, and the drug, antibody, protein, peptide, ligand, imaging marker, targeting ligand, delivery agent, nanoparticle or carrier comprises a 1,3-dipole or a 1,3-(hetero)diene, preferably containing an azide, a nitrone or a nitrile oxide, more preferably an azide. Alternatively, the drug, antibody, protein, peptide, ligand, imaging marker, targeting ligand, delivery agent, nanoparticle or carrier comprises a cycloalkyne compound according to the present invention, and the nanoparticle comprises a 1,3-dipole or a 1,3-(hetero)diene, preferably containing an azide, a nitrone or a nitrile oxide, more preferably an azide.

[0040] Description of the drawings

[0041] Figure 1 : Dihydrazone 2 analysis data

[0042] GCMS: Agilent 6890N / column: RXi-5MS 20m, ID 180μm, df 0.18μm. Average velocity 50cm / s / carrier gas: He. Initial temperature 100℃ / initial time: 1.5min / solvent delay: 1.3min. Rate 75℃ / min, final temperature: 250℃, final time: 4.5min. Split ratio 20:1 / injection temperature: 250℃, injection volume: 1μl. Detection: MSD (EI-positive) / detection temperature: 280℃ / mass range: 50-550. Detection: FID / detector temperature: 300℃.

[0043] Figure 2 : Dihydrazone 2 analysis data

[0044] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.7411 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0045] Figure 3 [Example 2 Compound 3] TMTHSI analysis data

[0046] Column: Waters XSelect CSH C18 (30x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: 95% acetonitrile + 5% 10 mM ammonium bicarbonate in water. Eluent B: 10 mM ammonium bicarbonate in water. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0047] Figure 4 [Example 2 Compound 3] TMTHSI analysis data

[0048] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~297.5603 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0049] Figure 5 [Example 2 Compound 3] TMTHSI analysis data

[0050] Bruker BioSpin GmbH; Methods Pulse sequence: zgpg30. Relaxation delay: 8 s. Solvent: CDCl3. Temperature ~300.5658 K. Scan number: 1024. Frequency: 100.622829328806 MHz. Nuclear: 13C.

[0051] Figure 6 [Example 2 Compound 3] TMTHSI analysis data

[0052] Bruker BioSpin GmbH; Method Pulse sequence: jmod. Relaxation delay: 8 s. Solvent: CDCl3. Temperature: ~301.3172 K. Scan number: 1024. Frequency 100.622829802853 MHz. Nuclear: 13C.

[0053] Figure 7 [Example 3 Compound 4] TMTHSI-Suc-NHS analysis data

[0054] Column: Waters XSelect CSH C18 (30x2.1 mm, 3.5μ). Flow rate: 1 ml / min; column temperature: 35°C. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% formic acid in water. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection PDA (210-400 nm). Detection: MSD ESI pos / neg (mass range: 100-1000). Detection: ELSD (Alltech 3300): gas flow rate 1.5 ml / min, gas temperature 40°C.

[0055] Figure 8 [Example 3 Compound 4] TMTHSI-Suc-NHS analysis data.

[0056] Bruker BioSpin GmbH: Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.2043 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0057] Fig. 9 [Example 3 Compound 4] TMTHSI-Suc-NHS analysis data

[0058] Column: Waters XSelect CSH C18 (30x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0059] Fig.10 [Example 3 Compound 4] TMTHSI-Suc-NHS analysis data

[0060] Bruker BioSpin GmbH: Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.5264 K. Number of scans: 64. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0061] Fig.11 [Example 5 Compound 6] PoC TMTHSI analysis data

[0062] Column: Waters XSelect CSH C18 (30x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: 95% acetonitrile + 5% 10 mM ammonium bicarbonate in water. Eluent B: 10 mM ammonium bicarbonate in water. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm) detection PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0063] Fig.12 [Example 5 Compound 6] PoC TMTHSI analysis data

[0064] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~297.6677 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0065] Fig.13 [Example 6 Compound 9] PoC TMTHSI-Suc-NHBn analysis data

[0066] Column: Waters XSelect CSH C18 (30x2.1mm, 3.5μ). Flow rate: 1 ml / min; Column temperature: 35°C. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% formic acid in water. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-400 nm). Detection: MSD ESI pos / neg (mass range: 100-1000). Detection: ELSD (Alltech 3300): gas flow rate: 1.5 ml / min, gas temperature: 40°C.

[0067] Fig.14 [Example 6 Compound 9] PoC TMTHSI-Suc-NHBn analysis data

[0068] Column: Waters XSelect CSH C18 (30x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0069] Fig.15 [Example 6 Compound 9] PoC TMTHSI-Suc-NHBn analysis data

[0070] Bruker BioSpin GmbH: Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.097 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0071] Fig.16 [Example 8 Compound 8] TMTHSI-HER2 analysis data

[0072] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 0.8ml / min; Column temperature: 25°C. Eluent A: acetonitrile. Eluent B: 10mM aqueous ammonium bicarbonate. Lin. Gradient: t=0min 5%A, t=3.5min 98%A, t=3min 98%A. Post-run time: 2min. Detection DAD (220-320nm, 210nm and 220nm). Detection PDA (210-320nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0073] Figure 17: Fig.17A Comparison of the reaction kinetics of TMTHSI and BCN-OH. TMTHSI (upper curve) is significantly faster than BCN-OH (lower curve). Fig. 17B Amplification of TMTHSI measurements.

[0074] Fig.18 :[Example 11 Compound 11] N 1 -(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N 4 -(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of succinamide.

[0075] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=3.5 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0076] Fig.19 :[Example 11 Compound 11] N 1 -(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N 4 -(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiol-1-ylidene) succinamide NMR data.

[0077] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.9557 K. Number of scans: 16. Frequency 400.132470966543 MHz. Nucleus: 1H.

[0078] Fig. 20 :[Example 12 Compound 12] (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectral data of 2,5-dioxopyrrolidin-1-yl-thiol-1-ylidene)carbamic acid ester.

[0079] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0080] Fig.21 :[Example 12 Compound 12] (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 NMR data of 2,5-dioxopyrrolidin-1-yl-thiol-1-ylidene)carbamic acid ester.

[0081] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.8484 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0082] Fig. 22 :[Example 14 Compound 14] 1-(2-(2-hydroxyethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of thiol-1-thiol-1-ylidene) urea.

[0083] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0084] Fig.23 :[Example 14 Compound 14] 1-(2-(2-hydroxyethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiol-1-ylidene) urea NMR data.

[0085] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~299.7071 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0086] Fig.24 :[Example 15 Compound 15] 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectral data of methyl thiophene-1-ylidene)sulfamoyl)benzoate.

[0087] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0088] Fig.25 :[Example 15 Compound 15] 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6NMR data of methyl thiophene-1-ylidene)sulfamoyl)benzoate.

[0089] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~299.8145 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0090] Fig.26 :[Example 16 Compound 16] 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of 1-thiophene-1-ylidene)sulfamoyl)benzoic acid.

[0091] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0092] Fig. 27 :[Example 16 Compound 16] 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -NMR data of thiol-1-ylidene)sulfamoyl)benzoic acid.

[0093] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~300.1365 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0094] Fig.28 :[Example 17 Compound 17] 3,3,6,6-tetramethyl-1-(methylimino)-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -LCMS and mass spectrometry data of thiol-1-oxide.

[0095] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0096] Fig.29 :[Example 17 Compound 17] 3,3,6,6-tetramethyl-1-(methylimino)-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -NMR data of Thiepiphene 1-oxide.

[0097] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~300.1365 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0098] Fig.30 :[Example 18 Compound 18] 4-((3,3,6,6-tetramethyl-1-(methylimino)-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 LCMS and mass spectral data of methyl thiophen-2-yl)methyl)benzoate.

[0099] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0100] Fig.31 :[Example 18 Compound 18] 4-((3,3,6,6-tetramethyl-1-(methylimino)-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6NMR data of methyl thiophen-2-yl)methyl)benzoate.

[0101] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~299.9218 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0102] Fig.32 :[Example 19 Compound 19] 4-(((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectral data of methyl thiophene-1-ylidene)amino)methyl)benzoate.

[0103] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0104] Fig.33 :[Example 19 Compound 19] 4-(((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 NMR data of methyl thiophene-1-ylidene)amino)methyl)benzoate.

[0105] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~299.9218 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0106] Fig.34 :[Example 13 Compound 13] 2,2,2-trifluoro-N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of thiol-1-thiol)acetamide.

[0107] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0108] Fig.35 :[Example 13 Compound 13] 2,2,2-trifluoro-N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -NMR data of thiol-1-ylidene)acetamide.

[0109] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~299.1845 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0110] Fig.36 : LCMS and mass spectrum data of [Example 20 Compound 21] tert-butyl (2-((2-aminoethyl)disulfanyl)ethyl)carbamate.

[0111] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0112] Fig.37 : NMR data of [Example 20 Compound 21] tert-butyl (2-((2-aminoethyl)disulfanyl)ethyl)carbamate.

[0113] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~295.7355 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0114] Fig.38 : LCMS and mass spectrum data of [Example 21 Compound 22] 2,2-dimethyl-4,13-dioxo-3-oxa-8,9-dithia-5,12-diazahexadecane-16-oic acid.

[0115] Column: Waters XSelect CSH C18 (30x2.1mm, 3.5μ). Flow rate: 1 ml / min; Column temperature: 35°C. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% formic acid in water. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-400 nm). Detection: MSD ESI pos / neg (mass range: 100-1000). Detection: ELSD (Alltech 3300): gas flow rate: 1.5 ml / min, gas temperature: 40°C.

[0116] Fig.39 : [Example 21 Compound 22] NMR data of 2,2-dimethyl-4,13-dioxo-3-oxa-8,9-dithia-5,12-diazahexadecane-16-oic acid.

[0117] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~295.6282 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0118] Fig.40 : NMR data of [Example 22 Compound 23] 4-((2-((2-aminoethyl)disulfanyl)ethyl)amino)-4-oxobutanoic acid trifluoroacetate.

[0119] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: MeOD. Temperature: ~298.097 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nuclear: 1H.

[0120] Fig.41:[Example 23 Compound 24] 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectral data of 1-thiophene-1-ylidene)ureido)ethyl)disulfanyl)ethyl)amino)butyric acid.

[0121] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0122] Fig.42 :[Example 23 Compound 24] 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 NMR data of 1-thiophene-1-ylidene)ureido)ethyl)disulfanyl)ethyl)amino)butyric acid.

[0123] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.097 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0124] Fig.43 :[Example 24 Compound 25] 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectral data of 2,5-dioxopyrrolidin-1-yl (thiepin-1-ylidene)ureido)ethyl)disulfanyl)ethyl)amino)butyric acid 2,5-dioxopyrrolidin-1-yl ester.

[0125] Column: Waters XSelect CSH C18 (30x2.1mm, 3.5μ). Flow rate: 1 ml / min; Column temperature: 35°C. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% formic acid in water. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-400 nm). Detection: MSD ESI pos / neg (mass range: 100-1000). Detection: ELSD (Alltech 3300): gas flow rate: 1.5 ml / min, gas temperature: 40°C.

[0126] Fig.44 :[Example 24 Compound 25] 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 NMR data for 2,5-dioxopyrrolidin-1-yl-thiol-1-ylidene)ureido)ethyl)disulfanyl)ethyl)amino)butyric acid 2,5-dioxopyrrolidin-1-yl ester.

[0127] Bruker BioSpin GmbH; Method Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: ~298.097 K. Number of scans: 16. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0128] Fig.45 : LCMS and mass spectrum data of [Example 29 Compound 30] (S)-15-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoylamino)-2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazahexadecane-16-oic acid.

[0129] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0130] Fig.46 : [Example 29 Compound 30] (S) -15-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazahexadecane-16-oic acid NMR data.

[0131] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: ~295.7355 K. Number of scans: 64. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0132] Fig.47 :[Example 30 Compound 31]N 2 -(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -NMR data for (2-(2-aminoethoxy)ethyl)-L-glutamine.

[0133] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: ~298.097 K. Number of scans: 64. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0134] Fig.48 :[Example 31 Compound 32]N 2 -(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(2-(2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of 1-thiophene-1-ylidene)ureido)ethoxy)ethyl)-L-glutamine.

[0135] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0136] Fig.49 :[Example 31 Compound 32]N 2 -(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(2-(2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 NMR data of 1-thiol-1-ylidene)ureido)ethoxy)ethyl)-L-glutamine.

[0137] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: ~298.097 K. Number of scans: 128. Frequency: 400.132470966543 MHz. Nucleus: 1H.

[0138] Fig.50 : [Example 33 Compound 34] 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of thiol-1-thiol-1-ylidene) urea.

[0139] Column: Waters XSelect CSH C18 (50x2.1mm 3.5μ). Flow rate: 1 ml / min; Column temperature: 25°C. Eluent A: Acetonitrile. Eluent B: 10 mM aqueous ammonium bicarbonate. Lin. Gradient: t=0 min 5% A, t=1.6 min 98% A, t=3 min 98% A. Post-run time: 1.3 min. Detection: DAD (220-320 nm, 210 nm and 220 nm). Detection: PDA (210-320 nm). Detection: MSD ESI pos / neg (mass range 100-1000).

[0140] Fig.51 : [Example 33 Compound 34] 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiol-1-ylidene) urea NMR data.

[0141] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: ~297.86 K. Number of scans: 128. Frequency: 400.232471584084 MHz. Nucleus: 1H.

[0142] Fig.52 : [Example 34 Compound 35] 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 LCMS and mass spectrometry data of thiol-1-thiol) urea-Cy7 adduct formate salt.

[0143] Column: Waters XSelect CSH C18 (50x2.1mm, 3.5μ). Flow rate: 0.8ml / min; Column temperature: 35°C. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% formic acid in water. Lin. Gradient: t=0min 5%A, t=3.5min 98%A, t=6min 98%A. Post-run time: 2min. Detection DAD (220-320nm, 210nm and 220nm). Detection: PDA (210-800nm). Detection: MSD ESI pos / neg (mass range: 100-1000). Detection: ELSD (Alltech3300): gas flow rate: 1.5ml / min, gas temperature: 40°C.

[0144] Fig.53 : [Example 34 Compound 35] 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 NMR data of thiol-1-thiol) urea-Cy7 adduct formate.

