Strained multi-functionalized trans-cyclooctenes

Abstract

The present invention relates to novel multi-functionalized trans-cyclooctenes of formula (I). It also relates to conjugates, compositions, medical uses of such compounds, and methods for producing such compounds as well as conjugates of such compounds.

Description

[0001] Strained multi-functionalized trans-cyclooctenes

[0002] Field of the invention

[0003] The present invention relates to novel multi-functionalized trans-cyclooctenes of formula (I). It also relates to conjugates, compositions, medical uses of such compounds, and methods for producing such compounds as well as conjugates of such compounds.

[0004] Background art

[0005] Click chemistry such as in vivo click chemistry has been demonstrated as an attractive platform for diagnostic and therapeutic uses, especially when used in antibody-drug conjugates (ADCs). For example, the in vivo click reaction between radiolabelled tetrazines and transcyclooctene (TCO) functionalized with an antibody has been shown for this purpose (Fang et al, Tetrahedron. 2019 Aug 9;75(32):4307-4317). TCOs with multiple reactive handles are known from WC2023281040.

[0006] To further enhance the clinical capability of trans-cyclooctenes, however, they would have to comprise additional reactive handles, and / or show an even faster click reaction, whereby the click reaction induces a release of a releasable moiety. Currently available trans-cyclooctenes with more than one handle require several purification steps during their synthesis.

[0007] As a result, there is a need for multi-functionalized trans-cyclooctenes that satisfy these criteria. There is a need for multi-functional TCOs that are synthetically accessible via straightforward routes. There is a need for muti-functional TCOs wherein at least one group releases after a click. There is a need for multi-functional TCOs that have fast click kinetics. There is a need for multi-functional TCOs that have fast release kinetics after a click.

[0008] Summary of the invention

[0009] The inventors surprisingly found compounds that can have an increased level of symmetry, obviating the need for the separation of stereoisomers during preparation. These compounds allowed for easier preparation through reduced purification requirements. The compounds can have improved release properties, improved polarity that can for instance improve solubility, and the compounds can offer more reactive handles for further modification. Thus the invention provides a compound of general formula (I), or a salt thereof: wherein one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H; Y1and Y2are in each instance independently -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2or -CEH, or Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring; Z1, Z2, and Z3are in each instance independently O, S, Se, or NQN; Ra, Rb, Rc, and Rdare in each instance independently -O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, or a leaving group; Pr is in each instance independently -H, a linear, branched, or cyclic C1 -6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, -NO2, alkoxy, - C(=0)0(CH2)O-4H, or haloalkoxy; Q, QN, QN1, and QN2are in each instance independently -H or a linear, branched or cyclic C1 -6 alkyl or acyl, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1 , 2, 3 or 4. Preferably m is 0. In preferred embodiments X2is -Raor -Z1-C(=Z2)-Rb, and X1is -H. In preferred embodiments Y1and Y2are in each instance independently -C(=O)-RCor -CH2-Rd. Preferably Y1and Y2both represent the same moiety, or wherein at most one of Y1and Y2is C5-10(hetero)aryl.

[0010] Preferably the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p- methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Preferably the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is

[0011] Preferably the compound is of general formula (V-OO), (V-HO), (V-OH) or (V-HH):

[0012] (V-OO), (V-HO), (V-OH), (V-HH).

[0013] In some embodiments the compound is of general formula (ll-j) or (Vl-a) or an enantiomer thereof:

[0014] Preferably the compound is of general formula (IX-H1) or (IX-H2) or an enantiomer thereof, wherein preferably Rcand Rdare in each instance chosen independently from -OH or -O-C1-2 alkyl or a leaving group:

[0015] (IX-H1), (IX-H2).

[0016] Also provided is a conjugate comprising a moiety conjugated to a compound as defined above, preferably wherein said moiety is a small organic molecule, a pharmaceutically active substance, a macromolecule, a particle, a surface, or a biomolecule, more preferably wherein said moiety is a protein, most preferably wherein said moiety is an antibody.

[0017] Also provided is a method for producing a conjugate as defined above, the method comprising the steps of: i) providing a compound as defined above; ii) providing a second compound, preferably wherein said second compound is a small organic molecule, a pharmaceutically active substance, a macromolecule, or a biomolecule, more preferably wherein said second compound is a protein, most preferably wherein said second compound is an antibody; iii) reacting the compound provided in step i) with the second compound to form a conjugate; and optionally iv) isolating the conjugate obtained in step iii).

[0018] Also provided is a composition comprising a compound as defined above, or a conjugate as defined above, and a pharmaceutically acceptable excipient, preferably wherein the composition comprises a tetrazine, more preferably dipyridyl tetrazine.

[0019] The compounds and compositions can be for use as a medicament, wherein preferably at least one of Ra, Rb, Rc, and Rdis a pharmaceutically active substance, wherein preferably at least one of Ra, Rb, Rc, and Rdis a targeting moiety, wherein the pharmaceutically active substance is preferably a chemotherapeutic agent, and the targeting moiety is preferably an antibody or a fragment thereof.

[0020] Also provided is a method for releasing a pharmaceutically active substance, the method comprising the steps of: i) providing a conjugate as defined above wherein at least one of Ra, Rb, Rc, and Rdis the pharmaceutically active substance; ii) contacting the provided conjugate with a 1 ,2,4,5-tetrazine; wherein the conjugate as defined above is preferably of general formula (ll-a) or (Vl-a), more preferably of general formula (Vl-a). Also provided is a compound of general formula (Intermediate-1), (Intermediate-2), or (Intermediate-3), or a salt thereof:

[0021] (Intermediate-1) (Intermediate-2) (Intermediate-3) wherein hal is -SePr, -SPr, or a halogen, preferably a halogen such as iodine; one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H; Y1and Y2are in each instance independently - C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2or -CEH, or Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring; Z1, Z2, and Z3are in each instance independently O, S or NQN; Ra, Rb, Rc, and Rdare in each instance independently - O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, or a leaving group; Pr is in each instance independently -H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, -NO2, alkoxy, -C(=0)0(CH2)o-4H, or haloalkoxy; Q, QN, QN1, and QN2are in each instance independently -H or a linear, branched or cyclic C1-6 alkyl or acyl, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1 , 2, 3 or 4.

[0022] Description of embodiments

[0023] Compound

[0024] The invention relates to compounds of general formula (I), or a salt thereof: wherein one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H;

[0025] Y1and Y2are in each instance independently -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2or -C=H, or Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring;

[0026] Z1, Z2, and Z3are in each instance independently O, S, Se, or NQN;

[0027] Ra, Rb, Rc, and Rdare in each instance independently -O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, or a leaving group;

[0028] Pr is in each instance independently -H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, -NO2, alkoxy, -C(=0)0(CH2)o-4H, or haloalkoxy; Q, QN, QN1, and QN2are in each instance independently -H or a linear, branched or cyclic C1-6 alkyl or acyl, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1 , 2, 3 or 4.

[0029] Such a compound is called a compound according to the invention in the context of this application. Compounds according to the invention comprise a (4E)-bicyclo[6.1 .0]non-4-ene moiety. In other words, the carbon-carbon double bond depicted in the general formulae (I) through (VIII) and all subformulae thereof has an E configuration. Compounds according to the invention can be considered multi-functionalized trans-cyclooctenes, at least because Y1, Y2, and one of the X moieties can all allow functionalisation. The intermediates according to the invention do not have an E configuration.

[0030] X1andX2moieties of the compound

[0031] Compounds according to the invention comprise moieties X1and X2attached to the eightmembered ring in general formula (I). One of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is - H. Because only one of X1and X2is not H, these moieties can sometimes together be referred to as X. Most preferably it is X1that is H and X2that is as defined elsewhere. Herein, Z1and Z2are in each instance chosen independently from O, S, Se, or NQN, preferably O, S, or NQN; Raand Rbare in each instance chosen independently from -O-Pr, -S-Pr, -SePr, -NQN1QN2, or a leaving group, or optionally a small organic molecule, a pharmaceutically active substance, or a targeting moiety, preferably they are chosen from -O-Pr, -S-Pr, -SePr, -NQN1QN2, or a leaving group; Pr is in each instance chosen independently from -H, a linear, branched, or cyclic C1 -6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, -NO2, -C(=0)0(CH2)o-4H, or haloalkoxy; and QN, QN1and QN2are in each instance chosen independently from -H or a linear, branched or cyclic C1 -6 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy, and optionally - C(=0)0(CH2)O-4H.

[0032] When comprised in X1or X2, C1-6 alkyl can be C1-4 alkyl, more preferably C2-4 alkyl, most preferably C2 alkyl. When comprised in X1or X2, the alkyl is preferably not unsaturated. When comprised in X1or X2, the alkyl is preferably not optionally substituted.

[0033] In preferred embodiments, X1is -Raor -Z1-C(=Z2)-Rband X2is -H. More preferably, m is 0 in these preferred embodiments.

[0034] In preferred embodiments, X2is -Raor -Z1-C(=Z2)-Rband X1is -H. More preferably, m is 0 in these preferred embodiments.

[0035] In preferred embodiments, X1is -Z1-C(=Z2)-Rband X2is -H, wherein Z1and Z2are in each instance chosen independently from O, S or NQN, more preferably from O and S, even more preferably Z1and Z2are O. Most preferably, m is 0 in these preferred embodiments.

[0036] In preferred embodiments, X2is -Z1-C(=Z2)-Rband X1is -H, wherein Z1and Z2are in each instance chosen independently from O, S or NQN, more preferably from O and S, even more preferably Z1and Z2are O. Most preferably, m is 0 in these embodiments. In preferred embodiments, X1is-Z1-C(=Z2)-Rb, wherein -Z1-C(=Z2)-Rbis chosen from -O- C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, -NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH-C(=O)- Rb, and -NH-C(=S) -Rb. More preferably, m is 0 in these embodiments.

[0037] In preferred embodiments, X2is-Z1-C(=Z2)-Rb, wherein -Z1-C(=Z2)-Rbis chosen from -O- C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, -NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH-C(=O)- Rb, and -NH-C(=S) -Rb. More preferably, m is 0 in these embodiments.

[0038] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Rbis chosen from -O-Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetra hydro pyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Even more preferably, -Z1-C(=Z2)-Rbis chosen from -O-C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, -NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH-C(=O)-Rb, and -NH-C(=S) -Rbin these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0039] As used herein, a protecting group has its customary meaning of a group that is linked to the heteroatom in such a way as to reduce its reactivity, or to protect it from certain conditions. After having served their function, protecting groups can generally be removed again using routine chemistry. A skilled person is well aware of protecting groups and can select useful protecting groups for particular groups or atoms to be protected, reaction conditions, storage conditions, purification techniques, or available deprotection techniques. A helpful reference in this regard is Greene's Protective Groups in Organic Synthesis, Wuts & Greene, DOI:10.1002 / 0470053488.

[0040] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Rbis a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG-2), (LG- 3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Even more preferably, -Z1-C(=Z2)-Rbis chosen from -O-C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, -NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH- C(=O)-Rb, and -NH-C(=S) -Rbin these preferred embodiments. Most preferably, m is 0 and / or X1is H in these preferred embodiments.

[0041] Leaving groups have their customary meaning here. A skilled person is capable of selecting appropriate leaving groups. Preferred leaving groups leave as anions via heterolysis, and can stabilize the additional electron density that results from bond heterolysis. Preferred leaving groups are active esters and halogens, and preferred active esters are (halo)phenyl ester such as pentafluorophenyl ester, N-hydroxysuccinimidyl ester, thio(halo)phenyl ester, and nitrophenyl ester such as paranitrophenyl ester.

