FIELD OF THE INVENTION The invention disclosed herein relates to compounds, combinations, kits, and methods using same, for use in bioorthogonal release reactions. BACKGROUND

Selective chemical reactions that are orthogonal to the diverse functionality of biological systems are called bio-orthogonal reactions and occur between two abiotic groups with exclusive mutual reactivity. These can be used to selectively modify biochemical structures, such as proteins or nucleic acids, which typically proceed in water and at near-ambient temperature, and may be applied in complex chemical environments, such as those found in living organisms.

Bio-orthogonal reactions are broadly useful tools with applications that span chemical synthesis, materials science, chemical biology,

diagnostics, and medicine. Especially prominent application areas for bioorthogonal reactions include drug delivery agents and prodrugs for pharmaceutical applications, as well as various reversible bioconjugates and sophisticated spectroscopic bioprobes for applications in the field of biological analysis.

One prominent bioorthogonal reaction is the inverse-electron-demand Diels Alder (IEDDA) reaction between a trans-cyclooctene (TCO) and a tetrazine (TZ). In previous studies the IEDDA reaction was used for pretargeted radioimmunoimaging, treating tumor-bearing mice with trans- cyclooctene(TCO)-tagged antibody or antibody fragments, followed one or more days later by administration and selective conjugation of a

radiolabeled tetrazine probe to the TCO tag of the tumor-bound antibody [R. Rossin, M. S. Robillard, Curr. Opin. Chem. Biol.2014, 21, 161-169].

Based on the IEDDA conjugation a release reaction has been developed, which was termed the IEDDA pyridazine elimination, a“click-to- release” approach that affords instantaneous and selective release upon conjugation [R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen, M. S. Robillard, Angew. Chem. Int. Ed.2013, 52, 14112-14116]. IEDDA reactions between tetrazines (i.e. diene) and alkenes (i.e. dienophile) afford 4,5-dihydropyridazines, which usually tautomerize to 1,4- and 2,5- dihydropyridazines. It was demonstrated that the 1,4-dihydropyridazine product derived from a TCO containing a carbamate-linked doxorubicin (Dox) at the allylic position and tetrazine is prone to eliminate CO ₂ and Dox via an electron cascade mechanism eventually affording aromatic

pyridazine. The triggered release has been demonstrated in PBS (phosphate buffered saline), serum, cell culture and in mice and holds promise for a range of applications in medicine, chemical biology, and synthetic chemistry, including triggered drug release, biomolecule uncaging and capture&release strategies.

In general the IEDDA pyridazine elimination enables the controlled manipulation of a wide range of substrates in relatively complex

environments, in the presence of a range of other chemical functional groups. This control can be temporal and, optionally, also spatial. The manipulation can be versatile, e.g. for a variety of purposes including but not limited to activating, deactivating, releasing, trapping, or otherwise altering a Construct attached to a chemically cleavable group.

The IEDDA pyridazine elimination has been applied in triggered drug release from antibody-drug conjugates (ADCs) capable of participating in an IEDDA reaction (Figure 1). ADCs are a promising class of

biopharmaceuticals that combine the target-specificity of monoclonal antibodies (mAbs) or mAb fragments with the potency of small molecule toxins. Classical ADCs are designed to bind to an internalizing cancer cell receptor leading to uptake of the ADC and subsequent intracellular release of the drug by enzymes, thiols, or lysosomal pH. Routing the toxin to the tumour, while minimizing the peripheral damage to healthy tissue, allows the use of highly potent drugs resulting in improved therapeutic outcomes. The use of the IEDDA pyridazine elimination for ADC activation allows the targeting of non-internalizing receptors, as the drug is cleaved chemically instead of biologically.

In general prodrugs, which may comprise ADCs, are an interesting application for the IEDDA pyridazine elimination reaction, in which a drug is deactivated, bound or masked by a moiety, and is reactivated, released or unmasked after an IEDDA reaction has taken place.

Background art for the aforementioned technology further includes WO ₂ 012/156919, WO ₂ 012156918A1, WO 2014/081303, and

US20150297741. Herein a dienophile, the aforementioned TCO, is used as a chemically cleavable group in e.g. a protecting group in synthetic chemistry, a cleavable linker or mask in chemical biology, in vitro diagnostics, and in vivo prodrug activation. The group is attached to a Construct (e.g. molecule, protein, peptide, polymer, dye, surface) in such a way that the release of the dienophile from the Construct can be provoked by allowing the dienophile to react with a diene, the aforementioned TZ. The dienophile is an eight- membered non-aromatic cyclic alkenylene or alkenyl group, particularly a TCO group.

In some applications, the TCO is part of prodrug which is first injected in the blood stream of a subject and may be targeted to a certain part of the body, e.g. a tumor. Then, a certain percentage of the prodrug is immobilized at the targeted spot, while another percentage is cleared by the body. After several hours or days, an activator comprising a tetrazine is administered to release a drug from the prodrug, preferably only at the targeted spot. The tetrazine itself is also subject to clearance by the body at a certain clearance rate. In general, the tetrazine reacts in an initial step with a dienophile- containing Construct (e.g. a dienophile-containing prodrug) to form a conjugate. This is referred to as the click conjugation step. Next, via one or multiple mechanisms, the Construct is preferably released from the

Construct-dienophile (e.g. prodrug). It will be understood that a high yield in the click conjugation step, i.e. a high click conjugation yield, does not necessarily result in a high yield of released Construct, i.e. a high drug release yield. From the viewpoint of bio-orthogonality the chemistry works well. However, it is desired that better IEDDA reactions are developed. Achieving high Construct release yields in IEDDA reactions remains a challenge both in vivo and in vitro. In particular, it is desired that the reaction between a Construct-bearing dienophile and a diene results in a high Construct release yield in vitro and/or in vivo.

Furthermore, achieving fast Construct release in IEDDA reactions remains a challenge both in vivo and in vitro. In particular, it is desired that the reaction between a Construct-bearing dienophile and a diene results in a fast Construct release in vitro and/or in vivo. Typically, the tetrazine motives that typically give high release are less reactive than the tetrazines that have successfully been used for click conjugations in vivo. These more reactive tetrazines give a good click conjugation yield, but result in a poor click release yield. In the

abovementioned study [R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen, M. S. Robillard, Angew. Chem. Int. Ed.2013, 52, 14112-14116], it was shown that for example 3,6-bis-(2-pyridyl)-1,2,4,5-tetrazine gives high click conjugation rate and yield, but a very poor click release yield of only 7% in PBS/MeCN (3:1) or only 12% in serum. In addition, for tetrazines that give a high release yield, for example 3,6-bis-methyl-1,2,4,5-tetrazine, this release typically takes several hours and typically exhibits a maximum 80-90 % release yield R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen, M. S. Robillard, Angew. Chem. Int. Ed.2013, 52, 14112-14116].

For at least those reasons, it is a desire to improve the click release rate and yield of tetrazine motifs with relatively high reactivity towards dienophiles (i.e. high click conjugation yield) in vitro and/or in vivo.

Another desire is to improve the click release rate and possibly the click release yield of tetrazines with relatively good click conjugation and/or release yields in vitro and/or in vivo.

Another desire is to improve the click release rate, and preferably also the click release yield, of tetrazines with low reactivity towards dienophiles in vitro and/or in vivo.

Another desire is to achieve a combination of a high click conjugation yield with a Construct-bearing dienophile and a high Construct release yield both in vitro and in vivo.

Another desire is to achieve a combination of a high click conjugation reaction rate with a dienophile, a high click conjugation yield between a Construct-bearing dienophile and the tetrazine and a high Construct release yield and a high Construct release rate, preferably both in vitro and in vivo. Previous studies have attempted to address said desires by engineering the diene, rather than the dienophile, to afford higher conjugation yields, or increased release yields / rates, or both.

A previous study ([R. Rossin, S.M.J. van Duijnhoven, W. ten Hoeve, H.M. Janssen, L.H.J. Kleijn, F.J.M. Hoeben, R.M. Versteegen, M.S.

Robillard, Bioconj.Chem., 2016, 27, 1697-1706]) has shown that linking a 10 kDa dextran to a 3-methyl-6-(2-pyridyl)-tetrazine or a 3-methyl-6- (methylene)-tetrazine resulted in high click conjugation yields with a TCO in vivo, but to suboptimal drug release yields both in vitro and vivo.

Another publication ([Fan et al., Angew. Chem. Int. Ed., 2016, 55, 14046-14050]) aimed at in vitro reactions, showed that small, asymmetrical tetrazines give a higher conjugation reactivity with TCOs than symmetrical bis-alkyl-tetrazines, but the click-release yields were still not quantitative.

Two other publications [Carlson et al., J. Am. Chem. Soc.2018, 140, 3603-3612] and [Sarris et al, Chemistry 2018, 24, 18075-18081] showed that bis-alkyl tetrazines containing carboxylate or amine groups can enhance the click-release rate, but these tetrazine remain relatively unreactive in the click-conjugation with TCO. It is desired that compounds are developed that address one or more of the abovementioned problems and/or desires. SUMMARY OF THE INVENTION In one aspect, the present invention pertains to a compound satisfying Formula (19):

Formula (19), and pharmaceutically acceptable salts thereof, wherein R48 is selected from the group consisting of -OH, -OC(O)Cl, -OC(O)O-N- succinimidyl, -OC(O)O-4-nitrophenyl, -OC(O)O-tetrafluorophenyl, -OC(O)O- pentafluorophenyl, -OC(O)-(S ^P ) _k C ^A , -OC(S)-(S ^P ) _k C ^A , -SC(O)-(S ^P ) _k C ^A , -SC(S)- (S ^P ) _k C ^A , -O-(L ^C ((S ^P ) _k C ^A ) _s ((S ^P ) _k C ^A ) _s ((S ^P ) _i -C ^B ) _j ) _r -(S ^P ) _k C ^A , -S- (L ^C ((S ^P )kC ^A )s((S ^P )kC ^A )s((S ^P )i-C ^B )j)r-(S ^P )kC ^A , and -(S ^P )kC ^A ; r is an integer in range of from 0 to 2; each s is independently 0 or 1; each i is independently an integer in a range of from 0 to 4, preferably 0 or 1; j is an integer in range of from 0 to 4, preferably 0 or 1; each k is

independently 0 or 1; L ^C is a self-immolative linker and S ^P is a spacer; each C ^A and C ^B are independently selected from the group consisting of organic molecules and inorganic molecules;

wherein at least one of conditions (a)-(c) is met: (a) at least one of X ¹ , X ² , X ³ , X ⁴ , X ⁵ , is CR ₄₇ Y ^T1 ; and Y ^T1 is positioned cis relative to H ^a ; (b) X ³ is Y ^T3 ; and (c) two adjacent X moieties selected from X ¹ , X ² , X ³ , X ⁴ , X ⁵ , are part of a fused ring satisfying one of the Formulae (20a)-(20g):

Y ^T2 is positioned syn relative to the 8-membered dienophile ring;

wherein X ^a and X ^b are part of the 8-membered ring of Formula (19), such that X ^a -X ^b or X ^b -X ^a is X ¹ -X ² , X ² -X ³ , X ³ -X ⁴ , or X ⁴ -X ⁵ ; for Formulae (20a)-(20c), X ^a and X ^b are CR ₄₇ , preferably CH;

for Formulae (20d)-(20g), X ^a and X ^b are independently CR47 or N, preferably CR47, more preferably CH;

X ⁶ and X ⁸ are each independently selected from the group consisting of Y ^T3 , C(R47)Y ^T2 , C(R47)2, O, S, C(O), C(S), and S(O)2;

X ⁷ is selected from the group consisting of Y ^T3 and Z ^T ;

when X ⁶ is Y ^T3 , then X ⁷ is Z ^T ;

when X ⁷ is Y ^T3 , then X ⁶ and X ⁸ are each independently selected from the group consisting of C(R ₄₇ )Y ^T2 _, C(R ₄₇ ) ₂ , O, and S; preferably C(R ₄₇ )Y ^T2 or C(R ₄₇ ) ₂ ;

wherein the fused ring satisfying Formula (20g) comprises at least one Y ^T2 or Y ^T3 moiety;

for Formulae (20b) and (20f), two R47 directly connected to the same carbon may together be =C-(R47)2, =S, or =O;

with the proviso that X ¹ -X ² , X ² -X ³ , X ³ -X ⁴ , and X ⁴ -X ⁵ are not -O-O-, -N-N-, -O- N- or -N-O-;

the direct bonds from X ^a and X ^b to the remainder of the ring fused to the 8- membered dienophile ring of Formulae (20a)-(20g) are cis relative to one another, and cis relative to H ^a ;

preferably no adjacent pairs of atoms being -O-O- are part of the fused rings of Formulae (20a)-(20g);

Y ^T1 is selected from the group consisting of OH, SH, N(R38)2, C(O)OH, C(S)OH, C(O)SH, C(S)SH, O-N(R ₃₈ ) ₂ , SO ₄ H, SO ₃ H, SO ₂ H, PO ₄ H ₂ , PO ₃ H, PO ₂ H, and C(N)N(R ₃₈ ) ₂ ;

Y ^T2 is selected from the group consisting of OH, SH, and N(R38)2;

Y ^T3 is NR ₃₈ and is not flanked by C(O), C(S), S(O), or S(O) ₂ ;

the remaining X ¹ , X ² , X ³ , X ⁴ , X ⁵ , are independently selected from the group consisting of C(R47)2 and O, such that at most two of X ¹ , X ² , X ³ , X ⁴ , X ⁵ are O, NR ₃₈ or N;

wherein, when R48 is -OC(O)-(S ^P )kC ^A , -OC(S)-(S ^P )kC ^A , -SC(O)-(S ^P )kC ^A , or -SC(S)-(S ^P ) _k C ^A , S ^P (when k>0) or C ^A (when k=0) is bound to the -OC(O)-, -OC(S)-, -SC(O)-, or -SC(S)- of R48 via an atom selected from the group consisting of O, C, S, and N, preferably a secondary or a tertiary N, wherein this atom is part of S ^P or C ^A ; preferably R ₄₈ is -OC(O)-(S ^P ) _k C ^A and S ^P or C ^A is bound to the -OC(O)- of R48 via an N atom;

wherein, when R48 is -O-(L ^C ((S ^P )kC ^A )s((S ^P )kC ^A )s((S ^P )i-C ^B )j)r-(S ^P )kC ^A or

-S-(L ^C ((S ^P ) _k C ^A ) _s ((S ^P ) _k C ^A ) _s ((S ^P ) _i -C ^B ) _j ) _r -(S ^P ) _k C ^A and r is 0, S ^P (when k>0) or C ^A (when k=0) is bound to the -O- or -S- moiety of R48 on the allylic position of the trans-cyclooctene ring of Formula (19) via a group selected from the group consisting of -C(O)-, and -C(S)-, wherein this group is part of S ^P or C ^A , wherein, when R48 is -O-(L ^C ((S ^P )kC ^A )s((S ^P )kC ^A )s((S ^P )i-C ^B )j)r-(S ^P )kC ^A or -S- (L ^C ((S ^P ) _k C ^A ) _s ((S ^P ) _k C ^A ) _s ((S ^P ) _i -C ^B ) _j ) _r -(S ^P ) _k C ^A and r is 1, L ^C is bound to the -O- or -S- moiety on the allylic position of the trans-cyclooctene ring of Formula (19) via a group selected from the group consisting of -C(Y ^C2 )Y ^C1 -, and a carbon atom, preferably an aromatic carbon, wherein this group is part of L ^C ,

wherein Y ^C1 is selected from the group consisting of -O-, -S-, and -NR36-, wherein Y ^C2 is selected from the group consisting of O and S,

wherein, when R48 is -O-(L ^C ((S ^P )kC ^A )s((S ^P )kC ^A )s((S ^P )i-C ^B )j)r-(S ^P )kC ^A , or

-S-(L ^C ((S ^P )kC ^A )s((S ^P )kC ^A )s((S ^P )i-C ^B )j)r-(S ^P )kC ^A and r is 1, then S ^P (when k>0) or C ^A (when k=0) is bound to L ^C via a moiety selected from the group

consisting of -O-, -S-, and -N-, preferably a secondary or a tertiary N, wherein said moiety is part of S ^P or C ^A ,

wherein, when R ₄₈ is -(S ^P ) _k C ^A , then S ^P (when k>0) or C ^A (when k=0) is bound to the allylic position of the trans-cyclooctene of Formula (19) via an -O- or an -S- atom, wherein this atom is part of S ^P or C ^A ,

wherein Z ^T is selected from the group consisting of C ₁ -C ₁₂ alkylene groups, C ₂ -C ₁₂ alkenylene groups, C ₇ -C ₁₂ alkynylene groups, C ₆ arylene groups, C ₄ - C ₅ heteroarylene groups, C ₃ -C ₈ cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₁₂ alkyl(hetero)arylene groups, C ₅ -C ₁₂ (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, C ₄ -C ₁₂ cycloalkylalkylene groups, wherein the alkylene groups, alkenylene groups, alkynylene groups,

(hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups, cycloalkylalkylene groups, are optionally substituted with a moiety selected from the group consisting of -(S ^P )i-C ^B with i independently being an integer in a range of from 0 to 4, preferably i is 0 or 1, -Cl, -F, -Br, -I, -OR ₃₇ , - N(R ₃₇ )2, =O, =NR ₃₇ , -SR ₃₇ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ and -Si(R ₃₇ )3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR ₃₇ -, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized;

wherein each R ₃₇ and R ₃₆ are independently selected from the group consisting of hydrogen, -(S ^P )i-C ^B , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 0 or 1;

wherein the R ₃₇ and R ₃₆ groups not being hydrogen are optionally

substituted with a moiety selected from the group consisting of -Cl, -F, -Br, - I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H _, -PO ₄ H _2, -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized, wherein R38 is independently selected from the group listed for R ₃₇ and R36, with the proviso that R38 is not attached to the remainder of the molecule via C(O), C(S), S(O), or S(O) ₂ ; wherein each R ₄₇ is independently selected from the group consisting of hydrogen, -(S ^P )i-C ^B , -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , - SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ ,

NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ ,

NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2, SC(=O)N(R ₃₇ )2,

OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2, NR ₃₇ C(=O)N(R ₃₇ )2, NR ₃₇ C(=S)N(R ₃₇ )2,

C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ ) ₂ , C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, - NR ₃₇ OR ₃₇ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ - C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄

(cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups; wherein i independently being an integer in a range of from 0 to 4, preferably i is an integer ranging from 0 to 1,

wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,

(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups,

(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,

(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , - N(R ₃₇ )2, -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ )2, -PO ₄ (R ₃₇ )2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized, wherein two R _37, R _38, R ₄₇ groups are optionally comprised in a ring, wherein two R _37, R _38, R ₄₇ groups are optionally comprised in a ring so as to form a ring fused to the eight-membered trans-ring. In a further aspect, the invention relates to a combination comprising the compound according to the invention, and a diene, preferably a tetrazine. In yet another aspect, the invention relates to the compound of the invention, or the combination according to the invention for use as a medicament. In another aspect still, the invention pertains to the compound of the invention, or the combination according to the invention for use in the treatment of a disease in a subject, preferably a human, wherein the disease is selected from the group consisting of cancer, central nervous system (CNS) diseases, infection, inflammation, cardiovascular diseases In another aspect still, the invention pertains to a non-therapeutic method for releasing a molecule from a compound according to the invention, said non-therapeutic method comprising the step of contacting a compound according to Formula (19) as defined herein with a diene as defined herein.

In another aspect, the invention relates to a non-therapeutic method for imaging a compound according to the invention in a subject, preferably a human, said non-therapeutic method comprising the steps of

(a) administering a compound according to Formula (19) as defined herein comprising a label, to the subject;

(b) imaging the compound according to Formula (19) present in the subject; wherein the label is selected from the group consisting of radionuclides, fluorescent dyes, and phosphorescent dyes. In another aspect, the invention relates to a non-therapeutic use of a compound according to the invention, or a combination according to the invention, for imaging in a subject, preferably a human, wherein the compound, or in case of the combination at least one of the compound according to Formula (19) and the diene, comprises a label selected from the group consisting of radionuclides, fluorescent dyes, and phosphorescent dyes. In another aspect still, the invention pertains to a non-therapeutic use of a compound according to the invention, or a combination according to the invention, for releasing a molecule, preferably in vitro. BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts a preferred embodiment of this invention. In both panels an ADC is administered to a cancer patient, and is allowed to circulate and bind to a target on the cancer cell. After the freely circulating ADC has sufficiently cleared from circulation, for example after 2 days post injection, the Activator, is administered and distributes systemically, allowing the reaction with the Trigger of cancer-bound Prodrug or ADC, releasing the Drug, after which the Drug can penetrate and kill

neighbouring cancer cells. Panel A depicts the cleavage of a carbamate- linked Drug and Panel B depicts the cleavage of an ether-linked Drug. Figure 2 depicts a preferred embodiment of this invention. An antibody construct comprising a bi-specific (anti-tumor and anti-CD3) antibody and a masking moiety (blocking protein) is administered to a cancer patient, and is allowed to circulate and bind to a target on the cancer cell. After the freely circulating construct has sufficiently cleared from cicrulation, for example after 2 days post injection, the Activator, is administered and distributes systemically, allowing the reaction with the Trigger of cancer-bound Prodrug, releasing the mask, after which T-cells bind the bi-specific antibody resulting in tumor killing. Figure 3 depicts the in vivo assembly of a functional cell penetration peptide (CPP) at the target site, leading to triggered CPP-induced drug internalization. Figure 4 depicts the in vivo unmasking of a functional cell penetration peptide (CPP) at the target site, leading to triggered CPP-induced drug internalization. Figure 5 depicts the use of the invention in radioimmunotherapy. A radiolabelled antibody is administered, allowed to circulate and bind an internalizing cancer receptor, and after sufficient internalization has occurred a Activator is administered that cleaves the radiolabel (e.g. a moiety comprising a radiometal-chelate complex) from the antibody, resulting in rapid renal clearance of the radioactivity from blood and non- target tissues, but not of the tumor cell-internalized radioactivity. Figure 6 depicts the use of the compounds of this invention for the site specific antibody conjugation with e.g. a drug , for ADC production. Figure 7 depicts the results of an in vitro experiment performed with a TCO-construct, containing a quenched fluorophore, and an Activator objects of this invention. The construct and Activator react and release and dequench the fluorophore very rapidly in PBS, cell culture medium and human plasma, producing a detectable increase of fluorescence in solution. DETAILED DESCRIPTION OF THE INVENTION The invention is based on the judicious insight that compounds, combinations, and kits according to the invention better address one or more of the abovementioned desires. This is surprising, for at least the reason that these desires are met by specific features of a dienophile, rather than of a diene where the background art focussed on.

In one aspect, the use of compounds, combinations, and kits according to the invention results in a high release yield and/or a fast release.