[0145] Bruker BioSpin GmbH; Methods Pulse sequence: zg30. Relaxation delay: 1 s. Solvent: MeOD. Temperature: ~296.1649 K. Number of scans: 64. Frequency: 400.132470966543 MHz. Nuclear: 1H.

[0146] Fig.54 : 1-Imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ produced according to the present invention 6 - Overview of thiophene 1-oxide (TMTHSI) derivatives.

[0147] Fig.55 : Kinetics of the reaction of TMTHSI with benzyl azide to determine the reaction rate constant value kt.

[0148] Figure 56: UPLC chromatogram of:

[0149] A) siRNA PLK1 oligonucleotide reference

[0150] B) Amino-modified siRNA PLK1 is completely converted into TMTHSI-functionalized siRNA PLK1 oligonucleotide, Example 25, Compound 26

[0151] C) Complete conversion of TMTHSI-functionalized siRNA oligonucleotides in reaction with azide-functionalized CPP1 peptides to produce CPP1-siRNA PLK1 conjugates, Example 26, Compound 27

[0152] D) Complete conversion of the acid-labile linker NHS ester in the reaction with the amine-CPP1-siRNA conjugate, resulting in L7-CPP1-siRNA PLK1 conjugate, Example 27, Compound 28. DETAILED DESCRIPTION

[0153] definition

[0154] As used in this specification and claims, the verb "to comprise" and its conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0155] Furthermore, the indefinite article "a" or "an" modifying an element does not exclude the possibility that more than one of the element is present, unless the context clearly requires the presence of one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one".

[0156] The compounds disclosed in the present specification and claims may be described as thiocycloheptyne compounds, i.e., cycloheptyne compounds in which there is a combination of sulfur and a triple bond in the ring structure. The triple bond of the cycloheptyne moiety may be located at any of three possible positions relative to sulfur (numbered according to "IUPAC Nomenclature of Organic Chemistry" rule A31.2). The description of any cycloheptyne compound in the present specification and claims is meant to include all three individual regioisomers of the cycloheptyne moiety.

[0157] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers of the compounds may exist. The description of any compound in this specification and claims is intended to include all diastereomers and mixtures thereof, unless otherwise stated. In addition, the description of any compound in this specification and claims is intended to include a single enantiomer, as well as any mixture of enantiomers (racemic mixtures or other mixtures), unless otherwise stated. When the structure of a compound is described as a specific enantiomer, it should be understood that the invention of the present application is not limited to the specific enantiomer.

[0158] Compounds may exist in different tautomeric forms. The compounds according to the invention are intended to include all tautomeric forms, unless stated otherwise.

[0159] The compounds disclosed in the specification and claims may also exist as exo- and endo-regioisomers. Unless otherwise indicated, the description of any compound in the specification and claims is meant to include individual exo-regioisomers and individual endo-regioisomers of the compound, as well as mixtures thereof.

[0160] In addition, the compounds disclosed in the present specification and claims may exist as cis-isomers and trans-isomers. Unless otherwise stated, the description of any compound in the specification and claims is intended to include a single cis-isomer and a single trans-isomer of the compound and mixtures thereof. As an example, when the structure of a compound is depicted as a cis-isomer, it should be understood that the corresponding trans-isomer or a mixture of a cis-isomer and a trans-isomer is not excluded from the invention of the present application.

[0161] Unsubstituted alkyl groups have the general formula C n H 2n+1 and may be straight chain or branched. Unsubstituted alkyl groups may also contain cyclic moieties and thus have the accompanying general formula C n H 2n-1 Optionally, the alkyl group is substituted with one or more substituents as further specified herein. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, tert-butyl, 1-hexyl, 1-dodecyl, and the like.

[0162] Unsubstituted alkenyl groups have the general formula C n H 2n-1 and may be straight or branched. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, pentenyl, decenyl, octadecenyl, and eicosene, etc. Unsubstituted alkenyl groups may also contain cyclic moieties and thus have the accompanying general formula C n H 2n-3 .

[0163] Unsubstituted alkenes have the general formula C n H 2n , while unsubstituted alkynes have the general formula C n H 2n-2 .

[0164] Aryl comprises at least six carbon atoms and can include monocyclic, bicyclic and polycyclic structures.Optionally, aryl can be substituted by one or more substituents further specified herein.Examples of aryl include groups such as phenyl, naphthyl, anthracenyl etc.

[0165] Arylalkyl and alkylaryl contain at least seven carbon atoms and can include monocyclic and bicyclic structures. Optionally, aryl can be substituted with one or more substituents further specified herein. Arylalkyl is, for example, benzyl, etc. Alkylaryl is, for example, 4-tert-butylphenyl, etc.

[0166] In the case where aryl is represented as (hetero)aryl, the notation is meant to include aryl and heteroaryl. Similarly, alkyl (hetero)aryl is meant to include alkyl aryl and alkyl heteroaryl, and (hetero)arylalkyl is meant to include arylalkyl and heteroarylalkyl.

[0167] The heteroaryl group contains from one to four heteroatoms selected from oxygen, phosphorus, nitrogen and sulfur.

[0168] Compound

[0169] Taking Almeida's thiocycloheptyne compound as a starting point, the inventors propose to have found an improved compound that can be used for copper-free click reactions. It was found that TMTH itself is not suitable for practical use due to its inherent instability (Dommerholt et al., Nature Communications 5, Article number: 5378 (2014)). The sulfur in the TMTH ring structure has been described in King et al. (Chem. Comm., 2012, 9308-9309) as being derivatized via direct alkylation. Since King has observed that the ring strain should not be stretched beyond its limit to prevent the reactivity of the alkyne bond, other derivatizations are described as indirect. In addition, any derivatization should provide a compound suitable for reaction under aqueous conditions (high reactivity) and for tolerating biological media in subsequent applications to produce a suitable bioorthogonal reagent.

[0170] The inventors have now surprisingly discovered that other derivatizations at sulfur are possible. The resulting compounds combine high reactivity in cycloadditions with good relative stability. The sulfonimides (>S(=O)(=NH) and / or sulfonyl diimides (>S(=NR)2) of the present invention are a new class of compounds that can be used as bioorthogonal labels or conjugates. The sulfonimides and / or sulfonyl diimides of the present invention can be easily derivatized with other functional groups and linkers using known chemistry and can be further functionalized, for example for bioorthogonal labeling, imaging or modification (e.g. surface modification of target molecules). The compounds of the present invention can be conjugated to a variety of biologically active compounds and / or drug delivery systems.

[0171] TMTH and TMTH derivatives disclosed in King et al. (Chem. Comm., 2012, 9308-9309) have shown poor stability and cannot be used as is for further synthesis (Li, Molecules, 2016, 21, 1393, Krebs, Tet. Lett., 1970, 761-764). However, the TMTHSI compounds and derivatives according to the present invention can be synthesized and isolated, and are stable to both alkaline and acidic purification, and can therefore be used for further synthesis. TMTHSI and its TMTHSI derivatives according to the present invention have demonstrated an extended storage life of one year, as shown in the Examples. TMTHSI and TMTHSI derivatives can react with azides in acidic / neutral / alkaline aqueous environments and in combinations with organic solvents.

[0172] In a first aspect, the present invention therefore relates to compounds of formula (I)

[0173]

[0174] in:

[0175] n and m are independently 0, 1 or 2, and n+m is 2;

[0176] X is O or NR 9 ,

[0177] Y is NR 10 ,

[0178] R 1 , R 2 , R 3 , R 4 independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24(Hetero)arylalkyl, wherein O, N, P and S are further independently hydrogen, halogen (F, Cl, Br, I), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 The alkyl radicals are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the alkyl radicals, (hetero)aryl radicals, alkyl (hetero)aryl radicals and (hetero)arylalkyl radicals are independently optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkyloxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkyloxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkyloxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the silyl group is represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy and C3-C 12 Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S;

[0179] R 5 , R 6 , R 7 , R 8independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl, wherein O, N, P and S are further independently hydrogen, halogen (F, Cl, Br, I), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 The alkyl radicals are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the alkyl radicals, (hetero)aryl radicals, alkyl (hetero)aryl radicals and (hetero)arylalkyl radicals are independently optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkyloxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkyloxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkyloxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the silyl group is represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy and C3-C 12 Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S;

[0180] Wherein, optionally, R 1 and R 7 , R 1 With R 8 , R 2 With R 7 , R 2 With R 8 , R 3 With R 5 , R 3 With R 6 , R 4 With R 5 and / or R 4 With R 6 independently form a fused ring system, such as a cycloalkyl system, a cyclo(hetero)aryl system, a cycloalkyl(hetero)aryl system, a cyclo(hetero)arylalkyl system,

[0181] wherein the alkyl group of the fused ring system is optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups of the fused ring system are independently optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkyloxy, halogen, amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkyloxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkyloxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, wherein the silyl group is represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy and C3-C 12Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S;

[0182] R 9 , R 10 are independently selected from hydrogen, halogen (F, Cl, Br, I), R 12 、-CH=C(R 12 2. -C≡CR 12 、-[C(R 12 )2C(R 12 )2O] q -R 12 (where q is 1 to 200), -CN, -N3, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12 、-OS(O)2R 12 、-OS(O)OR 12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12 )C(X)XR 12 and -N(R 12 )C(X)N(R12 )2, wherein X is oxygen or sulfur, and wherein R 12 independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl.

[0183] In a preferred embodiment, the integers n and m are both 1, and the compound has formula (II):

[0184]

[0185] R 1 -R 8 , X, Y are as defined elsewhere herein. Preferably, the alkyne is located at the 4-5 position relative to sulfur (1-position). The resulting thiocycloheptyne is a symmetrical compound having an axis of symmetry through the sulfur and alkyne bonds. Compared to the known BCN compounds containing cis-trans isomerism and being chiral (E / Z), the current TMTHSI-compounds only present E / Z isomerism, but remain identical. The use of the symmetrical thiocycloheptynes ​​in the present application as click reaction reagents avoids mixtures of isomeric compounds with possible physicochemical or biologically variable behavior.

[0186] In a preferred embodiment, X is O. The resulting sulfonyl imines were successfully prepared with good stability and were successfully derivatized as shown in the examples.

[0187] In a preferred embodiment, R 10 is H or contains alcohol, amine, ester, C 1-4 Alkyl, carboxylic acid, trifluoroacetyl and n-hydroxysuccinimide (NHS) ester functional groups.

[0188] In a preferred embodiment, R 10 is H. The resulting S(=O)NH functionality is stable and has good reactivity at the NH position for further derivatization.

[0189] In a preferred embodiment, R 1 , R 2 , R 3 , R 4 independently selected from H, halogen (F, Cl, Br, I) and C1-C4 alkyl, preferably methyl or ethyl, or R 1 With R 2 and / or R 3 With R 4 In other preferred embodiments, R1 , R 2 , R 3 , R 4 Independently selected from C1-C4 alkyl, preferably methyl or ethyl.

[0190] In other preferred embodiments, R 1 , R 2 , R 3 , R 4 are the same and are selected from H, halogen and C1-C4 alkyl, or R 1 With R 2 and R 3 With R 4 In other preferred embodiments, R 1 , R 2 , R 3 , R 4 are the same and are selected from H, C1-C4 alkyl, preferably methyl or ethyl (preferably methyl), or R 1 With R 2 and R 3 With R 4 In other preferred embodiments, R 1 , R 2 , R 3 , R 4 The same and selected from C1-C4 alkyl, preferably methyl or ethyl, most preferably methyl.

[0191] In one embodiment, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 are independently H, halogen (F, Cl, Br, I) or C1-C 24 alkyl, and is preferably independently H or lower alkyl, i.e. C1-C4 alkyl, i.e. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or tert-butyl. In a particularly preferred embodiment, R 1 -R 4 In a more preferred embodiment, R 1 -R 4 All are methyl.

[0192] In a preferred embodiment, R 5 , R 6 , R 7 , R 8is independently selected from H, halogen (F, Cl, Br, I) and C1-C4 alkyl. More preferably, R 5 , R 6 , R 7 , R 8 are the same and are selected from H, halogen (F, Cl, Br, I) and C1-C4 alkyl, preferably methyl or ethyl. More preferably, R 5 , R 6 , R 7 , R 8 are the same and are selected from H and C1-C4 alkyl, preferably methyl or ethyl. 5 -R 8 All are H.

[0193] In a particularly preferred embodiment, the compound of formula (I) is 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -Thiophene 1-oxide:

[0194]

[0195] synthesis

[0196] In another aspect, the present invention relates to the synthesis of the compounds of the present invention, and in particular to a process for preparing a compound of formula (I), said process comprising the steps of:

[0197] a. converting the dihydrazone (2) into the iminodihydrosulfonylimino group (3);

[0198] b. Isolate the obtained iminodihydrosulfonylimino group (3).

[0199] The synthesis is based in part on the synthesis of TMTH by Almeida et al., using thiopanedione 1 as an intermediate in the synthesis of TMTH, followed in the present invention by a one-step conversion of the bishydrazone 2 to the sulfonimide 3.

[0200]

[0201] The synthesis of diketone 1 and dihydrazone 2 has been described in detail in the literature [Almeida et al.]. The synthesis begins with the conversion of diketone 1 to dihydrazone 2. The reaction is carried out in the presence of one or more hydrazines, preferably hydrazine sulfate and / or hydrazine monohydrate, and preferably under pressure.