[0042] In preferred embodiments, the leaving group is (LG-1), (LG-2), (LG-3), (LG-4), (LG-5), (LG- 6), (LG-7), or (LG-8):

[0043] The above leaving groups are particularly suited for Rband Rc. For Raand Rd, leaving groups are preferably linked via a carbonyl moiety, for instance via -O-C(=O)-P’, wherein P’ represents for instance a moiety as depicted above.

[0044] Particularly preferred are LG-1 , LG-2, and LG-8, even more so LG-1 and LG-2, and LG-1 is most preferred.

[0045] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Rbis chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Here, most preferably, m is 0 and / or X1is H.

[0046] In preferred embodiments, X1is -Raand X2is -H, wherein more preferably m is 0. In other preferred embodiments, X2is -Raand X1is -H, wherein more preferably m is 0. In preferred embodiments, one of X1and X2is -Ra, and the other is -H, wherein Rais chosen from -O-Pr and -

[0047] S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4- methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p- methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetra hydro pyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0048] In preferred embodiments, one of X1and X2is -Ra, and the other is -H, wherein Rais a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG-2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Most preferably, m is 0 and / or X1is H in these preferred embodiments.

[0049] In preferred embodiments, one of X1and X2is -Ra, and the other is -H, wherein Rais chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Most preferably, m is 0 and / or X1is H in these preferred embodiments. In preferred embodiments, the compounds according to the invention are of general formula (ll-a), (ll-b), (ll-c), (ll-d), (ll-e), (ll-f), (ll-g), (ll-h), (ll-i), or (ll-j), or enantiomers thereof, preferably wherein m is 0 and / or X1is -H:

[0050] Of the above, ll-a, ll-b, ll-c, ll-d, and ll-j are preferred. In other embodiments, the compound is of one of formulas ll-e, ll-f, ll-g, ll-h, or ll-i. Of the above, ll-a and ll-b and ll-j are most preferred, wherein ll-a and ll-b are more preferred and ll-a is most preferred.

[0051] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Z1and Z2are in each instance chosen independently from O, S or NQN, more preferably from O and S, even more preferably Z1and Z2are O. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0052] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein -Z1-C(=Z2)-Rbis chosen from -O-C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, - NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH-C(=O)-Rb, and -NH-C(=S) -Rb. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0053] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Rbis chosen from -O-Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p- methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Even more preferably, -Z1-C(=Z2)-Rbis chosen from -O-C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, -NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH- C(=O)-Rb, and -NH-C(=S) -Rbin these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0054] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Rbis a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG- 2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Even more preferably, -Z1-C(=Z2)-Rbis chosen from -O-C(=O)-Rb, -S-C(=O)-Rb, -O-C(=S)-Rb, -S-C(=S)-Rb, -NQN-C(=O)-Rb, -NQN-C(=S) -Rb, -NH- C(=O)-Rb, and -NH-C(=S) -Rbin these preferred embodiments. Most preferably, m is 0 and / or X1is H in these preferred embodiments.

[0055] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Z1-C(=Z2)-Rb, and the other is -H, wherein Rbis chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Most preferably, m is 0 and / or X1is H in these preferred embodiments.

[0056] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Ra, and the other is -H, more preferably wherein m is 0 and / or X1is -H.

[0057] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Ra, and the other is -H, wherein Rais chosen from -O-Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert- butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0058] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Ra, and the other is -H, wherein Rais a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG-2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0059] In preferred embodiments, the compounds according to the invention are of general formula (ll-j) or (ll-a), preferably (ll-a), wherein one of X1and X2is -Ra, and the other is -H, wherein Rais chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Most preferably, m is 0 and / or X1is H in these preferred embodiments.

[0060] In preferred embodiments -NQN1QN2is -N(CH3)(CH2C(=O)OCH3) or -NHCH2C(=O)OCH3.

[0061] Y moieties of the compound

[0062] Compounds according to the invention comprise two moieties Y attached to the threemembered ring in general formula (I). Each Y, being Y1and Y2, is in each instance independently chosen from -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2or -C=H, preferably from -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl, more preferably from -C(=Z3)-RCor - CH2-Rd, most preferably it is -C(=Z3)-RC.

[0063] A skilled person will appreciate that -CH2-Rdis easily synthetically accessible from -C(=Z3)- Rc, for instance through reduction when Z3is O. -C(=Z3)-H or -CH=CH2or -C=H are in turn accessible from -CH2-Rd, particularly when Rdis -OH. For instance, using DIBAL-H, esters in Y can be converted into the corresponding aldehydes. Subsequently, the aldehydes can be converted into alkenes using a Wittig Olefination or a Horner-Wadsworth-Emmons reaction. Using the Ohira- Bestmann reagent, the aldehydes can be converted into the corresponding alkynes.

[0064] Herein, Z3is chosen from O, S, Se, or NQN, preferably O, S, or NQN. When one or two of Y1and Y2is -C(=Z3)-H, Z3is preferably O. Rcis chosen from -O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, a leaving group, a small organic molecule, a pharmaceutically active substance, or a targeting moiety; Pr is in each instance chosen independently from -H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy, and optionally -C(=0)0(CH2)o-4H; and QN, QN1and QN2are in each instance chosen independently from -H or a linear, branched or cyclic C1 -6 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy, and optionally -C(=0)0(CH2)o-4H.

[0065] C5-10(hetero)aryl for Y1and Y2can be any known (hetero)aryl, and is preferably C6-aryl, most preferably phenyl. In other embodiments it is C6-10-aryl, or C5-10-heteroaryl such as imidazolyl or pyridinyl.

[0066] Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring. Preferably this ring is a 4-8-membered ring, more preferably a 5-6-membered ring. Such rings can be formed by for instance using glycerol to form a diester with two carboxylic acids at Y1and Y2. Such rings can also be formed using a diamine and subsequent reduction. In such cases, Y1and Y2can be seen as forming a bridging moiety, and examples of suitable bridging moieties are - O-C(CH3)2-O-, -O-CH2-CH2-O-, -O-CH2-CH2-CH2-O-, -NH-CH2-CH2-NH-, -NH-CH2-CH2-CH2-NH-, - C(=O)-O-CH2-CH2-O-C(=O)-, -C(=O)-O-CH2-CH2-CH2-O-C(=O)-, -C(=O)-NH-CH2-CH2-NH-

[0067] C(=O)-, and -C(=O)-NH-CH2-CH2-CH2-NH-C(=O)-. For a 3-membered ring, Y1and Y2represent - CH2-CH2-.

[0068] In preferred embodiments, Y1and Y2represent the same moiety. This simplifies synthesis of the compounds by eliminating a source of divergent stereochemistry. When Y1and Y2do not represent the same moiety, it is preferred that one of Y1and Y2represent C5-10(hetero)aryl and the other represents -C(=Z3)-RCor -CH2-Rd. It was found that these compounds are still conveniently synthesised because surprisingly only one isomer is formed, for instance the following compound was obtained as such:

[0069] Therefore, when one of Y1and Y2represent C5-10(hetero)aryl, it is preferred that the compound is of general formula (lll-c) and Y1is C5-10(hetero)aryl, and Y2is -C(=Z3)-RCor -CH2-Rd. It was found that the reactivity of Y1and Y2is not identical, and therefore the groups could be selectively reacted (see the Examples section). For instance, when both Y1and Y2represent C(=O)- O-CH3, and the compound is of general formula (lll-c), Y2could be selectively deprotected. This allows the selective deprotection and reprotection of both Y1and Y2to obtain a variety of possible configurations. Accordingly in preferred embodiment Y1and Y2are in each instance independently -C(=O)-RCor -CH2-Rd.

[0070] When comprised in Y1or Y2, C1-6 alkyl can be C1-4 alkyl, more preferably C2-4 alkyl, most preferably C2 alkyl. When comprised in Y1or Y2, the alkyl is preferably not unsaturated. When comprised in Y1or Y2, the alkyl is preferably not optionally substituted.

[0071] In preferred embodiments, at least one of Y1and Y2, preferably both, are -C(=Z3)-RC. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0072] In preferred embodiments, at least one of Y1and Y2, preferably both, are -C(=Z3)-RC, wherein Z3is chosen from O, S or NQN, more preferably from O and S, even more preferably Z3is O. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0073] In preferred embodiments, at least one of Y1and Y2, preferably both, are -C(=Z3)-RC, wherein Rcis chosen from -O-Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p- methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Even more preferably, Z3is chosen from O and S in these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0074] In preferred embodiments, at least one of Y1and Y2, preferably both, are -C(=Z3)-RC, wherein Rcis a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG- 2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Even more preferably, Z3is chosen from O and S in these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0075] In preferred embodiments, at least one of Y1and Y2, preferably both, are -C(=Z3)-RC, wherein Rcis chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Even more preferably, Z3is chosen from O and S in these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0076] In preferred embodiments, at least one of Y1and Y2, preferably both, are -CH2-Rd. More preferably, m is 0 and / or X1is H in these preferred embodiments.

[0077] In preferred embodiments, at least one of Y1and Y2, preferably both, are -CH2-Rd, wherein Rdis chosen from -O-Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetra hydro pyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxy methyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0078] In preferred embodiments, at least one of Y1and Y2, preferably both, are -CH2-Rd, wherein Rdis a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG-2), (LG- 3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0079] In preferred embodiments, at least one of Y1and Y2, preferably both, are -CH2-Rd, wherein Rdis chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Most preferably, m is 0 and / or X1is - H in these preferred embodiments.

[0080] In preferred embodiments, the compounds according to the invention are of general formula (lll-a) or (lll-b) or (lll-c), preferably wherein m is 0 and / or X1is -H:

[0081] (lll-a), (lll-b) (lll-c)

[0082] Of the above, (lll-b) and (lll-c) are preferred, particularly (lll-c). As used herein, when a compound is of a general formula wherein stereochemistry is indicated, reference to that stereochemistry is primarily intended. A compound of general formula lll-a is thus primarily characterised by the stereochemistry of the two bridgehead hydrogen atoms. It should be clear that a compound can be of both general formula lll-b and ll-a, for instance - a combination that is particularly preferred.

[0083] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -C(=Z3)-RC. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0084] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -C(=Z3)-RC, wherein Z3is chosen from O, S or NQN, more preferably from O and S, even more preferably Z3is O. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0085] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -C(=Z3)-RC, wherein Rcis chosen from -O- Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis- (4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p- methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetra hydro pyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Even more preferably, Z3is chosen from O and S in these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0086] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -C(=Z3)-RC, wherein Rcis a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG-2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Even more preferably, Z3is chosen from O and S in these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0087] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -C(=Z3)-RC, wherein Rcis chosen from -O- Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Even more preferably, Z3is chosen from O and S in these preferred embodiments. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0088] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -CH2-Rd. More preferably, m is 0 and / or X1is H in these preferred embodiments.

[0089] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -CH2-Rd, wherein Rdis chosen from -O-Pr and -S-Pr, preferably wherein Pr a protecting group, more preferably wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4- methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p- methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetra hydro pyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0090] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -CH2-Rd, wherein Rdis a leaving group, more preferably wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is (LG-1), (LG-2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), or (LG-8). Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0091] In preferred embodiments, the compounds according to the invention are of general formula (lll-b) or (lll-c), preferably (lll-c), wherein Y1and Y2are -CH2-Rd, wherein Rdis chosen from -O-Pr, -S-Pr and a leaving group, more preferably from -O-Pr and a leaving group, even more preferably from -O-H and a leaving group. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0092] (Q)mmoiety of the compound

[0093] Compounds according to the invention may comprise 0, 1 , 2, 3 or 4 Q moieties attached to the eight-membered ring in general formula (I). The number of Q moieties attached to the eightmembered ring is m.