In another aspect, the use of compounds, combinations, and kits according to the invention results in a fast release.

In another aspect, the use of compounds, combinations, and kits according to the invention results in a fast click-conjugation and a high release yield and /or release rate. The invention, in a broad sense, is based on the judicious insight that a compound according to Formula (19) as described herein meets one or more of the abovementioned desires and/or resolves one or more of the abovementioned problems.

In another aspect, it is found that a combination of a compound according to Formula (19) as described herein and a diene as defined herein meets one or more of the abovementioned desires and/or resolves one or more of the abovementioned problems.

In yet another aspect, it is found that a kit comprising a combination according to the invention meets one or more of the abovementioned desires and/or resolves one or more of the abovementioned problems.

In a further aspect still, it is found that a combination according to the invention, and a kit according to the invention is useful in the treatment or imaging of patients or animals.

In yet a further aspect, it is found that a compound according to the invention, a combination according to the invention and a kit according to the invention are useful in bioorthogonal reactions in vitro and/or in vivo. Without wishing to be bound by theory, the inventors believe that the presence of the Y ^T1 , Y ^T2 and/or Y ^T3 groups as defined herein in the structures according to Formula (19) results in a higher click release yield and / or click release rate when contacted with a diene, as compared to known TCOs, in particular as compared to the same TCO lacking the said Y ^T1 , Y ^T2 and/or Y ^T3 groups. Still without wishing to be bound by theory, the inventors currently believe that this is the result of a destabilizing effect on the dihydropyridazine tautomer intermediates, in particular the 4,5- and/or the 1,4-dihydropyridazine tautomer intermediate that is formed upon conjugation of the TCO to a tetrazine. Furthermore, it was found that compounds according to Formula (19) as described herein give high click release yields and release rates when being contacted with dienes that give high click conjugation yields and rates. With reference to an example in Scheme 1A and without wishing to be bound to theory, the inventors believe that upon formation of the 4,5-dihydropyridazine tautomer 3, the Y ^T1 moiety (for example OH, NH ₂ ) engages in an hydrogen bond with cis-positioned Ha, inducing deprotonation and tautomerization to specifically the 1,4-dihydropyridazine intermediate 4, which then eliminates C ^A through pathway A and / or B. Pathway A comprises an 1,4-elimination affording free C ^A , 5 and

subsequently 6. Pathway B comprises a nucleophilic attack of Y ^T1 on the carbon to which C ^A is attached via a carbamate linker, resulting in cyclization-mediated release of C ^A and formation of 7 and 8. The inventors believe that suitably positioned Y ^T1 , Y ^T2 , and Y ^T3 moieties result in rapid and high yielding release through one or both of these pathways.

Scheme 1A. Proposed IEDDA pyridazine elimination mechanisms of this invention. Definitions The present invention will further be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

The verb "to comprise", and its conjugations, as used in this description and in the claims 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, 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 is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

The compounds disclosed in this description and in the claims may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds. The description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise. Thus, it will be understood that if herein a specific stereoisomer is indicated, the compound is limited to this specific stereoisomer. For example, in Formula (19) Y ^T2 is positioned syn relative to the 8-membered dienophile ring. While this includes enantiomers that are still syn, it does not include

stereoisomers in which Y ^T2 is positioned anti relative to the 8-membered dienophile ring.

In addition, the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer, unless stated otherwise. When the structure of a compound is depicted as a specific diastereomer, it is to be understood that the invention of the present application is not limited to that specific diastereomer, unless stated otherwise.

The compounds may occur in different tautomeric forms. The compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer, unless stated otherwise.

The compounds disclosed in this description and in the claims may further exist as exo and endo diastereomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual exo and the individual endo

diastereomers of a compound, as well as mixtures thereof. When the structure of a compound is depicted as a specific endo or exo diastereomer, it is to be understood that the invention of the present application is not limited to that specific endo or exo diastereomer, unless stated otherwise.

The compounds disclosed in this description and in the claims may further exist as anti and syn diastereomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual anti and the individual syn

diastereomers of a compound, as well as mixtures thereof. When the structure of a compound is depicted as a specific syn or anti diastereomer, it is to be understood that the invention of the present application is not limited to that specific syn or anti diastereomer, unless stated otherwise.

With respect to Formula (19), wherein Y ^T2 is positioned syn relative to the 8-membered dienophile ring; with "syn" it is meant that Y ^T2 is on the same side of the ring fused to the 8-membered dienophile ring as the 8-membered dienophile ring. Persons skilled in the art will understand that the syn-positioned Y ^T2 is facing towards the 8-membered dienophile as opposed to facing away, and can also be defined as being positioned endo relative to the 8-membered dienophile ring. In particular it is meant that Y ^T2 is facing towards the X moieties that flank Xa and Xb , and that are selected from X ¹ , X ² , X ³ , X ⁴ , X ⁵ . For the sake of clarity , "facing towards" will be understood as "being closer to". Further for the sake of clarity with "positioned syn" it is meant that Y ^T2 is positioned cis relative to the X moieties that flank X _a and X _b and that are selected from X ¹ , X ² , X ³ , X ⁴ , X ⁵ . Further for the sake of clarity it is meant that Y ^T2 is positioned cis relative to the direct bonds from X ^a and X ^b to the remainder of the 8-membered dienophile ring.

Unless stated otherwise, the compounds of the invention and/or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is deprotonated.

In several formulae, groups or substituents are indicated with reference to letters such as“A”,“B”,“X”,“Y”, and various (numbered)“R” groups. In addition, the number of repeating units may be referred to with a letter, e.g. n in -(CH ₂ ) _n -. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.

In several chemical formulae and texts below reference is made to "alkyl", "heteroalkyl", "aryl", “heteroaryl”,“alkenyl”,“alkynyl”,“alkylene”, “alkenylene”,“alkynylene”, "arylene",“cycloalkyl”,“cycloalkenyl”,

“cycloakynyl”, arenetriyl, and the like. The number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents as defined below, can be indicated by a designation preceding such terms (e.g.“C ₁ -C ₈ alkyl” means that said alkyl may have from 1 to 8 carbon atoms). For the avoidance of doubt, a butyl group substituted with a -OCH ₃ group is designated as a C ₄ alkyl, because the carbon atom in the substituent is not included in the carbon count.

Unsubstituted alkyl groups have the general formula CnH ₂ n+1 and may be linear or branched. Optionally, the alkyl groups are substituted by one or more substituents further specified in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1- dodecyl, etc. Unless stated otherwise, an alkyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR ₅ , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized. In preferred embodiments, up to two heteroatoms may be consecutive, such as in for example -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . In some preferred embodiments the heteroatoms are not directly bound to one another. Examples of heteroalkyls include - CH ₂ CH ₂ -O-CH ₃ , -CH ₂ CH ₂ -NH-CH ₃ , -CH ₂ CH ₂ -S(O)-CH ₃ , -CH=CH-O-CH ₃ , - Si(CH ₃ ) ₃ . In preferred embodiments, a C ₁ -C ₄ alkyl contains at most 2 heteroatoms.

A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkyl groups comprise at least three carbon atoms and have the general formula CnH ₂ n-1. Optionally, the cycloalkyl groups are substituted by one or more substituents further specified in this document. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Unless stated otherwise, a cycloalkyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR ₅ , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.

An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one C-C double bond have the general formula CnH ₂ n-1.

Unsubstituted alkenyl groups comprising two C-C double bonds have the general formula CnH ₂ n-3. An alkenyl group may comprise a terminal carbon- carbon double bond and/or an internal carbon-carbon double bond. A terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain. An alkenyl group may also comprise two or more carbon-carbon double bonds. Examples of an alkenyl group include ethenyl, propenyl, isopropenyl, t-butenyl, 1,3- butadienyl, 1,3-pentadienyl, etc. Unless stated otherwise, an alkenyl group may optionally be substituted with one or more, independently selected, substituents as defined below. Unless stated otherwise, an alkenyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR5, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.

An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one C-C triple bond have the general formula CnH ₂ n-3. An alkynyl group may comprise a terminal carbon-carbon triple bond and/or an internal carbon-carbon triple bond. A terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain. An alkynyl group may also comprise two or more carbon- carbon triple bonds. Unless stated otherwise, an alkynyl group may optionally be substituted with one or more, independently selected, substituents as defined below. Examples of an alkynyl group include ethynyl, propynyl, isopropynyl, t-butynyl, etc. Unless stated otherwise, an alkynyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR5, S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.

An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl.

Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures.

Optionally, the arylalkyl groups and alkylaryl may be substituted by one or more substituents further specified in this document. An arylalkyl group is for example benzyl. An alkylaryl group is for example 4-tert-butylphenyl. Preferably, heteroaryl groups comprise five to sixteen carbon atoms and contain between one to five heteroatoms. Heteroaryl groups comprise at least two carbon atoms (i.e. at least C2) and one or more heteroatoms N, O, P or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document. Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.

Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C ₃ ) and may include monocyclic and bicyclic structures. Optionally, the heteroaryl groups may be substituted by one or more substituents further specified in this document.

Where an aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group.

Similarly, an alkyl(hetero)aryl group is meant to include an alkylaryl group and an alkylheteroaryl group, and (hetero)arylalkyl is meant to include an arylalkyl group and a heteroarylalkyl group. A C ₂ -C ₂₄ (hetero)aryl group is thus to be interpreted as including a C ₂ -C ₂₄ heteroaryl group and a C ₆ -C ₂₄ aryl group. Similarly, a C ₃ -C ₂₄ alkyl(hetero)aryl group is meant to include a C ₇ -C ₂₄ alkylaryl group and a C ₃ -C ₂₄ alkylheteroaryl group, and a C ₃ -C ₂₄ (hetero)arylalkyl is meant to include a C ₇ -C ₂₄ arylalkyl group and a C ₃ -C ₂₄ heteroarylalkyl group.

A cycloalkenyl group is a cyclic alkenyl group. An unsubstituted cycloalkenyl group comprising one double bond has the general formula CnH ₂ n-3. Optionally, a cycloalkenyl group is substituted by one or more substituents further specified in this document. An example of a

cycloalkenyl group is cyclopentenyl. Unless stated otherwise, a cycloalkenyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR ₅ , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.

A cycloalkynyl group is a cyclic alkynyl group. An unsubstituted cycloalkynyl group comprising one triple bond has the general formula CnH ₂ n-5. Optionally, a cycloalkynyl group is substituted by one or more substituents further specified in this document. An example of a

cycloalkynyl group is cyclooctynyl. Unless stated otherwise, a cycloalkynyl group optionally contains one or more heteroatoms independently selected from the group consisting of O, NR ₅ , S, P, and Si, wherein the N, S, and P atoms are optionally oxidized and the N atoms are optionally quaternized.

When referring to a (hetero)aryl group the notation is meant to include an aryl group and a heteroaryl group. An alkyl(hetero)aryl group refers to an alkylaryl group and an alkylheteroaryl group. A

(hetero)arylalkyl group refers to an arylalkyl group and a heteroarylalkyl group. In general, when (hetero) is placed before a group, it refers to both the variant of the group without the prefix hetero- as well as the group with the prefix hetero-.

Herein, the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, S, P, and Si. It will be understood that groups with the prefix hetero- by definition contain heteroatoms. Hence, it will be understood that if a group with the prefix hetero- is part of a list of groups that is defined as optionally containing heteroatoms, that for the groups with the prefix hetero- it is not optional to contain heteroatoms, but is the case by definition.

Herein, it will be understood that when the prefix hetero- is used for combinations of groups, the prefix hetero- only refers to the one group before it is directly placed. For example, heteroarylalkyl denotes the combination of a heteroaryl group and an alkyl group, not the combination of a heteroaryl and a heteroalkyl group. As such, it will be understood that when the prefix hetero- is used for a combination of groups that is part of a list of groups that are indicated to optionally contain heteroatoms, it is only optional for the group within the combination without the prefix hetero- to contain a heteroatom, as it is not optional for the group within the

combination with the prefix hetero- by definition (see above). For example, if heteroarylalkyl is part of a list of groups indicated to optionally contain heteroatoms, the heteroaryl part is considered to contain heteroatoms by definition, while for the alkyl part it is optional to contain heteroatoms.

Herein, the prefix cyclo- denotes that groups are cyclic. It will be understood that when the prefix cyclo- is used for combinations of groups, the prefix cyclo- only refers to the one group before it is directly placed. For example, cycloalkylalkenylene denotes the combination of a cycloalkylene group (see the definition of the suffix -ene below) and an alkenylene group, not the combination of a cycloalkylene and a cycloalkenylene group.

In general, when (cyclo) is placed before a group, it refers to both the variant of the group without the prefix cyclo- as well as the group with the prefix cyclo-.

Herein, the suffix -ene denotes divalent groups, i.e. that the group is linked to at least two other moieties. An example of an alkylene is propylene (-CH ₂ -CH ₂ -CH ₂ -), which is linked to another moiety at both termini. It is understood that if a group with the suffix -ene is substituted at one position with -H, then this group is identical to a group without the suffix. For example, an alkylene substituted with -H is identical to an alkyl group. I.e. propylene, -CH ₂ -CH ₂ -CH ₂ -, substituted with -H at one terminus, - CH ₂ -CH ₂ -CH ₂ -H, is logically identical to propyl, -CH ₂ -CH ₂ -CH ₃ .

Herein, when combinations of groups are listed with the suffix - ene, it refers to a divalent group, i.e. that the group is linked to at least two other moieties, wherein each group of the combination contains one linkage to one of these two moieties. As such, for example alkylarylene is understood as a combination of an arylene group and an alkylene group. An example of an alkylarylene group is -phenyl-CH ₂ -, and an example of an arylalkylene group is -CH ₂ -phenyl-.

Herein, the suffix -triyl denotes trivalent groups, i.e. that the group is linked to at least three other moieties. An example of an arenetriyl is depicted below:

,

wherein the wiggly lines denote bonds to different groups of the main compound.

It is understood that if a group with the suffix -triyl is substituted at one position with -H, then this group is identical to a divalent group with the suffix -ene. For example, an arenetriyl substituted with -H is identical to an arylene group. Similarly, it is understood that if a group with the suffix - triyl is substituted at two positions with -H, then this group is identical to a monovalent group. For example, an arenetriyl substituted with two -H is identical to an aryl group.

It is understood that if a group, for example an alkyl group, contains a heteroatom, then this group is identical to a hetero-variant of this group. For example, if an alkyl group contains a heteroatom, this group is identical to a heteroalkyl group. Similarly, if an aryl group contains a heteroatom, this group is identical to a heteroaryl group. It is understood that“contain” and its conjugations mean herein that when a group contains a heteroatom, this heteroatom is part of the backbone of the group. For example, a C ₂ alkylene containing an N refers to -NH-CH ₂ -CH ₂ -, -CH ₂ -NH- CH ₂ -, and -CH ₂ -CH ₂ -NH-.

Unless indicated otherwise, a group may contain a heteroatom at non-terminal positions or at one or more terminal positions. In this case, “terminal” refers to the terminal position within the group, and not necessarily to the terminal position of the entire compound. For example, if an ethylene group contains a nitrogen atom, this may refer to

-NH-CH ₂ -CH ₂ -, -CH ₂ -NH-CH ₂ -, and -CH ₂ -CH ₂ -NH-. For example, if an ethyl group contains a nitrogen atom, this may refer to -NH-CH ₂ -CH ₃ , -CH ₂ -NH- CH ₃ , and -CH ₂ -CH ₂ -NH ₂ .

Herein, it is understood that cyclic compounds (i.e. aryl, cycloalkyl, cycloalkenyl, etc.) are understood to be monocyclic, polycyclic or branched. It is understood that the number of carbon atoms for cyclic compounds not only refers to the number of carbon atoms in one ring, but that the carbon atoms may be comprised in multiple rings. These rings may be fused to the main ring or substituted onto the main ring. For example, C ₁ 0 aryl optionally containing heteroatoms may refer to inter alia a naphthyl group (fused rings) or to e.g. a bipyridyl group (substituted rings, both containing an N atom).

Unless stated otherwise, (hetero)alkyl groups, (hetero)alkenyl groups, (hetero)alkynyl groups, (hetero)cycloalkyl groups,

(hetero)cycloalkenyl groups, (hetero)cycloalkynyl groups,

(hetero)alkylcycloalkyl groups, (hetero)alkylcycloalkenyl groups,

(hetero)alkylcycloalkynyl groups, (hetero)cycloalkylalkyl groups,

(hetero)cycloalkenylalkyl groups, (hetero)cycloalkynylalkyl groups,

(hetero)alkenylcycloalkyl groups, (hetero)alkenylcycloalkenyl groups, (hetero)alkenylcycloalkynyl groups, (hetero)cycloalkylalkenyl groups, (hetero)cycloalkenylalkenyl groups, (hetero)cycloalkynylalkenyl groups, (hetero)alkynylcycloalkyl groups, (hetero)alkynylcycloalkenyl groups, (hetero)alkynylcycloalkynyl groups, (hetero)cycloalkylalkynyl groups, (hetero)cycloalkenylalkynyl groups, (hetero)cycloalkynylalkynyl groups, (hetero)aryl groups, (hetero)arylalkyl groups, (hetero)arylalkenyl groups, (hetero)arylalkynyl groups, alkyl(hetero)aryl groups, alkenyl(hetero)aryl groups, alkynyl(hetero)aryl groups, cycloalkyl(hetero)aryl groups,

cycloalkenyl(hetero)aryl groups, cycloalkynyl(hetero)aryl groups,

(hetero)arylcycloalkyl groups, (hetero)arylcycloalkenyl groups, (hetero)arylcycloalkynyl groups, (hetero)alkylene groups, (hetero)alkenylene groups, (hetero)alkynylene groups, (hetero)cycloalkylene groups,

(hetero)cycloalkenylene groups, (hetero)cycloalkynylene groups,

(hetero)arylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, (hetero)arylalkenylene groups, (hetero)arylalkynylene groups, alkenyl(hetero)arylene, alkynyl(hetero)arylene, (hetero)arenetriyl groups, (hetero)cycloalkanetriyl groups, (hetero)cycloalkenetriyl and

(hetero)cycloalkynetriyl groups are optionally substituted with one or more substituents independently selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR ₅ , -SR ₅ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄

cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)arylalkyl groups, C ₄ -C ₂₄ (hetero)arylalkenyl groups, C ₄ -C ₂₄ (hetero)arylalkynyl groups, C ₄ -C ₂₄ alkenyl(hetero)aryl groups, C ₄ -C ₂₄ alkynyl(hetero)aryl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, C ₆ -C ₂₄

alkylcycloalkenyl groups, C ₁ 3-C ₂₄ alkylcycloalkynyl groups, C ₄ -C ₂₄

cycloalkylalkyl groups, C ₆ -C ₂₄ cycloalkenylalkyl groups, C ₁₃ -C ₂₄

cycloalkynylalkyl groups, C ₅ -C ₂₄ alkenylcycloalkyl groups, C ₇ -C ₂₄

alkenylcycloalkenyl groups, C ₁ 4-C ₂₄ alkenylcycloalkynyl groups, C ₅ -C ₂₄ cycloalkylalkenyl groups, C ₇ -C ₂₄ cycloalkenylalkenyl groups, C ₁₄ -C ₂₄ cycloalkynylalkenyl groups, C ₅ -C ₂₄ alkynylcycloalkyl groups, C ₇ -C ₂₄ alkynylcycloalkenyl groups, C ₁₄ -C ₂₄ alkynylcycloalkynyl groups, C ₅ -C ₂₄ cycloalkylalkynyl groups, C ₇ -C ₂₄ cycloalkenylalkynyl groups, C ₁₄ -C ₂₄ cycloalkynylalkynyl groups, C ₅ -C ₂₄ cycloalkyl(hetero)aryl groups, C ₇ -C ₂₄ cycloalkenyl(hetero)aryl groups, C ₁₄ -C ₂₄ cycloalkynyl(hetero)aryl groups, C ₅ - C ₂₄ (hetero)arylcycloalkyl groups, C ₇ -C ₂₄ (hetero)arylcycloalkenyl groups, and C ₁ 4-C ₂₄ (hetero)arylcycloalkynyl groups. Unless stated otherwise, the substituents disclosed herein optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. Preferably, these substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, and NR5.