[0202] The obtained bishydrazine 2 is converted to the target compound 3 in a two-step oxidation reaction. One step of the conversion includes an oxidation reaction (addition), in which the bishydrazone (2) is reacted with an oxidant to oxidize the sulfur (>S) functional group to a sulfonyl imide (>S(=O)(=NR) functional group. The other step of the conversion is an oxidation reaction (elimination), in which the bishydrazone (2) is reacted with an oxidant to oxidize the (bis)hydrazine (>C=N-NH2)2 functional group to an alkyne (-C≡C-) functional group. In a preferred embodiment, this two-step reaction is carried out in a one-step synthesis, preferably with the same oxidant, preferably iodobenzene diacetate:

[0203]

[0204] Coupling of linkers and functional groups

[0205] In another aspect of the invention, the linker and / or functional group is coupled to the functionalized thiocycloheptyne of the invention. Thus, in another aspect, the invention relates to compounds wherein X and / or Y and / or an atom adjacent to the S atom in the thiocycloheptyne ring of the compound of formula (I) are independently coupled to an optional linker (L) and a functional group (Q) to produce a compound of formula (III):

[0206] (Formula I)-LQ;

[0207] (III)

[0208] Wherein the linking group (L) is absent or selected from a linear or branched C1-C 24 Alkylene, C2-C 24 Alkenylene, C2-C 24 Alkynylidene, C3-C 24 Cycloalkylene, C5-C 24 Cycloalkenylene, C5-C 24 Cycloalkynylene, C7-C 24 Alkyl (hetero) arylene, C7-C 24 (Hetero)aryl alkylene, C5-C 24 (Hetero)arylalkenylene, C9-C 24 (hetero)arylalkynylene, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl(hetero)arylene, (hetero)arylalkylene, (hetero)arylalkenylene and (hetero)arylalkynylene are optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 Cycloalkynyl, C8-C 12Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the silyl group can be represented by the formula (R 11 )3Si- represents, where R 11 As defined above;

[0209] The functional group Q is selected from hydrogen, halogen (F, Cl, Br, I), R 12 、-CH=C(R 12 2. -C≡CR 12 、-[C(R 12 )2C(R 12 )2O] q -R 12 (where q is 1 to 200), -CN, -N3, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12 、-OS(O)2R 12 、-OS(O)OR 12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12 )C(X)XR 12and -N(R 12 )C(X)N(R 12 )2, wherein X is oxygen or sulfur, and wherein R 12 independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl. Preferably, R 12 It is hydrogen or C1-C6 alkyl.

[0210] If the optional linking group (L) and the functional group (Q) are attached to an atom adjacent to the S atom in the thiocycloheptyne ring, then R 5 , R 6 , R 7 or R 8 One of the R is replaced by an optional linking group (L) connected to the functional group (Q). If the optional linking group (L) and the functional group (Q) are connected to X or Y, respectively, then R 9 or R 10 are respectively replaced by an optional linking group (L) connected to the functional group (Q).

[0211] Preferably, the optional linking group (L) and the functional group (Q) are connected to one of X or Y, preferably to Y, and / or to an atom in the thiocycloheptyne ring adjacent to the S atom. More preferably, the optional linking group (L) and the functional group (Q) are connected to one of X or Y, preferably to Y.

[0212] In one embodiment, a compound is provided which is constituted as follows: a compound of formula (I) according to the present invention is coupled at atom X or Y, preferably at Y and / or at an atom adjacent to the S atom in the thiocycloheptyne ring with an optional linker (L) and a functional group (Q) to produce a compound of formula (III):

[0213] (Formula I)-LQ

[0214] (III)

[0215] Wherein the linking group (L) is absent or selected from a linear or branched C1-C 24 Alkylene, C2-C 24 Alkenylene, C2-C 24 Alkynylidene, C3-C 24 Cycloalkylene, C5-C 24 Cycloalkenylene, C5-C 24 Cycloalkynylene, C7-C 24 Alkyl (hetero) arylene, C7-C 24(Hetero)aryl alkylene, C5-C 24 (Hetero)arylalkenylene, C9-C 24 (hetero)arylalkynylene, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl(hetero)arylene, (hetero)arylalkylene, (hetero)arylalkenylene and (hetero)arylalkynylene are optionally substituted by one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 Cycloalkynyl, C8-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the silyl group can be represented by the formula (R 11 )3Si- represents, where R 11 As defined above;

[0216] The functional group Q is selected from hydrogen, halogen (F, Cl, Br, I), R 12 、-CH=C(R 12 2. -C≡CR 12 、-[C(R 12 )2C(R 12 )2O] q -R 12 (where q is 1 to 200), -CN, -N3, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12 、-OS(O)2R 12 、-OS(O)OR12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12 )C(X)XR 12 and -N(R 12 )C(X)N(R 12 )2, wherein X is oxygen or sulfur, and wherein R 12 independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C 24 Alkyl, C6-C 24 (Hetero)aryl, C7-C 24 Alkyl (hetero) aryl and C7-C 24 (Hetero)arylalkyl.

[0217] A variety of linkers may be used, and the selection of a linker may be based on other criteria, such as those generally related to the end-use application as disclosed elsewhere herein.

[0218] In a preferred embodiment, L is absent or selected from -[C(R 12 )2C(R 12 )2O] q -R 12 (where q is 1 to 200), -CN, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12、-OS(O)2R 12 、-OS(O)OR 12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12 )C(X)XR 12 and -N(R 12 )C(X)N(R 12 )2, wherein X is oxygen or sulfur, and wherein R 12 As defined elsewhere.

[0219] In other preferred embodiments, L is absent or is a chain of 1-25 atoms, preferably 1-15 atoms in length, and comprises one or more, preferably 1-6, moieties independently selected from -S(O)2-, -S-, -SS-, -C(O)NH-, -NHC(O)-, -C(O)O- and phenylene, whereby the chain length is determined by the number of atoms in the longest straight chain of atoms. The longest straight chain may comprise one or more further heteroatoms, such as O, N, S and P. Preferably, it may comprise one or more further O atoms. In preferred embodiments, L comprises 1, 2, 3, 4 or 5 moieties independently selected from -S(O)2-, -S-, -SS-, -C(O)NH-, -NHC(O)-, -C(O)O- and phenylene. More preferably, the chain having a length of 1 to 25 atoms, preferably 1 to 15 atoms, is not branched, i.e. the chain is linear, optionally with the exception of heteroatoms which are not contained in one or more moieties (-S(O)2-, -C(O)NH-, -NHC(O)-, -C(O)O-) in the linear chain.

[0220] In one embodiment, Q is selected from -OR 12 、-N(R 12 )2.- + N(R 12 )3. -C(O)N(R 12 )2. -C(O)OR 12 、-OC(O)R 12 、-OC(O)OR 12 、-OC(O)N(R12 )2、-N(R 12 )C(O)R 12 、-N(R 12 )C(O)OR 12 and -N(R 12 )C(O)N(R 12 )2, where R 12 As defined elsewhere herein.

[0221] In a preferred embodiment, Q comprises alcohol, amine, thiol, carboxylate, carboxylic acid or activated ester, ketone, aldehyde, nitrile, maleimide, olefin, alkyne, heteroaromatic acid ester, leaving group and phosphoramidite. In a particularly preferred embodiment, Q is alcohol, amine, carboxylate, carboxylic acid or N-hydroxysuccinimide (NHS) ester.

[0222] In one embodiment, the coupling of the linker L and / or the functional group Q is at the functionalized sulfur of the thiocycloheptyne, i.e. at X and / or Y of the S(=X)(=Y) portion of the molecule. The coupling is performed with the functional group, optionally via a linker. The functional group Q can be attached to any molecule of interest.

[0223] In certain embodiments, Q can be connected to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, a carrier, or a combination thereof. Chemical reactions for such couplings are well developed, and those skilled in the art can select appropriate coupling chemical reactions to couple Q to a target molecule.

[0224] Click React

[0225] The alkynyl groups of the compounds of the present invention are reactive and can be functionalized using, for example, cycloaddition reactions. In embodiments, the alkynyl groups can react with compounds comprising 1,3-dipoles or 1,3-(hetero)diene. In certain embodiments, the compounds comprising 1,3-dipoles or 1,3-(hetero)diene are compounds containing azides, compounds containing nitrone, or compounds containing nitrile oxides. Most preferably, compounds containing azides. In embodiments, compounds containing azides can be coupled using a copper-free click reaction. Coupling produces a triazole-type structure.

[0226] The compounds of the present invention are bifunctional compounds with reactive sites at the functionalized sulfur position and the alkyne functional group. The two functional groups can be functionalized independently, one via an optional linker (L) and a functional group (Q), and the other via click chemistry. Functionalization can be performed independently with drugs (e.g., small molecules, genetic materials, etc.), antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles, carriers.

[0227] Thus, in one embodiment, the functional group Q is optionally coupled via a linker L to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, a carrier.

[0228] In another embodiment, the alkyne can be coupled to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, a carrier. The coupling of the alkyne is preferably carried out via the reaction of the alkyne with a compound comprising a 1,3-dipole or a 1,3-(hetero)diene. In certain embodiments, the compound comprising a 1,3-dipole or a 1,3-(hetero)diene is an azide-containing compound, a nitrone-containing compound, or a nitrile oxide-containing compound, more preferably an azide-containing compound.

[0229] Therefore, the compounds of the present invention are suitable for generating a combination of two target molecules / functional groups via 1,3-dipole / 1,3 (hetero)diene / thiepine derivative / (linker) / functional group. Schematically, the general principle of this application of the thiepine of the present invention is exemplified in the following table:

[0230]

[0231] The linker used may be biodegradable, ie initially stable but cleavable over time or under certain circumstances (eg physiological circumstances).

[0232] In certain embodiments, such combinations are preferred, i.e., wherein the nanoparticles are coupled to a functional group / target molecule selected from a drug, an antibody, a protein, a peptide, a ligand, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, a carrier via an optional linker / 1,3-dipole or 1,3-heterodienes / thiocycloheptynes / optional linker. The nanoparticles themselves may include drugs, antibodies, proteins, peptides. The preferred 1,3-dipole or 1,3-heterodienes for coupling the nanoparticles to the thiocycloheptynes ​​derivatives of the present invention are azides (N3).

[0233] Nanoparticles

[0234] Drug delivery systems are increasingly used in pharmaceutical science. Through well-designed derivatization, drugs can be delivered, targeted, monitored and provided to patients with increased effectiveness and efficacy. The use of these drug delivery systems (such as nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates, drug polymer conjugates) is developing rapidly in pharmaceutical science.

[0235] Pharmaceutical science uses these systems to reduce drug toxicity and side effects, improve delivery and add imaging ligands to aid in monitoring effectiveness.

[0236] Nanoparticles, which are typically <100 nm in diameter, are made of a variety of materials, and their intended applications in medicine include drug delivery (including in vitro and in vivo diagnostics), nutraceuticals, and the production of improved biocompatible materials. Nanoparticles can be customized for specific purposes, and a variety of materials are available. Most drug-targeted nanoparticles are based on (bio) polymer materials, which can be in a variety of forms. The source material can be biologically derived (such as phospholipids, lipids, lactic acid, dextran, chitosan), or have more "chemical" properties (such as various (co) polymers). These nanoparticles are typically functionalized by connecting, coupling, (covalently) binding active ingredients (drugs, ligands, imaging ligands, etc.). The functionalization of many of these nanoparticles can be achieved by various chemical methods, such as copper-free click chemistry using the thiocycloheptane derivatives of the present invention.

[0237] In certain embodiments, the nanoparticles are self-assembled polymer micelles, preferably from thermosensitive block copolymers. In particular, copolymers based on PEG-b-poly(N-hydroxyalkylmethacrylamide-oligomeric lactates) with partially methacrylated oligomeric lactate units are preferred, but other (meth)acrylamide esters can also be used to construct thermosensitive blocks of HPMAm (hydroxypropylmethacrylamide) and HEMAm (hydroxyethylmethacrylamide) and N-(meth)acryloyl amino acid esters, such as esters and optional (oligomeric) lactates. Also preferred thermosensitive block copolymers are derived from monomers containing functional groups that can be modified by derivatized and underivatized methacrylate groups, such as HPMAm-lactate polymers; that is, such modifications include the incorporation of linker moieties.

[0238] Other types of functional thermosensitive (co)polymers that can be used are hydrophobically modified poly(N-hydroxyalkyl)(meth)acrylamides, copolymer compositions of N-isopropylacrylamide (NIPAAm) with monomers containing reactive functional groups (e.g. acidic acrylamide and other moieties such as N-acryloyloxysuccinimide) or similar copolymers of poly(alkyl)2-oxazines, etc. Also preferred thermosensitive groups can be based on NIPAAm and / or alkyl-2-oxazolines, which can react with monomers containing reactive functional groups, such as (meth)acrylamides or (meth)acrylates containing hydroxyl, carboxyl, amine or succinimide groups.

[0239] Suitable thermosensitive polymers are described in US Pat. No. 7,425,581 and EP-A-1 776 400. Also in WO 2010 / 033022 and WO 2013 / 002636.

[0240] WO2012 / 039602 describes drug-polymer matrix particles using such polymers. Furthermore, in WO2012 / 039602, biodegradable linker molecules are described that can be used in these known polymer matrix particles.

[0241] Typically, the thermosensitive block copolymer-based nanoparticles outlined above can be linked to the compounds of the present invention using azide-alkyne copper-free coupling. Examples of this are described in WO2017086794.

[0242] Thus, in certain embodiments of the invention, nanoparticles are prepared wherein the thiocycloheptyne derivatives of the invention are coupled to azide-containing nanoparticles. In certain embodiments, the nanoparticles are self-assembled polymer micelles, preferably from thermosensitive block copolymers.

[0243] The present invention also relates to the use of the thiocycloheptyne derivatives of the present invention in a method for coupling two target molecules, wherein optionally, the molecules are independently selected from drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles and carriers. The present invention also relates to the use of the thiocycloheptyne derivatives of the present invention in bioorthogonal, optional copper-free click reactions, and the use of the method of coupling nanoparticles to one or more of drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles and carriers using copper-free click reactions. In particular, the coupling of azide-containing thermosensitive polymers to the compounds of the present invention is advantageous because the click chemistry between the alkyne-containing compounds of the present invention and azides is faster than that of conventional alkynes used in click chemistry, especially in the preparation of biodegradable nanoparticles and conjugated / embedded drugs.

[0244] For the purpose of clear and concise description, features may be described herein as part of the same or separate aspects or embodiments of the present invention. It will be appreciated by those skilled in the art that the scope of the present invention may include embodiments having a combination of all or some features described herein as part of the same or separate embodiments.

[0245] The invention will be explained in more detail in the following non-limiting examples.

[0246] Example

[0247] Fig.54 Provided is a 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ produced according to the present invention. 6 -An overview of thiol-1-oxide (TMTHSI) derivatives, the synthesis of which is described in the following examples.

[0248] The core structure of TMTHSI (Example 2) can be functionalized in various ways.