[0094] In preferred embodiments, the compounds according to the invention are of general formula (IV-0), (IV-1 a), (IV- 1 b), (IV-2a), (IV-2b), (IV-2c), (IV-3a), (IV-3b), or (IV-4), preferably wherein X1is -H:

[0095] (IV-4).

[0096] In preferred embodiments, the compounds according to the invention are of general formula (IV-1 a), (IV-1 b), (IV-2a), (IV-2b), (IV-2c), (IV-3a), (IV-3b), or (IV-4), wherein Q is in each instance chosen independently from a linear, branched or cyclic C1 -6 alkyl, wherein alkyl is optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy, preferably wherein X1is -H.

[0097] In some embodiments, m is 0, 1 , 2, or 3. In some embodiments, m is 1 , 2, or 3. In some embodiments, m is 1 or 2. In some preferred embodiments, m is 0 or 1 . In some embodiments, m is 1 . In some embodiments, m is 2. Most preferably m is 0.

[0098] Preferred substitutions and configurations of the compound

[0099] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -C(=Z3)-RC. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0100] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -CH2-Rd. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0101] In preferred embodiments, one of X1and X2is -Ra, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -C(=Z3)-RC. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0102] In preferred embodiments, one of X1and X2is -Ra, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -CH2-Rd. More preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0103] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -C(=Z3)-RC, wherein Z1, Z2, and Z3are in each instance chosen independently from O, S or NQN, more preferably from O and S, even more preferably Z1, Z2, and Z2are O. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0104] In preferred embodiments, one of X1and X2is -Z1-C(=Z2)-Rb, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -CH2-Rd, wherein Z1, Z2, and Z3are in each instance chosen independently from O, S or NQN, more preferably from O and S, even more preferably Z1, Z2, and Z2are O. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0105] In preferred embodiments, one of X1and X2is -Ra, and the other of X1and X2is -H, and at least one of Y1and Y2, preferably both, are -C(=Z3)-RC, wherein Z1, Z2, and Z3are in each instance chosen independently from O, S or NQN, more preferably from O and S, even more preferably Z1, Z2, and Z2are O. Most preferably, m is 0 and / or X1is -H in these preferred embodiments.

[0106] In preferred embodiments, X2is -O-C(=O)-Rb, and at least one of Y1and Y2, preferably both, are -C(=O)-RC. More preferably, m is 0 in these preferred embodiments and the compound is thus of general formula (V-OO). Most preferably, Rband Rcare in each instance chosen independently from -O-Pr, -S-Pr and a leaving group, or from -O-Pr and a leaving group, or from - O-H and a leaving group.

[0107] In preferred embodiments, X2is -O-C(=O)-Rb, and at least one of Y1and Y2, preferably both, are -CH2-Rd. More preferably, m is 0 in these preferred embodiments and the compound is thus of general formula (V-OH). Most preferably, Rband Rdare in each instance chosen independently from -O-Pr, -S-Pr and a leaving group, or from -O-Pr and a leaving group, or from - O-H and a leaving group.

[0108] In preferred embodiments, X2is -Ra, and at least one of Y1and Y2, preferably both, are - C(=O)-RC. More preferably, m is 0 in these preferred embodiments and the compound is thus of general formula (V-HO). Most preferably, Raand Rcare in each instance chosen independently from -O-Pr, -S-Pr and a leaving group, or from -O-Pr and a leaving group, or from -O-H and a leaving group.

[0109] In preferred embodiments, X2is -Ra, and at least one of Y1and Y2, preferably both, are - CH2-Rd. More preferably, m is 0 in these preferred embodiments and the compound is thus of general formula (V-HH). Most preferably, Raand Rdare in each instance chosen independently from -O-Pr, -S-Pr and a leaving group, or from -O-Pr and a leaving group, or from -O-H and a leaving group.

[0110] In preferred embodiments, the compounds according to the invention are of general formula

[0111] In preferred embodiments, the compounds of the invention are of general formula (ll-c), (ll-d), (ll-e), (ll-f), (ll-g), (ll-h), (ll-i), (ll-j), (lll-a), (lll-b), (lll-c), (IV-0), (IV-1 a), ( (IV-2b), (IV-2c), (IV-3a), (IV-3b), (IV-4), (V-OOH), (V-HOH), (V-OHH), (V-HHH), (V-O (V-OHO), (V-HHO), (Vl-a), (Vl-b), (Vl-c), (Vl-d), (VII-OOO), (VII-HOO), (VII-OHO), ( OOH), (VII-HOH), (VII-OHH), (VII-HHH), (VIII-OOO), (VIII-HOO), (VIII-OHO), (V OOH), (VIII-HOH), (VIII-OHH), (VIII-HHH), (IX-H1), (IX-H2), (IX-H3), (IX-H4), (IX-H H7), (IX-H8), (IX-H9), (IX-H10), (IX-H11), (IX-H12), wherein at least one of Ra, Rb, small organic molecule or a pharmaceutically active substance and / or wherein at least one of Ra, Rb, Rc, and Rdis a targeting moiety, more preferably wherein the pharmaceutically active substance is a chemotherapeutic agent, more preferably wherein and the targeting moiety is preferably an antibody or a fragment thereof. Most preferably, compounds according to these preferred embodiments are for use a medicament, preferably for use in the treatment or diagnosis of cancer.

[0112] In preferred embodiments, the compounds according to the invention are of general formula (ll-a) and (lll-a), or (I l-a) and (lll-b), or (ll-a) and (lll-c), or (ll-b) and (lll-a), or (ll-b) and (lll-b), or (II- b) and (lll-c), or (ll-c) and (lll-a), or (I l-c) and (lll-b), or (ll-c) and (lll-c), or (ll-d) and (lll-a), or (I l-d) and (lll-b), or (ll-d) and (lll-c), or (I l-e) and (lll-a), or (ll-e) and (lll-b), or (ll-e) and (lll-c), or (ll-f) and (lll-a), or (ll-f) and (lll-b), or (ll-f) and (lll-c), or (ll-g) and (lll-a), or (ll-g) and (lll-b), or (I l-g) and (III- c), or (ll-h) and (lll-a), or (ll-h) and (lll-b), or (ll-h) and (lll-c), or (ll-i) and (lll-a), or (ll-i) and (lll-b), or (ll-i) and (lll-c), or (ll-j) and (lll-a), or (ll-j) and (lll-b), or (ll-j) and (lll-c), more preferably (ll-a) and (lll-b) or (ll-a) and (lll-c) or (ll-j) and (lll-b) or (ll-j) and (lll-c), most preferably (ll-a) and (lll-c); or enantiomers thereof, preferably wherein m is 0 and / or X1is -H. Herein, it is understood that “a compound of general formula (XX) and (YY)” should be interpreted as a compound which can be represented by both general formula (XX) and general formula (YY), wherein (XX) and (YY) are placeholder names.

[0113] In preferred embodiments, the compounds according to the invention are of general formula (Vl-a) or (Vl-b) or (Vl-c) or (Vl-d), preferably (Vl-a) or (Vl-c), most preferably (Vl-c); or an enantiomer thereof, more preferably wherein m is 0 and / or X1is -H:

[0114] (Vl-a) (Vl-b) (Vl-c), (Vl-d).

[0115] In preferred embodiments, the compounds according to the invention are of general formula (VII-OOO), (VII-HOO), (VII-OHO), (VII-HHO), (VII-OOH), (VII-HOH), (VII-OHH), (VII-HHH), (VIII- OOO), (VIII-HOO), (VIII-OHO), (VIII-HHO), (VIII-OOH), (VIII-HOH), (VIII-OHH), or (VIII-HHH), or an enantiomer thereof, more preferably wherein Ra, Rb, Rcand Rdare in each instance chosen independently from -O-Pr, -S-Pr and a leaving group, even more from -O-Pr and a leaving group, most preferably from -0-H and a leaving group:o

[0116] (VII-OOO), (VII-HOO), (VII-OHO), (VII-HHO),

[0117] (VIII-OOH), (VIII-HOH), (VIII-OHH), (VIII-HHH),

[0118] In preferred embodiments, the compounds according to the invention are of general formula (VII-OO), (VII-HO), (VII-OH), or (VII-HH), or an enantiomer thereof, more preferably wherein Ra, Rb, Rcand Rdare in each instance chosen independently from -O-Pr, -S-Pr and a leaving group, even more from -O-Pr and a leaving group, most preferably from -O-H and a leaving group.

[0119] In preferred embodiments, when stereochemistry is indicated for atoms in the double bond of the cyclooectene ring or adjacent to that double bond, reference is made to both the structure as drawn and its enantiomer. A skilled person will appreciate that stereocenters in compounds of the invention can have an effect on the steric or electronic organisation of these compounds, yet that full mirror images (enantiomers) lead to compounds with identical steric and electronic organisation.

[0120] The compounds are preferably of general formula (IX-H1), (IX-H2), (IX-H3), (IX-H4), (IX- H5), (IX-H6), (IX-H7), (IX-H8), (IX-H9), (IX-H10), (IX-H11), or (IX-H12). In some embodiments they are general formula (IX-H7), (IX-H8), (IX-H9), (IX-H10), (IX-H11), or (IX-H12). In preferred embodiments they are general formula (IX-H1), (IX-H2), (IX-H3), (IX-H4), (IX-H5), or (IX-H6). (IX- H4) is most preferred. Herein, preferably Rcand Rdare in each instance chosen independently from

[0121] -OH or C1-2 alkyl or a leaving group, more preferably -OH or -CH3 or -CH2-CH3, most preferably - CH3or -CH2-CH3.

[0122] (IX-H9), (IX-H10) (IX-H11) (IX-H12)

[0123] Click and release

[0124] In preferred embodiments, a compound according to the invention is able to undergo a click reaction, more preferably wherein the click reaction is between the compound and a 1 , 2,4,5- tetrazine, most preferably a tetrazine of general formula (Tz), wherein QTzis in each instance chosen independently from -H, phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6-pyrimidyl, 2,5-pyrimidyl, 3,5-pyrmidyl, and 2,4-pyrimidyl, or optionally a derivative thereof that is further substituted at QTz.

[0125] In preferred embodiments, QTzis in each instance chosen independently from 2-pyridyl, 3-pyridyl, 4-pyridyl. In more preferred embodiments, QTzis 4-pyridyl. In preferred embodiments, a compound according to the invention is able to undergo a click reaction, wherein the click reaction can be represented by the general scheme (I) + (Tz) (I- Tz): In preferred embodiments, the rate ofthe click reaction is at least 50 M-1s-1, 100 M-1s-1, 150 M'1S'1, or 200 M'1s1for a 10 pM concentration of (I) at 20 °C, preferably under the conditions specified in Example 2.

[0126] In preferred embodiments, the rate of the click reaction is at least 250 M1s1, 300 M1s1, 350 M'1S'1, or 400 M1s1for a 50 pM concentration of (I) at 20 °C, preferably under the conditions specified in Example 2.

[0127] In preferred embodiments, a product of the click reaction of general formula (l-Tz) is able to undergo a release reaction, wherein X2or a part thereof is detached from the rest of (l-Tz), more preferably wherein said part is a leaving group.