In preferred embodiments, the substituents are selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , - CF ₃ , =O, =NR5, -SR5, C ₁ -C ₁₂ alkyl groups, C ₂ -C ₁₂ alkenyl groups, C ₂ -C ₁₂ alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C ₁₂ cycloalkyl groups, C ₅ -C ₁₂ cycloalkenyl groups, C ₁₂ cycloalkynyl groups, C ₃ - C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ -C ₁₂

(hetero)arylalkenyl groups, C ₄ -C ₁₂ (hetero)arylalkynyl groups, C ₄ -C ₁₂ alkenyl(hetero)aryl groups, C ₄ -C ₁₂ alkynyl(hetero)aryl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₆ -C ₁₂ alkylcycloalkenyl groups, C ₁₃ -C ₁₆

alkylcycloalkynyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₆ -C ₁₂

cycloalkenylalkyl groups, C ₁ 3-C ₁ 6 cycloalkynylalkyl groups, C ₅ -C ₁₂ alkenylcycloalkyl groups, C ₇ -C ₁₂ alkenylcycloalkenyl groups, C ₁₄ -C ₁₆ alkenylcycloalkynyl groups, C ₅ -C ₁₂ cycloalkylalkenyl groups, C ₇ -C ₁₂ cycloalkenylalkenyl groups, C ₁₄ -C ₁₆ cycloalkynylalkenyl groups, C ₅ -C ₁₂ alkynylcycloalkyl groups, C ₇ -C ₁₂ alkynylcycloalkenyl groups, C ₁₄ -C ₁₆ alkynylcycloalkynyl groups, C ₅ -C ₁₂ cycloalkylalkynyl groups, C ₇ -C ₁₂ cycloalkenylalkynyl groups, C ₁₄ -C ₁₆ cycloalkynylalkynyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups, C ₇ -C ₁₂ cycloalkenyl(hetero)aryl groups, C ₁₄ - C ₁ 6 cycloalkynyl(hetero)aryl groups, C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, C ₇ -C ₁₂ (hetero)arylcycloalkenyl groups, and C ₁₄ -C ₁₆ (hetero)arylcycloalkynyl groups. In preferred embodiments, the substituents are selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ , -NO ₂ , - CF ₃ , =O, =NR5, -SR5, C ₁ -C ₇ alkyl groups, C ₂ -C ₇ alkenyl groups, C ₂ -C ₇ alkynyl groups, C ₆ -C ₇ aryl groups, C ₂ -C ₇ heteroaryl groups, C ₃ -C ₇ cycloalkyl groups, C ₅ -C ₇ cycloalkenyl groups, C ₁₂ cycloalkynyl groups, C ₃ -C ₇

alkyl(hetero)aryl groups, C ₃ -C ₇ (hetero)arylalkyl groups, C ₄ -C ₇ (hetero)arylalkenyl groups, C ₄ -C ₇ (hetero)arylalkynyl groups, C ₄ -C ₇ alkenyl(hetero)aryl groups, C ₄ -C ₇ alkynyl(hetero)aryl groups, C ₄ -C ₇ alkylcycloalkyl groups, C ₆ -C ₇ alkylcycloalkenyl groups, C ₁ 3-C ₁ 6

alkylcycloalkynyl groups, C ₄ -C ₇ cycloalkylalkyl groups, C ₆ -C ₇

cycloalkenylalkyl groups, C ₁ 3-C ₁ 6 cycloalkynylalkyl groups, C ₅ -C ₇

alkenylcycloalkyl groups, C ₇ -C ₇ alkenylcycloalkenyl groups, C ₁₄ -C ₁₆ alkenylcycloalkynyl groups, C ₅ -C ₇ cycloalkylalkenyl groups, C ₇ -C ₈ cycloalkenylalkenyl groups, C ₁₄ -C ₁₆ cycloalkynylalkenyl groups, C ₅ -C ₇ alkynylcycloalkyl groups, C ₇ -C ₈ alkynylcycloalkenyl groups, C ₁₄ -C ₁₆ alkynylcycloalkynyl groups, C ₅ -C ₇ cycloalkylalkynyl groups, C ₇ -C ₈ cycloalkenylalkynyl groups, C ₁₄ -C ₁₆ cycloalkynylalkynyl groups, C ₅ -C ₇ cycloalkyl(hetero)aryl groups, C ₇ -C ₈ cycloalkenyl(hetero)aryl groups, C ₁₄ -C ₁₆ cycloalkynyl(hetero)aryl groups, C ₅ -C ₇ (hetero)arylcycloalkyl groups, C ₇ -C8 (hetero)arylcycloalkenyl groups, and C ₁₄ -C ₁₆ (hetero)arylcycloalkynyl groups, C ₄ -C ₈ (hetero)arylalkenyl groups, C ₄ -C ₈ (hetero)arylalkynyl groups, C ₄ -C8 alkenyl(hetero)aryl groups, C ₄ -C8 alkynyl(hetero)aryl groups, C ₅ -C9 cycloalkyl(hetero)aryl groups, C ₇ -C ₁ 1 cycloalkenyl(hetero)aryl groups, C ₁ 4- C ₁₈ cycloalkynyl(hetero)aryl groups, C ₅ -C ₉ (hetero)arylcycloalkyl groups, C ₇ - C ₁ 1 (hetero)arylcycloalkenyl groups, and C ₁ 4-C ₁ 8 (hetero)arylcycloalkynyl groups.

Unless stated otherwise, any group disclosed herein that is not cyclic is understood to be linear or branched. In particular, hetero)alkyl groups, (hetero)alkenyl groups, (hetero)alkynyl groups, (hetero)alkylene groups, (hetero)alkenylene groups, (hetero)alkynylene groups, and the like are linear or branched, unless stated otherwise.

The general term "sugar" is herein used to indicate a

monosaccharide, for example glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" is herein used to indicate a derivative of a monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and/or functional groups. Examples of a sugar derivative include amino sugars and sugar acids, e.g. glucosamine (GlcNH ₂ ), galactosamine (GalNH ₂ ) N-acetylglucosamine (GlcNAc), N- acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (ldoA).

A sugar may be without further substitution, and then it is understood to be a monosaccharide. A sugar may be further substituted with at one or more of its hydroxyl groups, and then it is understood to be a disaccharide or an oligosaccharide. A disaccharide contains two

monosaccharide moieties linked together. An oligosaccharide chain may be linear or branched, and may contain from 3 to 10 monosaccharide moieties.

The term "protein" is herein used in its normal scientific meaning. Herein, polypeptides comprising about 10 or more amino acids are

considered proteins. A protein may comprise natural, but also unnatural amino acids. The term“protein” herein is understood to comprise antibodies and antibody fragments.

The term“peptide” is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acids in a range of from 2 to 9.

The term“peptoids” is herein used in its normal scientific meaning.

An antibody is a protein, typically generated by the immune system, that is capable of recognizing and binding to a specific antigen.

While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (IgG1, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact

immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, antibody fusions, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'- SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem/bis- scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as Nanobody ^TM ), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DART ^TM ), and multimers and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri-scFvs) including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol.2012, 30, 575–582], and [Canc. Gen. Prot.201310, 1- 18], and [BioDrugs 2014, 28, 331–343], the contents of which are hereby incorporated by reference. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region. Other embodiments use antibody mimetics as Drug or Targeting Agent T ^T , such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference. For the avoidance of doubt, in the context of this invention the term "antibody" is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.

A linker is herein defined as a moiety that connects two or more elements of a compound. For example in a bioconjugate, a biomolecule and a targeting moiety are covalently connected to each other via a linker.

A biomolecule is herein defined as any molecule that can be isolated from nature or any molecule composed of smaller molecular building blocks that are the constituents of macromolecular structures derived from nature, in particular nucleic acids, proteins, glycans and lipids. Examples of a biomolecule include an enzyme, a (non-catalytic) protein, a polypeptide, a peptide, an amino acid, an oligonucleotide, a monosaccharide, an oligosaccharide, a polysaccharide, a glycan, a lipid and a hormone.

As used herein, an organic molecule is defined as a molecule comprising a C-H bond. It will be understood that“organic molecule” as used herein includes biomolecules, such as nucleic acids (oligonucleotides, polynucleotides, DNA, RNA), peptides, proteins (in particular antibodies), carbohydrates (monosaccharides, oligosaccharides, and polysaccharides), aptamers, hormones, toxins, steroids, cytokines, and lipids; small organic molecules as defined herein; polymers (in particular polyethylene glycol); LNA and PNA; amino acids; peptoids; molecules comprising a radionuclide; fluorescent dyes; drugs; resins (in particular polystyrene and agarose);

beads; particles (in particular polymersomes, liposomes, and beads); gels; surfaces; organometallic compounds; metal complexes; cells; and

combinations thereof.

As used herein, an inorganic molecule is defined as any molecule not being an organic molecule, i.e. not comprising a C-H bond. It will be understood that“inorganic molecule” as used herein includes surfaces (in particular chips, wafers, gold, metal, silica-based surfaces such as glass); particles such as beads (in particular magnetic beads, gold beads), silica- based particles, polymer-based materials, iron oxide particles; carbon nanotubes; allotropes of carbon (in particular fullerenes such as

Buckminsterfullerene; graphite, graphene, diamond, Lonsdaleite, Q-carbon, linear acetylenic carbon, amorphous carbon, and carbon nanotubes); drugs (in particular cisplatin); metal complexes; and combinations thereof.

As used herein,“particle” is preferably defined as a microparticle or a nanoparticle.

The term "salt thereof' means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. The term "salt thereof' also means a compound formed when an amine is protonated. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

The term "pharmaceutically accepted" salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter-ions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.

"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium,

tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

The logarithm of the partition-coefficient, i.e. Log P, is herein used as a measure of the hydrophobicity of a compound. Typically, the Log P is defined as

The skilled person is aware of methods to determine the partition-coefficient of compounds without undue experimentation. Alternatively, the skilled person knows that software is available to reliably estimate the Log P value, for example as a function within ChemDraw® software or online available tools.

The unified atomic mass unit or Dalton is herein abbreviated to Da. The skilled person is aware that Dalton is a regular unit for molecular weight and that 1 Da is equivalent to 1 g/mol (grams per mole).

It will be understood that herein, the terms“moiety” and“group” are used interchangeably when referring to a part of a molecule.

It will be understood that when a heteroatom is denoted as - X(R’)2-, wherein X is the heteroatom and R’ is a certain moiety, then this denotes that two moieties R’ are attached to the heteroatom.

It will be understood that when a group is denoted as, for example, -((R51)2-R52)2- or a similar notation, in which R51 and R52 are certain moieties, then this denotes that first, it should be written as -R ₅₁ - R51-R52-R51-R51-R52- before the individual R51 and R52 moieties are selected, rather than first selecting moieties R51 and R52 and then writing out the formula. The Inverse Electron-Demand Diels-Alder reaction (IEDDA) The established IEDDA conjugation chemistry generally involves a pair of reactants that comprise, as one reactant (i.e. one Bio-orthogonal Reactive Group), a suitable diene, such as a derivative of tetrazine, e.g. an electron- deficient tetrazine and, as the other reactant (i.e. the other Bio-orthogonal Reactive Group), a suitable dienophile, such as a trans-cyclooctene (TCO). The exceptionally fast reaction of (substituted) tetrazines, in particular electron-deficient tetrazines, with a TCO moiety results in an intermediate that rearranges to a dihydropyridazine Diels-Alder adduct by eliminating N2 as the sole by-product. The initially formed 4,5-dihydropyridazine product may tautomerize to a 1,4- or a 2,5-dihydropyridazine product, especially in aqueous environments. Below a reaction scheme is given for a [4+2] IEDDA reaction between (3,6)-di-(2-pyridyl)-s-tetrazine diene and a trans-cyclooctene dienophile, followed by a retro Diels Alder reaction in which the product and dinitrogen is formed. Because the trans-cyclooctene derivative does not contain electron withdrawing groups as in the classical Diels Alder reaction, this type of Diels Alder reaction is distinguished from the classical one, and frequently referred to as an“inverse-electron-demand Diels Alder (IEDDA) reaction”. In the following text the sequence of both reaction steps, i.e. the initial Diels-Alder cyclo-addition (typically an inverse electron-demand Diels Alder cyclo-addition) and the subsequent retro Diels Alder reaction will be referred to in shorthand as the“inverse electron- demand Diels Alder reaction” or“inverse electron-demand Diels Alder conjugation” or“IEDDA”. The product of the reaction is then the IEDDA adduct or conjugate. This is illustrated in Scheme 1 below.

Scheme 1: the IEDDA conjugation reaction The two reactive species are abiotic and do not undergo fast metabolism or side reactions in vitro or in vivo. They are bio-orthogonal, e.g. they selectively react with each other in physiologic media. Thus, the compounds and the method of the invention can be used in a living organism. Moreover, the reactive groups are relatively small and can be introduced in biological samples or living organisms without significantly altering the size of biomolecules therein. References on the inverse electron demand Diels Alder reaction, and the behavior of the pair of reactive species include:

[Thalhammer et al., Tetrahedron Lett., 1990, 31, 47, 6851-6854], [Wijnen et al., J. Org. Chem., 1996, 61, 2001-2005], [Blackman et al., J. Am. Chem. Soc., 2008, 130, 41, 13518-19], Rossin et al., Angew. Chem. Int. Ed.2010, 49, 3375], [Devaraj et al., Angew. Chem. Int. Ed.2009, 48, 7013], [Devaraj et al., Angew. Chem. Int. Ed., 2009, 48, 1-5]. The IEDDA Pyridazine Elimination Reaction

Below, the dienophile, a TCO, that is comprised in combinations and kits of the invention may be referred to as a“Trigger”. The dienophile is connected at the allylic position to a Construct-A. Moreover, tetrazines that are used in the IEDDA pyridazine elimination reaction may be referred to as

“Activators”. The term Construct-A in this invention is used to indicate any substance, carrier, biological or chemical group, of which it is desired to have it first in a bound (or masked) state, and being able to provoke release from that state.

The inventors previously demonstrated that the

dihydropyridazine product derived from a tetrazine (the Activator) and a TCO containing a carbamate-linked drug (doxorubicin, the Construct-A) at the allylic position is prone to eliminate CO ₂ and the amine-containing drug, eventually affording aromatic pyridazine.

Without wishing to be bound by theory, the inventors believe that the Activator provokes Construct-A release via a cascade mechanism within the IEDDA adduct, i.e. the dihydropyridazine. The cascade

mechanism can be a simple one step reaction, or it can be comprised in multiple steps that involves one or more intermediate structures. These intermediates may be stable for some time or may immediately degrade to the thermodynamic end-product or to the next intermediate structure. In any case, whether it be a simple or a multistep process, the result of the cascade mechanism is that the Construct-A gets released from the IEDDA adduct. Without wishing to be bound by theory, the design of the diene is such that the distribution of electrons within the IEDDA adduct is unfavorable, so that a rearrangement of these electrons must occur. This situation initiates the cascade mechanism, and it therefore induces the release of the Construct-A. Specifically, and without wishing to be bound by theory, the inventors believe that the NH moiety comprised in the various dihydropyridazine tautomers, such as the 1,4-dihydropyridazine tautomer, of the IEDDA adduct can initiate an electron cascade reaction, a concerted or consecutive shift of electrons over several bonds, leading to release of the Construct-A. Occurrence of the cascade reaction in and /or Construct-A release from the Trigger is not efficient or cannot take place prior to the IEDDA reaction, as the Trigger-Construct-A conjugate itself is relatively stable as such. The cascade can only take place after the Activator and the Trigger-Construct conjugate have reacted and have been assembled in the IEDDA adduct.

With reference to Scheme 2 below, and without wishing to be bound by theory, the inventors believe that the pyridazine elimination occurs from the 1,4-dihydropyridazine tautomer 4. Upon formation of the 4,5-dihydropyridazine 3, tautomerization affords intermediates 4 and 7, of which the 2,5-dihydropyridazine 7 cannot eliminate the C ^A . Instead it can slowly convert into aromatic 8, which also cannot eliminate C ^A or it can tautomerize back to intermediate 3. Upon formation of 4 the C ^A is eliminated near instantaenously, affording free C ^A 8 as an amine, and pyridazine elimination products 5 and 6. This elimination reaction has been shown to work equally well in the cleavage of carbonates, esters and ethers from the TCO trigger [Versteegen et al., Angew. Chem. Int. Ed., 2018, 57, 10494]. The Trigger in Scheme 2 is also optionally bound to a Construct-B (C ^B ) , which in this case cannot release from the Trigger. Thereby Construct A can be seperated from Construct B by means of the IEDDA pyridazine elimination.

2,5-tautomer,

7 Scheme 2. Proposed IEDDA pyridazine elimination mechanism. In preferred embodiments, the dienophile trigger moiety used in the present invention comprises a trans-cyclooctene ring. Herein, this eight-membered ring moiety will be defined as a trans-cyclooctene moiety, for the sake of legibility, or abbreviated as“TCO” moiety. It will be understood that the essence resides in the possibility of the eight-membered ring to act as a dienophile and to be released from its conjugated Construct-A upon reaction.

The dienophiles of the invention and tetrazines are capable of reacting in an inverse electron-demand Diels-Alder reaction (IEDDA).

IEDDA reaction of the Trigger with the Activator leads to release of the Construct-A through an electron-cascade-based elimination, termed the “pyridazine elimination”. When an Activator reacts with a Trigger capable of eliminating Construct-A, the combined proces of reaction and Construct-A elimination is termed the“IEDDA pyridazine elimination”.

This invention provides a Construct-A-conjugated Trigger that reacts with an Activator, resulting in the cleavage of the Trigger from the Construct-A. In one prominent embodiment this results in the cleavage of Construct-A from Construct-B. In another embodiment the Trigger cleavage results in cleavage of one Construct A from another Construct A, wherein both can release from a self-immolative linker attached to the Trigger. In another embodiment, Trigger cleavage results in the cleavage of one or more Construct-A from one or more Construct-B. Construct-B is the Construct that is bound to the dienophile, and cannot be released from the dienophile, unless it is bound to the allylic position via a spacer or self-imolative linker that also binds Construct-A. In preferred embodiments, the Trigger is used as a reversible covalent bond between two molecular species.

Scheme 3a below is a general scheme of Construct release according to this invention, wherein the Construct being released is termed Construct-A (C ^A ), and wherein another Construct, Construct-B (C ^B ) can optionally be bound to the dienophile, but not via the allylic position, wherein Construct-B cannot be released from the dienophile.

Scheme 3a: General scheme of IEDDA pyridazine elimination reaction for the release of Construct-A according to this invention Scheme 3b below is a general scheme of Construct release according to another embodiment of this invention, wherein Construct-B (C ^B ) is bound to the dienophile via a spacer or self-imolative linker that also binds

Construct-A and, wherein when the spacer or self-immolative linker is released from the allylic position then Construct-B and Construct A are released from the Trigger and from each other.

Scheme 3b: General scheme of IEDDA pyridazine elimination reaction for the release of Construct-A from Construct-B according to a another embodiment of this invention The Construct release occurs through a powerful, abiotic, bio-orthogonal reaction of the dienenophile (Trigger) with the diene (Activator), viz. the aforementioned IEDDA. The masked or bound Construct is a Construct- dienenophile conjugate. Possibly the Construct-A is linked to one or more additional Constructs A linked via a self-immolative linker. It will be understood that in Scheme 3 in the IEDDA adduct as well as in the end product after release, the indicated dienophile group and the indicated diene group are the residues of, respectively, the dienophile and diene groups after these groups have been converted in the IEDDA reaction.

The difference between C ^A and C ^B is that the bond between C ^B and the moiety holding C ^B is not broken upon reaction of the Trigger with the diene, whereas the bond between C ^A and the moiety holding C ^A is broken upon reaction of the Trigger with the diene. A person skilled in the art will understand that the moiety holding C ^A and C ^B refers to the Trigger, or a self immolative linker L ^C bound to the Trigger. For the sake of clarity, when C ^B is bound to a L ^C that is bound to the Trigger, the L ^C holding C ^B will release from the Trigger upon reaction with the diene but the C ^B will not release from the released L ^C . Likewise if C ^B is bound directly to the Trigger, C ^B will not release from the Trigger upon reaction with the diene. A person skilled in the art will understand that when it is required to seperate one Construct (1) from another Construct (2), that one of the following

requirements have to be met:

1) one C ^A is Construct 1 and another C ^A is Construct 2

2) Construct 1 is C ^A and Construct 2 is C ^B

3) Construct 1 and 2 are both C ^B , provided that one C ^B is bound directly to the Trigger and the other is bound to a L ^C , or provided that one C ^B moiety is bound to a different L ^C moiety than the other C ^B moiety.

The invention provides, in one aspect, the use of a tetrazine as an Activator for the release, in a chemical, biological, or physiological environment, of a Construct linked to a TCO. In connection herewith, the invention also pertains to a tetrazine as an Activator for the release, in a chemical, biological, or physiological environment, of a substance linked to a TCO. The fact that the reaction is bio-orthogonal, and that many structural options exist for the reaction pairs, will be clear to the skilled person. E.g., the IEDDA reaction is known in the art of bioconjugation, diagnostics, pre- targeted medicine. Reference is made to, e.g., WO 2010/119382, WO

2010/119389, and WO 2010/051530. Whilst the invention presents an entirely different use of the reaction, it will be understood that the various structural possibilities available for the IEDDA reaction pairs as used in e.g. pre-targeting, are also available in the field of the present invention.

Other than is the case with e.g. medicinally active substances, where the in vitro or in vivo action is often changed with minor structural changes, the present invention first and foremost requires the right chemical reactivity combined with sufficient stability for the intended application. Thus, the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienophiles. Compounds according to Formula (19) In Formula (19), r is an integer in range of from 0 to 2. In a preferred embodiment, r is 0. In a preferred embodiment, r is 1. In a preferred embodiment, r is 2. In Formula (19), each s is independently 0 or 1. In a preferred embodiment, s is 0. In a preferred embodiment, s is 1. In Formula (19), each i is independently an integer in a range of from 0 to 4, preferably 0 or 1. In Formula (19), j is an integer in range of from 0 to 4, preferably from 0 to 2, more preferably 0 or 1. In Formula (19), each k is independently 0 or 1.

In a preferred embodiment, in Formula (19) only condition (a) is met.

In a preferred embodiment, in Formula (19) only condition (b) is met.

In a preferred embodiment, in Formula (19) only condition (c) is met.

In a preferred embodiment, when in Formula (19) two R ₃₇ , R38, R47 groups are optionally comprised in a ring so as to form a ring fused to the eight- membered trans-ring, this ring fused to the eight-membered trans-ring is as defined in WO 2012/156919 A1 which is hereby incorporated by reference in its entirety. More preferably the ring fused to the eight-membered trans- ring is as defined in WO 2012/156919 A1 on page 15, line 25 to page 18, line 9. Z ^T Z ^T is selected from the group consisting of C ₁ -C ₁₂ alkylene groups, C ₂ -C ₁₂ alkenylene groups, C ₇ -C ₁₂ alkynylene groups, C ₆ arylene groups, C ₄ -C ₅ heteroarylene groups, C ₃ -C ₈ cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₁₂ alkyl(hetero)arylene groups, C ₅ -C ₁₂ (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, C ₄ -C ₁₂ cycloalkylalkylene groups, wherein the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups, cycloalkylalkylene groups, are optionally substituted with a moiety selected from the group consisting of -(S ^P ) _i -C ^B with i independently being an integer in a range of from 0 to 4, preferably i is 0 or 1, -Cl, -F, -Br, -I, -OR ₃₇ , - N(R ₃₇ )2, =O, =NR ₃₇ , -SR ₃₇ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ and -Si(R ₃₇ )3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR ₃₇ -, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

Preferably, Z ^T is selected from the group consisting of C ₁ -C ₆ alkylene groups, C2-C ₆ alkenylene groups, C ₇ alkynylene groups, C ₆ arylene groups, C ₄ -C ₅ heteroarylene groups, C ₃ -C ₆ cycloalkylene groups, C ₅ -C ₈

cycloalkenylene groups, C ₅ -C8 alkyl(hetero)arylene groups, C ₅ -C8

(hetero)arylalkylene groups, C ₄ -C8 alkylcycloalkylene groups, C ₄ -C8 cycloalkylalkylene groups, wherein the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups,

cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups, cycloalkylalkylene groups, are optionally substituted with a moiety selected from the group consisting of -(S ^P )i-C ^B with i independently being an integer in a range of from 0 to 4, preferably i is 0 or 1, -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , =O, =NR ₃₇ , -SR ₃₇ , -SO ₃ H, -PO ₃ H _, - PO ₄ H ₂ , -NO ₂ and -Si(R ₃₇ )3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR ₃₇ -, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

Preferably, Z ^T is selected from the group consisting of C ₁ -C ₃ alkylene groups, C ₂ -C ₃ alkenylene groups, C ₃ alkynylene groups, C ₃ heteroarylene groups, and C ₃ cycloalkylene groups, wherein the alkylene groups, alkenylene groups, alkynylene groups, heteroarylene groups, and

cycloalkylene groups, are optionally substituted with a moiety selected from the group consisting of -(S ^P ) _i -C ^B with i independently being an integer in a range of from 0 to 4, preferably i is 0 or 1, -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, =O, =NR ₃₇ , -SR ₃₇ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ and -Si(R ₃₇ )3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, - S-, -NR ₃₇ -, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. In preferred embodiments, the Z ^T groups are not substituted. In preferred embodiments, the Z ^T groups do not contain heteroatoms. R ₄₈ Preferably, R ₄₈ is -OC(O)-(S ^P ) _k C ^A , and S ^P (when k>0) or C ^A (when k=0) is bound to the -OC(O)- of R48 via an atom selected from the group consisting of O, C, S, and N, preferably a secondary or a tertiary N, wherein this atom is part of S ^P or C ^A . Preferably, R48 is -O-L ^C -(S ^P )kC ^A and S ^P (when k>0) or C ^A (when k=0) is bound to L ^C via a moiety selected from the group consisting of -O-, -S-, and -N-, preferably a secondary or a tertiary N, wherein said moiety is part of S ^P or C ^A . It is preferred that R48 is axially positioned on the 8- membered dienophile ring. It is preferred that R48 is positioned trans relative to H ^a . Other preferred embodiments In preferred embodiments, Y ^T1 is OH, N(R ₃₈ ) ₂ , C(O)OH, O-N(R ₃₈ ) ₂ , SH, more preferably OH, N(R ₃₈ ) ₂ , O-N(R ₃₈ ) ₂ , more preferably OH, N(R ₃₈ ) ₂ , most preferably Y ^T1 is OH. In other preferred embodiments, Y ^T1 is N(R38)2, more preferably NR ₃₈ H, most preferably NH ₂ . In preferred embodiments, no Y ^T2 and Y ^T3 are present and Y ^T1 is OH, N(R38)2, C(O)OH, O-N(R38)2, more preferably OH, N(R38)2, most preferably Y ^T1 is OH. In preferred

embodiments, Y ^T2 is OH. In preferred embodiments, no Y ^T1 and Y ^T3 are present and Y ^T2 is OH. It is preferred that X ¹ and X ⁵ are not O.