[0249] Example 2, X = O, Y = N, core TMTHSI structure, R1-R4 = methyl, R5-R8 = hydrogen

[0250] Example 3, X = O, Y = N, is Example 2, wherein L = succinic acid linker, Q = activated NHS ester, R1-R4 = methyl, R5-R8 = hydrogen

[0251] Example 5 is Example 3, wherein the NHS ester is replaced in the reaction with benzylamine, R1-R4 = methyl, R5-R8 = hydrogen

[0252] Example 8, X = O, Y = N, is Example 3, wherein the NHS ester is replaced in the reaction with the HER2 peptide, R1-R4 = methyl, R5-R8 = hydrogen

[0253] Example 11, X=O, Y=N, is Example 3, wherein the NHS ester is replaced in the reaction with the diamino-PEG spacer (=L), Q is an amine, R1-R4=methyl, R5-R8=hydrogen

[0254] Example 12, X=O, Y=N,

[0255]

[0256] R1-R4 = methyl, R5-R8 = hydrogen

[0257] Example 13, X = O, Y = N-trifluoroacetamide, L = no linker, Q = no functional group. R1-R4 = methyl, R5-R8 = hydrogen. This compound is intended for α-sulfur substitution

[0258] Example 14, X=O, Y=N, is Example 12, wherein the NHS ester is replaced in the reaction with the amino-PEG-hydroxy spacer (=L), Q is hydroxy, R1-R4=methyl, R5-R8=hydrogen

[0259] Example 15, X = O, Y = N, is Example 2, wherein L = -S(O2)phenyl-, Q = 3-methoxycarbonyl, R1-R4 = methyl, R5-R8 = hydrogen

[0260] Example 16, X = O, Y = N, is Example 2, wherein L = -S(O2)phenyl, Q = 3-hydroxycarbonyl, R1-R4 = methyl, R5-R8 = hydrogen

[0261] Example 17, X = O, Y = N-methyl, L = no linker, Q = no functional group. R1-R4 = methyl, R5-R8 = hydrogen. This compound is intended for α-sulfur substitution

[0262] Example 18, X = O, Y = N-methyl, L = no linker, Q = no functional group. R1-R4 = methyl, R5-R8 = one hydrogen is replaced by L = -methylenephenyl-, Q = 4-methoxycarbonyl. This compound is an α-sulfur substituted variant of TMTHSI

[0263] Example 19, X = O, Y = N, is Example 2, wherein L = -methylenephenyl-, Q = 4-methoxycarbonyl, R1-R4 = methyl, R5-R8 = hydrogen

[0264] Example 23, X=O, Y=N, is Example 12, wherein the NHS moiety is replaced by a cystamine-based amine linker, L=-C(O)NHC2H4SSC2H4NHC(O)C3H6-, Q=COOH,

[0265]

[0266] R1-R4 = methyl, R5-R8 = hydrogen

[0267] Example 24, X=O, Y=N, is Example 23 activated as an NHS ester, L=-C(O)NHC2H4SSC2H4NHC(O)C3H6-, Q=((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl, C(O)OSu.

[0268]

[0269] R1-R4 = methyl, R5-R8 = hydrogen

[0270] Example 25, X=O, Y=N, is Example 24, wherein the NHS ester is replaced in the reaction with the amino-C6 functionalized siRNA oligonucleotide, R1-R4=methyl, R5-R8=hydrogen

[0271] Example 31, X = O, Y = N, is Example 12, wherein the NHS ester is replaced in the reaction with the aminoPEG-functionalized folic acid, R1-R4 = methyl, R5-R8 = hydrogen

[0272] Example 33, X=O, Y=N, is Example 12, wherein the NHS ester is replaced in the reaction with the diamino-PEG spacer (=L), Q is an amine, R1-R4=methyl, R5-R8=hydrogen

[0273] Example 34, X = O, Y = N, is Example 33 reacted with Cy7 NHS labeling. L = diamino-PEG spacer, R1-R4 = methyl, R5-R8 = hydrogen

[0274] Those skilled in the art will be able to readily provide other derivatives having other variations of R1-R8, L and / or Q, and in addition to the structures and / or Fig.54 In addition to the structures shown in , the PCR products can be coupled to other target molecules such as drugs, antibodies, proteins, peptides, ligands, imaging markers, targeting ligands, delivery agents, nanoparticles or carriers using reactions known in the art.

[0275] Example 1: Synthesis of (3,3,6,6-tetramethylthiazane-4,5-diylidene)bis(hydrazine).

[0276]

[0277] 3,3,6,6-tetramethylthiaheptane-4,5-dione (6.17 g, 30.8 mmol), ethanol (2.4 ml) and ethylene glycol (60 ml) were loaded into a glass autoclave (300 ml). Hydrazine sulfate (16.03 g, 123 mmol, 4 eq) and hydrazine monohydrate (3 ml, 61.6 mmol, 2 eq) were added to the stirred mixture. The autoclave was closed and heated in an oil bath at 140° C. for 20 hours.

[0278] After cooling to room temperature, the mixture was layered between water (500 ml) and ether (200 ml) and transferred to a separatory funnel while filtering through a piece of cotton to remove some white solid residue (probably hydrazine sulfate, as it is slowly soluble in water). The layers were separated and the aqueous layer was extracted with diethyl ether (2×100 ml). The combined ether layers were washed with water (2×75 ml) and brine (100 ml), dried over Na2SO4, and concentrated under reduced pressure to give 5.75 g of the crude product as a viscous yellow oil.

[0279] The residue was purified by flash column chromatography (silica 40 g; 30% EtOAc in heptane); the product fractions were combined and concentrated under reduced pressure. The residue was co-evaporated with diethyl ether to give 0.9 g (12%) of the product as an off-white solid.

[0280] GC / MS (Method_A)t R 4.47min, M + =228.

[0281] * 1 H NMR (400 MHz, CHLOROFORM-d) δ 5.24 (s, 4H), 2.54 (d, J = 14.4 Hz, 2H), 2.48 (d, J = 14.4 Hz, 2H), 1.34 (s, 6H), 1.21 (s, 6H).

[0282] Example 2: 1-Imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -Synthesis of Thiepiphene 1-oxide (TMTHSI).

[0283]

[0284] Load (3,3,6,6-tetramethylthiazane-4,5-diylidene) bis(hydrazine) (100mg, 0.44mmol) and ammonium acetate (270mg, 3.5mmol, 8 equivalents) into 8ml screw cap vials and suspend in methanol (0.4ml). The mixture is cooled in ice / water. Add a mixture solution of MeOH (0.4ml) and dichloromethane (0.6ml) of iodobenzene diacetate (494mg, 1.53mmol, 3.5 equivalents) dropwise to control heat release and gas formation (reaction shows gas evolution when adding each drop). After addition is complete, the mixture is stirred at room temperature for 1 hour.

[0285] The mixture is diluted with dichloromethane (2ml) and quenched with saline (1ml). The organic layer is taken out by pipette and passed through a phase separator. The aqueous residue is extracted with dichloromethane (2x), and the organic phase is passed through a phase separator and merged with the previous extract each time. The organic extract is concentrated under reduced pressure, and the evaporation is carefully monitored because the product may be volatile, to obtain 427mg of the crude product in the form of a yellow oil. The residue is purified by preparative RP-MPLC (Reveleris, XSelect; 10-50% MeCN aqueous solution, 10mM NH4HCO3, pH=9.5). The product fractions are merged and extracted with dichloromethane (3x), and each organic extract is passed through a phase separator and concentrated under reduced pressure. The residue is co-evaporated with heptane and diethyl ether to obtain 33mg (37%) of crystalline residue.

[0286] LC / MS(SC_BASE)t R 1.628min, purity 98.1%, mass measured value [M+H] + 200.

[0287] 1 H NMR (400 MHz, CHLOROFORM-d) δ 3.26 (d, J = 14.1 Hz, 2H), 3.18 (d, J = 14.1 Hz, 2H), 2.76 (s, 1H), 1.46 (s, 6H), 1.30 (s, 6H).

[0288] TMTHSI was shown to be stable during storage. Compound 3 was stored as a powder in both light and dark locations at atmospheric pressure. 1H-NMR and LC / MS (SC_BASE) confirmed that the compound was still intact after 10 months of storage.

[0289] Example 3: Coupling to a linker. 4-oxo-4-((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of 2,5-dioxopyrrolidin-1-yl (thiophene)amino)butyric acid ester.

[0290]

[0291] Diisopropylethylamine (266 μl, 1.52 mmol, 2 equiv.) was added to 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -thiophene 1-oxide (152 mg, 0.76 mmol) and succinic anhydride (114 mg, 1.14 mmol, 1.5 eq.) were added to a mixture of dichloromethane (5 ml), and the resulting mixture was stirred at room temperature for 20 hours.

[0292] The reaction mixture was concentrated under reduced pressure and redissolved in dichloromethane, and concentrated under reduced pressure again. The residue was redissolved in dichloromethane (5ml) and N-hydroxysuccinimide (220mg, 1.91mmol, 2.5 equivalents) was added. The mixture was stirred for 5 minutes, and then a dichloromethane (0.5ml) suspension of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (366mg, 1.91mmol, 2.5 equivalents) was added via a pipette. The resulting solution was stirred at room temperature for 4 hours.

[0293] The mixture was quenched with 2M aqueous KHSO4 and the organic phase was passed through a phase separator. The filtrate was concentrated under reduced pressure to give 420 mg of the crude mixture as a foam.

[0294] The residue was purified by flash column chromatography (silica 12 g, 50-75% EtOAc in heptane), the product fractions were pooled and concentrated under reduced pressure to give 141 mg (46%) of the product as a white solid.

[0295] LC / MS(SC_ACID)t R 1.84min, purity 85%, mass measured value [M+H] + 397.

[0296] 1H NMR (400 MHz, CHLOROFORM-d) δ 3.79 (d, J = 14.1 Hz, 2H), 3.63 (d, J = 14.2 Hz, 2H), 2.97-2.90 (m, 2H), 2.88-2.81 (m, 4H), 2.79-2.71 (m, 2H), 1.55 (s, 6H), 1.28 (s, 6H).

[0297] Example 4: Coupling to a linker. 1 -Benzyl-N 4 -(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -Synthesis of thiol-1-yl) succinamide.

[0298]

[0299] 4-oxo-4-((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 To a solution of 2,5-dioxopyrrolidin-1-yl (15.8 mg, 0.040 mmol) in dichloromethane (1 ml) was added benzylamine (8.71 μl, 0.080 mmol, 2 equiv). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then purified by flash column chromatography (silica, 12 g; 70-100% EtOAc in heptane), and the product fractions were combined and concentrated under reduced pressure to give 13.1 mg of product with a purity of ~83% by LC / MS.

[0300] The material was purified by preparative RP-MPLC (Reveleris, XSelect; 20-60% MeCN in water, 10 mM NH4HCO3, pH = 9.5) and the product fractions were pooled and concentrated under reduced pressure to give 6.2 mg (40%) of the title compound as a white solid.

[0301] LC / MS(SC_BASE)t R 2.01min, purity 96.8%, mass measured value [M+H] + 389.

[0302] 1H NMR (400MHz, chloroform-d) δ7.36–7.22 (m, 5H), 6.25 (s, 1H), 4.44 (d, J = 5.7Hz, 2H), 3.60 (d, J = 14.1Hz, 2H), 3.5 3 (d, J=14.1Hz, 2H), 2.73 (dd, J=6.8, 6.2Hz, 2H), 2.52 (dd, J=6.6, 6.4Hz, 2H), 1.49 (s, 6H), 1.24 (s, 6H).

[0303] Example 5: Click reaction. 1-Benzyl-6-imino-4,4,8,8-tetramethyl-1,4,5,6,7,8-hexahydro-6λ 4 -Synthesis of thiol[4,5-d][1,2,3]triazole 6-oxide.

[0304]

[0305] LC / MS:

[0306] Load 1-Imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ into an LC / MS vial 6 -Thiophene 1-oxide (5 mg, 0.025 mmol) was dissolved in MeCN (1 ml), benzyl azide was added and the reaction mixture was analyzed by LC / MS. LC / MS analysis showed complete consumption of starting material and formation of product as a mixture with unreacted benzyl azide.

[0307] LC / MS(SC_BASE)t R 1.728min, purity 64%, mass measured value [M+H] + 333.

[0308] 1 H-NMR:

[0309] 1-Imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -Thiophene 1-oxide (5 mg, 0.025 mmol) was dissolved in CDCl3 (0.6 ml) and benzyl azide was added. After about 15 minutes of reaction time, the reaction temperature was recorded. 1 H-NMR:

[0310] 1H NMR (400 MHz, CHLOROFORM-d) δ 7.40–7.29 (m, 3H), 7.06–7.01 (m, 2H), 5.73 (s, 2H), 3.45 (d, J=8.9 Hz, 4H), 2.69 (s, 1H), 1.70 (d, J=2.1 Hz, 6H), 1.44 (d, J=2.7 Hz, 6H).

[0311] The starting material was completely consumed and there was -35 mol% excess of benzyl azide.

[0312] Example 6: Click reaction. 1 -Benzyl-N 4 -(1-benzyl-4,4,8,8-tetramethyl-6-oxo-4,5,7,8-tetrahydro-1H-6λ 4 Synthesis of thiol-[4,5-d][1,2,3]triazole-6-ylidene)succinamide.

[0313]

[0314] To a solution containing N dissolved in CDCl3 1 -Benzyl-N 4 -(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiophene-1-ylidene) succinamide (4.6 mg, 12 μmol) was added to a CDCl3 solution of benzyl azide (10 vol%, 15.5 μL, 1.05 equiv). The mixture was shaken and after about 5 minutes of reaction time, 1 H-NMR analysis.

[0315] 1 H NMR (400MHz, chloroform-d) δ7.43–7.27(m, 8H), 7.06–7.00(m, 2H), 6.18(s, 1H), 5. 72 (s, 2H), 4.42 (d, J = 5.8Hz, 2H), 3.96 (d, J = 15.5Hz, 1H), 3.82 (d, J = 15.0H z, 1H), 3.70 (d, J = 15.3Hz, 1H), 3.57 (d, J = 15.1Hz, 1H), 2.74–2.67 (m, 2H), 2.49 (t, J=6.7Hz, 2H), 1.70 (s, 3H), 1.67 (s, 3H), 1.44 (s, 3H), 1.36 (s, 3H).

[0316] The mixture was concentrated to dryness and the residue was analyzed by LC / MS:

[0317] LC / MS (SC_BASE) showed a product peak with the target mass (t R 1.947min, purity 75%, [M+H] + 522).

[0318] LC / MS (SC_ACID) showed two product peaks [t R 1.90 (57%) and 1.94 min (23%)], target mass ([M+H]+522).

[0319] Example 7: Coupling of TMTHSI-containing linkers to HER2-peptide. Preparation of TMTHSI-succinyl-Fcycl[CGDGFYAC]YMDV.