[0128] In preferred embodiments, the release reaction can be represented by general scheme (l-Tz) X2#+ (l-Tz#), wherein X2#represents one or more moieties derived from X2:

[0129] (l-Tz) (l-Tz#)

[0130] In some embodiments the release is of X1 instead.

[0131] For l-Tz it is preferred that X1or X2is -Z1-C(=Z2)-Rb, preferably that X1or X2comprises a carbamate or a carbonate. In such embodiments, the release of X2 releases Rbunder loss of Z1=C=Z2, which when Z1and Z2are O equates to loss of CO2. It is therefore highly preferred that X1or X2is -O-C(=O)-Rb, most preferably X2is -O-C(=O)-Rb.

[0132] In preferred embodiments, the release reaction is spontaneous at 20 °C, preferably under the conditions specified in Example 5, wherein the release reaction at 20 °C results in at least 95% conversion after 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,

[0133] 26, 27, 28, 29, or 30 minutes.

[0134] In preferred embodiments, the release reaction is spontaneous at 20 °C, preferably under the conditions specified in Example 5, wherein the release reaction at 20 °C results in at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% conversion after 20 minutes.

[0135] In preferred embodiments, a compound according to the invention is of general formula (V-OO), and is able to undergo a click reaction, represented by the general scheme (V-OO) + (Tz) (V-OO-Tz), more preferably wherein (V-OO-Tz) is able to undergo a spontaneous release reaction, represented by general scheme (V-OO-Tz) -> Rb+ CO2 + (V-OO-Tz#), most preferably wherein the release reaction at 20 °C results in at least 95% conversion after 20 minutes:

[0136] (V-OO-Tz) (V-OO-Tz#)

[0137] In preferred embodiments, a compound according to the invention is of general formula (V- OH), and is able to undergo a click reaction, represented by the general scheme (V-OH) + (Tz) (V-OH-Tz), more preferably wherein (V-OH-Tz) is able to undergo a spontaneous release reaction, represented by general scheme (V-OH-Tz) -> Rb+ CO2 + (V-OH-Tz#), most preferably wherein the release reaction at 20 °C results in at least 95% conversion after 20 minutes:

[0138] (V-OH-Tz) (V-OH-Tz#)

[0139] In preferred embodiments, a compound according to the invention is of general formula (VII-OO), and is able to undergo a click reaction, represented by the general scheme (VII-OO) + (Tz) (VII-OO-Tz), more preferably wherein (VII-OO-Tz) is able to undergo a spontaneous release reaction, represented by general scheme (VII-OO-Tz) -> Rb+ CO2 + (VII-OO-Tz#), most preferably wherein the release reaction at 20 °C results in at least 95% conversion after 20 minutes:

[0140] (VII-OO-Tz) (VII-OO-Tz#)

[0141] In preferred embodiments, a compound according to the invention is of general formula (VII-OH), and is able to undergo a click reaction, represented by the general scheme (VII-OH) + (Tz) (VII-OH-Tz), more preferably wherein (VII-OH-Tz) is able to undergo a spontaneous release reaction, represented by general scheme (VII-OH-Tz) -> Rb+ CO2 + (VII-OH-Tz#), most preferably wherein the release reaction at 20 °C results in at least 95% conversion after 20 minutes:

[0142] (VII-OH-Tz) (VII-OH-Tz#)

[0143] In the context of the release reaction, it is clear that an isolated Rb, as drawn in the general schemes above, is meant to include all relevant ions, salts and protonation forms or the Rbmoiety defined as part of a general formula (I). Preferably, Rbis a leaving group in this context.

[0144] From the above, it follows that also provided is a method for releasing a a small organic molecule or a pharmaceutically active substance, the method comprising the steps of: i) providing a conjugate according to the invention wherein at least one of Ra, Rb, Rc, and Rdis the small organic molecule or the pharmaceutically active substance; ii) contacting the provided conjugate with a 1 ,2,4,5-tetrazine; wherein the conjugate is preferably of general formula (I l-a) or (Vl-a) or (VII-OOO), (VII- HOO), (VII-OHO), (VII-HHO), (VII-OOH), (VII-HOH), (VII-OHH), (VII-HHH), (VIII-OOO), (VIII-HOO), (VIII-OHO), (VIII-HHO), (VIII-OOH), (VIII-HOH), (VIII-OHH), or (VIII-HHH), more preferably of general formula (Vl-a). Preferably in these cases a small organic molecule or a pharmaceutically active compound is comprised in Raor Rb, more preferably in Rb.

[0145] Further definitions

[0146] In the context of the invention, a salt of a compound according to the invention is preferably a pharmaceutically acceptable salt. Such salts include salts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Zn and Mn; salts of organic bases such as N,N’-diacetylethylenediamine, glucamine, triethylamine, choline, dicyclohexylamine, benzylamine, trialkylamine, thiamine, guanidine, diethanolamine, alpha-phenylethylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, hydroxyethylpiperidine, and the like. Such salts also include amino acid salts such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine, etc. Such salts may include acid addition salts where appropriate, which are for example sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides such as HCI or HBr salts, acetates, trifluoroacetates, tartrates, maleates, citrates, succinates, palmoates, methanesulphonates, tosylates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like. Preferred salts are HCI salts, formic acid salts, acetic acid salts, and trifluoroacetic acid salts. More preferred salts are HCI salts, acetic acid salts and formic acid salts, most preferably HCI salts.

[0147] The compound according to the invention can be a hydrate or a solvate. In the context of the invention a hydrate refers to a solvate wherein the solvent is water. The term solvate, as used herein, refers to a crystal form of a substance which contains solvent. Solvates are preferably pharmaceutically acceptable solvates and may be hydrates or may comprise other solvents of crystallization such as alcohols, ether, and the like.

[0148] In the context of this invention, the number of carbon atoms in a moiety such as a linear, branched, or cyclic alkyl, or acyl is indicated as for example C1 -6, in this non-limiting case indicating that from 1 to 6 carbon atoms are envisaged, such as 1 , 2, 3, 4, 5, or 6 carbon atoms. Similarly C2- 4alkyl has 2, 3, or 4 carbon atoms. The number of carbon atoms can be expressed as the total number of carbon atoms not counting further substitutions, the total number of carbon atoms, or as the number of carbon atoms that can be found in the longest continuous internal sequence of carbon atoms. Preferably, the number of carbon atoms is expressed as the total number of carbon atoms not counting further substitutions.

[0149] In the context of this invention, unsubstituted alkyl groups have the general formula CnH2n+i and may be linear or branched. Unsubstituted alkyl groups may also be cyclic, and thus have the concomitant general formula CnH2n-i. Optionally, the alkyl groups are substituted by one or more substituents chosen independently from halogen, alkoxy, and haloalkoxy. Examples of suitable alkyl groups include, but are not limited to, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, - CH(CH3)CH2CH3, -CH2CH(CH3)2, -CH2CH2CH2CH3, -C(CH3)3, 1 -hexyl and the like. Preferred alkyl groups are linear or branched, most preferably linear. Preferred cyclic alkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, most preferably cyclopentyl.

[0150] Each instance of acyl, or alkyl individually is optionally unsaturated, and optionally substituted with halogen, alkoxy, and haloalkoxy. In the context of this invention, acyl moieties are alkyl moieties wherein the proximal carbon atom is substituted by an oxo moiety (=0). In the context of the invention, halogen is fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Preferred halogens for compounds according to the invention are fluorine, chlorine, and bromine, more preferred halogens are chlorine or bromine, a most preferred halogen is chlorine. In the context of this invention, alkoxy is -O-alkyl, preferably wherein said alkyl is a C1 -C6 linear or branched alkyl, more preferably a C1-C4 linear or branched alkyl. In the context of this invention, haloalkoxy is an alkoxy, wherein said alkyl comprised in said -O-alkyl is substituted with one or more halogens. A most preferred alkoxyl is methoxy. -C(=0)0(CH2)o-4H is preferably -C(=0)0(CH2)o-3H, more preferably - C(=0)0(CH2)O-IH, most preferably -C(=O)O(CH3.

[0151] Alkyl and acyl groups of the invention are optionally unsaturated. In preferred embodiments, alkyl is not unsaturated. Unsaturated alkyl groups are preferably alkenyl or alkynyl groups. In the context of this invention, unsubstituted alkenyl groups have the general formula CnH2n-i, and may be linear or branched. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, pentenyl and the like. Unsubstituted alkenyl groups may also contain a cyclic moiety, and thus have the concomitant general formula CnH2n-3. Preferred alkenyl groups are linear or branched, most preferably, linear. Highly preferred unsaturated cyclic alkyl groups are aryl groups, such as phenyl. In the context of this invention, unsubstituted alkynyl groups have the general formula CnH2n-3 and may be linear or branched. Unsubstituted alkynyl groups may also contain a cyclic moiety, and thus have the concomitant general formula CnH2n-5. Optionally, the alkynyl groups are substituted by one or more substituents further specified in this document. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propargyl, n-but-2-ynyl, and n-but-3-ynyl. Preferred alkyl groups are linear or branched, most preferably linear.

[0152] In preferred embodiments, C1-6 alkyl when optionally unsaturated and optionally substituted can be C1 -6 alkyl, C1- 6acyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cyclic alkyl, or C5-6 aryl, optionally substituted with one or more moieties selected from halogen, methyl, ethyl, propyl, methoxy, ethoxy, and tri fluoro methyl. In preferred embodiments, C1 -4alkyl when optionally unsaturated and optionally substituted can be C1 -4 alkyl, C1-4 acyl, C2-4 alkenyl, C2-4 alkynyl, or C3-4 cyclic alkyl, optionally substituted with one or more moieties selected from halogen, methyl, ethyl, propyl, methoxy, ethoxy, and trifluoromethyl.

[0153] A leaving group comprised in a compound is defined in the context of this application as a moiety that is attached to the rest of the compound by a covalent bond that can be cleaved heterolytically under mild conditions. Preferred leaving groups are (LG-1), (LG-2), (LG-3), (LG-4), (LG-5), (LG-6), (LG-7), and (LG-8).

[0154] A pharmaceutically active substance is defined in the context of this application as a compound or a moiety that is biologically active when it is introduced in a subject, preferably wherein said subject is an animal or a human, more preferably a human. A skilled person understands which pharmaceutically active substances can advantageously be used in this invention. Preferably, the substance has at least one heteroatom through which it is linked to general formula I. Optionally the pharmaceutically active substance can be linked to general formula I via a linker, which is preferably a linker of at most 10 atoms in length, wherein the atoms are preferably selected from C, O, and N, more preferably the atoms are C with at most one O and at most one N, wherein the linker is optionally substituted with one or more O. For instance a preferred linker is -NH-CH2-CH2- CH2-CH2-CH2-C(=O)-, also known as aminohexanoic acid. Preferred pharmaceutically active substances are small molecules with a molecular weight of at most 750 Da, more preferably at most 500 Da. Preferred classes of pharmaceutically active substances are antibiotics and chemotherapeutic agents. Preferred chemotherapeutic agents are anthracyclines (aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin), anthracenediones (mitoxantrone, pixantrone), streptomyces (actinomycin, bleomycin, mitomycin, plicamycin), topoisomerase inhibitors: camptotheca (camptothecin, topotecan, irinotecan, rubitecan, belotecan), podophyllum (etoposide, teniposide); tyrosine kinase inhibitors: axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, neratinib, nilotinib, semaxanib, sorafenib, sunitinib, vandetanib; cyclin-dependent kinase inhibitors: alvocidib, seliciclib; photosensitizers: aminolevulinic acid / methyl aminolevulinate, efaproxiral, porphyrin derivatives (porfimer sodium, talaporfin, temoporfin, verteporfin) of which anthracyclines are most preferred, and a preferred anthracycline is doxorubicin. Doxorubicin is preferably linked to the remainder of general formula (I) via its nitrogen atom, more preferably while forming a carbamate.