In preferred embodiments, when a fused ring is present satisfying any of the Formulae (20a)-(20f) that there are no other rings fused to the 8- membered dienophile ring.

In preferred embodiments, R ₃₇ , R38, R47 groups are not OH, SH, N(R ₃₈ ) ₂ , O-N(R ₃₈ ) ₂ , C(N)N(R ₃₈ ) ₂ . In preferred embodiments, R _37, R _38, R ₄₇ groups are not OH, SH, N(R38)2, C(O)OH, C(S)OH, C(O)SH, C(S)SH, O- N(R38)2, SO ₄ H, SO ₃ H, SO ₂ H, PO ₄ H ₂ , PO ₃ H, PO ₂ H, C(N)N(R38)2.

In preferred embodiments, the compound of Formula (19) comprises at most three moieties each independently selected from the group consisting of Y ^T1 , Y ^T2 , Y ^T3 , more preferably at most two moieties, even more preferably one moiety.

In preferred embodiments, one of X ² , X ³ , X ⁴ is CR47Y ^T1 , most preferably X ³ or X ⁴ is CR ₄₇ Y ^T1 . In preferred embodiments, one of X ² , X ³ , X ⁴ is CR47Y ^T1 , most preferably X ³ or X ⁴ is CR47Y ^T1 , and the remaining X ¹ , X ² , X ³ , X ⁴ , X ⁵ are C(R47)2, preferably CH ₂ . In preferred embodiments, two of X ² , X ³ , X ⁴ are CR ₄₇ Y ^T1 , and the remaining X ¹ , X ² , X ³ , X ⁴ , X ⁵ are C(R ₄₇ ) ₂ , preferably CH ₂ . In preferred embodiments, X ³ is Y ^T3 , and the remaining X ¹ , X ² , X ³ , X ⁴ , X ⁵ are C(R47)2, preferably CH ₂ . In preferred embodiments, X ² and X ³ are part of a fused ring satisfying one of the Formulae (20a)-(20g), and the remaining X ¹ , X ² , X ³ , X ⁴ , X ⁵ are C(R47)2, preferably CH ₂ .

In preferred embodiments, X ³ and X ⁴ are part of a fused ring satisfying one of the Formulae (20a)-(20g), and the remaining X ¹ , X ² , X ³ , X ⁴ , X ⁵ are C(R47)2, preferably CH ₂ .

In preferred embodiments, the fused ring satisfies Formula (20a). In preferred embodiments, the fused ring satisfies Formula (20b). In preferred embodiments, the fused ring satisfies Formula (20c).

For Formula (20g) it is preferred that when X ⁶ and X ⁸ are both Y ^T3 that X ⁷ is not C ₁ alkylene. For Formula (20g) it is preferred that X ⁶ and X ⁸ are both Y ^T3 , preferably NR38, and that X ⁷ is C2 alkylene. For Formula (20g) it is preferred that X ⁶ and X ⁸ are both C(R ₄₇ ) ₂ , preferably CH _2, and X ⁷ is Y ^T3 , preferably NR38. In preferred embodiments, X ^a and X ^b are CH. In preferred embodiments, the fused ring satisfies Formula (20a), and that X ^a and X ^b are CH.

In a preferred embodiment, at most 4 of R ₃₇ , R38, R47 comprised in X ¹ , X ² , X ³ , X ⁴ , and X ⁵ (i.e. in total for X ¹ -X ⁵ , not per moiety X) are not H, preferably at most 3 are not H, more preferably at most 2 are not H, most preferably at most 1 is not H.

It is preferred that when two R _37, R _38, R ₄₇ groups comprised in X ¹ , X ² , X ³ , X ⁴ , X ⁵ are comprised in a ring so as to form a ring fused to the eight- membered trans-ring, that these rings fused to the eight-membered trans- ring are C ₃ -C ₇ cycloalkylene groups and C ₄ -C ₇ cycloalkenylene groups, optionally substituted and containing heteroatoms as described for R47.

In preferred embodiments C ^A and/or C ^B are bound to the remainder of the molecule via a residue of R32 as defined herein, wherein preferably said residue of R32 equals or is comprised in a Spacer.

A person skilled in the art will understand that "residue of R ₃₂ " means the conjugation reaction product of R32 with another chemical group so as to form a conjugate between C ^A and / or C ^B with the Trigger, a Spacer, or L ^C .

In other embodiments, C ^A and/or C ^B are bound to the remainder of the molecule via C ^M2 as defined herein, wherein preferably C ^M2 equals or is comprised in a Spacer.

In yet other embodiments, C ^A and/or C ^B are bound to the remainder of the molecule via C ^X as defined herein, wherein preferably C ^X equals or is comprised in a Spacer.

In preferred embodiments, moiety C ^X , C ^M2 and the said residue of R32 are comprised in C ^A and/or C ^B .

In preferred embodiments, C ^M2 is selected from the group consisting of amine, amide, thioamide, aminooxy, ether, carbamate, thiocarbamate, urea, thiourea, sulfonamide, and sulfoncarbamate.

In preferred embodiments C ^M2 equals R10. In preferred embodiments C ^M2 equals C ^X . In preferred embodiments, C ^M2 is:

wherein the dashed line denotes a bond to or towards C ^A or C ^B and the wiggly line denotes a bond to the remaining part of the dienophile. In other embodiments the wiggly line denotes a bond to or towards C ^A or C ^B and the dashed line denotes a bond to the remaining part of the dienophile.

In preferred embodiments, C ^X is:

wherein the dashed line denotes a bond to or towards C ^A or C ^B and the wiggly line denotes a bond to the remaining part of the dienophile. In other embodiments the wiggly line denotes a bond to or towards C ^A or C ^B and the dashed line denotes a bond to the remaining part of the dienophile.

With reference to above schemes with examples of C ^M2 and C ^X , in preferred embodiments, when C ^A or C ^B is a protein, such as an antibody, the dashed line denotes a bond to or towards C ^A or C ^B .

In preferred embodiments, when k or i is 0, C ^A and /or C ^B is linked to the remaining part of Formula 19 via a moiety selected from the group consisting of -O-, -C(R ⁶ )2-, -NR ⁶ -, -C(O)-, and -S-, wherein said moieties are part of C ^A and /or C ^B .

In preferred embodiments, when k or i is at least 1, then C ^A and /or C ^B is linked to S ^P via a moiety selected from the group consisting of -O-, - C(R ⁶ ) ₂ -, -NR ⁶ -, -C(O)-, and -S-, wherein said moieties are part of C ^A and /or C ^B , and S ^P is linked to the remaining part of Formula 19 via a moiety selected from the group consisting of -O-, -C(R ⁶ ) ₂ -, -NR ⁶ -, -C(O)- and -S-, wherein said moieties are part of S ^P .

In preferred embodiments, at most three C ^B is comprised in the structure of Formula (19), more preferably at most two, most preferably at most one C ^B is comprised in the structure of Formula (19).

In a preferred embodiment, C ^A and /or C ^B before conjugation to the remainder of the compound of Formula (19) comprises at least one moiety selected from the group consisting of -OH, -NHR’, -CO ₂ H, -SH, -S-S-, -SCH ₃ - -N ₃ , terminal alkynyl, terminal alkenyl, -C(O)R', C ₈ -C ₁₂ (hetero)cycloalkynyl, C ₃ -C ₄ cycloalkenyl, nitrone, nitrile oxide, (imino)sydnone, isonitrile,

(oxa)norbornene, and tetrazine, said moiety used for conjugation to a moiety comprising the dienophile and R ₃₂ so as to form the compound satisfying Formula (19), and comprising a C ^M2 or C ^X moiety.

In preferred embodiments the C ^A and /or C ^B is bound to the

remainder of the compound of Formula (19) via a C ^M2 selected from the group consisting of amine, amide, thioamide, aminooxy, carbamate, thiocarbamate, urea, thiourea, sulfonamide, and sulfoncarbamate.

In preferred embodiments C ^M2 equals R ₁₀ .

In a preferred embodiment, when C ^A or C ^B is conjugated via -SH or - S-S-, then C ^M2 is selected from the group consisting of

wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A or C ^B , In a preferred embodiment, when moiety C ^A , C ^B , or the Administration Agent is conjugated via -SMe-, then C ^M2 is:

wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A , C ^B , or the Administration Agent.

In a preferred embodiment, when C ^A or C ^B is conjugated via -NR’-, then C ^M2 is selected from the group consisting of wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A or C ^B .

In a preferred embodiment, when C ^A or C ^B is conjugated via -C- derived from a moiety that was -C(O)R’ or -C(O)R’-, then C ^M2 is selected from the group consisting of wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A or C ^B .

In a preferred embodiment, when C ^A or C ^B is conjugated via -C(O)- derived from a moiety that was -C(O)OH, then C ^M2 is selected from the group consisting of

wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A or C ^B .

In a preferred embodiment, when C ^A or C ^B is conjugated via -O-, then C ^M2 is selected from the group consisting of

wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A or C ^B .

In a preferred embodiment, when C ^A or C ^B is conjugated via -N ₃ that was reacted with an R ₃₂ that comprised an alkyne group, then the resulting C ^X comprises a triazole ring, wherein each C ^X is independently selected from the group consisting of

wherein the wiggly line denotes a bond to the remaining part of the molecule, and wherein the dashed line denotes a bond to C ^A or C ^B . R ⁶

Preferably, each R ⁶ is independently selected from the group consisting of hydrogen, -(S ^P )i-C ^B , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 0 or 1;

the R ⁶ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, - PO ₃ H _, -PO ₄ H _2, -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each R ⁶ is individually selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl groups, C ₂ -C ₈ alkenyl groups, C ₂ - C8 alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂

(hetero)arylalkyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂

cycloalkylalkyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein the R ⁶ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of - Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NH, and - SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, R ⁶ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C2-C ₄ alkynyl groups, C ₆ -C8 aryl, C2-C8 heteroaryl, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁₀ alkyl(hetero)aryl groups, C ₃ -C ₁₀ (hetero)arylalkyl groups, C ₄ - C8 alkylcycloalkyl groups, C ₄ -C8 cycloalkylalkyl groups, C ₅ -C ₁ 0 cycloalkyl(hetero)aryl groups and C ₅ -C ₁₀ (hetero)arylcycloalkyl groups, wherein the R ⁶ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , - SO ₃ H, -PO ₃ H _, -PO ₄ H _2, -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, R ⁶ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C ₂ -C ₄ alkenyl groups, and C _4-6 (hetero)aryl groups, wherein for R ⁶ the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, -SH, -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ and - NO ₂ and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized.

In preferred embodiments, R ⁶ is selected from the group consisting of hydrogen, C ₁ -C ₃ alkyl groups, C ₂ -C ₃ alkenyl groups, and C ₄ -6 (hetero)aryl groups, wherein for R ⁶ the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, -SH, -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ and - NO ₂ and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized.

In preferred embodiments, the R ⁶ groups not being hydrogen are not substituted. In preferred embodiments, the R ⁶ groups not being hydrogen do not contain heteroatoms. In preferred embodiments, the R ⁶ groups are hydrogen. R ⁷

In preferred embodiments, each R ⁷ is independently selected from the group consisting of hydrogen, -(S ^P )i-C ^B , -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, - NO ₂ , -CF ₃ , -SR ₃₇ , S(=O) ₂ N(R ₃₇ ) ₂ , OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ ,

SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O- R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2, SC(=O)N(R ₃₇ )2, OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ ,

C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, - NR ₃₇ OR ₃₇ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ - C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄

(cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups; wherein i is an integer in a range of from 0 to 4, preferably i is 0 or 1, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups,

(cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups,

(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,

(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , - N(R ₃₇ )2, -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ )2, -PO ₄ (R ₃₇ )2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each R ⁷ is independently selected from the group consisting hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ ,

NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2, SC(=O)N(R ₃₇ )2, OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, - NR ₃₇ OR ₃₇ , C ₁ -C ₈ alkyl groups, C ₂ -C ₈ alkenyl groups, C ₂ -C ₈ alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂

(hetero)arylalkyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂

cycloalkylalkyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and

(hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ ) _2, -PO ₄ (R ₃₇ ) _2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each R ⁷ is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, - NO ₂ , -CF ₃ , -SR ₃₇ , S(=O) ₂ N(R ₃₇ ) ₂ , OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ ,

SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O- R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O) ₂ R ₃₇ , NR ₃₇ S(O) ₂ R ₃₇ , -ON(R ₃₇ ) _{2, -} NR ₃₇ OR ₃₇ , C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C2-C ₄ alkynyl groups, C ₆ -C8 aryl groups, C2-C8 heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁₀ alkyl(hetero)aryl groups, C ₃ -C ₁₀

(hetero)arylalkyl groups, C ₄ -C ₁ 0 alkylcycloalkyl groups, C ₄ -C ₁ 0 cycloalkylalkyl groups, C ₅ -C ₁₀ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₀ (hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and

(hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ )2, -PO ₄ (R ₃₇ )2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each R ⁷ is independently selected from the group consisting of hydrogen and C ₁ -C ₃ alkyl groups, C ₂ -C ₃ alkenyl groups, and C ₄ -6 (hetero)aryl groups, wherein the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, =NH, -N(CH ₃ )2, - S(O)2CH ₃ , and -SH, and are optionally interrupted by at most one

heteroatom selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, R ⁷ is preferably selected from the group consisting of hydrogen, methyl, -CH ₂ -CH ₂ -N(CH ₃ )2, and -CH ₂ -CH ₂ -S(O)2- CH ₃ . In preferred embodiments, the R ⁷ groups not being hydrogen are not substituted. In preferred embodiments, the R ⁷ groups not being hydrogen do not contain heteroatoms. In preferred embodiments, the R ⁷ groups are hydrogen. R ⁸ and R ⁹ Preferably, R ⁸ and R ⁹ are as defined for R ⁷ . In preferred embodiments, at least one or all R ⁸ are -H. In preferred embodiments, at least one or all R ⁸ are -CH ₃ . In preferred embodiments, at least one or all R ⁹ are -H. In preferred embodiments, at least one or all R ⁹ are -CH ₃ . R32 R ₃₂ is a conjugation moiety, which is chemical group that can be used for binding, conjugation or coupling of a Construct, such as Construct-B, or a Spacer, a Linker L ^C , the Trigger , or another molecule or construct of interest. The person skilled in the art is aware of the myriad of strategies that are available for the chemoselective or -unselective or enzymatic coupling or conjugation of one molecule or construct to another.

In preferred embodiments, R ₃₂ is a moiety that allows conjugation to a protein comprising natural and/or non-natural amino acids. Moieties suitable for conjugation are known to the skilled person. Conjugation strategies are for example found in [O. Boutureira, G.J.L. Bernardes, Chem. Rev., 2015, 115, 2174-2195].

In particularly favourable embodiments, R ₃₂ is selected from the group consisting of N-maleimidyl groups, halogenated N-alkylamido groups, sulfonyloxy N-alkylamido groups, vinyl sulfone groups, (activated) carboxylic acids, benzenesulfonyl halides, ester groups, carbonate groups, sulfonyl halide groups, thiol groups or derivatives thereof, C2-6 alkenyl groups, C _2-6 alkynyl groups, C _7-18 cycloalkynyl groups, C _5-18

heterocycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl] groups, C _3-12 cycloalkenyl groups, azido groups, phosphine groups, nitrile oxide groups, nitrone groups, nitrile imine groups, isonitrile groups, diazo groups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazine groups, halogenated N- maleimidyl groups , aryloxymaleimides, dithiophenolmaleimides, bromo- and dibromopyridazinediones, 2,5-dibromohexanediamide groups, alkynone groups, 3-arylpropiolonitrile groups, 1,1-bis(sulfonylmethyl)-methylcarbonyl groups or elimination derivatives thereof, carbonyl halide groups,

allenamide groups, 1,2-quinone groups, isothiocyanate groups, isocyanate groups, aldehyde groups, triazine groups, tetrazine groups, squaric acids, 2- imino-2-methoxyethyl groups, (oxa)norbornene groups, (imino)sydnones, methylsulfonyl phenyloxadiazole groups, aminooxy groups, 2-amino benzamidoxime groups, ethynylphosphonamidates, groups reactive in the Pictet- Spengler ligation and hydrazino- Pictet- Spengler (HIPS) ligation, DNA intercalators and photocrosslinkers.

In preferred embodiments, R ₃₂ is an N-maleimidyl group connected to the remaining part of the compound according to Formula (19) via the N atom of the N-maleimidyl group.

In other preferred embodiments, R ₃₂ is selected from the group consisting of, hydroxyl groups, amine groups, halogens, vinyl pyridine groups, disulfide groups, pyridyl disulfide groups, sulfonyloxy groups, mercaptoacetamide groups, anhydride groups, sulfonylated

hydroxyacetamido groups, sulfonyl chlorides, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In other embodiments R32 is group that can be connected to another group by means of an enzyme, for example sortase or Tubulin tyrosine ligase. R36 In a preferred embodiment, R ₃₆ is as defined for R ₃₇ .

In Formula (19) R ₃₆ is preferably selected from the group consisting of hydrogen, C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, and C ₄ -6 (hetero)aryl groups, wherein the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, -SH, -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ and -NO ₂ and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized.

In preferred embodiments, the R ₃₆ groups not being hydrogen are not substituted. In preferred embodiments, the R ₃₆ groups not being hydrogen do not contain heteroatoms. R ₃₇ Preferably each R ₃₇ is independently selected from the group consisting of hydrogen, -(S ^P )i-C ^B , C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C ₈ cycloalkyl groups, C ₅ -C ₈ cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂

(hetero)arylalkyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂

cycloalkylalkyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein i is an integer in the range of from 0 to 4, preferably 1, wherein the R ₃₇ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, - I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

Preferably each R ₃₇ is independently selected from the group consisting of hydrogen, -(S ^P )i-C ^B , C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C ₂ -C ₄ alkynyl groups, C ₆ -C ₈ aryl, C ₂ -C ₈ heteroaryl, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁₀ alkyl(hetero)aryl groups, C ₃ -C ₁₀

(hetero)arylalkyl groups, C ₄ -C8 alkylcycloalkyl groups, C ₄ -C8 cycloalkylalkyl groups, C ₅ -C ₁₀ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₀

(hetero)arylcycloalkyl groups, wherein i is an integer in the range of from 0 to 4, preferably 1, wherein the R ₃₇ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, - I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. In preferred embodiments, the R ₃₇ groups not being hydrogen are not substituted. In preferred embodiments, the R ₃₇ groups not being hydrogen do not contain heteroatoms. R ₄₇ In preferred embodiments, each R47 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, - NO ₂ , -CF ₃ , -SR ₃₇ , S(=O) ₂ N(R ₃₇ ) ₂ , OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ ,

SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O- R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O) ₂ R ₃₇ , NR ₃₇ S(O) ₂ R ₃₇ , -ON(R ₃₇ ) _{2, -} NR ₃₇ OR ₃₇ , -(S ^P )i-C ^B , C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C8 cycloalkyl groups, C ₅ -C ₈ cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂

cycloalkylalkyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein i is an integer in the range of from 0 to 4, preferably 1; wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of - Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ ) _2, -PO ₄ (R ₃₇ ) _2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally

quaternized.

In preferred embodiments, each R ₄₇ is independently selected from the group consisting of hydrogen -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , -SO ₃ , -PO ₃ - _, - NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ ,

SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O- R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2, NR ₃₇ C(=O)N(R ₃₇ )2, NR ₃₇ C(=S)N(R ₃₇ )2, C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O) ₂ R ₃₇ , NR ₃₇ S(O) ₂ R ₃₇ , -ON(R ₃₇ ) _{2, -} NR ₃₇ OR ₃₇ , -(S ^P )i-C ^B , C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C2-C ₄ alkynyl groups, C ₆ -C ₈ aryl groups, C ₂ -C ₈ heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁₀ alkyl(hetero)aryl groups, C ₃ -C ₁₀

(hetero)arylalkyl groups, C ₄ -C ₁ 0 alkylcycloalkyl groups, C ₄ -C ₁ 0

cycloalkylalkyl groups, C ₅ -C ₁₀ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₀ (hetero)arylcycloalkyl groups, wherein i is an integer in the range of from 0 to 4, preferably 1; wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups, (hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of - Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ )2, -PO ₄ (R ₃₇ )2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally

quaternized.

In a preferred embodiment, R ₄₇ is a moiety satisfying any one of Formulae (2x) and (2y):

wherein in both Formula (2x) and (2y) the wiggly line denotes a bond to the remainder of the molecule. In Formula (2y), C ^M2 is coupled to a Construct-B (C ^B ), preferably a targeting agent, preferably selected from the group consisting of proteins, antibodies, peptoids and peptides.