[0320]

[0321] Fcycl[CGDGFYAC]YMDV (20 mg, 13 μmol) was loaded into an 8 ml screw cap vial and dissolved in DMSO (1 ml). 4-Oxo-4-((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 2,5-dioxopyrrolidin-1-yl thiophene-1-ylidene)amino)butyric acid 2,5-dioxopyrrolidin-1-yl ester (6.5 mg, 14 μmol, 1.04 equiv.) was added, followed by diisopropylethylamine (13 μl, 75 μmol, 5.6 equiv.). The mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative RP-MPLC (Reveleris, LUNA-C18, 20-60% MeCN in water, 0.1% formic acid), and the product fractions were pooled and lyophilized to give 16.6 mg (69%) of product.

[0322] LC / MS(AN_BASE_M1800)t R 2.57min, purity 96%, mass measured value [MH] - 1768, [M-2H] 2- 883.

[0323] Analytical methods

[0324] GC / MS Method:

[0325] Method A, instrument: GC: Agilent 6890N, FID: detection temperature: 300°C and MS: 5973MSD, EI-positive, detection temperature: 280°C mass range: 50-550; column: RXi-5MS 20m, ID 180μm, df 0.18μm; average velocity: 50cm / s; injection volume: 1μl; injection temperature: 250°C; split ratio: 20 / 1; carrier gas: He; initial temperature: 100°C; initial time: 1.5min; solvent delay: 1.3min; rate 75°C / min; final temperature 250°C; final time 2.5min.

[0326] LC / MS Method:

[0327] SC_ACID, equipment: Agilent 1260Bin. Pump: G1312B, degasser; autosampler, ColCom, DAD: Agilent G1315D, 220-320nm, MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-1000, ELSDAlltech 3300 gas flow rate 1.5ml / min, gas temperature 40°C; column: Waters XSelectTM C18, 30x2.1mm, 3.5μ, temperature: 35°C, flow rate: 1mL / min, gradient: t0=5%A, t 1.6min =98%A,t 3min =98% A, post-run time: 1.3 min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water).

[0328] SC_BASE, equipment: Agilent 1260Bin. Pump: G1312B, degasser; autosampler, ColCom, DAD: Agilent G1315C, 220-320nm, MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-1000; column: Waters XSelectTM CSH C18, 30x2.1mm, 3.5μ, temperature: 25°C, flow rate: 1mL / min, gradient: t0=5%A, t 1.6min =98%A,t 3min =98% A, post-running time: 1.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate aqueous solution (pH=9.5).

[0329] AN_BASE_M1800, equipment: Agilent 1260Bin. Pump: G1312B, degasser; autosampler, ColCom, DAD: Agilent G1315C, 220-320nm, MSD: Agilent LC / MSD G6130B ESI, pos / neg800-1800; column: Waters XSelectTM CSH C18, 50x2.1mm, 3.5μ, temperature: 25°C, flow rate: 0.8mL / min, gradient: t0=5%A, t 3.5min =98%A,t 6min =98% A, post-running time: 2 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate aqueous solution (pH=9.5).

[0330] 1 H-NMR:

[0331] All data were recorded on a Bruker Avance-400 Ultra-Shielded NMR spectrometer using CDCl3 or DMSO-d6 as solvent. 1 H-NMR spectra and are reported in ppm using TMS (0.00 ppm) as an internal standard.

[0332] Example 8: Coupling of HER2-peptide containing linker TMTHSI to azide containing nanoparticles (CriPec)

[0333]

[0334] HER2 peptide-labeled core-crosslinked polymer micelles were produced by conjugating HER2 peptide to the surface of the polymer micelle shell and used for targeting studies. In order to allow HER2 peptide conjugation via click chemistry, azide-functionalized polymer micelles were made essentially according to the reported protocol [Hu et al., Biomaterials. 2015; 53: 370-8], but with the addition of azide-functionalized block copolymer fractions (7.5 kDa, derivatized with 10 mol% crosslinker L2 [1], 5w% of the total block copolymer), instead of (95w%) non-functionalized block copolymers (7.5 kDa, derivatized with 10 mol% crosslinker L2 [Biomaterials. 2015; 53: 370-8], 95w% of the total block copolymer). The two block copolymers were synthesized according to the same procedure, but for the former, azide-PEG was used. 5000 -OH synthesized (azide-PEG 5000)2-ABCPA initiator was replaced. The obtained azide-functionalized polymer micelles were then purified in 20mM ammonium acetate pH 5 buffer containing 130mM NaCl, the buffer was exchanged into 20mM sodium phosphate pH 7.4 buffer containing 130mM NaCl and concentrated to about 30mg / mL polymer equivalent using tangential flow filtration (TFF) equipped with a modified polyethersulfone (mPES) 100kDa module (Spectrumlabs). Then, the HER2 peptide was conjugated to the concentrated azide-functionalized core-crosslinked polymer micelles via copper-free click chemistry using the following method:

[0335] At room temperature, ACN (86 μL) was added to 200 μL of azide-functionalized modified core cross-linked polymer micelles (0.4 μmol azide equivalent), while stirring (300 rpm) in an amber UPLC vial. When the heat dissipated, HER2 peptide (1.0 equivalent, 0.4 μmol, 86 μL, Mercachem) was added dropwise to the reaction mixture. The process of the conjugation reaction was monitored by UPLC-UV for 24 hours, and the reaction was stopped thereafter. Based on UPLC, the conversion rate of the HER2 peptide was determined to be 43%, which was converted into 2.2% HER2 peptide-labeled core cross-linked polymer micelles. After the conjugation reaction, the polymer micelles labeled with HER2 peptides were purified by TFF for 10 v% ethanol to remove unreacted HER2 peptides.

[0336] Example 9: Coupling of HER2-peptide containing linker TMTHSI to azide containing nanoparticles (CriPec) at acidic pH.

[0337] To allow for HER2 peptide conjugation via click chemistry, azide-functionalized polymer micelles were made essentially as reported [Hu et al., Biomaterials. 2015; 53: 370-8], but with the addition of a fraction of azide-functionalized block copolymer (7.5 kDa, derivatized with 10 mol% cross-linker L2 [1], 5 w% of total block copolymer) instead of (95 w%) non-functionalized block copolymer (7.5 kDa, derivatized with 10 mol% cross-linker L2 [Biomaterials. 2015; 53: 370-8], 95 w% of total block copolymer). The two block copolymers were synthesized following the same procedure, but for the former, azide-PEG was used. 5000 -OH synthesized (azide-PEG 5000)2-ABCPA initiator was substituted. The obtained azide-functionalized polymer micelles were then purified and concentrated to about 60 mg / mL polymer equivalent in 20 mM ammonium acetate pH 5 buffer containing 130 mM NaCl using tangential flow filtration (TFF) equipped with a modified polyethersulfone (mPES) 100 kDa module (Spectrumlabs). The HER2 peptide was then conjugated to the concentrated azide-functionalized core-crosslinked polymer micelles via copper-free click chemistry using the following method:

[0338] At room temperature, 150mM ammonium acetate pH 5 (130 μ L) is added to 1500 μ L azide functionalized modified core cross-linked polymer micelles (0.57 μ mol azide equivalent), while stirring (300 rpm) in amber UPLC vials. When heat dissipates, HER2 peptide (5.0 equivalents, 2.85 μ mol, 842 μ L 2mg / ml HER2-TMTH stock solution) is added dropwise to the reaction mixture. The process of conjugation reaction is monitored by UPLC-UV for 24 hours, and the reaction is stopped thereafter. Based on UPLC, the conversion rate of measuring HER2 peptide is 23%, which is converted into 5% HER2 peptide-labeled core cross-linked polymer micelles. After conjugation reaction, unreacted HER2 peptide is removed by TFF for 10v% ethanol to purify the polymer micelles of HER2 peptide labeling.

[0339] Example 10: Comparison of reaction kinetics of model click reactions between TMTHSI and BCNOH with benzyl azide.

[0340] Alkynes (TMTSHI or BCN-OH) were reacted with 1.3 equivalents of benzyl azide, and the reaction kinetics were measured using NMR (CDCl3). The conversion of TMTHSI to benzyl azide reached 79% after 225 seconds, and the reaction with BCN-OH took about 40 times longer (9312 seconds). The reaction conversion (%) was calculated based on the triazole signal, and the level of the triazole signal at the end of the reaction was considered to be 100% level = when all TMTHSI or BCN-OH was completely reacted with benzyl azide. The measurement results are included in Fig.17A TMTHSI (upper curve) is significantly faster than BCN-OH (lower curve). Fig. 17B A zoom-in of the measurement of TMTHSI is shown.

[0341] Example 11: N 1 -(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N 4 -(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -Synthesis of thiol-1-thiol) succinamide.

[0342]

[0343] 4-oxo-4-((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiophene-1-ylidene)amino)butyric acid 2,5-dioxopyrrolidin-1-yl ester (89 mg, 0.22 mmol) in dichloromethane (2 ml) solution was added dropwise to 1,2-bis (2-aminoethoxy) ethane (131 μl, 0.90 mmol, 4 equivalents) in dichloromethane (5 ml) solution over 5 minutes. After the addition was complete, the mixture was stirred at room temperature for 15 minutes. The mixture was then quenched with saturated NH4Cl aqueous solution (5 ml), and the organic phase was moved to a phase separator with a pipette. Each time the aqueous residue was extracted with dichloromethane (2x), the organic phase was moved to a phase separator with a pipette. The combined filtrate was concentrated under reduced pressure to obtain 47.7 mg of crude product. The residue was purified by preparative RP-MPLC (Reveleris, XSelect 10-50% MeCN in water, 10 mM NH4HCO3, pH = 9.5), the product fractions were combined and concentrated to ~15 ml under reduced pressure and transferred to a 50 ml round-bottom flask and lyophilized to give 26 mg (27%) of the product as a white solid.

[0344] LC / MS(SC_BASE)t R 1.77min, purity 99%, mass measured value [M+H] + 430. Fig.18

[0345] 1 H NMR (400MHz, chloroform-d) δ6.73 (t, J=5.4Hz, 1H), 3.68 (d, J=14.1Hz, 2H), 3.63 (s, 4H), 3.59 (d, J=10.3Hz, 2H), 3.57–3.52 (m, 4H), 3.4 5 (q, J=5.3Hz, 2H), 2.90 (t, J=5.1Hz, 2H), 2.69 (t, J=6.9Hz, 2H), 2.48 (t, J=6.9Hz, 2H), 2.16 (bs, 2H), 1.51 (s, 6H), 1.27 (s, 6H).

[0346] Example 12: (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of 2,5-dioxopyrrolidin-1-yl (thiol-1-thiazol-1-ylidene)carbamic acid ester.

[0347]

[0348] N,N'-disuccinimidyl carbonate (52 mg, 0.20 mmol, 2 eq) was charged into an 8 ml screw cap vial and dissolved in acetonitrile (2 ml). To this end, 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ was added dropwise using a syringe. 6 -Thiophene 1-oxide (20mg, 0.1mmol) in acetonitrile (1ml) solution. The mixture was stirred at room temperature for one hour. The mixture was diluted with a mixture of diethyl ether (15ml) and ethyl acetate (15ml) and quenched with water. The organic layer was washed with water again, then washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain 117mg of crude product as a white solid. The residue was purified by flash column chromatography (silicon dioxide 4g; 20-50% EtOAc in heptane solution), the product fractions were combined and concentrated under reduced pressure to obtain 49mg (43%) of the product as a white fluffy solid.

[0349] LC / MS(SC_BASE)t R 1.97min, purity 95%, mass measured value [M+Na] + 363, [2M+Na] + 703.

[0350] 1 H NMR (400 MHz, CHLOROFORM-d) δ 3.92 (d, J = 14.2 Hz, 2H), 3.45 (d, J = 14.2 Hz, 2H), 2.80 (s, 4H), 1.52 (s, 6H), 1.29 (s, 6H).

[0351] TMTHSI was shown to be stable during storage. Compound 12 was stored as a powder in both light and dark locations at atmospheric pressure. 1 H-NMR and LC / MS (SC_BASE) confirmed that the compound was still intact after 4 months of storage.

[0352] Example 13: 2,2,2-trifluoro-N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of thiol-1-yl)acetamide

[0353]

[0354] TMTHSI (50 mg, 0.25 mmol) was loaded into 8 ml screw cap vials, and the material was dissolved in acetonitrile (2 ml). KCO (69 mg, 0.50 mmol, 2 equivalents) was added, followed by trifluoroacetic anhydride (38 μl, 0.27 mmol, 1.1 equivalents). The mixture was stirred at room temperature for one hour. The mixture was diluted with diethyl ether and washed with water (2x). The organic layer was washed with salt water, dried over NaSO and concentrated under reduced pressure to obtain 56 mg of the crude product as a white solid.

[0355] The residue was purified by preparative RP-MPLC (Reveleris Prep, LUNA-C18, 20-60% MeCN in water, 0.1% (v / v) formic acid). The product fractions were combined and lyophilized to give 35 mg (47%) of the product as a white solid.

[0356] LC / MS(SC_BASE)t R 2.18min, purity 99.0%, mass measured value [M+H] + 296.

[0357] 1 H NMR (400 MHz, CHLOROFORM-d) δ 3.86 (d, J = 14.1 Hz, 2H), 3.66 (d, J = 14.1 Hz, 2H), 1.57 (s, 6H), 1.33 (s, 6H).

[0358]

[0359] Example 14: 1-(2-(2-hydroxyethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -Synthesis of thiol-1-yl)urea.

[0360] 2-(2-amino-ethoxy)ethanol (29 μl, 0.29 mmol, 2 eq) and diisopropylethylamine (63 μl, 0.36 mmol, 2.5 eq) were loaded into an 8 ml screw cap vial and dissolved in dichloromethane (2 ml). (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ) was slowly added via pipette. 6The mixture was diluted with water and extracted with dichloromethane (3x), and each organic layer was filtered through a phase separator. The combined organic extracts were concentrated under reduced pressure to give 43 mg of crude product. The residue was purified by preparative RP-MPLC (Reveleris, XSelect 10-50% MeCN in water, 10 mM NH4HCO3, pH = 9.5), and the product fractions were combined and lyophilized to give 12 mg (25%) of the product as a fluffy solid.

[0361] LC / MS(SC_BASE)t R 1.73min, purity 94%, mass measured value [M+H] + 331.