[0155] A targeting moiety is preferably an antibody, wherein an antibody refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab, F(ab)2, Fv, Fab, F(ab)2, scFv, minibody, biabody, scFv-Fc, and other fragments that retain the antigen binding function of the parent antibody. As such, a targeting moiety may preferably refer to an immunoglobulin or glycoprotein, or fragment or portion thereof, or to a construct comprising an antigen-binding portion comprised within a modified immunoglobulin-like framework, or to an antigen-binding portion comprised within a construct comprising a non- immunoglobulin-like framework or scaffold. A targeting moiety can also be an aptamer or a lectin.

[0156] A small organic molecule preferably has a molecular weight of at most 750 Da, more preferably at most 500 Da. A preferred small organic molecule comprises at least one heteroatom. Examples of small organic molecules are small fluorophores such as coumarins, preferably aminocoumarins such as 7-aminocoumarins. Preferred coumarins are methylcoumarins such as 4- methylcoumarins. A most preferred coumarin is 7-amino-4-methylcoumarin.

[0157] In a further aspect, the invention provides a conjugate comprising a moiety conjugated to a compound according to the invention. Such a conjugate is called a conjugate according to the invention in the context of this application. The moiety is called the conjugated moiety in this context. The moiety can be released from the compound according to the invention after a click reaction, as demonstrated in the Examples. This enables a variety of medical applications in the field of targeted treatment, localized treatment, and diagnostics. Other applications can be in the field of analytics or other forms of testing, when for instance surfaces are coated with a compound according to the invention.

[0158] In preferred embodiments, the conjugated moiety is a pharmaceutically active substance, a macromolecule, a particle, a surface, or a biomolecule, more preferably a protein, most preferably an antibody. Suitable particles are nanoparticles such as detectable beads or quantum dots. Further suitable particles are resin beads such as magnetic beads or beads suitable for packing a bed. Suitable surfaces are reactor surfaces, reactor channel surfaces, microchannel surfaces, microplate surfaces, reactor well surfaces, cuvette surfaces, and surfaces of disposables.

[0159] In the context of this application, a macromolecule is preferably defined as a molecule having an molecular mass of at least 1000 Daltons. Preferably the mass is not larger than 100 kDa. A biomolecule is preferably defined as a molecule obtained or derived from a naturally occurring organism. A protein refers to a polymer of amino acids, which may be optionally substituted (e.g. glycosylated) or modified (e.g. crosslinked). A preferred protein comprises from 10 to 1000 amino acids. A highly preferred protein is an antibody or a fragment thereof, preferably an antibody.

[0160] An antibody can refer to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab, F(ab)2, Fv, Fab, F(ab)2, scFv, minibody, biabody, scFv-Fc, and other fragments that retain the antigen binding function of the parent antibody. As such, an antibody may refer to an immunoglobulin or glycoprotein, or fragment or portion thereof, orto a construct comprising an antigen-binding portion comprised within a modified immunoglobulin-like framework, or to an antigen-binding portion comprised within a construct comprising a non-immunoglobulin-like framework or scaffold.

[0161] When the conjugated moiety is a small organic molecule or a pharmaceutically active substance it is preferably conjugated at X1or X2, more preferably X1. When the conjugated moiety is a macromolecule, a particle, a surface, or a biomolecule, it is preferably conjugated at Y1or Y2. To combine functionalities such as targeting and release, preferred conjugates can comprise a plurality of moieties, preferably at least one small organic molecule or pharmaceutically active substance, and at least one further moiety such as a targeting moiety or a protein.

[0162] Methods for producing a conjugate

[0163] In a further aspect, the invention provides a method for producing a conjugate according to the invention, the method comprising the steps of: i) providing a compound according to the invention; ii) providing a second compound, preferably wherein said second compound is a small organic molecule, a pharmaceutically active substance, a macromolecule, a particle, a surface, or a biomolecule, more preferably wherein said second compound is a pharmaceutically active substance, a macromolecule, or a biomolecule, more preferably wherein said second compound is a pharmaceutically active substance of a protein, even more preferably wherein said second compound is a protein, most preferably wherein said second compound is an antibody; iii) reacting the compound provided in step i) with the second compound to form a conjugate; and optionally iv) isolating the conjugate obtained in step iii).

[0164] In a further aspect, the invention provides a composition comprising at least one compound or composition according to the invention, and a pharmaceutically acceptable excipient. Such a composition is referred to herein as a composition according to the invention. Preferred compositions according to the invention are pharmaceutical compositions. In preferred embodiments, the composition according to the invention is formulated for oral, sublingual, parenteral, intravascular, intravenous, subcutaneous, or transdermal administration, optionally for administration by inhalation; preferably for oral administration. Suitable excipients are water such as water for injection, and pharmaceutically acceptable buffers such as PBS. Further suitable pharmaceutically acceptable excipients include processing agents and delivery modifiers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences, " Mack Pub. Co., New Jersey (1991), and "Remington: The Science and Practice of Pharmacy, " Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003), 21stedition (2005) and 22ndedition (2012), and a skilled person can select a suitable one based on the intended application.

[0165] In preferred embodiments, a composition according to the invention comprises a tetrazine, more preferably 1 ,2,4,5-tetrazine, even more preferably a tetrazine of general formula (Tz), wherein QTzis in each instance chosen independently from -H, phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,6- pyrimidyl, 2,5-pyrimidyl, 3,5-pyrmidyl, and 2,4-pyrimidyl, most preferably wherein QTzis in each instance chosen independently from 2-pyridyl, 3-pyridyl, 4-pyridyl. In the context of this application, dipyridyl tetrazine refers to a compound of general formula (Tz), wherein QTzis in each instance chosen independently from 2-pyridyl, 3-pyridyl, and 4-pyridyl.

[0166] Medical use

[0167] In a further aspect, the invention provides a compound according to the invention, a conjugate according to the invention, or a composition according to the invention, for use as a medicament, preferably for use in the treatment or diagnosis of cancer, more preferably treatment.

[0168] In a further aspect, the invention provides in vivo, in vitro, or ex vivo method, or a method of treatment, comprising the administration of a compound according to the invention, a conjugate according to the invention, or a composition according to the invention. More preferably, the method of treatment is for the treatment or diagnosis of cancer, more preferably treatment.

[0169] Compounds for use as a medicament preferably comprise at least one of a pharmaceutically active substance and a targeting moiety at Ra, Rb, Rc, or Rd. Compounds for use as a medicament can also comprise both of a pharmaceutically active substance and a targeting moiety at Ra, Rb, Rc, or Rd. Preferably the pharmaceutically active substance is at Raor Rb. Preferably the targeting moiety is at Rcor Rd. When the medicament is for treatment, a pharmaceutically active substance is preferably comprised, more preferably both a pharmaceutically active substance and a targeting moiety are comprised. When the medicament is for diagnosis, a targeting moiety is preferably comprised.

[0170] Intermediate Products

[0171] Various compounds can be of interest for the provision of other compounds and conjugates according to the invention. In particular the invention provides a compound of general formula (Intermediate-1), (Intermediate-2), or (Intermediate-3), or a salt thereof:

[0172] (Intermediate-1) (Intermediate-3) wherein hal is -SePr, -SPr, or a halogen, preferably a halogen such as iodine; one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H; Y1and Y2are in each instance independently -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2 or -C=H, or Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring;

[0173] Z1, Z2, and Z3are in each instance independently O, S or NQN;

[0174] Ra, Rb, Rc, and Rdare in each instance independently -O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, or a leaving group;

[0175] Pr is in each instance independently -H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, -NO2, alkoxy, -C(=0)0(CH2)o-4H, or haloalkoxy;

[0176] Q, QN, QN1, and QN2are in each instance independently -H or a linear, branched or cyclic C1-6 alkyl or acyl, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1 , 2, 3 or 4.

[0177] Herein, hal is preferably -SeAc, -SAc, or a halogen, and a preferred halogen is iodine, which is most preferred for hal. For Intermediate-1 and Intermediate-2, none of X1and X2are -H because only one of X1and X2are present. For Intermediate-3 one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H. Features and definitions for Y1and Y2and Z1, Z2, and Z3and Ra, Rb, Rc, and Rdand Pr and Q, QN, QN1, and QN2are preferably as provided elsewhere herein. Preferred intermediates are of general formula (Intermediate-1) or (Intermediate-3). Preferred compounds of general formula (Intermediate-1) have iodine for hal and -OAc for X2. A preferred example is compound 5 from the Examples. Preferred compounds of general formula (Intermediate-3) have H for X1and -OAc or -OH for X2. Preferred examples are compounds 6 and 7 from the Examples.

[0178] Preferred compounds of general formula (Intermediate-1), (Intermediate-2), or (Intermediate-3), are of formula (Intermediate-1 a), (lntermediate-2a), or (lntermediate-3a):

[0179] (Intermediate-1 a) (lntermediate-2a) (Intermediated).

[0180] In formula (Intermediate-1 a), (Intermediated), or (Intermediated), it is preferred that m is 0, and it is preferred that Y1and Y2are in each instance independently -C(=Z3)-RC, preferably -C(=O)-RC, more preferably -C(=O)-O-Pr. Herein, Pr is preferably C1 -C6 alkyl such as -CH3 or -CH2-CH3.

[0181] General Definitions

[0182] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a combination or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0183] When a structural formula or chemical name is understood by the skilled person to have chiral centers, yet no chirality is indicated, for each chiral center individual reference is made to all three of either the racemic mixture, the pure R enantiomer, or the pure S enantiomer.

[0184] The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.

[0185] In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

[0186] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value.

[0187] Each embodiment as identified herein may be combined together unless otherwise indicated. The invention has been described above with reference to a number of embodiments. A skilled person could envision trivial variations for some elements of the embodiments. These are included in the scope of protection as defined in the appended claims. All patent and literature references cited are hereby incorporated by reference in their entirety.

[0188] Legends to the Figures

[0189] Fig. 1 - Click-to-release reaction of trans-cyclooctenes (TCOs) 12 and 13 with tetrazine 10. Conditions for (i.) are 4-nitrophenyl isocyanate (2 equiv), triethylamine (3 equiv), 4-dimethylaminopyridine (DMAP, cat), dichloromethane (DCM), 40 °C, 18 h, 33-52%. The axial isomer 12 revealed particularly rapid release of the payload (p-NA) and the respective elimination adduct (E1).

[0190] Examples

[0191] Example 1 — Synthesis of compounds and intermediates

[0192] Example 1.1 - synthetic strategy

[0193] Scheme E1. Synthesis of compounds 8 and 9 starting from commercially available cis,cis-1,5- cyclooctadiene (1). i. p-ABSA (1.2 equiv), DBU (1.2 equiv), 21 °C, 18 h, 92%. ii. Cu (0.05 equiv), 3 (0.15 equiv), 130 °C, 18 h, 58%. Hi. NIS (1.1 equiv), acetic acid (5 equiv), DCM, 21 °C, 18 h, 88%. iv. DBU (5 equiv), toluene, 100 °C, 18 h, 84%. v. potassium carbonate (2.0 equiv), methanol, 21 °C, 18 h, 86%. vi. methyl benzoate (2 equiv), IPA:MTBE 1:19, hr, 21 °C, 18 h, 8 / 9 1:2.6, 58%.