In a preferred embodiment, in Formula (19), if X ³ is CR ₄₇ Y ^T1 and X ¹ , X ² , X ⁴ , and X ⁵ are CH ₂ , then R ₄₇ in X ³ does not satisfy Formula (2x) or (2y). R33 In preferred embodiments, each individual R ₃₃ is selected from the group consisting of C ₁ -C ₁₂ alkylene groups, C ₂ -C ₁₂ alkenylene groups, C ₂ -C ₁₂ alkynylene groups, C ₆ arylene groups, C ₄ -C ₅ heteroarylene groups, C ₃ -C8 cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₁₂ alkyl(hetero)arylene groups, C ₅ -C ₁₂ (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, C ₄ -C ₁₂ cycloalkylalkylene groups, wherein the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups,

cycloalkylalkylene groups, are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R’) ₂ , =O, =NR’, -SR’, - SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ and -Si(R’)3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR’-, -P-, and - Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In particularly favourable embodiments, each individual R ₃₃ is selected from the group consisting of C ₁ -C ₆ alkylene groups, C2-C ₆ alkenylene groups, and C2-C ₆ alkynylene groups, more preferably from the group consisting of C ₁ -C ₃ alkylene groups, C ₂ -C ₃ alkenylene groups, and C ₂ - C ₃ alkynylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,

cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. R35 In preferred embodiments, each individual R ₃₅ is selected from the group consisting of C ₁ -C ₈ alkylene groups, C ₂ -C ₈ alkenylene groups, C ₂ -C ₈ alkynylene groups, C ₆ arylene groups, C ₄ -C ₅ heteroarylene groups, C ₃ -C ₆ cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₁₂

alkyl(hetero)arylene groups, C ₅ -C ₁₂ (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, C ₄ -C ₁₂ cycloalkylalkylene groups, wherein for the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups,

cycloalkylalkylene groups, are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR’, -N(R’) ₂ , =O, =NR’, -SR’, - SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ and -Si(R’)3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR’-, -P-, and - Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each individual R ₃₅ is selected from the group consisting of C ₁ -C ₄ alkylene groups, C ₂ -C ₄ alkenylene groups, C ₂ -C ₄ alkynylene groups, C ₆ arylene groups, C ₄ -C ₅ heteroarylene groups, C ₃ -C ₆ cycloalkylene groups, wherein the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, and cycloalkylene groups, are optionally substituted with a moiety selected from the group consisting of - Cl, -F, -Br, -I, -OR’, -N(R’) ₂ , =O, =NR’, -SR’, -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ , -NO ₂ and -Si(R’)3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR’-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. R’ In preferred embodiments, each R’ is independently selected from the group consisting of hydrogen, C ₁ -C ₆ alkylene groups, C2-C ₆ alkenylene groups, C2- C ₆ alkynylene groups, C ₆ arylene, C ₄ -C ₅ heteroarylene, C ₃ -C ₆ cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₁₂ alkyl(hetero)arylene groups, C ₅ -C ₁₂ (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, and C ₄ -C ₁₂ cycloalkylalkylene groups.

In preferred embodiments, each R’ is independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkylene groups, C2-C ₄ alkenylene groups, C ₂ -C ₄ alkynylene groups, C ₆ arylene, C ₄ -C ₅ heteroarylene, C ₃ -C ₆ cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₈ alkyl(hetero)arylene groups, C ₅ -C8 (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, and C ₄ -C8 cycloalkylalkylene groups.

Unless stated otherwise, for R’ the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups, cycloalkylalkylene groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, - I, -OH, -NH ₂ , =O, -SH , -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ , -NO ₂ , and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si, wherein the N, S, and P atoms are optionally oxidized.

In a preferred embodiment, R’ is as defined for R ₃₇ .

In a preferred embodiment, in relation to C ^M2 , C ^X , and the conjugation of C ^A and C ^B , R’ is as defined for R ₃₇ . R” In preferred embodiments, each R” is independently selected from the group consisting of

, wherein the wiggly line depicts a bond to an ethylene glycol group or optionally to the R33 adjacent to R32 when t4 is 0, and the dashed line depicts a bond to R33 or G.

In preferred embodiments, R” is -CH ₂ -C(O)NR’- or -CH ₂ -NR’, N, C ₅ -C ₆ arenetriyl, C ₄ -C ₅ heteroarenetriyl, C ₃ -C ₆ cycloalkanetriyl, and C ₄ -C ₆ cycloalkenetriyl, wherein the arenetriyl, heteroarenetriyl, cycloalkanetriyl, and cycloalkenetriyl are optionally further substituted with groups selected from the group consisting of -Cl, -F, -Br, -I, -OR’, -N(R’)2, -SR’, -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , and -CF ₃ , and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR’-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. Preferably, G is CR’. L In preferred embodiments, L is selected from the group consisting of -CH ₂ - OCH ₃ , -CH ₂ -OH, -CH ₂ -C(O)OH, -C(O)OH. In preferred embodiments, L is preferably -CH ₂ -OCH ₃ . t1, t2, t3, t4, t5 In preferred embodiments, t ₁ is 0. In other embodiments, t ₁ is 1. In preferred embodiments, t2 is 0. In other embodiments, t2 is 1. In preferred

embodiments, t ₃ is an integer in a range of from 0 to 12. Preferably, t ₃ is an integer in a range of from 1 to 10, more preferably in a range of from 2 to 8. In particularly favourable embodiments, t3 is 4 and y is 1. In preferred embodiments, t ₄ is 0. In other embodiments, t ₄ is 1. In preferred

embodiments, t5 is an integer in a range of from 6 to 48, preferably from 15 to 40, more preferably from 17 to 35, even more preferably from 20 to 30, most preferably from 22 to 28. In particularly preferred embodiments, t ₅ is 23. Trans-cyclooctenes In a preferred embodiment, the dienophile Trigger moiety used in the present invention comprises a trans-cyclooctene ring, and particularly refers to a structure satisfying Formula (19), the ring optionally including one or more hetero-atoms. The skilled person is familiar with the fact that the dienophile activity is not necessarily dependent on the presence of all carbon atoms in the ring, since also heterocyclic monoalkenylene eight-membered rings are known to possess dienophile activity.

Thus, in general, the invention is not limited to strictly trans- cyclooctene. The person skilled in organic chemistry will be aware that other eight-membered ring-based dienophiles exist, which comprise the same endocyclic double bond as the trans-cyclooctene, but which may have one or more heteroatoms elsewhere in the ring. I.e., the invention generally pertains to eight-membered non-aromatic cyclic alkene moieties, preferably a cyclooctene moiety, and more preferably a trans-cyclooctene moiety.

It should be noted that, depending on the choice of nomenclature, the TCO dienophile may also be denoted E-cyclooctene. With reference to the conventional nomenclature, it will be understood that, as a result of substitution on the cyclooctene ring, depending on the location and molecular weight of the substituent, the same cyclooctene isomer may formally become denoted as a Z-isomer. In the present invention, any substituted variants of the invention, whether or not formally“E” or“Z,” or “cis” or“trans” isomers, will be considered derivatives of unsubstituted trans-cyclooctene, or unsubstituted E-cyclooctene. The terms”trans- cyclooctene” (TCO) as well as E-cyclooctene are used interchangeably and are maintained for all dienophiles according to the present invention, also in the event that substituents would formally require the opposite

nomenclature. I.e., the invention relates to cyclooctene in which carbon atoms 1 and 6 as numbered below in Formula 4b are in the E (entgegen) or trans position.

The TCO may consist of multiple isomers, also comprising the equatorial vs. axial positioning of substituents, such as R48, on the TCO. In this respect, reference is made to Whitham et al. J. Chem. Soc. (C), 1971, 883-896, describing the synthesis and characterization of the equatorial and axial isomers of trans-cyclo-oct-2-en-ol, identified as (1RS, 2RS) and (1SR, 2RS), respectively. In these isomers the OH substituent is either in the equatorial or axial position. In a preferred embodiment, for TCO structures where the R ₄₈ can be either in the axial or the equatorial position, the R ₄₈ is in the axial position.

Trans-cyclooctene or E-cyclooctene derivatives are very suitable as Triggers, especially considering their high reactivity. Optionally, the trans- cyclooctene (TCO) moiety comprises at least two exocyclic bonds fixed in substantially the same plane, and/or it optionally comprises at least one substituent in the axial position, and not the equatorial position. The person skilled in organic chemistry will understand that the term“fixed in substantially the same plane” refers to bonding theory according to which bonds are normally considered to be fixed in the same plane. Typical examples of such fixations in the same plane include double bonds and strained fused rings. E.g., the at least two exocyclic bonds can be the two bonds of a double bond to an oxygen (i.e. C=O). The at least two exocyclic bonds can also be single bonds on two adjacent carbon atoms, provided that these bonds together are part of a fused ring (i.e. fused to the TCO ring) that assumes a substantially flat structure, therewith fixing said two single bonds in substantially one and the same plane. Examples of the latter include strained rings such as cyclopropyl and cyclobutyl. Without wishing to be bound by theory, the inventors believe that the presence of at least two exocyclic bonds in the same plane will result in an at least partial flattening of the TCO ring, which can lead to higher reactivity in the IEDDA reaction. A background reference providing further guidance is WO 2013/153254.

The dienophiles for use in the invention can be synthesized by the skilled person, on the basis of known synthesis routes to cyclooctenes and corresponding hetero atom(s)-containing rings. The skilled person further is aware of the wealth of cyclooctene derivatives that can be synthesized via the ring closing metathesis reaction using Grubbs catalysts. As mentioned above, the TCO possibly includes one or more heteroatoms in the ring. This is as such sufficiently accessible to the skilled person [e.g. WO ₂ 016025480]. Reference is made, e.g., to the presence of a thioether in TCO: [Cere et al. J. Org. Chem. 1980, 45, 261]. Also, e.g., an - O-SiR2-O moiety in TCO: [Prevost et al. J. Am. Chem. Soc.2009, 131, 14182]. References to TCO syntheses wherein the allylic positioned leaving group (R48) is an ether, ester, carbonate, carbamate or a thiocarbamate are: [Versteegen et al Angew. Chem. Int. Ed.2018, 57, 10494], and [Steiger et al Chem Comm 2017, 53, 1378]. Exemplary compounds include the following structures, indicated below with literature references. Where a cyclooctene derivative is depicted as a Z-cyclooctene it is conceived that this can be converted to the E- cyclooctene analog.

Preferred Triggers of this invention include:

Further preferred Triggers of this invention include: Construct-A (C ^A ) and Construct-B (C ^B ) The Constructs A and Constructs B include but are not limited to small molecules, organic molecules, metal coordination compounds, molecules comprising a radionuclide, chelates comprising a radiometal, inorganic molecules, organometallic molecules, biomolecules, polymers, resins, particles (e.g. micro- and nanoparticles), liposomes, micelles, polymersomes, gels, surfaces, cells, biological tissues, and pathogens.

In preferred Prodrug or in vivo embodiments, C ^A is selected from the group consisting of Drugs, Targeting Agents, Labels, Administration

Agents, and Masking Moieties. Preferably, C ^A is a Drug, preferably a Drug as defined herein.

In some preferred Prodrug or in vivo embodiments, C ^B is selected from the group consisting of Drugs, Targeting Agents, Labels,

Administration Agents, and Masking Moieties. Preferably, C ^B is selected from the group consisting of Targeting Agents, and Masking Moieties.

In preferred embodiments, the compounds of Formula (19) comprise at least one Label and at least one Administration Agent, and preferably satisfy at least one of the conditions (i)-(iii):

(i) at least one C ^A is a Label, and at least one C ^A is an Administration Agent; (ii) at least one C ^A is a Label, and at least one C ^B is an Administration Agent;

(iii) at least one C ^A is an Administration Agent, and at least one C ^B is a Label;

for all conditions (i)-(iii): with the proviso that if X ¹ -X ⁵ contain the

Administration Agent and at least one C ^A is a Label, X ¹ -X ⁵ do not contain the same Label as the at least one C ^A ; with the proviso that if at least one C ^A is a Label and X ¹ -X ⁵ contain the same Label as the at least one C ^A , X ¹ -X ⁵ do not contain an Administration Agent; with the preference that if at least one C ^B comprised in R ₄₈ is an Administration Agent, then the other C ^B comprised in R48 are not a Label. In a preferred embodiment, only condition (i) is met. In a preferred embodiment, only condition (ii) is met. In a preferred embodiment, only condition (iii) is met. In a preferred

embodiment, both conditions (i) and (ii) are met. In a preferred embodiment, both conditions (i) and (iii) are met. In a preferred embodiment, both conditions (ii) and (iii) are met. In a preferred embodiment, all three conditions (i)-(iii) are met.

In preferred in vitro embodiments C ^A and C ^B include but are not limited to small molecules, organic molecules (including fluorescent dyes), metal coordination compounds, molecules comprising a radionuclide, chelates comprising a radiometal, inorganic molecules, organometallic molecules, biomolecules, drugs, polymers, resins (e.g. polystyrene, agarose), particles (e.g. beads, magnetic beads, gold, silica-based particles and materials, polymers and polymer-based materials, glass, iron oxide particles, micro- and nanoparticles such as liposomes and polymersomes), gels, surfaces (e.g. glass slides, chips, wafers, gold, metal, silica-based, polymer, plastic, resin), cells, biological tissues, pathogens (viruses, bacteria, fungi, yeast). The Constructs may for example comprise a combination of the aforementioned Constructs. Examples of biomolecules include:

carbohydrates, biotin, peptides, peptoids, lipids, proteins, enzymes, oligonucleotides, DNA, RNA, PNA, LNA, aptamers, hormones, toxins, steroids, cytokines, antibodies, antibody fragments (e.g. Fab2, Fab, scFV, diabodies, triabodies, VHH), antibody (fragment) fusions (e.g. bi-specific and trispecific mAb fragments).

In preferred in vitro embodiments C ^A and C ^B can also be R ₃₂ or a moiety comprising R32, as defined herein, wherein R32 can be used to bind to a further C ^A and C ^B . For example, C ^A can be R ₃₂ being a maleimide or photocrosslinker that is bound to the T ^R via a Spacer S ^P . The maleimide or photocrosslinker can be used to further conjugate the T ^R to a protein. In this particular embodiment C ^A and C ^B are a biomolecule-binding moiety. In preferred embodiments, each C ^A and C ^B are independently selected from the group consisting of organic molecules, inorganic molecules, organometallic molecules, resins, beads, glass, microparticles, nanoparticles, gels, surfaces, and cells. Preferably, each C ^A and C ^B are independently selected from the group consisting of organic molecules, and inorganic molecules.

In preferred embodiments, each C ^A and C ^B are independently selected from the group consisting of small molecules, proteins, carbohydrates, peptides, peptoids, oligosaccharides, molecules comprising a radionuclide, fluorescent dyes, inorganic molecules, organometallic molecules, polymers, lipids, oligonucleotides, DNA, RNA, PNA, LNA, drugs, resins, beads, glass, microparticles, nanoparticles, gels, surfaces, and cells.

Preferably, a small molecule is a small organic molecule. Preferably, a small molecule has a molecular weight of at most 2 kDa, more preferably at most 1 kDa, more preferably at most 750 Da, more preferably at most 500 Da, and most preferably at most 300 Da. Preferably, a small molecule has a molecular weight of at least 15 Da, more preferably at least 50 Da, more preferably at least 75 Da, and most preferably at least 100 Da.

It will be understood that“molecules comprising a radionuclide” include chelators that chelate a radionuclide.

In another preferred embodiment, each C ^A and C ^B are independently a moiety according to Formula (5) as defined herein. Construct-Trigger assemblies A Construct-Trigger comprises a conjugate of the Construct or Constructs C ^A and the Trigger T ^R . Optionally the Trigger is further linked to Construct or Constructs C ^B .

The general formula of the Construct-Trigger is shown below in Formula (5a) and (5b). For the avoidance of doubt, as Y ^C is part of L ^C and C ^A , Y ^C is not separately denoted in Formula (5a) and (5b).

C ^A is Construct A, C ^B is Construct B, S ^P is Spacer; T ^R is Trigger, and L ^C is Linker. Formula (5a): b,c,e,f,g,h ³ 0; a,d ³ 1. Formula (5b): c,e,f,g,h ³ 0; a,b,d ³ 1.

In the Trigger-Construct conjugate, the Construct C ^A and the Trigger T ^R - the TCO derivative- can be directly linked to each other. They can also be bound to each other via a self-immolative linker L ^C , which may consist of multiple (self-immolative, or non immolative) units. With reference to Formula 5a and 5b, when L ^C contains a non immolative unit, this unit equals a Spacer S ^P and c ³ 1. It will be understood that the invention encompasses any conceivable manner in which the diene Trigger is attached to the one or more Construct C ^A . The same holds for the attachment of one or more Construct C ^B to the Trigger or the linker L ^C . The same holds for the optional attachment of one or more Spacer S ^P to the Trigger or the linker L ^C . Methods of affecting conjugation, e.g. through reactive amino acids such as lysine or cysteine in the case of proteins, are known to the skilled person. Exemplary conjugation methods are outlined in the section on Spacers herein below.

It will be understood that the Construct C ^A is linked to the TCO in such a way that the Construct C ^A is eventually capable of being released after formation of the IEDDA adduct. Generally, this means that the bond between the Construct C ^A and the TCO, or in the event of a self-immolative Linker L ^C , the bond between the Linker and the TCO and between the Construct C ^A and the Linker, should be cleavable. Predominantly, the Construct C ^A and the optional Linker is linked via a hetero-atom, preferably via O, N, NH, or S. The cleavable bond is preferably selected from the group consisting of carbamate, thiocarbamate, carbonate, ester, ether, thioether, amide, thioester bonds.

It shall be understood that one C ^B can be modified with more than one Trigger. For example, an antibody can be modified with 4 TCO-drug constructs by conjugation to 4 amino acid residues, wherein C ^A is drug.

Likewise, it shall be understood that one C ^A can be modified with more than one Trigger. For example, a protein drug can be masked by conjugation of 4 amino acid residues to 4 TCO-polyethylene glycol constructs, wherein polyethylene glycol is C ^B .

Furthermore, it shall be understood that one C ^A can be modified with more than one Trigger, wherein at least one Trigger links to a Targeting Agent, being C ^B , and at least one Trigger links to a Masking Moiety being C ^B , wherein C ^A can be a Drug, preferably a protein. Spacers S ^P It will be understood that when herein, it is stated that“each individual S ^P is linked at all ends to the remainder of the structure” this refers to the fact that the spacer S ^P connects multiple moieties within a structure, and therefore the spacer has multiple ends by definition. The spacer S ^P may be linked to each individual moiety via different or identical moieties that may be each individually selected. Typically, these linking moieties are to be seen to be part of spacer S ^P itself. In case the spacer S ^P links two moieties within a structure,“all ends” should be interpreted as“both ends”. As an example, if the spacer connects a trans-cyclooctene moiety to a Construct A, then“the remainder of the molecule” refers to the trans-cylooctene moiety and Construct A, while the connecting moieties between the spacer and the trans-cyclooctene moiety and Construct A (i.e. at both ends) may be individually selected.

In a preferred embodiment, Spacers S ^P may consist of one or multiple Spacer Units S ^U arranged linearly and/or branched and may be connected to one or more C ^B moieties and / or one or more L ^C or T ^R moieties. The Spacer may be used to connect C ^B to one T ^R (Example A below; with reference to Formula 5a and 5b: f, e, a = 1) or more T ^R (Example B and C below; with reference to Formula 5a and 5b: f, e = 1, a ³ 1), but it can also be used to modulate the properties, e.g. pharmacokinetic properties, of the C ^B -T ^R -C ^A conjugate (Example D below; with reference to Formula 5a and 5b: one or more of c,e,g,h ³ 1). Thus a Spacer unit does not necessarily connect two entities together, it may also be bound to only one component, e.g. the T ^R or L ^C . Alternatively, the Spacer may comprise a Spacer Unit linking C ^B to T ^R and in addition may comprise another Spacer Unit that is only bound to the Spacer and serves to modulate the properties of the conjugate (Example F below; with reference to Formula 5a and 5b: e ³ 1). The Spacer may also consist of two different types of S ^U constructs, e.g. a PEG linked to a peptide, or a PEG linked to an alkylene moiety (Example E below; with reference to Formula 5a and 5b: e ³ 1). For the sake of clarity, Example B depicts a S ^U that is branched by using a multivalent branched S ^U . Example C depicts a S ^U that is branched by using a linear S ^U polymer, such as a peptide, whose side chain residues serve as conjugation groups.

The Spacer may be bound to the Activator in similar designs such as depicted in above examples A- F. The Spacer Units include but are not limited to amino acids, nucleosides, nucleotides, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. Exemplary preferred biopolymer S ^U are peptides. Yet other examples are alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, aryl, arylene, alkylaryl, alkylarylene, arylalkyl, arylalkylene, arylalkenyl, arylalkenylene, arylalkynyl, arylalkynylene , polyethyleneamino,

polyamine, which may be substituted or unsubstituted, linear or branched, may contain further cyclic moieties and / or heteroatoms, preferably O, N, and S, more preferably O; wherein In preferred embodiments these example S ^U comprise at most 50 carbon atoms, more preferably at most 25 carbon atoms, more preferably at most 10 carbon atoms. In some preferred embodiments the S ^U is independently selected from the group consisting of (CH ₂ )r, (C ₃ -C8 carbocyclo), O-(CH ₂ )r, arylene, (CH ₂ )r-arylene, arylene-(CH ₂ )r, (CH ₂ ) _r -(C ₃ -C ₈ carbocyclo), (C ₃ -C ₈ carbocyclo)-(CH ₂ ) _r , (C ₃ -C ₈ heterocyclo), (CH ₂ )r -(C ₃ -C8 heterocyclo), (C ₃ -C8 heterocyclo)-(CH ₂ )r,

-(CH ₂ )rC(O)NR4(CH ₂ )r, (CH ₂ CH ₂ O)r, (CH ₂ CH ₂ O)rCH ₂ ,(CH ₂ )rC(O)NR4(CH ₂ CH ₂ O) _r , (CH ₂ ) _r C(O)NR ₄ (CH ₂ CH ₂ O) _r CH _2, (CH ₂ CH ₂ O) _r C(O)NR ₄ (CH ₂ CH ₂ O) _r , (CH ₂ CH ₂ O)r C(O)NR4(CH ₂ CH ₂ O)rCH ₂ , (CH ₂ CH ₂ O)rC(O)NR4CH ₂ ; wherein r is independently an integer from 1 -10, and R ₄ is as defined herein.

Other examples of Spacer Units S ^U are linear or branched

polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CO ₂ H. Other polymeric Spacer Units are polymers and copolymers such as poly-(2-oxazoline), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(1-hydroxymethylethylene hydroxymethyl-formal (PHF). Other exemplary polymers are

polysaccharides, glycopolysaccharides, glycolipids, polyglycoside,

polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring polysaccharides that can be used as S ^U are cellulose, amylose, dextran, dextrin, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin. In yet other exemplary embodiments, the polymeric S ^U comprises a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Exemplary preferred polymeric S ^U are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides.