[0362] 1 H NMR (400 MHz, CHLOROFORM-d) δ 5.41-5.30 (m, 1H), 3.84-3.75 (m, 2H), 3.75-3.70 (m, 2H), 3.60-3.53 (m, 4H), 3.48 (d, J=14.1 Hz, 2H), 3.43-3.34 (m, 2H), 1.49 (s, 6H), 1.27 (s, 6H).

[0363]

[0364] Example 15: 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of methyl thiophene-1-yl)sulfamoyl)benzoate.

[0365] An 8 ml screw cap vial was charged with 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6-Thiophene 1-oxide (57mg, 0.28mmol) and dissolved in dichloromethane (1.5ml), pyridine (50μl, 0.62mmol, 2.16 equivalents) was added, and the mixture was stirred for 15 minutes. 3-(chlorosulfonyl) methyl benzoate (81mg, 0.34mmol, 1.5 equivalents) was added, and the mixture was stirred at room temperature for 3 hours. The mixture was quenched in 0.1N HCl aqueous solution (15ml) and extracted with dichloromethane (3x), and each organic extract was filtered through a phase separator. The combined extracts were concentrated under reduced pressure to obtain 100mg of crude product in an oily residue. The residue was purified by preparative RP-MPLC (Reveleris, LUNA-C18, 30-70% MeCN aqueous solution, 0.1% (v / v) formic acid). The product fractions were merged and freeze-dried to obtain 39mg (34%) of the product in the form of a white fluffy solid.

[0366] LC / MS(SC_BASE)t R 2.16min, purity 100%, mass measured value [M+H] + 398.

[0367] 1 H NMR (400MHz, chloroform-d) δ 8.65–8.61 (m, 1H), 8.21 (d, J = 7.8Hz, 1H), 8.17 (d, J = 7.9Hz, 1H), 7.58 (t, J = 7 .8Hz, 1H), 4.08 (d, J=14.3Hz, 2H), 3.95 (s, 3H), 3.41 (d, J=14.2Hz, 2H), 1.49 (s, 6H), 1.28 (s, 6H).

[0368]

[0369] Example 16: 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of thiol-1-yl)sulfamoyl)benzoic acid.

[0370] An 8 ml screw cap vial was charged with 3-(N-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6-thiophene-1-ylidene)sulfamoyl)benzoic acid methyl ester (29 mg, 73 μmol) was added and the material was dissolved in THF (0.5 ml). A solution of LiOH·H2O (7 mg, 167 μmol, 2.3 equiv) in water (0.5 ml). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by preparative RP-MPLC (Reveleris, LUNA-C18, 30-70% MeCN in water, 0.1% (v / v) formic acid). The product fractions were combined and lyophilized to give 26 mg (93%) of the product as a white fluffy solid.

[0371] LC / MS(SC_BASE)t R 1.70min, purity 98%, mass measured value [M+H] + 384.

[0372] 1 H NMR (400MHz, chloroform-d) δ8.70 (t, J=1.8Hz, 1H), 8.26 (d, J=7.7Hz, 1H), 8.22 (d, J=7.8Hz, 1H), 7.6 2 (t, J=7.8Hz, 1H), 4.10 (d, J=14.3Hz, 2H), 3.42 (d, J=14.2Hz, 2H), 1.49 (s, 6H), 1.29 (s, 6H).

[0373]

[0374] Example 17: 3,3,6,6-Tetramethyl-1-(methylimino)-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 -Synthesis of Thiephedrine 1-oxide.

[0375] An 8 ml screw cap vial was charged with 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6-Thiophene 1-oxide (50mg, 0.25mmol) and dissolved in THF (1ml), the solution was treated with KOtBu (1.7M in THF; 200μl, 1.35 equivalents). The resulting suspension was stirred for 5 minutes, then iodomethane (30μl, 0.48mmol, 1.9 equivalents) was added, and the mixture was stirred at room temperature for 2 hours. The mixture was diluted with diethyl ether and quenched with water. The layers were separated and the aqueous layer was extracted with diethyl ether (2x). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain 44mg of a crude product in the form of a transparent oil. The residue was purified by preparative RP-MPLC (Reveleris, XSelect 10-50% MeCN aqueous solution, 10mM NH4HCO3, pH=9.5), and the product fractions were combined and concentrated under reduced pressure. The residue was diluted with acetonitrile and lyophilized to give 31.3 mg (58%) of the product as a white solid.

[0376] LC / MS(SC_BASE)t R 1.79min, purity 99%, mass measured value [M+H] + 214.

[0377] 1 H NMR (400MHz, chloroform-d) δ 3.32 (d, J=13.9Hz, 2H), 3.07 (d, J=14.0Hz, 2H), 2.84 (s, 3H), 1.43 (s, 6H), 1.28 (s, 6H).

[0378]

[0379] Example 18: 4-((3,3,6,6-tetramethyl-1-(methylimino)-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6 Synthesis of methyl thiophene-2-yl)methyl)benzoate.

[0380] An 8 ml screw cap vial was charged with 3,3,6,6-tetramethyl-1-(methylimino)-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6-Thiophene 1-oxide (31mg, 0.14mmol) and dissolved in anhydrous THF (1ml). LiHMDS (1M in THF; 174μl, 0.17mmol, 1.2 equivalents) was added, and the mixture was stirred at room temperature for 15 minutes, then 4-(bromomethyl) methyl benzoate (50mg, 0.22mmol, 1.5 equivalents) was added and the mixture was stirred at room temperature for 20 hours. The mixture was quenched in water and extracted with diethyl ether (3×10ml). The combined organic layer was washed with brine and concentrated under reduced pressure to obtain a semi-solid residue. The residue was purified by preparative RP-MPLC (Reveleris, LUNA-C18, 30-70% MeCN aqueous solution, 0.1% (v / v) formic acid). The product fractions were combined and freeze-dried to obtain 9mg (17%) of the product in the form of a white fluffy solid.

[0381] LC / MS(SC_BASE)t R 2.20min, purity 97%, mass measured value [M+H] + 362.

[0382] 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.97 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 15.7 Hz, 1H), 6.25 (d, J = 15.8 Hz, 1H), 3.91 (s, 3H), 3.81-3.69 (m, 1H), 3.02 (d, J = 12.8 Hz, 1H), 2.83-2.76 (m, 4H), 1.44 (s, 3H), 1.39 (s, 3H), 1.37 (s, 6H).

[0383]

[0384] Example 19: 4-(((3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of methyl thiophene-1-yl)amino)methyl)benzoate.

[0385] An 8 ml screw cap vial was charged with 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ 6-Thiophene 1-oxide (75 mg, 0.37 mmol) and dissolved in anhydrous THF (1.5 ml). KOtBu (1.7 M in THF; 280 μl, 0.47 mmol, 1.26 eq.) was added and the resulting mixture was stirred at room temperature for 15 minutes. Methyl 4-(bromomethyl)benzoate (129 mg, 0.56 mmol, 1.5 eq.) was added to the resulting suspension and the mixture was stirred at room temperature for 6 hours. The mixture was quenched in water and extracted with diethyl ether (3×15 ml), the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give 128 mg of crude product as a semi-solid residue. The residue was purified by preparative RP-MPLC (Reveleris, LUNA C18, 30-70% MeCN aqueous solution, 0.1% (v / v) formic acid). The product fractions were combined and lyophilized to give 28 mg (21%) of the product as an oily residue.

[0386] LC / MS(SC_BASE)t R 2.14min, purity 86%, mass measured value [M+H] + 348.

[0387] 1 H NMR (400MHz, chloroform-d) δ7.99 (d, J=8.3Hz, 2H), 7.46 (d, J=8.0Hz, 2H), 4.39 (s, 2H), 3.9 1 (s, 3H), 3.35 (d, J=14.0Hz, 2H), 3.18 (d, J=14.1Hz, 2H), 1.45 (s, 6H), 1.28 (s, 6H).

[0388]

[0389] Example 20: Synthesis of tert-butyl (2-((2-aminoethyl)disulfanyl)ethyl)carbamate.

[0390] A 250 ml round bottom flask was charged with bis-(2-aminoethyl) disulfide dihydrochloride (1.49 g, 6.62 mmol) and MeOH (75 ml). Triethylamine (2.89 ml, 20.8 mmol, 3.15 eq) was added, and when all solids were dissolved, di-tert-butyl dicarbonate (1.45 g, 6.64 mmol, 1 eq) was added. The mixture was stirred at room temperature for about 1 hour.

[0391] The mixture was quenched by adding 1M NaH2PO4 aqueous solution (50ml), and the organic solvent was removed by evaporation. The aqueous residue was washed with diethyl ether (3×30ml) and then made alkaline using 1N NaOH aqueous solution (~70ml). The aqueous mixture was extracted with diethyl ether (3×60ml). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure to obtain 0.71g of colorless oil. The aqueous layer was further extracted with dichloromethane (3×50ml), the combined organic extracts were dried over Na2SO4, and concentrated with the amount previously separated to obtain 0.78g (46%) of the product in a transparent viscous oily state.

[0392] LC / MS(SC_BASE)t R 1.83min, purity 98%, mass measured value [M+H] + 253.

[0393] 1 H NMR (400 MHz, CHLOROFORM-d) δ 4.93 (s, 1H), 3.53-3.39 (m, 2H), 3.03 (t, J=6.2 Hz, 2H), 2.84-2.74 (m, 4H), 1.62 (s, 2H), 1.45 (s, 9H).

[0394]

[0395] Example 21: Synthesis of 2,2-dimethyl-4,13-dioxo-3-oxa-8,9-dithia-5,12-diazahexadecane-16-oic acid.

[0396] A 250 ml round bottom flask was charged with tert-butyl (2-((2-aminoethyl)disulfanyl)ethyl)carbamate (0.78 g, 3.09 mmol) and the material was dissolved in dichloromethane (25 ml). DMAP (0.11 g, 0.93 mmol, 0.3 eq) was added followed by succinic anhydride (0.3 g, 3.09 mmol, 1 eq). The resulting mixture was stirred at room temperature for 1 hour. The mixture was quenched with 1 N KHSO4 aqueous solution (30 ml), the layers were separated, and the organic layer was washed with 1 N KHSO4 aqueous solution (~30 ml). The combined aqueous layers were extracted with dichloromethane (30 ml), the combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give 1.02 g (94%) of the crude product as a solid.

[0397] LC / MS(SC_ACID)t R 1.80min, purity 100%, mass measured value [M+Na] + 375, [MH]- 351.

[0398] 1 H NMR (400MHz, chloroform-d) δ 11.57 (bs, 1H), 7.02 (t, J = 5.9Hz, 1H), 5.07 (t, J = 6.3Hz, 1H), 3.59 (q, J = 5.9Hz, 2H), 3.53– 3.34 (m, 2H), 2.86 (t, J=6.0Hz, 2H), 2.77 (t, J=7.2Hz, 2H), 2.73–2.66 (m, 2H), 2.64–2.47 (m, 2H), 1.45 (s, 9H).

[0399]

[0400] Example 22: Synthesis of 4-((2-((2-aminoethyl)disulfanyl)ethyl)amino)-4-oxobutanoic acid trifluoroacetate.

[0401] 2,2-dimethyl-4,13-dioxo-3-oxa-8,9-dithiazole-5,12-diazahexadecane-16-acid (1.02g, 2.89mmol) is loaded into a 250ml round-bottom flask. The material is dissolved in dichloromethane (5ml) and treated with trifluoroacetic acid (3ml, 39.2mmol, 13.5 equivalents). The reactant is stirred at room temperature for about 1 hour. The reactant is concentrated under reduced pressure, co-evaporated with toluene, dichloromethane and diethyl ether, to obtain 1.4g (132%). The residue is dissolved in water and washed with dichloromethane (3x). The water layer is freeze-dried to obtain 1.04g (98%) of the product in a viscous transparent oily state.

[0402] 1 H NMR (400 MHz, methanol-d4) δ 3.51 (dt, J = 8.4, 6.4 Hz, 2H), 3.36–3.23 (m, 2H, consistent with the residual peak of methanol-d4), 2.99 (dq, J = 8.4, 6.1, 5.6 Hz, 2H), 2.86 (dt, J = 8.8, 6.6 Hz, 2H), 2.61 (dt, J = 8.5, 6.4 Hz, 2H), 2.49 (dt, J = 8.7, 6.5 Hz, 2H).

[0403]

[0404] Example 23: 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of thiol-1-yl)ureido)ethyl)disulfanyl)ethyl)amino)butyric acid.

[0405] An 8 ml screw cap vial was charged with (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiol-1-ylidene)carbamic acid 2,5-dioxopyrrolidin-1-yl ester (98 mg, 0.29 mmol) was added and the material was suspended / dissolved in acetonitrile (1 ml). DIPEA (115 μL, 0.66 mmol, 2.3 eq) was added followed by a solution of 4-((2-((2-aminoethyl)disulfanyl)ethyl)amino)-4-oxobutanoic acid TFA salt (113 mg, 0.31 mmol, 1.07 eq) in water (0.5 ml) to form a suspension. An additional amount of acetonitrile (0.5 ml) was added and the mixture was stirred at room temperature for 20 hours.

[0406] The reaction mixture was purified by preparative RP-MPLC (Reveleris Prep, LUNA-C18, 10-50% MeCN in water, 0.1% (v / v) formic acid). The product fractions were combined and lyophilized to give 38 mg (28%) of the product.

[0407] LC / MS(SC_ACID)t R 1.77min, purity 96%, mass measured value [M+H] + 478.

[0408] 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.73 (s, 1H), 6.52 (s, 0.2H), 6.00 (s, 0.8H), 3.91-3.76 (m, 2H), 3.58-3.38 (m, 6H), 2.92-2.73 (m, 4H), 2.59-2.36 (m, 4H), 1.49 (s, 6H), 1.31 (s, 6H).

[0409]

[0410] Example 24: 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of 2,5-dioxopyrrolidin-1-yl (thiop-1-ylidene)ureido)ethyl)disulfanyl)ethyl)amino)butyric acid 2,5-dioxopyrrolidin-1-yl ester.