[0194] Example 1.2 - intermediates

[0195] Dimethyl diazomalonate (3): 4-Acetamidobenzenesulfonyl azide (8.73 g,

[0196] 36.3 mmol) was dissolved in dry ACN (50 mL) in a flame-dried flask under JL

[0197] MeO2C CO2Me N2. Hereafter, dimethyl malonate (2, 3.48 mL, 30.3 mmol) and DBU (5.4 mL,

[0198] 36.3 mmol) were added dropwise at 0 °C and the reaction was stirred at rt for 18 h. The reaction mixture was quenched with aqueous sat. NH4CI (50 mL) and the solvent was removed in vacuo. The solid residue was triturated with heptane (3 x, 100 mL) and filtrated to separate the product from the insoluble ABSNH2. The filter was washed with cold heptane (50 mL) and the filtrate was concentrated in vacuo to afford 9 (4.68 g, 98 %) as a red liquid which could be directly used in the next step without further purification.1H NMR (500 MHz, CDCb) 6 3.75 (s, 6H).13C NMR (126 MHz, CDCb) 6 161.3, 52.4.

[0199] Dimethyl bicyclo[6.1.0]non-4-ene-9,9-dicarboxylate (4): 1 ,5- MeO2C„ .CO2Me

[0200] Cyclooctadiene (1 , 23.3 mL, 189.1 mmol) in a flame-dried flask under N2 was , / \%H stirred and copper powder (554 mg, 8.7 mmol) was added. The reaction [ mixture was stirred at 130 °C for 30 minutes before the dropwise addition of \ / dimethyl diazomalonate (3, 4.6 g, 29.0 mmol) via a syringe pump (20 pL / min) followed by stirring overnight. The reaction mixture was concentrated in vacuo and purified with silica gel column chromatography (0 — 20% EtOAc in heptane) to afford 4 (4.0 g, 58%) as a colorless oil. TLC (EtOAc / heptane, 1 :4 v / v): RF = 0.46.1H NMR (500 MHz, CDCb) 6 5.61 (t, J = 4.2 Hz, 1 H), 3.75 (s, 2H), 3.70 (s, 1 H), 2.35 (dddd, J = 10.8, 7.6, 5.5, 3.1 Hz, 1 H), 2.15 - 2.05 (m, 2H), 1.86 - 1.80 (m, 1 H), 1 .77 - 1 .66 (m, 1 H).13C NMR (126 MHz, CDCb) 6 171 .6, 168.2, 129.5, 52.8, 52.3, 26.9, 24.7. HRMS (m / z): [M + Na]+calcd. for Ci3Hi8O4Na: 261.1103, found 261.1074.

[0201] Dimethyl (1 R,4R,5R,8S)-4-acetoxy-5-iodobicyclo[6.1 .0]nonane-9,9- dicarboxylate (5): (Z)-Dimethyl bicyclo[6.1 .0]non-4-ene-9,9-dicarboxylate (4, 6.8 g, 28.7 mmol) was dissolved in dry DCM (100 mL) before the addition of N-iodosuccinimide (7.1 g, 31 .6 mmol) and acetic acid (8.2 mL, 143.6 mmol). The reaction mixture was stirred for 4 h after which it was diluted with DCM (100 mL). The organic layer was washed with 10% aqueous Na2S2O3(100 mL), aqueous sat. NaHCO3(100 mL) and brine (100 mL). The organic layer was dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (0 — 30% EtOAc in heptane) to afford 5 (10.7 g, 88%) as a colorless oil. TLC (EtOAc / heptane, 1 :4 v / v): RF = 0.24.1H NMR (500 MHz, CDCb) 6 4.95 (ddd, J = 7.8, 6.0, 3.6 Hz, 1 H), 4.74 (td, J = 7.7, 4.2 Hz, 1 H), 3.77 (s, 3H), 3.72 (s, 3H), 2.28 - 2.15 (m, 3H), 2.10 (s, 3H), 2.06 - 2.00 (m, 1 H), 1.82 (ddt, J = 18.5, 9.3, 2.9 Hz, 2H), 1.72 (ddd, J = 12.5, 9.3, 3.3 Hz, 1 H), 1.45 - 1.36 (m, 2H), 1.32 - 1.25 (m, 1 H).13C NMR (126 MHz, CDCb) 6 171.1 , 169.9, 167.4, 73.3, 53.0, 52.5, 36.8, 33.6, 32.3, 30.6, 30.4, 23.5, 21.6, 21.2. HRMS (m / z): [M + Na]+calcd. for Ci5H2ilO6Na: 447.0280, found 447.0269.

[0202] Dimethyl (1S,5R,8R,Z)-5-acetoxybicyclo[6.1.0]non-3-ene-9,9- dicarboxylate (6): (Z)-Dimethyl (1 R,4R,5R,8S)-4-acetoxy-5- iodobicyclo[6.1 .0]nonane-9,9-dicarboxylate (5, 1 .9 g, 4.5 mmol) was dissolved in toluene after which DBU (3.4 mL, 22.4 mmol) was added. The reaction mixture was stirred for 18 h at 100 °C before it was diluted with EtOAc (100 mL) and the organic layer was washed with aqueous sat. NH4CI (100 mL) and brine (100 mL). The organic layer was dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (0 30% EtOAc in heptane) to afford 6 (924 mg, 83%) as a colorless oil. TLC

[0203] (EtOAc / heptane, 2:3 v / v): RF = 0.51 .1H NMR (500 MHz, CDCb) 6 5.86 - 5.77 (m, 1 H), 5.62 (dd, J = 11 .1 , 5.2 Hz, 1 H), 5.34 - 5.28 (m, 1 H), 3.78 (s, 3H), 3.70 (s, 3H), 2.45 - 2.39 (m, 1 H), 2.28 (dddd, J = 15.6, 8.5, 5.2, 1.1 Hz, 1 H), 2.04 (s, 3H), 1.96 - 1.90 (m, 1 H), 1.87 - 1.84 (m, 2H), 1 .77 - 1.69 (m, 2H), 1.46 - 1.37 (m, 1 H).13C NMR (126 MHz, CDCb) 6 170.9, 170.3, 167.5, 134.5, 129.0, 75.1 , 52.8, 52.5, 39.1 , 35.6, 34.6, 29.4, 24.6, 23.6, 21.4. HRMS (m / z): [M + Na]+calcd. for Ci5H2o06Na: 319.1158, found 319.1144.

[0204] Dimethyl (1 S,5R,8R,Z)-5-hydroxybicyclo[6.1 .0]non-3-ene-9,9- dicarboxylate (7): (Z)-Dimethyl (1 S,5R,8R,Z)-5-acetoxybicyclo[6.1 .0]non-3- ene-9,9-dicarboxylate (6, 570 mg, 1 .7 mmol) was dissolved in dry MeOH (20 mL) before the addition of K2CO3 (471 mg, 3.4 mmol). The reaction mixture was stirred for 18 h at rt before it was quenched by the addition of acetic acid and diluted with EtOAc (100 mL). The organic layer was washed with aqueous sat. NaHCO3(100 mL), brine (100 mL) and dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (10 50% EtOAc in heptane) to afford 7 (371 mg, 86%) as a colorless oil. TLC

[0205] (EtOAc / heptane, 1 :1 v / v): RF = 0.29.1H NMR (500 MHz, CDCh) 5 5.82 - 5.71 (m, 1 H), 5.67 (dd, J = 11 .0, 4.8 Hz, 1 H), 4.37 - 4.30 (m, 1 H), 3.76 (s, 3H), 3.69 (s, 3H), 2.37 (dd, J = 10.4, 7.0 Hz, 1 H), 2.28 - 2.21 (m, 1 H), 1 .96 - 1 .87 (m, 1 H), 1 .85 - 1 .80 (m, 2H), 1 .80 - 1 .70 (m, 2H), 1 .33 (dtd, J = 15.4, 11.8, 1.2 Hz, 1 H).13C NMR (126 MHz, CDCh) 6 171.0, 167.8, 138.7, 128.2, 73.0, 52.8, 52.5, 39.0, 34.9, 29.6, 24.5, 23.7. HRMS (m / z): [M + Na]+calcd. for Ci3Hi8O5Na: 277.1052, found 277.1026.

[0206] Example 1.3 - Compounds

[0207] Dimethyl (1 S,5R,8R,Z)-5-hydroxybicyclo[6.1 .0]non-3-ene-9,9- dicarboxylate (8 & 9): (Z)-Dimethyl (1 S,5R,8R,Z)-5- hydroxybicyclo[6.1 .0]non-3-ene-9,9-dicarboxylate (7, 500 mg, 1.7 mmol) and methyl benzoate (539 pL, 4.3 mmol) in deoxygenated solvent (isopropanol / MTBE, 1 :19, 100 mL) were loaded into a UV irradiation setup containing a self-fabricated photoreactor consisting of a OSAGA RVS UV-C 55w pond filter with a Philips PL-L 55 W UV-C lamp (254 nm) and FEP tubing (i.d. 2.7 mm, o.d. 3.18 mm; distance between the tubing and the UV lamp = 0.7 - 1 .4 cm). During continuous irradiation the reaction was flushed trough a flash silica gel cartridge (12 gr) containing 10% w / w% AgNO3on silica on a thin layer of regular silica with cotton on top (covered with Al foil). After 18 h the reaction was stopped and the cartridge was removed and flushed with solvent

[0208] (isopropanol / MTBE, 1 :19, 100 mL) and dried with a stream of N2. The dried silica was suspended in ammonium hydroxide (25 Wt. %, 100 mL) and stirred for 5 min. DCM (200 mL) was added and the suspension was filtered. The organic layer was separated and the aqueous phase was extracted with DCM (3 x, 100 mL). The combined organic layers were brine (100 mL) and dried with MgSC , concentrated in vacuo and purified with silica gel column chromatography (50% Et3O in toluene) to afford 8 and 9 as colorless oils (1 :2.6) and mix fractions containing both isomers (total yield: 254 mg, 58%). HRMS (m / z): [M + Na]+calcd. for Ci3Hi8O5Na: 277.1052, found 277.1038.

[0209] 8 (axial): TLC (Et2O / toluene, 1 :1 v / v): RF = 0.33.1H NMR (500 MHz, CDCh) 5 5.94 (ddd, J = 16.8, 11 .5, 3.3 Hz, 1 H), 5.65 - 5.59 (m, 1 H), 4.26 (td, J = 7.2, 4.5 Hz, 1 H), 3.76 (s, 3H), 3.70 (s, 3H), 2.76 (ddd, J = 13.9, 8.5, 3.5 Hz, 1 H), 2.60 (td, J = 12.0, 7.8 Hz, 1 H), 2.50 - 2.42 (m, 1 H), 2.01 - 1.92 (m, 2H), 1 .68 - 1.62 (m, 1 H), 1 .55 - 1 .46 (m, 1 H), 1.28 (dt, J = 14.9, 10.4 Hz, 1 H).13C NMR

[0210] (126 MHz, CDCh) 6 171.4, 168.1 , 136.3, 132.1 , 67.0, 52.9, 52.4, 41 .8, 37.3, 30.8, 27.9, 19.6. 9 (equatorial): TLC (Et2O / toluene, 1:1 v / v): RF = 0.30.1H NMR (500 MHz, CDCb) 65.88-5.80 (m, 1H), 5.62 (ddd, J= 16.6, 9.4, 1.3 Hz, 1H), 4.13 (td, J = 9.7, 5.4 Hz, 1 H), 3.76 (s, 3H), 3.68 (s, 3H), 2.84 - 2.74 (m, 2H), 2.35 (td, J = 9.5, 2.7 Hz, 1H), 2.17 (ddd, J = 12.9,7.4,5.4 Hz, 1H), 2.10 (ddd, J= 15.1,7.4, 4.5 Hz, 1H), 1.60 (ddd, J = 11.9, 9.5, 4.5 Hz, 1H), 1.48 (ddd, J= 12.7, 11.2,

[0211] 9.8 Hz, 1H), 0.78 (dt, J= 15.1, 11.6 Hz, 1H).13C NMR (126 MHz, CDCb) 6171.4, 168.4, 139.7,

[0212] 131.6, 76.2, 52.8, 52.3, 41.8, 37.3, 32.1, 26.2, 25.9.