In some aspects of the invention polymers used in a S ^U have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.

Other exemplary S ^U are dendrimers, such as poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol based dendrimers.

The S ^U of the invention expressly include but are not limited to conjugates prepared with commercially available cross-linker reagents such as BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB, DTME, BMB, BMDB, BMH, BMOE, BM(PEO) ₃ and BM(PEO) ₄ .

To construct a branching Spacer one may use a S ^U based on one or several natural or non-natural amino acids, amino alcohol, aminoaldehyde, or polyamine residues or combinations thereof that collectively provide the required functionality for branching. For example serine has three

functional groups, i.e. acid, amino and hydroxyl groups and may be viewed as a combined amino acid an aminoalcohol residue for purpose of acting as a branching S ^U . Other exemplary amino acids are lysine and tyrosine.

In preferred embodiments, the Spacer consists of one Spacer Unit, therefore in those cases S ^P equals S ^U . In preferred embodiments the Spacer consists of two, three or four Spacer Units.

In some aspects of the S ^P has a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa. In some aspects of the invention, the S ^P has a mass of no more than 5000 daltons, no more than 4000 daltons, no more than 3000 daltons, no more than 2000 daltons, no more than 1000 daltons, no more than 800 daltons, no more than 500 daltons, no more than 300 daltons, no more than 200 daltons. In some aspects the S ^P has a mass from 100 daltons, from 200 daltons, from 300 daltons to 5000 daltons. In some aspects of the S ^P has a mass from 30, 50, or 100 daltons to 1000 daltons, from about 30, 50, or 100 daltons to 500 daltons.

In preferred embodiments, S ^P is a spacer selected from the group consisting of C ₁ -C ₁₂ alkylene groups, C ₂ -C ₁₂ alkenylene groups, C ₂ -C ₁₂ alkynylene groups, C ₆ arylene groups, C ₄ -C ₅ heteroarylene groups, C ₃ -C ₈ cycloalkylene groups, C ₅ -C ₈ cycloalkenylene groups, C ₅ -C ₁₂

alkyl(hetero)arylene groups, C ₅ -C ₁₂ (hetero)arylalkylene groups, C ₄ -C ₁₂ alkylcycloalkylene groups, C ₄ -C ₁₂ cycloalkylalkylene groups, wherein for S ^P the alkylene groups, alkenylene groups, alkynylene groups, (hetero)arylene groups, cycloalkylene groups, cycloalkenylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, alkylcycloalkylene groups,

cycloalkylalkylene groups, are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR’, -N(R ₃₇ ) ₂ , =O, =NR ₃₇ , -SR ₃₇ , and -Si(R ₃₇ )3, and optionally contain one or more heteroatoms selected from the group consisting of -O-, -S-, -NR ₃₇ -, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally

quaternized.

In preferred embodiments, S ^P comprises one or more moiety C ^M2 , C ^X or a residue of R ₃₂ , as described herein. In preferred embodiments, said C ^M2 , C ^X or a residue of R32 connects the S ^P to C ^B , C ^A , L ^C , T ^R , or another S ^P . Linker L ^C L ^C is an optional self-immolative linker, which may consist of multiple units arranged linearly and/or branched and may release one or more C ^A moieties. By way of further clarification, if r is 0 the species C ^A directly constitutes the leaving group of the release reaction, and if r > 0 , the self-immolative linker L ^C constitutes the leaving group of the release reaction. The possible L ^C structures, their use, position and ways of attachment of linkers L ^C , constructs C ^A and C ^B , and the T ^R are known to the skilled person, see for example [Papot et al., Anticancer Agents Med. Chem., 2008, 8, 618-637]. Nevertheless, preferred but non-limiting examples of self-immolative linkers L ^C are benzyl-derivatives, such as those drawn below. There are two main self-immolation mechanisms: electron cascade elimination and cyclization- mediated elimination. The preferred example below on the left functions by means of the cascade mechanism, wherein the bond between the allylic carbon of the Trigger and the -O- or -S- attached to said carbon is cleaved, and an electron pair of Y ^C1 , for example an electron pair of NR ⁶ , shifts into the benzyl moiety resulting in an electron cascade and the formation of 4- hydroxybenzyl alcohol, CO ₂ and the liberated C ^A . The preferred example in the middle functions by means of the cyclization mechanism, wherein cleavage of the bond to the NR ⁶ on the side of the Trigger leads to

nucleophilic attack of the amine on the carbonyl, forming a 5-ring 1,3- dimethylimidazolidin-2-one and liberating the C ^A . The preferred example on the right combines both mechanisms, this linker will degrade not only into CO ₂ and one unit of 4-hydroxybenzyl alcohol (when Y ^C1 is O), but also into one 1,3-dimethylimidazolidin-2-one unit.

wherein the wiggly line indicates a bond to -O- or -S- on the allylic position of the trans-cyclooctene, and the double dashed line indicates a bond to C ^A .

By substituting the benzyl groups of aforementioned self- immolative linkers L ^C , it is possible to tune the rate of release of the construct C ^A , caused by either steric and/or electronic effects on the cyclization and/or cascade release. Synthetic procedures to prepare such substituted benzyl-derivatives are known to the skilled person (see for example [Greenwald et al, J. Med. Chem., 1999, 42, 3657-3667] and

[Thornthwaite et al, Polym. Chem., 2011, 2, 773-790]. Some preferred substituted benzyl-derivatives with different release rates are drawn below.

Self-immolative linkers that undergo cyclization include but are not limited to substituted and unsubstituted aminobutyric acid amide, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring system, 2- aminophenylpropionic acid amides, and trimethyl lock-based linkers, see e.g. [Chem. Biol.1995, 2, 223], [J. Am. Chem. Soc.1972, 94, 5815], [J. Org. Chem.1990, 55, 5867], the contents of which are hereby incorporated by reference. Preferably, with an L ^C that releases C ^A by means of cyclization, the remainder of C ^A is bound to L ^C via an aromatic oxygen of sulfur. It will be understood that e.g. aromotic oxygen means an oxygen that is directly attached to an aromatic group.

Further preferred examples of L ^C can be found in WO ₂ 009017394(A1), US7375078, WO ₂ 015038426A1, WO2004043493, Angew. Chem. Int. Ed.2015, 54, 7492– 7509, the contents of which are hereby incorporated by reference.

In some aspects of the invention the L ^C has a mass of no more than 1000 daltons, no more than 500 daltons, no more than 400 daltons, no more than 300 daltons, or from 10, 50 or 100 to 1000 daltons, from 10, 50, 100 to 400 daltons, from 10, 50, 100 to 300 daltons, from 10, 50, 100 to 200 daltons, e.g., 10-1000 daltons, such as 50-500 daltons, such as 100 to 400 daltons.

A person skilled in the art will know that one L ^C may be connected to another L ^C that is bound to C ^A , wherein upon reaction of the Activator with the Trigger T ^R , L ^C -L ^C -C ^A is released from the T ^R , leading to self-immolative release of both L ^C moietes and the C ^A moiety. With respect to the L ^C formulas disclosed herein, the L ^C linking the T ^R to the other L ^C then does not release C ^A but an L ^C that is bound via Y ^C1 and further links to a C ^A .

In a preferred embodiment, L ^C is selected from the group consisting of linkers according to Group I, Group II, and Group III.

Linkers according to Group I are

, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein U, V, W, Z are each independently selected from the group consisting of -CR ⁷ -, and -N-; wherein e is either 0 or 1; wherein X is selected from the group consisting of -O-, -S- and -NR ⁶ - ;wherein preferably each R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, and C _4-6 (hetero)aryl groups; wherein for R ⁸ and R ⁹ the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, -SH, - SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ and -NO ₂ and optionally contain at most two

heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si- , wherein the N, S, and P atoms are optionally oxidized; wherein for linkers according to Group I C ^A is linked to L ^C via a moiety selected from the group consisting of -O-, -N-, -C-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of C ^A . Preferably, for linkers of Group I both R ⁸ and R ⁹ are hydrogen.

The linker according to Group II is

, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein m is an integer between 0 and 2, preferably m is 0; wherein e is either 0 or 1; wherein for linkers according to Group II C ^A is linked to L ^C via a moiety selected from the group consisting of -O-, -N-, -C-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of C ^A .

Preferably, for linkers of Group II both R ⁸ and R ⁹ are hydrogen. Preferably, for linkers of Group II R ⁷ is methyl or isopropyl.

Linkers according to Group III are

wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein for linkers according to Group III C ^A is linked to L ^C via a moiety selected from the group consisting of -O- and -S-, preferably -O- or -S- bound to a C _4-6 (hetero)aryl group, wherein said moieties are part of C ^A , wherein preferably each R ⁶ is independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, and C _4-6 (hetero)aryl groups, wherein for R ⁶ the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, - NH ₂ , =O, -SH, -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ and -NO ₂ and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, - P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein preferably each R ⁷ is independently selected from the group consisting of hydrogen and C ₁ -C ₃ alkyl groups, C ₂ -C ₃ alkenyl groups, and C ₄ -6 (hetero)aryl groups, wherein for R ⁷ the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, =NH, -N(CH ₃ )2, -S(O)2CH ₃ , and - SH, and are optionally interrupted by at most one heteroatom selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally

quaternized, wherein R ⁷ is preferably selected from the group consisting of hydrogen, methyl, -CH ₂ -CH ₂ -N(CH ₃ )2, and -CH ₂ -CH ₂ -S(O)2-CH ₃ .

Preferably, for linkers of Group III, R ⁶ is hydrogen. Preferably, for linkers of Group III, R ⁶ is methyl.

R ⁶ , R ⁷ , R ⁸ , R ⁹ comprised in said Group I, II and III, can optionally also be -(S ^P ) _i -C ^B .

For all linkers according to Group I and Group II Y ^C1 is selected from the group consisting of -O-, -S-, and -NR ⁶ -, preferably -NR ⁶ -. For all linkers according to Group III, Y ^C1 is -NR ⁶ -. For all linkers according to Group I, Group II, and Group III, Y ^C2 is selected from the group consisting of O and S, preferably O.

When two L ^C are linked to each other, then the L ^C attached to the -O- or -S- at the allylic position of the trans-cyclooctene is selected from the group consisting of linkers according to Group I and Group II, and the L ^C between the L ^C attached to the -O- or -S- at the allylic position of the trans- cyclooctene and C ^A is selected from Group III, and that the wiggly line in the structures of Group III then denotes a bond to the L ^C attached to the -O- or - S- at the allylic position of the trans-cyclooctene instead of a bond to the allylic -O- or -S- on the trans-cyclooctene ring, and that the double dashed line in the structures of Groups I and II then denotes a bond to the L ^C between the L ^C attached to the -O- or -S- at the allylic position of the trans- cyclooctene and the C ^A instead of a bond to C ^A .

In preferred embodiments, L ^C is selected from the group consisting of linkers according to Group IV, Group V, Group VI, and Group VII, wherein linkers according to Group IV are

, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein C ^A is linked to L ^C via a moiety selected from the group consisting of -O- and -S-, preferably from the group consisting of -O-C ₅ -8-arylene- and -S-C ₅ -8-arylene-, wherein said moieties are part of C ^A .

Linkers according to Group V are

, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein C ^A is linked to L ^C via a moiety selected from the group consisting of -O- and -S-, wherein said moieties are part of C ^A . In the first linker of Group V, R ⁷ is preferably–(CH ₂ ) ₂ -N(CH ₃ ) ₂ .

Linkers according to Group VI are

, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein C ^A is linked to L ^C via a moiety selected from the group consisting of -O-, -N-, and -S-, preferably a secondary or a tertiary amine, wherein said moieties are part of C ^A .

Linkers according to Group VII are

, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the trans-cyclooctene, wherein C ^A is linked to L ^C via a moiety selected from the group consisting of -O-, -N-, and -S-, preferably from the group consisting of secondary amines and tertiary amines, wherein said moieties are part of C ^A , wherein when multiple double dashed lines are shown within one L ^C , each C ^A moiety is independently selected.

For all linkers according to Group IV, Group V, Group VI, and Group VII, Y ^C1 is selected from the group consisting of -O-, -S-, and -NR ⁶ -.

For Groups IV-VII preferably R ⁶ and R ⁷ are as defined herein, more preferably as defined for Groups I-III. For Groups I-VII, i is an integer in a range of from 0 to 4, preferably 0 or 1, and wherein j is 0 or 1. Preferably, R ⁶ , R ⁷ , R ⁸ , R ⁹ used in any one of Groups I-VII are not substituted. R ⁶ , R ⁷ , R ⁸ , R ⁹ are as defined herein. Preferably, R ⁶ is hydrogen. Preferably, R ⁷ is hydrogen. Preferably, R ⁸ is hydrogen. Preferably, R ⁹ is hydrogen. Targeting The kits of the invention are very suitable for use in targeted imaging, targeted delivery of drugs and therapeutic radiation, and for selective biomolecule or tissue binding in vitro.

A "primary target" as used in the present invention preferably relates to a target for a targeting agent for therapy. In other embodiments it relates to a target for imaging theranostics, diagnostics, or in vitro studies. For example, a primary target can be any molecule, which is present in an organism, tissue or cell. Targets include cell surface targets, e.g. receptors, glycoproteins; structural proteins, e.g. amyloid plaques; abundant

extracellular targets such as stroma targets, tumor microenvironment targets, extracellular matrix targets such as growth factors, and proteases; intracellular targets, e.g. surfaces of Golgi bodies, surfaces of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways; and/or foreign bodies, e.g. pathogens such as viruses, bacteria, fungi, yeast or parts thereof. Examples of primary targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up regulated or down-regulated in a certain disorder. According to a particular embodiment of the present invention, the primary target is a protein such as a (internalizing or non- internalizing) receptor.

Furthermore, a preferred primary target is the blood, i.e. to prolong the blood circulation of a compound, and/or to reduce extravasation into other tissues. For example, the blood can be targeted by attaching a polyethylene glycol (PEG) to the compound. This typically increases blood circulation times. For example, by attaching a compound to a 500 nm PLGA particle, the conjugate will not readily extravaste from blood into other tissues (e.g. muscle), except for its rapid uptake and clearance through the liver and spleen. Organs, such as the liver, may also be primary targets. Hexoses can be attached to a compound to specifically target the liver, for example.

A further primary target is the immune system in general. According to the present invention, the primary target can be selected from any suitable targets within the human or animal body or on a pathogen or parasite, e.g. a group comprising cells such as cell membranes and cell walls, receptors such as cell membrane receptors, intracellular structures such as Golgi bodies or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral particles, antibodies, proteins, carbohydrates,

monosacharides, polysaccharides, cytokines, hormones, steroids,

somatostatin receptor, monoamine oxidase, muscarinic receptors,

myocardial sympatic nerve system, leukotriene receptors, e.g. on leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-)angiogenesis marker, gastrin receptor, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opoid receptors, GPIIb/IIIa receptor and other thrombus related receptors, fibrin, calcitonin receptor, tuftsin receptor, integrin receptor, fibronectin, VEGF/EGF and VEGF/EGF receptors, TAG72, CEA, A33, CD19, CD20,CD22, CD25, CD30, CD33, CD40, CD45, CD56, CD74, CD79, CD105, CD123, CD138, CD163, CD174, CD184, CD227, CD269, CD326, CD340, CD352, MUC ₁ , MUC ₁₆ , GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, CD44v6, G250, HLA DR, ED-A, ED-B, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix

metalloproteinase (MMP), ADAM-9, P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, herpes simplex virus tyrosine kinase, human tyrosine kinase, MSR1, FAP, CXCR, tumor endothelial marker (TEM), cMET, IGFR, FGFR, GPA33, hCG, HER2, HER3, CA19, TAM, LGALS3BP, nectin-4, IFGR, , PD1, PDL1, AGS-5, AGS-16, endosialin, ETBR, TM4SF1, BCMA, GPC2, TROP-2, AXL, HLA-DR, B7-H3, MTX3, MTX5, EFNA4, NOTCH, tissue factor (TF), PDGFR, GITR, OX40, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, NKG2A.

ccording to a further particular embodiment of the invention, the primary target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an

inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. This can be achieved by selecting primary targets with tissue-, cell- or disease- specific expression. For example, membrane folic acid receptors mediate intracellular accumulation of folate and its analogs, such as methotrexate. Expression is limited in normal tissues, but receptors are overexpressed in various tumor cell types.

In preferred embodiments the Primary Target equals a therapeutic target. It shall be understood that a therapeutic target is the entity that is targeted by the Drug to afford a therapeutic effect. Targeting Agents T ^T A Targeting Agent, T ^T , binds to a Primary Target. In order to allow specific targeting of the above-listed Primary Targets, the Targeting Agent T ^T can comprise compounds including but not limited to antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), proteins, peptides, e.g.

octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, cell penetrating peptide, membrane translocation moiety, bombesin, elastin, peptide mimetics, organic compounds, inorganic compounds, carbohydrates, monosaccharides, oligosacharides, polysaccharides, oligonucleotides, aptamers, viruses, whole cells, phage, drugs, polymers, liposomes, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, toxins. Examples of organic compounds envisaged within the context of the present invention are, or are derived from, dyes, compounds targeting CAIX and PSMA, estrogens, e.g. estradiol, androgens, progestins, corticosteroids, methotrexate, folic acid, and cholesterol.

According to a particular embodiment of the present invention, the Primary Target is a receptor and a Targeting Agent is employed, which is capable of specific binding to the Primary Target. Suitable Targeting Agents include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands. Other examples of Targeting Agents of protein nature include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, Affibody molecules such as for example ABY-025, Ankyrin repeat proteins, ankyrin-like repeat proteins, interferons, e.g. alpha, beta, and gamma interferon, interleukins, lymphokines, colony stimulating factors and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet-derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin. Alternative examples of targeting agents include DNA, RNA, PNA and LNA which are e.g. complementary to the Primary Target.

Examples of peptides as targeting agents include LHRH receptor targeting peptides, EC-1 peptide, RGD peptides, HER2-targeting peptides, PSMA targeting peptides, somatostatin-targeting peptides, bombesin. Other examples of targeting agents include lipocalins, such as anticalins. One particular embodiment uses Affibodies ^TM and multimers and derivatives.

In a preferred embodiment T ^T is an antibody. In a preferred

embodiment the T ^T is selected from antibodies and antibody derivatives such as antibody fragments, fragment fusions, proteins, peptides, peptide mimetics, organic molecules, dyes, fluoresencent molecules, enzyme substrates.

In a preferred embodiment the TT being an organic molecule has a molecular weight of less than 2000 Da, more preferably less than 1500 Da, more preferably less than 1000 Da, even more preferably less than 500 Da.

In another preferred embodiment the T ^T is selected from antibody fragments, fragment fusions, and other antibody derivatives that do not contain a Fc domain.

In another embodiment the T ^T is a polymer and accumulates at the Primary Target by virtue of the EPR effect. Typical polymers used in this embodiment include but are not limited to polyethyleneglycol (PEG), poly(N- (2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), polyvinylpyrrolidone (PVP), poly(1-hydroxymethylethylene hydroxymethyl-formal (PHF). Other examples are copolymers of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Other examples are oligopeptides, polypeptides, glycopolysaccharides, and polysaccharides such as dextran and hyaluronan.

Typically, a suitable polymer is polyethyleneglycol (PEG), preferably with a number of repeating units in a range of from 2 to 4000, and a molecular weight in a range of from 200 Da to 100,000 Da.

In addition reference is made to [G. Pasut, F.M. Veronese, Prog.

Polym. Sci.2007, 32, 933–961].

Other T ^T ' are nanoparticles, microparticles, liposomes, micelles, polymersomes, dendrimers, biomolecules, peptides, peptoids, proteins, carbohydrates, oligonucleotides, oligosaccharides, lipids, liposomes, albumin, albumin-binding moieties, dyes, fluoresencent molecules, and enzyme substrates.

In other embodiments, the T ^T is selected from amino acids,

nucleosides, nucleotides, carbohydrates, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, oligonucleotides, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units.

In some aspects of the invention polymeric T ^T moieties have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa.

Other exemplary T ^T Moieties are dendrimers, such as poly(propylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol based dendrimers.

In preferred embodiments the targeting agent T ^T localizes or has retention in a particular system, tissue, or organ in the body, for example, blood circulation, lymphatic system, the nervous system, the digestion system, RES system, or organs such as the heart or kidney. For example, microparticles will localize in the liver, large hydrophilic polymers will have retention in circulation. Likewise, use of an albumin binding moiety as T ^T will result in prolonged retention in circulation.

In preferred embodiments, T ^T is used to modify the pharmacokinetics of the moiety it is attached to. This can include, but is not limited to, delaying the blood clearance of said moiety, affecting the volume of distribution of said moiety (e.g. reducing or increasing the volume of distribution), affecting the metabolism of said moiety, and/or affecting (preferably avoiding) the sticking or uptake of said moiety to non-target tissues. Exemplary T ^T 's in this regard are polymer, peptide, peptoid, dendrimer, protein, carbohydrate, oligonucleotide, oligosaccharide, lipid, liposome, micelle, nanoparticle, microparticle, albumin, albumin-binding moiety, and small to medium sized organic molecules such as steroids and dyes. Typically, a suitable polymer is polyethyleneglycol (PEG) or

polypropyleneglycol (PPG).

According to a further particular embodiment of the invention, the Primary Target and Targeting Agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. This can be achieved by selecting Primary Targets with tissue-, cell- or disease- specific expression. For example, the CC ₄ 9 antibody targets TAG72, the expression of which is limited in normal tissues, but receptors are overexpressed in various solid tumor cell types.

In one embodiment the Targeting Agent specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Following specific binding or complexing of the T ^T with the receptor, the cell is permissive for uptake of the Prodrug, which then internalizes into the cell. The subsequently administered

Activator will then enter the cell and activate the Prodrug, releasing the Drug inside the cell. In another embodiment the Targeting Agent

specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given cell population. Following specific binding or complexing of the T ^T with the receptor, the cell is not permissive for uptake of the Prodrug. The subsequently administered Activator will then activate the Prodrug on the outside of the cell, after which the released Drug will enter the cell.

As used herein, a T ^T that "specifically binds or complexes with" or "targets" a cell surface molecule, an extracellular matrix target, or another target, preferentially associates with the target via intermolecular forces. For example, the ligand can preferentially associate with the target with a dissociation constant (Kd or KD) of less than about 50 nM, less than about 5 nM, or less than about 500 pM.

In some embodiments the T ^T can be a cell penetrating moiety, such as cell penetrating peptide. In a preferred embodiment T ^T can be non- functional that becomes functional upon reaction of the Trigger with the Activator. In a particularly preferred embodiment, said non-functional T ^T is a portion of a cell penetrating peptide that is bound to another portion of a cell penetrating peptide upon reaction of the Trigger with the Activator. In another preferred embodiment the T ^T , preferably a cell penetrating peptide is unmasked upon reaction of the Trigger with the Activator.