[0411] An 8 ml screw cap vial was charged with 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6-thiophene-1-ylidene)urea)ethyl)disulfanyl)ethyl)amino)butyric acid (38.8 mg, 0.081 mmol) and dichloromethane (1 ml) were added. N-hydroxysuccinimide (13 mg, 0.11 mmol, 1.4 equiv) was added followed by EDC (22 mg, 0.11, 1.4 equiv). The mixture was stirred at room temperature for about 2 hours. The mixture was concentrated under reduced pressure and the resulting white foam was subjected to preparative RP-MPLC (Reveleris Prep, LUNA-C18, 20-60% MeCN in water, 0.1% (v / v) formic acid). The product fractions were combined and lyophilized. The freeze-dried material was dissolved in dichloromethane and transferred to an 8 ml brown glass vial, the solvent was evaporated under a stream of N2 and co-evaporated with diethyl ether to give 35 mg (76%) of the product.

[0412] LC / MS(SC_ACID)t R 1.86min, purity 93%, mass measured value [M+H] + 575.

[0413] 1 H NMR (400 MHz, CHLOROFORM-d) δ 6.73 (s, 1H), 5.32 (t, J = 6.1 Hz, 1H), 3.78 (d, J = 14.1 Hz, 2H), 3.58 (q, J = 6.1 Hz, 2H), 3.53-3.41 (m, 4H), 3.00 (t, J = 7.1 Hz, 2H), 2.89-2.76 (m, 8H), 2.67 (t, J = 7.1 Hz, 2H), 1.47 (s, 6H), 1.28 (s, 6H).

[0414]

[0415] Example 25: Coupling of TMTHSI-disulfide NHS ester to siRNA oligonucleotides. Preparation of TMTHSI-NH-siRNAPLK1.

[0416] 2 ml UPLC vials were loaded with siRNA PLK1 oligonucleotides (5 mg, 0.36 μmol) and borate buffer pH 8.4 (250 μl) was added. 20 mM 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ was prepared. 65 equivalents (90 μl, 1.04 mg, 1.8 μmol) of 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ) ...3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ)-4-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ)-4-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ)-4-((2-(3-(3,3,6,6-tetramethyl-1-oxo- 6 After additional stirring, the reaction showed >90% conversion to TMTHSI-NH-siRNAPLK1. The excess TMTHSI NHS 4-oxo-4-((2-((2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ) was removed by purification by PD-10 (buffer exchange to phosphate buffer, pH 7.4) and Vivaspin (5000Da MWCO, 4000g, 3 times phosphate buffer pH 7.4, final concentration step). 6 The final solution of TMTHSI-NH-siRNA PLK1 (1000 μL) was transferred to a 2 ml UPLC vial and stored at +4C.

[0417]

[0418] Example 26: Coupling of Azide-CPP1 Peptide to TMTHSI-NH-siRNA Oligonucleotide Preparation of CPP1-TMTHSI-NH-siRNA PLK1 Conjugate.

[0419] First, prepare the acetonitrile / water (1:1, 100 μl volume) stock solution of azide-CPP1 peptide (0.49 mg, 0.35 μmol). Initially, 50 μl (0.5 equivalent, 0.25 mg, 0.18 μmol) of the stock solution was added to a 2 ml UPLC vial containing TMTHSI-NH-siRNAPLK1 (0.35 μmol in 1000 μl phosphate buffer pH 7.4). The mixture was stirred at room temperature and monitored by UPLC. After 30 minutes, all azide-CPP1 peptides were converted, and then the second half of the azide-CPP1 peptide stock solution (0.5 equivalent, 0.25 mg, 0.18 μmol) was added. After stirring for another 30 minutes, the reaction to CPP1-TMTHSI-NH-siRNA PLK1 reached complete conversion. The final crude product was washed (5000Da MWCO, 4000g, 3 times phosphate buffer pH 7.4, final concentration step) and concentrated in Vivaspin centrifuge tubes. The final solution of CPP1-TMTHSI-NH-siRNA PLK1 (750 μl) was transferred to a 2 ml UPLC vial.

[0420]

[0421] Example 27: Coupling of acid-labile linker 7 to CPP1-TMTHSI-siRNA oligonucleotide. Preparation of linker 7-CPP1-TMTHSI-NH-siRNA PLK1 conjugate.

[0422] A solution of CPP1-TMTHSI-NH-siRNA PLK1 (750 μl) was transferred to a Vivaspin centrifuge tube and the buffer was exchanged into borate buffer pH 8.4 (5000Da MWCO, 4000g, 3 times borate buffer pH 8.4, final concentration step). Final volume after Vivaspin: 1200 μl. A stock solution of 20 mM hydrazone MA linker NHS ester (linker 7) was prepared in DMSO. While stirring, 5 equivalents of hydrazone MA linker NHS ester stock solution (66 μl, 0.73 mg, 1.6 μmol) in DMSO were added to CPP1-TMTHSI-siRNA (0.32 μmol) in borate buffer (1000 μl) at room temperature and monitored by UPLC. After 45 minutes, the reaction showed complete conversion to linker 7-CPP1-TMTHSI-siRNAPLK1 conjugate. The final crude conjugate was purified by PD-10 (5000Da MWCO, 4000g, buffer exchange to phosphate buffer pH 7.4) and Vivaspin (5000Da MWCO cutoff, 4000g, 3 times phosphate buffer pH 7.4, final concentration step) to remove excess hydrazone MA linker NHS ester.

[0423]

[0424] Example 29: Synthesis of (S)-15-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazahexadecane-16-oic acid. (The product was isolated as a mixture of regioisomers)

[0425] Folic acid (1.84 g, 4.17 mmol) was suspended in anhydrous dimethyl sulfoxide (15 ml) and the flask was placed in a sand bath at 100°C until a solution was formed. The solution was then allowed to cool to room temperature and N-hydroxysuccinimide (503 mg, 4.37 mmol, 1.05 eq) was added, followed by a solution of N-Boc-2-(2-amino-ethoxy)-ethylamine (852 mg, 4.17 mmol) in anhydrous dimethyl sulfoxide (3 ml). The mixture was stirred for 10 minutes, then EDC (840 mg, 4.38 mmol, 1.05 eq) was added and the mixture was stirred at room temperature for 17 hours.

[0426] The reaction mixture was purified (in 7 batches) by preparative RP-MPLC (Reveleris, XSelect 5-30% MeCN in water, 10 mM NH4HCO3, pH = 9.5). The product fractions were combined and partially concentrated under reduced pressure. The resulting residue was lyophilized to give 0.91 g (34%) of the product.

[0427] LC / MS(SC_BASE)t R 1.37min, purity 100%, mass measured value [MH] - 626.

[0428] 1 H NMR (400MHz, DMSO-d6+D2O) δ8.66 (s, 1H), 7.99 (q, J=4.9Hz, 1H), 7.65 (dd, J=11.6, 8.5Hz, 2H), 6.75 (d, J=6.1Hz, 1H), 6.67 (dd, J=9.0, 2.7Hz, 2H), 4.52 (s, 2H), 4.33 (dd, J=8.9, 4.8H z, 0.4H), 4.19 (dd, J = 8.1, 4.7 Hz, 0.6H), 3.45-3.31 (m, 4H), 3.26-3.13 (m, 2H), 3.11-3.01 (m, 2H), 2.26 (t, J = 7.1, 6.4 Hz, 1H), 2.15 (t, J = 8.0 Hz, 1H), 2.08-1.82 (m, 2H), 1.35 and 1.33 (2x s, combined 9H).

[0429]

[0430] Example 30: N 2 -(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -Synthesis of (2-(2-aminoethoxy)ethyl)-L-glutamine. (The product is separated as a mixture of α / β regioisomers)

[0431] (S)-15-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazahexadecane-16-oic acid (403 mg, 0.64 mmol) was dissolved in trifluoroacetic acid (2 ml, 0.64 mmol) and the resulting mixture was stirred at room temperature for 25 minutes. The reaction mixture was concentrated under reduced pressure and the residue was co-evaporated with dichloromethane (2x). The residue was placed in N,N-dimethylformamide (1 ml) and warmed until completely dissolved, then allowed to cool to room temperature. The solution was treated with triethylamine (3×200 μl) to give an orange precipitate. The mixture was diluted with acetone and the solid was collected by filtration. The residue was washed with acetone (3x) and air-dried to give 346 mg (97%) of the product as an orange solid.

[0432] 1 H NMR (400MHz, DMSO-d6) δ8.66–8.57 (m, 1H), 8.24 (t, J=5.5Hz, 0.6H), 8.07 (t, J=5.6Hz, 0.4H), 7.72–7. 62(m, 1H), 7.62–7.51(m, 1H), 7.30(bs, 2H), 6.91(q, J=6.1Hz, 1H), 6.63(t, J=7.6Hz, 2H), 4.52–4.38(m , 2H), 4.30 (q, J=6.3Hz, 0.4H), 4.10 (q, J=6.4Hz, 0.6H), 3.55 (q, J=6.2, 5.6Hz, 3H), 3.43 (bs, 3H), 3.3 1–3.13 (m, 4H), 2.94 (q, J=6.2, 5.8Hz, 2H), 2.21–2.11 (m, 2H), 2.06–1.79 (m, 3H), 0.97 (t, J=7.2Hz, 1H).

[0433] NMR showed a very broad HOD signal at signal between 4.5 ppm and 2.5 ppm.

[0434]

[0435] Example 31: N 2 -(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(2-(2-(3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6Synthesis of (-thiol-1-yl)urea)ethoxy)ethyl)-L-glutamine. (The product is separated as a mixture of α / β regioisomers)

[0436] N 2 -(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzoyl)-N 5 -(2-(2-aminoethoxy)ethyl)-L-glutamine (90.3 mg, 0.13 mmol) was dissolved in hot dimethyl sulfoxide (1.5 ml) and allowed to cool to room temperature. Triethylamine (107 μl, 0.77 mmol, 6 equiv) was added and the resulting mixture was added to (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 The reaction mixture was stirred at room temperature for 4 days.

[0437] The reaction mixture was purified by preparative RP-MPLC (Reveleris, XSelect 5-40% MeCN in water, 10 mM NH4HCO3, pH = 9.5). The product fractions were combined and lyophilized to give 51 mg (53%) of the product.

[0438] LC / MS(SC_BASE)t R 1.45min, purity 99%, mass measured value [M+H] + 753.

[0439] 1 H NMR (400MHz, DMSO-d6+D2O) δ8.66 (s, 1H), 8.02–7.90 (m, 1H), 7.70–7.59 (m, 2H), 6.72–6.61 (m, 3H), 4.50 (s, 2H), 4.37-4.32 (m, 0.4H), 4.22–4.13 (m, 0.6H), 3.90–3.79 (m, 2H), 3.59 (dd, J=14. 0, 4.5Hz, 2H), 3.44–3.30 (m, 5H), 3.25–3.14 (m, 2H), 3.09 (t, J=5.8Hz, 2H), 2.26 (t, J=7.7Hz, 1 H), 2.15 (t, J=7.6Hz, 1H), 2.05-1.93 (m, 1H), 1.90 (d, J=13.5Hz, 1H), 1.33 (s, 6H), 1.19 (s, 6H).

[0440] Example 32: Coupling of TMTHSI-containing linker folate to azide-containing nanoparticles (CriPec).

[0441]

[0442] Folic acid-labeled core-crosslinked polymer micelles were produced by conjugating folic acid to the surface of the polymer micelle shell and used for targeting studies. In order to allow folic acid conjugation via click chemistry, azide-functionalized polymer micelles were made essentially according to the reported protocol [Hu et al., Biomaterials. 2015; 53; 370-8], but a fraction of azide-functionalized block copolymer (22 kDa, derivatized with 10 mol% crosslinker L2 [1], 5w% of the total block copolymer) was added instead of (95w%) non-functionalized block copolymer (22 kDa, derivatized with 10 mol% crosslinker L2 [Biomaterials. 2015; 53: 370-8], 95w% of the total block copolymer). The two block copolymers were synthesized according to the same procedure, but for the former, azide-PEG was used. 5000 -OH synthesized (azide-PEG 5000 )2-ABCPA initiator was replaced. The obtained azide-functionalized polymer micelles were then purified in 20 mM ammonium acetate pH 5 buffer containing 130 mM NaCl, the buffer was exchanged into 20 mM sodium phosphate pH 7.4 buffer containing 130 mM NaCl and concentrated to about 96 mg / mL polymer equivalent using tangential flow filtration (TFF) equipped with a modified polyethersulfone (mPES) 100 kDa module (Spectrumlabs). Folic acid was then conjugated to the concentrated azide-functionalized core-crosslinked polymer micelles via copper-free click chemistry using the following method:

[0443] At room temperature, DMSO (93 μL) was added to 200 μL of azide-functionalized modified core cross-linked polymer micelles (0.13 μmol azide equivalents), while stirring (300 rpm) in an amber UPLC vial. When the heat dissipated, folic acid (1.0 equivalent, 0.13 μmol, 5 μL, Mercachem) was added dropwise to the reaction mixture. The progress of the conjugation reaction was monitored by UPLC-UV for 4 hours, after which the reaction was stopped. Based on UPLC, the conversion rate of folic acid was determined to be 45%, which was converted into 2.3% folic acid-labeled core cross-linked polymer micelles.

[0444]

[0445] Example 33: 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -Synthesis of thiol-1-yl)urea.

[0446] Under N2 atmosphere, (3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1,6-thiaheptane-1-ylidene)carbamic acid 2,5-dioxopyrrolidin-1-yl ester (197mg, 0.58mmol) was dissolved in dichloromethane (12.5ml), and 2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-1-amine) (0.42ml, 2.89mmol, 5 equivalents) was added. The reactant was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was placed in a 1:1 mixture of acetonitrile / water (4ml) and purified by preparative RP-MPLC (Reveleris, XSelect 10-50% MeCN aqueous solution, 10mM NH4HCO3, pH=9.5) (in two batches). The product fractions were combined and lyophilized to give 156 mg (72%) of the product as a white fluffy solid.

[0447] LC / MS(SC_BASE)t R 1.67min, purity 99%, mass measured value [M+H] + 374.

[0448] 1 H NMR (400MHz, DMSO-d6+D2O) δ3.86 (d, J=14.0Hz, 2H), 3.61 (d, J=13.9Hz, 2H), 3.54–3.46 (m, 4H ), 3.40 (q, J=6.0Hz, 4H), 3.15–3.04 (m, 3H), 2.67 (t, J=5.7Hz, 1H), 1.34 (s, 6H), 1.21 (s, 6H).

[0449]

[0450] Example 34: 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 Synthesis of thiol-1-thiol) urea-Cy7 adduct formate.