[0213] Example 1.4 - Derivatives and conjugates

[0214] Dimethyl (1 S,5 / ?,8 / ?,Z)-5-(((4-nitrophenyl)carbamoyl)oxy)bicyclo[6.1.0]non-3-ene-9,9- toluene) to afford 12 (13 mg, 52%) as a white solid. TLC

[0215] (Et2O / toluene, 1:1 v / v): RF = 0.59.1H NMR (500 MHz, CDCb) 68.20 (d, J = 9.3 Hz, 2H), 7.55 (d, J

[0216] = 9.2 Hz, 2H), 5.87 (ddd, J= 15.3, 11.4, 3.3 Hz, 1H), 5.66 (dd, J= 16.8, 4.9 Hz, 1H), 5.11 -5.05 (m, 1 H), 3.79 (s, 3H), 3.72 (s, 3H), 2.87 - 2.76 (m, 1 H), 2.69 - 2.52 (m, 2H), 2.01 (s, 2H), 1.73 - 1.63 (m, 2H), 1.39 - 1.32 (m, 1 H).13C NMR (126 MHz, , CDCb) 6171.2, 152.3, 144.0, 134.1 , 131.3, 125.4, 117.8, 70.9, 53.0, 52.5, 38.5, 32.0, 30.4, 27.8, 19.4.

[0217] Dimethyl (1 S,5 / ?,8 / ?,Z)-5-(((4-nitrophenyl)carbamoyl)oxy)bicyclo[6.1.0]non-3-ene-9,9- in toluene) to afford 13 (183 mg, 33%) as a white solid. TLC

[0218] (Et2O / toluene, 1:1 v / v): RF = 0.64.1H NMR (500 MHz, CDCb) 68.23 - 8.16 (m, 2H), 7.59 - 7.51

[0219] (m, 2H), 6.04 (dt, J = 15.9, 7.7 Hz, 1H), 5.67 (dd, J= 16.7, 9.8 Hz, 1H), 5.07 (td, J= 9.9, 5.4 Hz,

[0220] 1H), 3.79 (s, 3H), 3.71 (s, 3H), 2.87 -2.83 (m, 2H), 2.42-2.29 (m, 2H), 2.19 (ddd, J= 15.4, 7.2, 4.6 Hz, 1 H), 1.67 - 1.61 (m, 1 H), 1.58 - 1.55 (m, 1 H), 0.89 (dt, J = 15.2, 11.7 Hz, 1 H).13C NMR (126 MHz, , CDCb) 6171.3, 168.2, 152.4, 144.0, 143.2, 135.1, 133.9, 125.4, 117.8, 79.0, 52.9, 52.4, 41.7, 34.2, 31.7, 26.0, 25.9. Dimethyl (1S,5R,8R,Z)-5-(((4-nitrophenoxy)carbonyl)oxy)bicyclo[6.1.0]non-3-ene-9,9- .

[0221] Hereafter, the reaction mixture was quenched with aqueous sat. NH4CI (2 mL) was added and extracted with DCM (2 x 20 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL) and dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (0 — 35% EtOAc in heptane) to afford 14 (20 mg, 49%) as a white solid mixed with a minor amount of 4-nitrophenol. TLC (EtOAc / heptane, 1 :1 v / v): R = 0.57.1H NMR (500 MHz, CDCb) 6 8.27 (d, J = 9.1 Hz, 2H), 7.39 (d, J = 9.2 Hz, 2H), 5.97 (ddd, J = 16.8, 11.7, 3.4 Hz, 1 H), 5.67 - 5.62 (m, 1 H), 5.06 (td, J = 7.7, 4.8 Hz, 1 H), 3.78 (s, 3H), 3.72 (s, 3H), 2.81 (ddd, J = 12.4,

[0222] 8.7, 3.4 Hz, 1 H), 2.70 - 2.55 (m, 2H), 2.12 - 1 .97 (m, 2H), 1 .84 - 1 .76 (m, 1 H), 1 .70 (ddd, J = 11 .8, 10.0, 5.7 Hz, 1 H), 1.36 (dt, J = 15.5, 10.9 Hz, 1 H).13C NMR (126 MHz, CDCb) 6 171 .2, 167.9,

[0223] 155.7, 151.9, 145.5, 134.8, 130.1 , 125.4, 121.9, 74.4, 53.1 , 52.5, 38.1 , 31.8, 30.0, 27.7, 19.2. HRMS (m / z): [M + Na]+calcd. for C20H2iNiO9Na: 442.1114, found 442.1131.

[0224] Dimethyl (1S,5R,8R,Z)-5-(((4-nitrophenoxy)carbonyl)oxy)bicyclo[6.1.0]non-3-ene-9,9-

[0225] Hereafter, the reaction mixture was quenched with aqueous sat. NH4CI (2 mL) was added and extracted with DCM (2 x 20 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL) and dried with MgSC , concentrated in vacuo and purified with silica gel column chromatography (0 — 40% EtOAc in heptane) to afford 16 (133 mg, 70%) as a white solid mixed with a minor amount of 4-nitrophenol. TLC (EtOAc / heptane, 1 :1 v / v): RF = 0.64.1H NMR (500 MHz, CDCb) 6 8.26 (d, J = 9.2 Hz, 2H), 7.37 (d, J = 9.2 Hz, 2H), 6.06 (ddd, J = 16.0, 8.6, 6.7 Hz, 1 H), 5.74 (ddd, J = 16.8, 9.7, 1.3 Hz, 1 H), 5.04 (td, J = 10.0, 5.4 Hz, 1 H), 3.78 (s, 3H), 3.70 (s, 3H), 2.92 - 2.79 (m, 2H), 2.45 - 2.35 (m, 2H), 2.25 - 2.17 (m, 1 H), 1.76 - 1 .62 (m, 2H), 0.96 - 0.83 (m, 1 H).13C NMR (126 MHz, CDCb) 6 171.3, 168.2, 155.6, 151.9, 145.4, 134.6, 134.3, 125.4, 121.9, 82.2, 53.0, 52.5, 41 .6, 33.7, 31 .6, 25.9, 25.8. HRMS (m / z): [M + Na]+calcd. for C2oH2iNi09Na: 442.1114, found 442.11 18. Dimethyl (1 S,5R,8R,Z)-5-(((4-fluorobenzyl)(methyl)carbamoyl)oxy)bicyclo[6.1 .0]non-3-ene- 9,9-dicarboxylate (Axial, 15): Dimethyl (1 S,5R,8R,Z)-5-(((4- nitrophenoxy)carbonyl)oxy)bicyclo[6.1 .0]non-3-ene-9,9-dicarboxylate (14, 25 mg, 59.6 pmol) was dissolved in dry DCM (5 mL) and DIPEA (31 pL, 178.8 pmol), 1 -(4-fluorophenyl)-A / -methylmethanamine (13 pL, 89.4 pmol) and a catalytic amount of DMAP were added and the reaction was stirred for 18 h at 40 °C. Hereafter, the reaction was diluted with DCM (20 mL) and washed with water (4 x 20 mL). The organic layer was washed with brine (20 mL), dried with MgSC , concentrated in vacuo and purified with silica gel column chromatography (0 — 50% EtOAc in heptane) to afford 15 (13 mg, 52%) as a colorless oil. TLC (EtOAc / heptane, 1 :1 v / v): RF = 0.47.1H NMR (500 MHz, CDCb) 6 7.24 - 7.18 (m, 2H), 7.06 - 6.99 (m, 2H), 5.88 - 5.59 (m, 2H), 5.02 (td, J = 7.5, 4.2 Hz, 1 H), 4.54 - 4.38 (m, 2H), 3.78 (s, 3H), 3.71 (s, 3H), 2.88 (s, 3H), 2.81 - 2.70 (m, 1 H), 2.67 - 2.47 (m, 2H), 2.08 - 1 .88 (m, 2H), 1 .69 - 1 .60 (m, 2H), 1 .38 - 1 .27 (m, 1 H).19F NMR (470 MHz, CDCb) 6 -115.19 (d, J = 65.9 Hz). HRMS (m / z): [M + Na]+calcd. for C22H26O6FNNa: 442.1642, found 442.1633.

[0226] Dimethyl (1 S,5R,8R,Z)-5-(((4-fluorobenzyl)(methyl)carbamoyl)oxy)bicyclo[6.1 .0]non-3-ene-

[0227] 9,9-dicarboxylate (Equatorial, 17): Dimethyl (1 S,5R,8R,Z)-5-(((4- nitrophenoxy)carbonyl)oxy)bicyclo[6.1 .0]non-3-ene-9,9-dicarboxylate (16, 100 mg, 238.4 pmol) was dissolved in dry DCM (10 mL) and DIPEA (125 pL, 715.3 pmol), 1-(4-fluorophenyl)- / V- methylmethanamine (47 pL, 357.7 pmol) and a catalytic amount of DMAP were added and the reaction was stirred for 18 h at 40 °C. Hereafter, the reaction was diluted with DCM (20 mL) and washed with water (4 x 20 mL). The organic layer was washed with brine (20 mL), dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (0 60% EtOAc in heptane) to afford 17 (68 mg, 68%) as a colorless oil. TLC (EtOAc / heptane,

[0228] 4:1 v / v): RF = 0.64.1H NMR (500 MHz, CDCb) 6 7.24 - 7.13 (m, 2H), 7.06 - 6.97 (m, 2H), 5.96 (dt, J = 15.9, 7.7 Hz, 1 H), 5.73 - 5.60 (m, 1 H), 5.07 - 4.96 (m, 1 H), 4.42 (s, 2H), 3.77 (s, 3H), 3.69 (d, J = 2.6 Hz, 3H), 2.88 - 2.78 (m, 5H), 2.39 - 2.24 (m, 2H), 2.15 (ddd, J = 15.2, 7.3, 4.5 Hz, 1 H), 1 .64 - 1.56 (m, 1 H), 1.55 - 1.46 (m, 1 H), 0.85 (dt, J = 15.3, 11.6 Hz, 1 H).13C NMR (126 MHz, CDCb) 6 171.3, 168.2, 163.3, 161.3, 136.2, 136.1 , 133.4, 133.4, 132.8, 132.7, 115.6, 1 15.5, 78.5, 52.8, 52.3, 51.8, 41.7, 37.4, 34.4, 31.9, 26.2, 25.9.19F NMR (470 MHz, CDCb) 6 -115.19 (d, J = 56.7 Hz). HRMS (m / z): [M + Na]+calcd. for C22H26O6FNNa: 442.1642, found 442.1643.