In other embodiments, the T ^T is a polymer, particle, gel, biomolecule or another above listed T ^T moiety and is locally injected to create a local depot of Prodrug or Activator, which can subsequently be reacted by with respectively the Activator or the Prodrug.

In another embodiment the targeting agent T ^T is a solid material such as but not limited to polymer, metal, ceramic, wherein this solid material is or is comprised in a cartridge, reservoir, depot, wherein preferably said cartridge, reservoir, depot is used for drug release in vivo.

In some embodiments, the targeting agent T ^T also acts as a Drug, denoted as D ^D . In a particularly preferred embodiment, the T ^T acts as a Drug D ^D by binding the primary target. In other preferred embodiments, the T ^T acts as a D ^D after the Trigger has been cleaved. It is preferred that when a T ^T is comprised in an embodiment of the invention, it equals C ^B . Masking moieties In order to avoid the drawbacks of current prodrug activation, such as low release yields and/or slow reactions, as discussed above, the IEDDA pyridazine elimination using the compounds of the invention can be used to provoke release of a Masking Moiety from a masked Drug. In this type of Prodrug, the Masking Moiety is attached to the Drug via a Trigger, and this Trigger is not activated endogeneously by e.g. an enzyme or a specific pH, but by a controlled administration of the Activator, i.e. a species that reacts with the Trigger moiety in the Prodrug, to induce release of the Masking Moiety or the Drug from the Trigger (or vice versa, release of the Trigger from the Masking Moiety or Drug, however one may view this release process), resulting in activation of the Drug.

The present invention provides a kit for the administration and activation of a Prodrug, the kit comprising a Masking Moiety, denoted as M ^M , linked covalently, directly or indirectly, to a Trigger moiety, which in turn is linked covalently, directly or indirectly, to a Drug, denoted as D ^D , and an Activator for the Trigger moiety, wherein the Trigger moiety comprises a dienophile satisfying Formula (19) and the Activator comprises a tetrazine satisfying any one of Formulae (4), (4a), or (6)-(14).

In another aspect, the invention presents a Prodrug comprising a Masking Moiety, M ^M , linked, directly or indirectly, to dienophile moiety satisfying above Formula (19).

In yet another aspect, the invention provides a method of modifying a Drug, D ^D , with one or more Masking Moieties M ^M affording a Prodrug that can be activated by an abiotic, bio-orthogonal reaction, the method comprising the steps of providing a Masking Moiety and a Drug and chemically linking the Masking Moiety and a Drug to a dienophile moiety satisfying Formula (19).

In a still further aspect, the invention provides a method of treatment wherein a patient suffering from a disease that can be modulated by a drug, is treated by administering, to said patient, a Prodrug comprising a Trigger moiety linked to a Masking Moiety M ^M and a Drug D ^D , after activation of which by administration of an Activator, satisfying any one of Formulae (4), (4a), or (6)-(14), the Masking Moiety will be released, activating the Drug, wherein the Trigger moiety comprises a dienophile structure satisfying Formula (19).

In a still further aspect, the invention is a compound comprising a dienophile moiety, said moiety comprising a linkage to a Masking Moiety M ^M , for use in prodrug therapy in an animal or a human being.

In another aspect, the invention is the use of a diene as an Activator for the release, in a physiological environment, of a substance covalently linked to a compound satisfying Formula (19). In connection herewith, the invention also pertains to a diene, for use as an Activator for the release, in a physiological environment, of a substance linked to a compound satisfying Formula (19), and to a method for activating, in a physiological

environment, the release of a substance linked to a compound satisfying Formula (19), wherein a tetrazine is used as an Activator.

In another aspect, the invention presents the use of the inverse electron-demand Diels-Alder reaction between a compound satisfying Formula (19) and a dienophile, preferably a trans-cyclooctene, as a chemical tool for the release, in a physiological environment, of a substance

administered in a covalently bound form, wherein the substance is bound to a compound satisfying Formula (19).

For the avoidance of doubt, in the context of this invention wherein a M ^M is removed from an antibody (i.e. Drug) the terms " activatable antibodies" and "Prodrug" mean the same.

For the avoidance of doubt, in the context of this invention wherein a M ^M is removed from a Drug, the Drug itself can optionally bind to one or more Primary Targets without the use of an additional Targeting Agent T ^T . In this context, the Primary target is preferably the therapeutic target.

In a preferred embodiment, the Drug comprises a Targeting Agent TT so that the Prodrug can bind a Primary Target. Following activation and M ^M removal the Drug then binds another Primary Target, which can be a therapeutic target.

In preferred embodiments, the Drug comprises one or more TT moieties, against one or different Primary Targets.

For the avoidance of doubt, in the context of the use of Masking Moieties, Primary target and therapeutic target are used interchangeably.

For the avoidance of doubt, one Drug construct can be modified by more than one Masking Moieties.

In preferred embodiments the activatable antibodies or Prodrugs of this invention are used in the treatment of cancer. In preferred

embodiments the activatable antibodies or Prodrugs of this invention are used in the treatment of an autoimmune disease or inflammatory disease such as rheumatoid arthritis. In preferred embodiments the activatable antibodies or Prodrugs of this invention are used in the treatment of a fibrotic disease such as idiopathic pulmonary fibrosis.

Exemplary classes of Primary Targets for activatable antibodies or Prodrugs of this invention include but are not limited to cell surface receptors and secreted proteins (e.g. growth factors), soluble enzymes, structural proteins (e.g. collagen, fibronectin) and the like. In preferred embodiments the Primary Target is an extracellular target. In preferred embodiments, the Primary Target is an intracellular target.

In another embodiment, the drug is a bi- or trispecific antibody derivative that serves to bind to tumor cells and recruit and activate immune effector cells (e.g. T-cells, NK cells), the immune effector cell binding function of which is masked and inactivated by being linked to a dienophile moiety as described above. The latter, again, serving to enable bio-orthogonal chemically activated drug activation.

When D ^D is C ^B it is preferred that D ^D is not attached to remainder of the Prodrug through its antigen-binding domain. Preferably D ^D is C ^A .

Masking moieties M ^M can for example be an antibody, protein, peptide, polymer, polyethylene glycol, polypropylene glycol carbohydrate, aptamers, oligopeptide, oligonucleotide, oligosaccharide, carbohydrate, as well as peptides, peptoids, steroids, organic molecule, or a combination thereof that further shield the bound drug D ^D or Prodrug. This shielding can be based on e.g. steric hindrance, but it can also be based on a non covalent interaction with the drug D ^D . Such Masking Moiety may also be used to affect the in vivo properties (e.g. blood clearance; biodistribution, recognition by the immune system) of the drug D ^D or Prodrug.

In preferred embodiments the Masking Moiety is an albumin binding moiety. In preferred embodiments, the Masking Moiety equals a Targeting Agent. In preferred embodiments , the Masking Moiety is bound to a

Targeting Agent. In a preferred embodiment the Drug D ^D , being C ^A , is modified with multiple M ^M , being C ^B _, wherein at least one of the bound M ^M is T ^T . In a preferred embodiment, when C ^A is D ^D then D ^D is not bound to T ^R via a Spacer S ^P .

In preferred embodiments the T ^R can itself act as a Masking Moiety, provided that C ^A is D ^D . For the sake of clarity, in these embodiments the size of the T ^R without the attachment of a M ^M is sufficient to shield the Drug D ^D from its Primary Target, which, in this context, is preferably the therapeutic target.

The M ^M of the modified D ^D can reduce the D ^D ’s ability to bind its target allosterically or sterically.

In specific embodiments, the M ^M is a peptide and does not comprise more than 50% amino acid sequence similarity to a natural protein-based binding partner of an antibody-based D ^D . In preferred embodiments M ^M is a peptide between 2 and 40 amino acids in length.

In one embodiment the M ^M reduces the ability of the D ^D to bind its target such that the dissociation constant of the D ^D when coupled to the M ^M towards the target is at least 100 times greater than the dissociation constant towards the target of the D ^D when not coupled to the M ^M . In another embodiment, the coupling of the M ^M to the D ^D reduces the ability of the D ^D to bind its target by at least 90%.

In preferred embodiments the M ^M in the masked D ^D reduces the ability of the D ^D to bind the target by at least 50 %, by at least 60 %, by at least 70 %, by at least 75 %, by at least 80 %, by at least 85 %, by at least 90 %, by at least 95 %, by at least 96 %, by at least 97 %, by at least 98 %, by at least 99 %, or by 100 %, as compared to the ability of the unmasked D ^D to bind the target. The reduction in the ability of a D ^D to bind the target can be determined , for example, by using an in vitro displacement assay, such as for example described for antibody D ^D in WO ₂ 009/025846 and

WO ₂ 010/081173.

In preferred embodiments the D ^D comprised in the masked D ^D is an antibody, which expressly includes full-length antibodies, antigen-binding fragments thereof, antibody derivatives antibody analogs, antibody mimics and fusions of antibodies or antibody derivatives.

In certain embodiments the M ^M is not a natural binding partner of the antibody. In preferred embodiments, the M ^M contains no or

substantially no homology to any natural binding partner of the antibody. In preferred embodiments the M ^M is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the antibody. In preferred embodiments the M ^M is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the antibody. In preferred embodiments, the M ^M is no more than 50% identical to any natural binding partner of the antibody. In preferred embodiments, the M ^M is no more than 25% identical to any natural binding partner of the antibody. In preferred embodiments, the M ^M is no more than 20% identical to any natural binding partner of the antibody. In preferred embodiments, the M ^M is no more than 10% identical to any natural binding partner of the antibody.

In the Prodrug, the M ^M and the Trigger T ^R - the dienophile derivative- can be directly linked to each other. They can also be bound to each other via a spacer S ^P or a self-immolative linker L ^C . It will be understood that the invention encompasses any conceivable manner in which the diene Trigger is attached to the M ^M . It will be understood that M ^M is linked to the dienophile in such a way that the M ^M is eventually capable of being released from the D ^D after formation of the IEDDA adduct. Generally, this means that the bond between the D ^D and the dienophile, or in the event of a self- immolative linker L ^C the bond between the L ^C and the dienophile and between the D ^D and the L ^C should be cleavable. Alternatively, this means that the bond between the M ^M and the dienophile, or in the event of a self- immolative linker L ^C the bond between the L ^C and the dienophile and between the M ^M and the L ^C should be cleavable.

In preferred embodiments, the antibody comprised in the masked antibody is a multi-antigen targeting antibody, comprising at least a first antibody or antigen-binding fragment or mimic thereof that binds a first Primary Target and a second antibody or antigen-binding fragment or mimic thereof that binds a second Primary Target. In preferred

embodiments, the antibody comprised in the masked antibody is a multi- antigen targeting antibody, comprising a first antibody or antigen-binding fragment or mimic thereof that binds a first Primary Target, a second antibody or antigen-binding fragment or mimic thereof that binds a second Primary Target, and a third antibody or antigen-binding fragment or mimic thereof that binds a third Primary Target. In preferred embodiments, the multi-antigen targeting antibodies bind two or more different Primary Targets. In preferred embodiments, the multi-antigen targeting antibodies bind two or more different epitopes on the same Primary Target. In preferred embodiments the multi-antigen targeting antibodies bind a combination of two or more different targets and two or more different epitopes on the same Primary Target. In preferred embodiments the masked multi-antigen targeting antibodies comprise one M ^M group, or two or more M ^M groups. It shall be understood that preferably at least one of the

Primary Targets is a therapeutic target.

In preferred embodiments of a multispecific activatable antibody, a scFv can be fused to the carboxyl terminus of the heavy chain of an IgG activatable antibody, to the carboxyl terminus of the light chain of an IgG activatable antibody, or to the carboxyl termini of both light and the heavy chain of an IgG activatable antibody. In preferred embodiments of a multispecific activatable antibody, a scFv can be fused to the amino terminus of the heavy chain of an IgG activatable antibody, to the amino terminus of the light chain of an IgG activatable antibody, or to the amino termini of both light and the heavy chain of an IgG activatable antibody. In preferred embodiments of a multispecific activatable antibody, a scFv can be fused to any combination of one or more carboxyl termini and one or more amino termini of an IgG activatable antibody. Methods of preparing multispecific antibodies are known to the person skilled in the art. In addition reference is made to [Weilde et al., Cancer Genomics & Proteomics 2013, 10, 1-18], [Weidle et al., Seminars in Oncology 2014, 41, 5, 653-660], [Jachimowicz et al., BioDrugs (2014) 28:331–343], the contents of which are hereby incorporated by reference.

In preferred embodiments, a M ^M linked to a T ^R is attached to and masks an antigen binding domain of the IgG. In preferred embodiments, a M ^M linked to a T ^R is attached to and masks an antigen binding domain of at least one scFv. In preferred embodiments, a M ^M linked to a T ^R is attached to and masks an antigen binding domain of the IgG and a M ^M linked to a T ^R is attached to and masks an antigen binding domain of at least one scFv.

In preferred embodiments, the M ^M has a dissociation constant, i.e., dissociation constant at an equilibrium state, K _d , for binding to the antibody that is greater than the Kd for binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has a Kd for binding to the antibody that is approximately equal to the K _d for binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has a Kd for binding to the antibody that is less than the K _d for binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has a K _d for binding to the antibody that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold greater than the K _d for binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has a Kd for binding to the antibody that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20- 1,000, or 100-1,000 fold greater than the K _d for binding of the antibody to its Primary Target.

In preferred embodiments, the M ^M has an affinity for binding to the antibody that is greater than the affinity of binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has an affinity for binding to the antibody that is approximately equal to the affinity of binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has an affinity for binding to the antibody that is less than the affinity of binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has an affinity for binding to the antibody that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold less than the affinity of binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has an affinity of binding to the antibody that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1,000, or 100-1,000 fold less than the affinity of binding of the antibody to its Primary Target. In preferred embodiments, the M ^M has an affinity of binding to the antibody that is 2 to 20 fold less than the affinity of binding of the antibody to its Primary Target.

In preferred embodiments, a M ^M not covalently linked to the antibody and at equimolar concentration to the antibody does not inhibit the binding of the antibody to its Primary Target. In preferred embodiments, the M ^M does not interfere of compete with the antibody for binding to the Primary Target when the Prodrug is in a cleaved state.

In preferred embodiments, the antibody has a dissociation constant of about 100 nM or less for binding to its Primary Target. In preferred embodiments, the antibody has a dissociation constant of about 10 nM or less for binding to its Primary Target. In preferred embodiments, the antibody has a dissociation constant of about 1 nM or less for binding to its Primary Target. In preferred embodiments, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the dissociation constant (K _d ) of the antibody when coupled to the M ^M towards its Primary Target is at least 20 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the Kd of the antibody when coupled to the M ^M towards its Primary Target is at least 40 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the K _d of the antibody when coupled to the M ^M towards its Primary Target is at least 100 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the Kd of the antibody when coupled to the M ^M towards its Primary Target is at least 1,000 times greater than the K _d of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the Kd of the antibody when coupled to the M ^M towards its Primary Target is at least 10,000 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, for example when using a non-binding steric M ^M as defined below, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the K _d of the antibody when coupled to the M ^M towards its Primary Target is at least 100,000 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, for example when using a non-binding steric M ^M as defined below, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the K _d of the antibody when coupled to the M ^M towards its Primary Target is at least 1,000,000 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target. In preferred embodiments, for example when using a non-binding steric M ^M as defined below, the coupling of the M ^M reduces the ability of the antibody to bind its Primary Target such that the Kd of the antibody when coupled to the M ^M towards its Primary Target is at least 10,000,000 times greater than the Kd of the antibody when not coupled to the M ^M towards its Primary Target.

Exemplary drugs that can be used in a Prodrug relevant to this invention using Masking Moieties include but are not limited to: antibodies, antibody derivatives, antibody fragments, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, carbohydrates, as well as peptides, peptoids, steroids, toxins, hormones, viruses, whole cells, phage. In preferred embodiments the drugs are low to medium molecular weight compounds, preferably organic compounds (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da, more preferably about 300 to about 1000 Da).

In one embodiment antibodies are used as the Drug. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this invention, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulins is of the class IgG including but not limited to IgG subclasses (IgG1, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as Fv, VHH, Fab, F(ab) ₂ , Fab', Fab'-SH, F(ab') ₂ , single chain variable fragment antibodies (scFv), tandem/bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibody (sdAb, also known as Nanobody ^TM ), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual- affinity retargeting proteins (DART ^TM ), and multimers and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri-scFvs) including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol.2012, 30, 575–582], and [Canc. Gen. Prot. 201310, 1-18], and [BioDrugs 2014, 28, 331–343], the contents of which is hereby incorporated by reference. Other embodiments use antibody mimetics as Drug, such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to

[Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region. Multimers may be linearly linked or may be branched and may be derived from a single vector or chemically connected, or non-covalently connected. Methods of making above listed constructs are known in the art. For the avoidance of doubt, in the context of this invention the term "antibody" is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.

Typical drugs for which the invention is suitable include, but are not limited to: proteins, peptides, oligosacharides, oligonucleotides,

monospecific, bispecific and trispecific antibodies and antibody fragment or protein fusions, preferably bispecific and trispecific. In preferred

embodiments the activatable antibody or derivative is formulated as part of a pro-Bispecific T Cell Engager (BITE) molecule.

Other embodiments use immunotoxins, which are a fusion or a conjugate between a toxin and an antibody. Typical toxins comprised in an immunotoxins are cholera toxin, ricin A, gelonin, saporin, bouganin, ricin, abrin, diphtheria toxin, Staphylococcal enterotoxin, Bacillus Cyt2Aa1 toxin, Pseudomonas exotoxin PE38, Pseudomonas exotoxin PE38KDEL, granule- associated serine protease granzyme B, human ribonucleases (RNase), or other pro-apoptotic human proteins. Other exemplary cytotoxic human proteins which may be incorporated into fusion constructs are caspase 3, caspase 6, and BH3-interacting domain death agonist (BID). Current immunotoxins have immunogenicity issues and toxicity issues, especially towards vascular endothelial cells. Masking the targeted toxin by a M ^M such as a PEG or peptide and removing the M ^M once the masked immunotoxin has bound to its target is expected to greatly reduce the toxicity and immunogenicity problems.

Other embodiments use immunocytokines, which are a fusion or a conjugate between a cytokine and an antibody. Typical cytokines used in cancer therapy include IL-2, IL-7, IL-12, IL-15, IL-21, TNF. A typical cytokine used in autoimmune diseases is the anti-inflammatory IL-10.

Masking the targeted cytokine by a M ^M such as a PEG or peptide and removing the M ^M once the masked immunocytokine has bound to its target is expected to greatly reduce the toxicity problems. Other embodiments use small to medium sized organic drugs.

In preferred embodiments the unmasked Drug is multispecific and binds to two or more same or different Primary Targets. In preferred embodiments the multispecific Drug comprises one or more (masked) antibodies (also referred to as binding moieties) that are designed to engage immune effector cells. In preferred embodiments the masked multispecific Prodrug comprises one or more (masked) antibodies that are designed to engage leukocytes. In preferred embodiments the masked multispecific Prodrug comprises one or more (masked) antibodies that are designed to engage T cells. In preferred embodiments the masked multispecific Prodrug comprises one or more (masked) antibodies that engage a surface antigen on a leukocyte such as on a T cell, natural killer (NK) cell, a myeloid

mononuclear cell, a macrophage and/or another immune effector cell. In preferred embodiments the immune effector cell is a leukocyte, a T cell, a NK cell, or a mononuclear cell.

In an exemplary multispecific masked Prodrug the Prodrug comprises an antibody (i.e. Targeting Agent) for a cancer receptor, e.g. TAG72, a antibody for CD3 on T cells, and an antibody for CD28 on T cells, wherein either the antibody for CD3 or for CD28 or both is masked by a M ^M . Another example is an activatable antibody that comprises an antibody for a cancer receptor, and an antibody for CD3 on T cells, wherein the antibody for CD3 is masked by a M ^M . Another example is a Prodrug that has an antibody for a cancer receptor, and an antibody for CD28 on T cells, wherein the antibody for CD28 is masked by a M ^M . Another example is a Prodrug that has an antibody for a cancer receptor, and an antibody for CD16a on NK cells, wherein the antibody for CD16a is masked by a M ^M . In yet another embodiment the unmasked Drug binds two different immune cells and optionally in addition a tumor cell. Said multispecific antibody derivatives can for example be prepared by fusing or conjugating antibodies, antibody fragments such as Fab, Fabs, scFv, camel antibody heavy chain fragments and proteins.

In some preferred embodiments the M ^M reduces the binding of the Drug to Primary Targets, equaling therapeutic targets, selected from CD3, CD28, PD-L1, PD-1, LAG-3, TIGIT, TIM-3, B7H4, Vista, CTLA-4 polysialic acids and corresponding lectins. In other preferred embodiments the M ^M masks a T-cell agonist, an NK cell agonist, an DC cell agonist.

In preferred embodiments of an immune effector cell engaging masked multispecific Prodrug such as a T-cell engaging multispecific activatable antibody, at least one antibody comprised in the Prodrug is a Targeting Agent and binds a Primary Target that is typically an antigen present on the surface of a tumor cell or other cell type associated with disease, such as, but not limited to, EGFR, erbB2, EpCAM, PD-L1, B7H3 or CD71 (transferrin receptor), and at least one other antibody comprised in the Prodrug binds Primary Target that is typically a stimulatory or inhibitory antigen present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and/or other immune effector cell, such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3 or VISTA. In preferred embodiments it is preferred that the targeted CD3 antigen is CD3e or CD3 epsilon.

One embodiment of the disclosure is a multispecific activatable antibody that includes an antibody Targeting Agent directed to a tumor target and another agonist antibody , the Drug, directed to a co-stimulatory receptor expressed on the surface of an activated T cell or NK cell, wherein the agonist antibody is masked. Examples of co-stimulatory receptors include but are not limited to CD27, CD137, GITR, HVEM, NKG2D, OX40. In this embodiment, once the Prodrug is tumor-bound and activated it would effectively crosslink and activate the T cell or NK cell expressed co- stimulatory receptors in a tumor dependent manner to enhance the activity of T cell or NK cells that are responding to any tumor antigen via their endogenous T cell or NK cell activating receptors. The activation dependent nature of these T cell or NK cell co-stimulatory receptors would focus the activity of the activated multispecific Prodrug to tumor specific T cells without activating all T cells independent of their antigen specificity.