[0451] An 8 ml screw cap vial was charged with Cy7-NHS ester (95 mg, 0.13 mol) and MeCN (1.5 ml), 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3,3,6,6-tetramethyl-1-oxo-4,5-didehydro-2,3,6,7-tetrahydro-1λ 6 -thiophene-1-ylidene) urea (66.5mg, 0.18mmol, 1.37 equivalents) in MeCN (1.5ml) solution, and the mixture was stirred at room temperature for 2 hours. The mixture was then stored overnight at -20°C. The mixture was allowed to warm to room temperature and then purified by preparative MPLC (Reveleris Prep, LUNA-C18, 20-60% MeCN aqueous solution, 0.1% (v / v) formic acid), the product fractions were concentrated under reduced pressure, and the residue was diluted with a small amount of MeCN and lyophilized overnight to obtain 95mg (77%) of the product as a dark green solid.

[0452] LC / MS(AN_ACID_DAY_UV800)t R 3.46min, purity 88%, mass measured value [M] + 905, [M+H] 2+ 453.

[0453] 1 H NMR (400MHz, methanol-d4) δ8.37 (s, 1H), 8.02-7.95 (m, 0.5H), 7.75 (dd, J=14.0, 8.5Hz, 1.5H), 7.47 (d, J=7.5Hz, 2H), 7.43–7.34 (m, 1H), 7. 31–7.20 (m, 3H), 7.15–6.92 (m, 1H), 6.81–6.34 (m, 2H), 6.24–6.09 (m, 1H), 4.11 (t, J=7.3Hz, 1H), 3.86 (d, J=14.1Hz, 2H), 3.66–3.55 (m , 8H), 3.55–3.45(m, 5H), 3.39–3.32(m, 3H), 3.29–3.21(m, 2H), 2.57(q, J=5.6Hz, 3H), 2.50–2.28(m, 1H), 2.27–2.15(m, 2H), 2.00–1. 89(m, 2H), 1.89–1.74(m, 3H), 1.75–1.61(m, 10H), 1.59–1.52(m, 2H), 1.51–1.42(m, 2H), 1.40(s, 7H), 1.33–1.20(m, 8H), 1.03(s, 1H).

[0454] Method: AN_ACID_DAY_UV800, Equipment: Agilent 1260Bin, Pump: G1312B, Degasser; Autosampler, ColCom, DAD: Agilent G1315D, 210–800 nm, MSD: Agilent LC / MSD G6130B ESI, pos / neg100-1000, ELSD Alltech 3300, Gas flow rate 1.5 ml / min, Gas temperature 40 °C; Column: Waters XSelect TM C18, 50x2.1 mm, 3.5 μ, temperature: 35 °C, flow rate: 0.8 mL / min, gradient: t0 = 5% A, t 3.5min =98%A,t 6min =98% A, post-run time: 2 min; eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water).

[0455] MS parameters

[0456] Source: ESI, capillary voltage: 3000 V, drying gas flow rate: 12 L / min, nebulizer pressure 60 psig, drying gas temperature: 350°C, Fragmentor 70, MS scan: MS range 100-1000 (positive and negative mode), scan speed: 0.84 sec / cycle.

[0457] Example 35: Reaction kinetics of TMTHSI with benzyl azide to determine the value of the reaction rate constant kt.

[0458] The kt values ​​of the conversion of benzyl azide with BCN-OH and TMTHSI were studied by monitoring the reaction by mass spectrometry. In this study, a QTOF mass spectrometer with an ESI source connected to it was used as the measurement method. In detail, alkynes (TMTSHI or BCN-OH) were reacted with 1.0 equivalents of benzyl azide in ACN / water / acetic acid (3:1:0.01%), and the reaction kinetics were measured using MS. The measurement results are included in Fig.55 In the reaction with benzyl azide, the kt value of TMTHSI was measured to be 0.83 M -1 S -1 , which is higher than BCN-OH (0.12M -1 S -1 ) is about 7 times faster.

[0459] References

[0460] -A. Krebs and H. Kimling, Tetrahedron Lett., 1970, 761–764.

[0461] -de Almeida, G., Sletten, E.M., Nakamura, H., Palaniappan, K.K., Bertozzi, C.R., Angew. Chem. Int. Ed., 2012, 51, 2443 - 2447.

[0462] -Li et al., Molecules, 2016, 21, 1393.

[0463] -Tota, A., Zenzola, M., Chawner, S.J., St John-Campbell, S., Carlucci, C., Romanazzi, G., Degennaro, L., Bull, J.A., Luisi, R., Chem. Commun., 2017, 53, 348 - 351.

[0464] -Hu et al., Biomaterials. 2015; 53:370 - 8.

[0465] -King et al., Chem Comm, 2012, 48, 9308 - 9309.

[0466] -Dommerholt et al., Nature Communications 5, Article number: 5378 (2014_).

[0467] -Dommerholt et al., Top Curr Chem(Z) 2016, 374:16.

Claims

1. Compounds of formula (II): in: X is O; Y is NR 10 , where R 10 It is H; R 1 , R 2 , R 3 and R 4 It is methyl; R 5 , R 6 , R 7 and R 8 It's hydrogen.

2. Compounds of formula (III): (Formula II)-LQ (III) wherein formula II is as defined in claim 1, wherein Y or an atom adjacent to the S atom in the thiocycloheptyne ring is coupled to the functional group (Q) or to the linker (L) and the functional group (Q) to produce the compound of formula (III), in L is absent or selected from a linear or branched C1-C 24 Alkylene, C2-C 24 Alkenylene, C2-C 24 Alkynylidene, C3-C 24 Cycloalkylene, C5-C 24 Cycloalkenylene, C5-C 24 Cycloalkynylene, C7-C 24 Alkyl arylene, C7-C 24 Alkyl heteroarylene, C7-C 24 Aryl alkylene, C7-C 24 Heteroarylalkylene, C5-C 24 Arylene, C5-C 24 Heteroarylalkenylene, C9-C 24 Aryl alkynylene and C9-C 24 wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, alkylheteroarylene, arylalkylene, heteroarylalkylene, arylalkenylene, heteroarylalkenylene, arylalkynylene and heteroarylalkynylene are unsubstituted or substituted with one or more substituents independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 Cycloalkynyl, C8-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy, C3-C 12 Cycloalkoxy, halogen, amino and silyl, wherein the silyl group can be represented by the formula (R 11 )3Si- represents, where R 11 Independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C 12 Cycloalkyl, C1-C 12 Alkoxy, C2-C 12 Alkenyloxy, C2-C 12 Alkynyloxy and C3-C 12 Cycloalkoxy, wherein the alkyl, the alkoxy, the cycloalkyl and the cycloalkoxy are optionally interrupted by one of a plurality of heteroatoms selected from O, N, P and S, and Q is selected from halogen, R 12 、-CH=C(R 12 2. -C≡CR 12 , wherein q is 1 to 200 -[C(R 12 )2C(R 12 )2O] q -R 12 , -CN, -N3, -NCX, -XCN, -XR 12 、-N(R 12 )2.- + N(R 12 )3. -C(X)N(R 12 )2、-C(R 12 )2XR 12 、-C(X)R 12 、-C(X)XR 12 、-S(O)R 12 、-S(O)2R 12 、-S(O)OR 12 、-S(O)2OR 12 、-S(O)N(R 12 )2、-S(O)2N(R 12 )2. -OS(O)R 12 、-OS(O)2R 12 、-OS(O)OR 12 、-OS(O)2OR 12 、-P(O)(R 12 )(OR 12 )、-P(O)(OR 12 )2.-OP(O)(OR 12 )2、-Si(R 12 )3. -XC(X)R 12 、-XC(X)XR 12 、-XC(X)N(R 12 )2、-N(R 12 )C(X)R 12 、-N(R 12 )C(X)XR 12 and -N(R 12 )C(X)N(R 12 )2, wherein X is oxygen or sulfur, and wherein R 12 independently selected from hydrogen, C1-C 24 Alkyl, C6-C 24 Aryl, C6-C 24 Heteroaryl, C7-C 24 Alkyl aryl, C7-C 24 Alkyl heteroaryl, C7-C 24 Arylalkyl and C7-C 24 Heteroarylalkyl.

3. Compounds of formula (III): (Formula II)-LQ (III) wherein formula II is as defined in claim 1, wherein Y or an atom adjacent to the S atom in the thiocycloheptyne ring is coupled to the functional group (Q) or to the linker (L) and the functional group (Q) to produce the compound of formula (III), in L does not exist, Q is selected from -OR 12 , -N(R 12 )2, - + N(R 12 )3, -C(O)N(R 12 )2, -C(O)OR 12 , -OC(O)R 12 , -OC(O)OR 12 , -OC(O)N(R 12 )2, -N(R 12 )C(O)R 12 , -N(R 12 )C(O)OR 12 and -N(R 12 )C(O)N(R 12 )2, Where R 12 independently selected from hydrogen, C1-C 24 Alkyl, C6-C 24 Aryl, C6-C 24 Heteroaryl, C7-C 24 Alkyl aryl, C7-C 24 Alkyl heteroaryl, C7-C 24 Arylalkyl and C7-C 24 Heteroarylalkyl.

4. A compound comprising the compound of claim 2 or 3, wherein the functional group Q is coupled to a target molecule.

5. The compound of claim 4, wherein the target molecule is one or more of a drug, a protein, a peptide and an oligonucleotide.

6. The compound of claim 4, wherein the target molecule is one or more of an antibody, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, and a carrier compound.

7. A compound according to any one of claims 1 to 3 coupled to a compound containing a 1,3-dipole, a 1,3-diene or a 1,3-heterodiene, wherein the alkynyl group of the compound of formula (II) is coupled to the compound containing a 1,3-dipole, a 1,3-diene or a 1,3-heterodiene.

8. The compound of claim 7, wherein the compound containing a 1,3-dipole or a 1,3-diene or a 1,3-heterodiene is an azide-containing compound, a nitrone-containing compound, or a nitrile-oxide-containing compound, and the compound containing a 1,3-dipole, a 1,3-diene, or a 1,3-heterodiene comprises a target molecule.

9. The compound of claim 7, wherein the 1,3-dipole, 1,3-diene or 1,3-heterodiene containing compound is an azide containing compound and the azide-alkyne coupling results in the formation of a triazole compound.

10. The compound of claim 8, wherein the target molecule is one or more of a drug, a protein, a peptide and an oligonucleotide.

11. The compound of claim 8, wherein the target molecule is one or more of an antibody, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, and a carrier compound.

12. A method for coupling a compound as claimed in any one of claims 1 to 6 with a compound containing a 1,3-dipole, a 1,3-diene or a 1,3-heterodiene, wherein the coupling is a copper-free click reaction, and wherein the compound containing a 1,3-dipole, a 1,3-diene or a 1,3-heterodiene comprises one or more of a drug, a protein, a peptide and an oligonucleotide.

13. A method for coupling a compound as claimed in any one of claims 1 to 6 with a compound containing a 1,3-dipole, a 1,3-diene or a 1,3-heterodiene, wherein the coupling is a copper-free click reaction, and wherein the compound containing a 1,3-dipole, a 1,3-diene or a 1,3-heterodiene comprises one or more of an antibody, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle and a carrier.

14. The method of claim 12 or 13, wherein the 1,3-dipole, 1,3-diene or 1,3-heterodiene containing compound is an azide containing compound, a nitrone containing compound or a nitrile oxide containing compound.

15. A method for preparing a construct comprising a nanoparticle and an active compound selected from the group consisting of a drug, a protein, a peptide, and an oligonucleotide, wherein the coupling of the nanoparticle and the active compound comprises coupling a compound as described in any one of claims 1 to 6 with an azide-containing compound to form a triazole compound, wherein the nanoparticle is the azide-containing compound and the active compound comprises the compound as described in any one of claims 1 to 6, or wherein the nanoparticle comprises the compound as described in any one of claims 1 to 6 and the active compound comprises the azide-containing compound.

16. A method for preparing a construct comprising a nanoparticle and an active compound selected from the group consisting of an antibody, an imaging marker, a targeting ligand, a delivery agent, a nanoparticle, and a carrier, wherein the coupling of the nanoparticle and the active compound comprises coupling a compound as described in any one of claims 1 to 6 with an azide-containing compound to form a triazole compound, wherein the nanoparticle is the azide-containing compound and the active compound comprises the compound as described in any one of claims 1 to 6, or wherein the nanoparticle comprises the compound as described in any one of claims 1 to 6 and the active compound comprises the azide-containing compound.

17. The method of claim 15 or 16, wherein the nanoparticles are self-assembled polymer micelles.

18. A method for preparing a compound of formula (II) as claimed in claim 1, comprising the following steps: a. Convert the dihydrazone (2) to the iminodihydrosulfonylimino group (3) b. Isolate the obtained iminodihydrosulfonylimino group (3).

19. The method of claim 18, wherein the conversion comprises an oxidation reaction in which the dihydrazone (2) is reacted with an oxidant to oxidize a sulfur functional group to a sulfonyl imide functional group, and wherein the conversion further comprises an oxidation reaction in which the dihydrazone (2) is reacted with an oxidant to oxidize a hydrazone functional group to an alkyne functional group.

20. The method of claim 19, wherein the oxidation is a two-step reaction.

21. The method of claim 20, wherein the oxidation is a one-pot reaction with an oxidizing agent, wherein the oxidizing agent is iodobenzene diacetate.

22. Use of a compound as claimed in any one of claims 1 to 6 in a method for coupling two target molecules, said coupling of two target molecules comprising coupling the two molecules via a compound as claimed in claim 2 or 3.

23. The use of claim 22, wherein the molecules are independently selected from drugs, proteins, peptides and oligonucleotides.

24. The use of claim 22, wherein the molecules are independently selected from antibodies, imaging labels, targeting ligands, delivery agents, nanoparticles and carriers.

25. Use of a compound according to any one of claims 1 to 6 in a bioorthogonal, copper-free click reaction.

26. Use of a compound as claimed in any one of claims 1 to 6 in a method for coupling nanoparticles to one or more of drugs, proteins, peptides and oligonucleotides using a copper-free click reaction.

27. Use of a compound as claimed in any one of claims 1 to 6 in a method for coupling nanoparticles to one or more of antibodies, imaging labels, targeting ligands, delivery agents, nanoparticles and carriers using a copper-free click reaction.

28. A compound selected from the group consisting of:

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