[0229] Example 1.5 - Selective reactivity of cyclopropyl handles

[0230] It was found that handles Y1and Y2could be selectively addressed, allowing their strategic deprotection and optional reprotection using different groups to tailor the conjugations as desired.

[0231] Scheme E2. Synthesis of partially deprotected TCO derivative 18. i. LiOH (5 equiv), THF:HzO (2:1), 40 °C, 18 h.

[0232] (1S,5R,8R,9R,Z)-5-(((4-fluorobenzyl)(methyl)carbamoyl)oxy)-9- (methoxycarbonyl)bicyclo[6.1 .0]non-3-ene-9-carboxylic acid

[0233] (Equatorial, 18): Dimethyl (1 S,5R,8R,Z)-5-(((4- fluorobenzyl)(methyl)carbamoyl)oxy)bicyclo[6.1 .0]non-3-ene-9,9- dicarboxylate (17, 33 mg, 78.8 pmol) was dissolved in THF:H2<D (2:1 , 5 mL) and LiOH (10 mg, 393.4 pmol) was added and the reaction was stirred for 18 h at 40 °C (covered in Al foil). Hereafter, the reaction was quenched with AcOH (23 pL, 393.4 pmol) and diluted with DCM (20 mL).

[0234] The organic layer was separated and the aqueous phase was extracted with DCM (10 mL, 4 x). The combined organic layers were washed with brine (20 mL), dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (0 — 10% MeOH in DCM) to afford 18 (10 mg, 32%) as a colorless oil. TLC (DCM / MeOH, 9:1 v / v): RF = 0.53.1H NMR (500 MHz, CDCb) 5 7.24 - 7.14 (m, 2H), 7.06 - 6.95 (m, 2H), 6.08 (dd, J = 16.5, 8.0 Hz, 1 H), 5.77 - 5.63 (m, 1 H), 5.12 - 5.05 (m, 1 H), 4.43 (s, 2H), 3.90 (s, 3H), 2.98 - 2.82 (m, 5H), 2.59 - 2.50 (m, 1 H), 2.40 - 2.27 (m, 1 H), 2.14 - 2.07 (m, 1 H), 1.96 - 1.86 (m, 1 H), 1.58 - 1.48 (m, 1 H), 1.18 (dt, J = 14.9, 11.7 Hz, 1 H).13C NMR (126 MHz, CDCb) 6 172.2, 163.3, 161.2, 155.6, 136.6, 133.3, 133.0, 115.7, 115.5, 78.4, 53.0, 51.9, 46.0, 39.0, 36.8, 34.2, 33.6, 26.4, 25.4.19F NMR (470 MHz, CDCb) 6 -115.08 (d, J = 70.6 Hz).

[0235] Example 2 — Characterisation of compounds and conjugates

[0236] 2.1 - Confirmation of stereochemistry

[0237] A 2D 1 H-1 H NOESY spectrum was obtained for the acetylated iodohydrin 5. As expected, a correlation was observed for the two protons indicated in the structure to the right. No correlation was observed for the other proton at the cyclopropyl ring and the proton that is on the same carbon atom as the -OAc moiety. This confirms the represented stereochemistry because the other possible diastereoisomer with acetate addition on the frontside of the molecule should not have this NOESY coupling of protons 3 and 1 b.

[0238] 2.2 - Kinetics study of click reaction The reaction rate in a TCO-Tz click reaction of 8 and 9 were measured under similar conditions as described in Versteegen et al., Angewandte Chemie International Edition 2013, 52 (52), 14112-14116. The second order reaction constant of the reaction between two different tetrazines (either 3,6- dimethyl-1 ,2,4,5-tetrazine (10) or 3,6-di(pyridin-2-yl)-1 ,2,4,5-tetrazine

[0239] (11)) and two TCOs (8 (X2= -OH, axial) and 9 (X2= -OH, equatorial)) were determined under second order conditions in MeCN at 20 °C by UV spectroscopy. Measurements were performed in triplo. A cuvette was filled with MeCN (800 pL) and equilibrated at 20 °C. A stock solution of tetrazine in DMSO (100 pl) was added to the cuvette, resulting in 1 mM of each of the compounds. The absorption of the tetrazine moiety was measured at 540 nm. Next, the cuvette was removed from the apparatus to add a stock solution of TCO in DMSO (100 pl) and briefly mixed the solution by shaking the cuvette before placing it back into the apparatus. The absorption at 540 nm was measured for 15 minutes. From this absorption at 540 nm, the concentration of tetrazine was calculated using a molar absorption coefficient of e=430 M'1cm_1. The second order rate constant fe was obtained from the slope of a plot of (1 / c - 1 / c0) versus time. The kinetics of TCO 8 and 3, 6-di(pyridin-2-yl)-1 ,2,4,5- tetrazine 11 at 20 °C in MeCN showed a reaction rate that with this technique can be classified as an instant reaction. The reaction between 8 and 10 showed a fe rate of 210.4 M1s-1(R2= 0.985). The reaction between 9 and 10 showed a fe rate of 6.9 M1s-1(R2= 0.960). The reaction between 9 and 11 showed a fe rate of 82.3 M1s-1(R2= 0.966).

[0240] TCO reactions known from the prior art (Versteegen et al., Angewandte Chemie International Edition 2013, 52 (52), 141 12-14116, DOI: 10.1002 / anie.201305969) were assessed at 83.3 pM and afforded a lower rate of 62.5 M-1s-1under similar conditions. The fastest TCOs known from W02023 / 281040 are referred to therein as E-02a and are reported as having rates of 437 M'1s_1with tetrazine 11 , which falls short of the ‘instant’ speed found in this study. E-02a can be seen as matching 8 except Y1is an ethyl ester where 8 has a methyl ester, and Y2is a proton in E-02a and not any moiety as defined for Y2herein. Thus, compounds of the invention outperform known close analogues in click reaction kinetics.

[0241] 2.3 — Kinetics study of release reaction

[0242] As shown in Fig. 1 , para-nitrophenyl carbamate conjugates of 8 and 9 were prepared, resulting in 12 and 13. These conjugates were tested with 4 equivalents of tetrazine 10 to assess the release kinetics. The axial isomer 12 revealed rapid release of the payload paranitroaniline (p-NA) and the appearance of the respective elimination adduct (E1). For the equatorial isomer 13 this process was slower. 12 showed almost complete release of the pay load after about 25 minutes. The fastest payload release observed by Versteegen et al. (cited above) was less than 20% release over 250 minutes.

Claims

Claims1 . A compound of general formula (I), or a salt thereof:wherein one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H;Y1and Y2are in each instance independently -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2 or -C=H, or Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring;Z1, Z2, and Z3are in each instance independently O, S, Se, or NQN;Ra, Rb, Rc, and Rdare in each instance independently -O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, or a leaving group;Pr is in each instance independently -H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, -NO2, alkoxy, -C(=0)0(CH2)o-4H, or haloalkoxy;Q, QN, QN1, and QN2are in each instance independently -H or a linear, branched or cyclic C1-6 alkyl or acyl, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1 , 2, 3 or 4.

2. The compound according to claim 1 , wherein m is 0.

3. The compound according to claim 1 or 2, wherein X2is -Raor -Z1-C(=Z2)-Rb, and X1is -H.

4. The compound according to any one of claims 1 -3, wherein Y1and Y2are in each instance independently -C(=O)-RCor -CH2-Rd.

5. The compound according to any one of claims 1 -4, wherein the protecting group is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4- methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p- methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE), or wherein the leaving group is a halogen, preferably chlorine, bromine, or iodine, more preferably chlorine, or wherein the leaving group is6. The compound according to any one of claims 1 -5, wherein the compound is of general formula (V-OO), (V-HO), (V-OH) or (V-HH):(V-OO), (V-HO), (V-OH), (V-HH).

7. The compound according to any one of claims 1-6, wherein Y1and Y2both represent the same moiety, or wherein at most one of Y1and Y2is C5-10(hetero)aryl.

8. The compound according to any one of claims 1 -7, wherein the compound is of general formula (ll-j) or (Vl-a) or an enantiomer thereof:

9. The compound according to any one of claims 1 -8, wherein the compound is of general formula (IX-H1) or (IX-H2) or an enantiomer thereof, wherein preferably Rcand Rdare in each instance chosen independently from -OH or -O-C1-2 alkyl or a leaving group:

10. A conjugate comprising a moiety conjugated to a compound as defined in any one of claims 1-9, preferably wherein said moiety is a small organic molecule, a pharmaceutically active substance, a macromolecule, a particle, a surface, or a biomolecule, more preferably wherein said moiety is a protein, most preferably wherein said moiety is an antibody.

11. A method for producing a conjugate as defined in claim 10, the method comprising the steps of: i) providing a compound as defined in any one of claims 1 -9; ii) providing a second compound, preferably wherein said second compound is a small organic molecule, a pharmaceutically active substance, a macromolecule, or a biomolecule, more preferably wherein said second compound is a protein, most preferably wherein said second compound is an antibody; iii) reacting the compound provided in step i) with the second compound to form a conjugate; and optionally iv) isolating the conjugate obtained in step iii).

12. A composition comprising a compound as defined in any one of claims 1 -9, or a conjugate as defined in claim 10, and a pharmaceutically acceptable excipient, preferably wherein the composition comprises a tetrazine, more preferably dipyridyl tetrazine.

13. A compound as defined in any one of claims 1 -9, or a conjugate as defined in claim 10, or a composition as defined in claim 12, for use as a medicament, wherein preferably at least one of Ra, Rb, Rc, and Rdis a pharmaceutically active substance, wherein preferably at least one of Ra, Rb, Rc, and Rdis a targeting moiety, wherein the pharmaceutically active substance is preferably a chemotherapeutic agent, and the targeting moiety is preferably an antibody or a fragment thereof.

14. A method for releasing a pharmaceutically active substance, the method comprising the steps of: i) providing a conjugate as defined in claim 10 wherein at least one of Ra, Rb, Rc, and Rdis the pharmaceutically active substance; ii) contacting the provided conjugate with a 1 ,2,4,5-tetrazine;wherein the conjugate as defined in claim 10 is preferably of general formula (ll-a) or (VI- a), more preferably of general formula (Vl-a).

15. A compound of general formula (Intermediate-1), (Intermediate-2), or (Intermediate-3), or a salt thereof:(Intermediate-1) (Intermediate-2) (Intermediate-3) wherein hal is -SePr, -SPr, or a halogen, preferably a halogen such as iodine; one of X1and X2is -Raor -Z1-C(=Z2)-Rb, and the other is -H;Y1and Y2are in each instance independently -C(=Z3)-RCor -CH2-Rdor C5-10(hetero)aryl or -C(=Z3)-H or -CH=CH2 or -C=H, or Y1and Y2together with the carbon atom to which they are attached form a 3-10-membered ring;Z1, Z2, and Z3are in each instance independently O, S or NQN;Ra, Rb, Rc, and Rdare in each instance independently -O-Pr, -S-Pr, -Se-Pr, -NQN1QN2, or a leaving group;Pr is in each instance independently -H, a linear, branched, or cyclic C1-6 alkyl or acyl, or a protecting group, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, -NO2, alkoxy, -C(=0)0(CH2)o-4H, or haloalkoxy;Q, QN, QN1, and QN2are in each instance independently -H or a linear, branched or cyclic C1-6 alkyl or acyl, wherein acyl or alkyl are optionally unsaturated and optionally substituted with halogen, alkoxy, or haloalkoxy; and m is 0, 1 , 2, 3 or 4.

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