One embodiment of the disclosure is a multispecific activatable antibody targeted to a disease characterized by T cell overstimulation, such as, but not limited to, an autoimmune disease or inflammatory disease microenvironment. Such a Prodrug includes an antibody, for example a IgG or scFv, directed to a target comprising a surface antigen expressed in a tissue targeted by a T cell in autoimmune or inflammatory disease and an antibody, for example IgG or scFv, directed to an inhibitory receptor expressed on the surface of a T cell or NK cell, wherein the T cell or NK cell inhibitory antibody is masked. Examples of inhibitory receptors include but are not limited to BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK- expressed KIRs. Examples of a tissue antigen targeted by T cells in autoimmune disease include but are not limited to a surface antigen expressed on myelin or nerve cells in multiple sclerosis or a surface antigen expressed on pancreatic islet cells in Type 1 diabetes. In this embodiment, the Prodrug localizes at the tissue under autoimmune attack or

inflammation, is activated by the Activator and co-engages the T-cell or NK cell inhibitory receptor to suppress the activity of autoreactive T cells responding to any disease tissue targeted antigens via their endogenous TCR or activating receptors.

Other non-limiting exemplary Primary Targets for the binding moieties comprised in Drugs of this invention are listed in the patent WO ₂ 015/013671, the contents of which are hereby incorporated by

reference.

In another embodiment, the Drug is a masked vaccine, which can be unmasked at a desired time and/or selected location in the body, for example subcutaneously and/or in the proximity of lymph nodes. In another embodiment, the Drug is a masked antigen, e.g. a masked peptide, which optionally is present in a Major Histocompatibility Complex (MHC) and which can be unmasked at a desired time and/or selected location in the body, for example subcutaneously and/or in the proximity of lymph nodes.

The Prodrug may further comprise another linked drug, which is released upon target binding, either by proteases, pH, thiols, or by

catabolism. Examples are provided in the review on Antibody-drug conjugates in [Polakis, Pharmacol. Rev.2016, 68, 3-19]. The invention further contemplates that the Prodrug can induce antibody-dependent cellular toxicity (ADCC) or complement dependent cytotoxicity (CDC) upon unmasking of one or more moieties of the Prodrug. The invention also contemplates that the Prodrug can induce antibody-dependent cellular toxicity (ADCC) or complement dependent cytotoxicity (CDC) independent of unmasking of one or more moieties of the Prodrug.

Some embodiments use as said additional drug

antiproliferative/antitumor agents, antibiotics, cytokines, anti-inflammatory agents, anti-viral agents, antihypertensive agents, chemosensitizing, radiosensitizing agents, DNA damaging agents, anti-metabolites, natural products and their analogs. It is preferred that the Drug is a protein or an antibody. Drugs Drugs (D ^D ) that can be used in a Prodrug or a Drug that can be deactivated relevant to this invention are pharmaceutically active compounds. In a preferred embodiment the pharmaceutically active compound is selected from the group consisting of cytotoxins, antiproliferative/antitumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti-angiogenic factors, enzyme inhibitors.

In preferred embodiments these pharmaceutically active compounds are selected from the group consisting of antibodies, antibody derivatives, antibody fragments, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, carbohydrates, as well as peptides, peptoids, steroids, toxins, hormones, cytokines, chemokines.

In preferred embodiments these drugs are low to medium molecular weight compounds, preferably organic compounds (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da, more preferably about 300 to about 1000 Da).

Exemplary cytotoxic drug types for use as conjugates to the TCO and to be released upon IEDDA reaction with the Activator, for example for use in cancer therapy, include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors.

Examples inlude but are not limited to colchinine, vinca alkaloids, anthracyclines (e.g. doxorubicin, epirubicin, idarubicin, daunorubicin), camptothecins, taxanes, taxols, vinblastine, vincristine, vindesine, calicheamycins, tubulysins, tubulysin M, cryptophycins, methotrexate, methopterin, aminopterin, dichloromethotrexate, irinotecans, enediynes, amanitins, deBouganin, dactinomycines, CC ₁ 065 and its analogs,

duocarmycins, maytansines, maytansinoids, dolastatins, auristatins, pyrrolobenzodiazepines and dimers (PBDs), indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycins (e.g. mitomycin C, mitomycin A, caminomycin), melphalan, leurosine, leurosideine,

actinomycin, tallysomycin, lexitropsins, bleomycins, podophyllotoxins, etoposide, etoposide phosphate, staurosporin, esperamicin, the pteridine family of drugs, SN-38 and its analogs, platinum-based drugs, cytotoxic nucleosides.

Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, and RNA polymerase inhibitors.

Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylene diamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ. Suitable auristatins are also described in U.S. Publication Nos.2003/0083263, 2011/0020343, and 2011/0070248; PCT Application Publication Nos. WO09/117531,

WO ₂ 005/081711, WO04/010957; WO02/088172 and WO01/24763, and U.S. Patent Nos.7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431;

6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety.

Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No.4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No.4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat No.4,986,988), dolastatin 14 (U.S. Pat No.5,138,036), dolastatin 15 (U.S. Pat No.4,879,278), dolastatin 16 (U.S. Pat No.6,239,104), dolastatin 17 (U.S. Pat No..6,239,104), and dolastatin 18 (U.S. Pat No..6,239,104), each patent incorporated herein by reference in their entirety.

Exemplary maytansines, maytansinoids, such as DM-1 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Patent Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441,163; 6,716,821 and 7,276,497. Other examples include mertansine and ansamitocin.

Pyrrolobenzodiazepines (PBDs), which expressly include dimers and analogs, include but are not limited to those described in [Denny, Exp. Opin. Ther. Patents, 10(4):459-474 (2000)], [Hartley et al., Expert Opin Investig Drugs.2011, 20(6):733-44], Antonow et al., Chem Rev.2011, 111(4), 2815- 64].Exemplary indolinobenzodiazepines are described in literature.

Exemplary pyridinobenzodiazepines are described in literature.

Calicheamicins include, e.g. enediynes, esperamicin, and those described in U.S. Patent Nos.5,714,586 and 5,739,116.

Examples of duocarmycins and analogs include CC ₁ 065,

duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C ₁ , duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin, CPI, CBI. Other examples include those described in, for example, US Patent No.5,070,092; 5,101,092; 5,187,186; 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,548,530; 6,586,618; 6,660,742; 6,756,397; 7,049,316; 7,553,816; 8,815,226; US20150104407;

61/988,011 filed may 2, 2014 and 62/010,972 filed June 11, 2014; the disclosure of each of which is incorporated herein in its entirety.

Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Publication Nos.2002/0103136 and 2010/0305149, and in U.S. Patent No.7,303,749, the disclosures of which are incorporated herein by reference in their entirety. Exemplary epothilone compounds include epothilone A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos.

6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO97/19086; WO98/08849; WO98/22461; WO98/25929; WO98/38192; WO99/01124; WO99/02514; WO99/03848; WO99/07692;

WO99/27890; and WO99/28324; the disclosures of which are incorporated herein by reference in their entirety.

Exemplary cryptophycin compounds are described in U.S. Patent Nos. 6,680,311 and 6,747,021; the disclosures of which are incorporated herein by reference in their entirety. Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin.

Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines and the like. Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones and cryptophycins. Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11, SN- 38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflometotecan, belotecan, lurtotecan and S39625. Other camptothecin compounds that can be used in the present invention include those described in, for example, J. Med. Chem., 29:2358- 2363 (1986); J. Med. Chem., 23:554 (1980); J. Med Chem., 30:1774 (1987). Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure. Exemplary cell cycle progression inhibitors include CDK

inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI 2536, BI6727, GSK461364, ON-01910; and KSP inhibitors such as, for example, SB 743921, SB 715992, MK-0731, AZD8477, AZ3146 and ARRY-520. Exemplary P13K/m-TOR/AKT signalling pathway inhibitors include phosphoinositide 3-kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors. Exemplary P13 kinases are disclosed in U.S. Patent No.6,608,053, and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615,

IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866,

SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765.

Exemplary AKT inhibitors include, but are not limited to AT7867.

Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B- Raf and p38 MAPK inhibitors. Exemplary MEK inhibitors are disclosed in U.S. Patent No.7,517,944 and include GDC-0973, GSK1120212,

MSC ₁ 936369B, AS703026, RO5126766 and RO ₄ 987655, PD0325901, AZD6244, AZD8330 and GDC-0973. Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885. Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190. Exemplary receptor tyrosine kinases inhibitors include but are not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), Gefitinib (Iressa), AP24534 (Ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (Dovitinib), Sunitinib (Sutent), Sorafenib (Nexavar), and Vatalinib. Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW- 2478. Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC ₁ 568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3- aminobenzamide, A-966492, and AZD2461. Exemplary Wnt/Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV- 939. Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and

proamanullin. Exemplary immunemodulators are APRIL, cytokines, including IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, TNF, interferon gamma, GMCSF, NDV-GMCSF, and agonists and antagonists of STING, agonists and antagonists of TLRs including TLR1/2, TLR3, TLR4 , TLR7/8, TLR9, TLR12, agonists and antagonists of GITR, CD3, CD28, CD40, CD74,

CTLA4, OX40, PD1, PDL1, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A. Other exemplary drugs include

puromycins, topetecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cemadotin, discodermolide, eleutherobin, mitoxantrone, pyrrolobenzimidazoles (PBI), gamma-interferon, Thialanostatin (A) and analogs, CDK11, immunotoxins, comprising e.g. ricin A, diphtheria toxin, cholera toxin.

In exemplary embodiments of the invention, the drug moiety is a mytomycin compound, a vinca alkaloid compound, taxol or an analogue, an anthracycline compound, a calicheamicin compound, a maytansinoid compound, an auristatin compound, a duocarmycin compound, SN38 or an analogue, a pyrrolobenzodiazepine compound, a indolinobenzodiazepine compound, a pyridinobenzodiazepine compound, a tubulysin compound, a non-natural camptothecin compound, a DNA binding drug, a kinase inhibitor, a MEK inhibitor, a KSP inhibitor, a P13 kinase inhibitor, a topoisomerase inhibitor, or analogues thereof.

In one preferred embodiment the drug is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor, (e.g. P13 kinase inhibitor: GDC- 0941 and PI-103), MEK inhibitor, KSP inhibitor, RNA polymerase inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, dolastatin,

calicheamicins, SN38, pyrrolobenzodiazepines, pyridinobenzodiazepines , indolinobenzodiazepines, DNA binding drugs, maytansinoids DM1 and DM4, auristatin MMAE, CC ₁ 065 and its analogs, camptothecin and its analogs, SN-38 and its analogs.

In another preferred embodiment the drug is selected from DNA binding drugs and microtubule agents, including pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines, maytansinoids, maytansines, auristatins, tubulysins, duocarmycins, anthracyclines, taxanes. In another preferred embodiment the drug is selected from colchinine, vinca alkaloids, tubulysins, irinotecans, an inhibitory peptide, amanitin and deBouganin.

In another preferred embodiment the drug is a radioactive moiety, said moiety comprising a radioactive isotope for radiation therapy. A radionuclide used for therapy is preferably an isotope selected from the group consisting of ²⁴ Na, ³² P, ³³ P, ⁴⁷ Sc, ⁵⁹ Fe, ⁶⁷ Cu, ⁷⁶ As, ⁷⁷ As, ⁸⁰ Br, ⁸² Br, ⁸⁹ Sr, ⁹⁰ Nb, ⁹⁰ Y, ¹⁰³ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹²¹ Sn, ¹²⁷ Te, ¹³¹ I, ¹⁴⁰ La, ¹⁴¹ Ce, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁴ Pr, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷² Tm, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹¹ Bi, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²¹⁴ Bi, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac and ²²⁷ Th.

When the radioactive moiety is intended to comprise a metal, such as ¹⁷⁷ Lu, such radiometal is preferably provided in the form of a chelate. In such a case the radioactive moiety preferably comprises a structural moiety capable of forming a coordination complex with such a metal. A good example hereof are macrocylic lanthanide(III) chelates derived from

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H4dota). In a preferred embodiment, the moiety is a chelating moiety selected from the group consisting of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), TETA (1,4,8,11- tetraazacyclotetradecane-N,N',N",N'-tetraacetic acid), OTTA (N1-(p- isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraac etic acid), deferoxamine or DFO (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4- dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N- hydroxybutanediamide), and HYNIC (hydrazinonicotinamide), DOTAM, TACN, sarcophagine, 3,4-HOPO-based chelators.

In other embodiments the radioactive moiety comprises a prostethic group (i.e. a phenol) that is bound by a non-metal radionuclide, such as ¹³¹ I.

In another embodiment, a combination of two or more different drugs are used. In preferred embodiments the released Drug is itself a prodrug designed to release a further drug. In preferred embodiments the released Drug is itself a prodrug designed to be coupled to another moiety, (such as another prodrug) to form an active Drug. In preferred embodiments the Drug has increased therapeutic efficacy after reaction of the Trigger with the Activator. In some embodiments the Drug has reduced therapeutic efficacy after reaction of the Trigger with the Activator.

Drugs optionally include a (portion of a) membrane translocation moiety (e.g. adamantine, poly-lysine/arginine, TAT, human lactoferrin) and/or a targeting agent (against e.g. a tumor cell receptor) optionally linked through a stable or labile linker. Exemplary references include: Trends in Biochemical Sciences, 2015,.40, 12, 749; J. Am. Chem. Soc.2015, 137, 12153-12160; Pharmaceutical Research, 2007, 24, 11, 1977.

It will further be understood that, in addition to one or more targeting agents (or C ^B ) that may be attached to the Trigger or Linker L ^C a targeting agent T ^T may optionally be attached to a drug, optionally via a spacer S ^P . In some embodiments, the targeting efficacy of said T ^T attached to a drug is increased after reaction of the Trigger with an Activator, wherein preferably the Drug is C ^B , wherein preferably the Activator comprises a T ^T , wherein optionally the targeting efficacy of said T ^T comprised in the Activator is increased after reaction of the Trigger with an Activator.

Alternatively, it will be further understood that the targeting agent (or C ^B ) may comprise one or more additional drugs which are bound to the targeting agent by other types of linkers, e.g. cleavable by proteases, pH, thiols, or by catabolism.

The invention further contemplates that when a targeting agent is a suitably chosen antibody or antibody derivative that such targeting agent can induce antibody-dependent cellular toxicity (ADCC) or complement dependent cytotoxicity (CDC).

Several drugs may comprise or be replaced by an imageable label to measure drug targeting and release.

It will be understood that chemical modifications may also be made to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.

Drugs containing an amine functional group for coupling to the TCO include mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetyl spermidine, 1-(2 chloroethyl)1,2-dimethanesulfonyl hydrazide, tallysomycin, cytarabine, dolastatins (including auristatins) and derivatives thereof.

Drugs containing a hydroxyl function group for coupling to the TCO include etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy- bicyclo[7.3.1]trideca-4-9-diene-2,6-diyne-13-one (U.S. Pat No.5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine- doxorubicin, n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof.

Drugs containing a sulfhydryl functional group for coupling to the TCO include esperamicin and 6-mecaptopurine, and derivatives thereof. It will be understood that the drugs can optionally be attached to the TCO derivative through a self-immolative linker L ^C , or a combination thereof, and which may consist of multiple (self-immolative, or non immolative) units.

According to a further particular embodiment of the invention, the Prodrug is selected so as to target and or address a disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.

In the Prodrug, the Construct-A and the TCO derivative can be directly linked to each other. They can also be bound to each other via a linker or a self-immolative linker L ^C . It will be understood that the invention encompasses any conceivable manner in which the dienophile TCO is attached to the Contruct-A. In preferred embodiments Construct-A is a Drug. Methods of affecting conjugation to these drugs, e.g. through reactive amino acids such as lysine or cysteine in the case of proteins, are known to the skilled person. Label

The compound of Formula (19) preferably comprises a Label that is capable of providing the desired diagnostic, imaging, and/or radiotherapeutic effect.

In a preferred embodiment, the Label is selected from the group consisting of MRI-imageable constructs, moieties comprising a spin label, moieties comprising an optical label, ultrasound-responsive constructs, X- ray-responsive moieties, moieties comprising a radionuclide, fluorescent dyes, luminescent dyes, FRET dyes, paramagnetic ions, superparamagnetic particles, and peptides.

Especially for imaging applications, it is preferred that the Label is a detectable label. A "detectable label" as used herein relates to the part of the compound of Formula (19) which allows detection of the compound of Formula (19) when present in a cell, tissue or organism. One type of detectable label envisaged within the context of the present invention is a contrast providing label. Different types of detectable labels are envisaged within the context of the present invention and are described hereinbelow.

Thus, according to a particular embodiment of the present invention, the compounds, combinations, kits, and methods of the present invention are used in imaging, especially medical imaging. In order to identify the Primary Target and/or to evaluate the biodistribution of the compound of Formula (19), use is made of a detectable Label.

Preferred detectable labels for imaging are contrast-providing moieties used in traditional imaging systems such as MRI-imageable constructs, moieties comprising spin labels, moieties comprising optical labels, ultrasound-responsive constructs, X-ray-responsive moieties, moieties comprising radionuclides, (bio)luminescent and FRET-type dyes.

Furthermore, preferred detectable labels envisaged within the context of the present invention include, and are not necessarily limited to, fluorescent molecules, e.g. autofluorescent molecules, molecules that fluoresce upon contact with a reagent, radioactive moieties or complexes; biotin, e.g., to be detected through binding of biotin by avidin; fluorescent tags, imaging constructs for MRI comprising a paramagnetic metal, imaging reagents, e.g., those described in U.S. Pat. Nos.4,741,900 and 5,326,856) and the like.

Preferably, the radionuclide comprised in a Label for imaging is an isotope selected from the group consisting of ³ H, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ¹⁹ F, ⁴⁴ Sc, ⁵¹ Cr, ⁵² Fe, ⁵² Mn, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Zn, ⁶² Cu, ⁶³ Zn, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁰ As, ⁷¹ As, ⁷² As, ⁷⁴ As, ⁷⁵ Se, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^8O Br, ⁸² Br, ⁸² Rb, ⁸⁶ Y, ⁸⁸ Y, ⁸⁹ Sr, ⁸⁹ Zr, ⁹⁷ Ru, ^99m Tc, ¹¹⁰ In, ¹¹¹ In, ¹¹³ In, ¹¹⁴ In, ¹¹⁷ Sn, ¹²⁰ I, ¹²² Xe, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁹³ Pt, ¹⁹⁵ Pt, ²⁰¹ Tl, and ²⁰³ Pb. More preferably, the radionuclide comprised in a Label for imaging is an isotope selected from the group consisting of ¹⁸ F, ⁴⁴ Sc, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹²³ I, ¹²⁴ I. Other elements and isotopes, such as being used for therapy may also be applied for imaging in certain applications.

In preferred embodiments, the MRI-imageable moiety is a

paramagnetic ion or a superparamagnetic particle. The paramagnetic ion preferably is an element selected from the group consisting of Gd, Fe, Mn, Cr, Co, Ni, Cu, Pr, Nd, Yb, Tb, Dy, Ho, Er, Sm, Eu, Ti, Pa, La, Sc, V, Mo, Ru, Ce, Dy, Tl. The ultrasound responsive moiety can comprise a microbubble, the shell of which consisting of a phospholipid, and/or (biodegradable) polymer, and/or human serum albumin. The microbubble can be filled with fluorinated gasses or liquids.

The X-ray-responsive moieties include but are not limited to iodine, barium, barium sulfate, gastrografin or can comprise a vesicle, liposome or polymer capsule filled with iodine compounds and/or barium sulfate.

Moreover, detectable labels envisaged within the context of the present invention also include peptides or polypeptides that can be detected by antibody binding, e.g., by binding of a detectable labeled antibody or by detection of bound antibody through a sandwich-type assay. In one embodiment the detectable labels comprise small size organic PET and SPECT radioisotopes, such as ¹⁸ F, ¹¹ C , ¹²³ I or ¹²⁴ I. Due to their small size, organic PET or SPECT radioisotopes are ideally suited for monitoring intracellular events as they do not greatly affect the properties of the

Targeting Agent in general and its membrane transport in particular.

In preferred embodiments, especially when the compound of Formula (19) is used in therapeutic applications, the Label is a therapeutic Label, said Label comprising a radioactive isotope for radiation therapy. A radionuclide used for therapy is preferably an isotope selected from the group consisting of ²⁴ Na, ³² P, ³³ P, ⁴⁷ Sc, ⁵⁹ Fe, ⁶⁷ Cu, ⁷⁶ As, ⁷⁷ As, ⁸⁰ Br, ⁸² Br, ⁸⁹ Sr, ⁹⁰ Nb, ⁹⁰ Y, ¹⁰³ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹²¹ Sn, ¹²⁷ Te, ¹³¹ I, ¹⁴⁰ La, ¹⁴¹ Ce, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁴ Pr, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷² Tm, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹¹ Bi, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²¹⁴ Bi, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac and ²²⁷ Th. More

preferably, the radionuclide comprised in a Label for therapy is an isotope selected from the group consisting of ⁹⁰ Y, ¹¹¹ In, ¹³¹ I, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, ²¹² Pb, ²¹³ Bi, ²²⁵ Ac, and ²²⁷ Th.

When the Label is intended to comprise a metal, such as ¹¹¹ In for SPECT imaging, such is preferably provided in the form of a chelate. In such a case the Label preferably comprises a structural moiety capable of forming a coordination complex with such a metal. A good example hereof are macrocylic lanthanide(III) chelates derived from 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (H ₄ dota).

In preferred embodiments, the Label is selected from the group consisting of -OR ₃₇ , -N(R ₃₇ ) ₂ , -CF ₃ , -SR ₃₇ , S(=O) ₂ N(R ₃₇ ) ₂ , OC(=O)R ₃₇ ,

SC(=O)R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ ,

NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ ,

NR ₃₇ C(=O)N(R ₃₇ )2, NR ₃₇ C(=S)N(R ₃₇ )2, C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ - C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups; the R ₃₇ groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, (cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups, (cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups, (cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with and/or chelating at least one isotope selected from the group consisting of ³ H, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ¹⁹ F, ⁴⁴ Sc, ⁵¹ Cr, ⁵² Fe, ⁵² Mn, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Zn, ⁶² Cu, ⁶³ Zn, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁰ As, ⁷¹ As, ⁷² As, ⁷⁴ As, ⁷⁵ Se, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^8O Br, ⁸² Br, ⁸² Rb, ⁸⁶ Y, ⁸⁸ Y, ⁸⁹ Sr, ⁸⁹ Zr, ⁹⁷ Ru, ^99m Tc, ¹¹⁰ In, ¹¹¹ In, ¹¹³ In, ¹¹⁴ In, ¹¹⁷ Sn, ¹²⁰ I, ¹²² Xe, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁹³ Pt, ¹⁹⁵ Pt, ²⁰¹ Tl, ²⁰³ Pb, ²⁴ Na, ³² P, ³³ P, ⁴⁷ Sc, ⁵⁹ Fe, ⁶⁷ Cu, ⁷⁶ As, ⁷⁷ As, ⁸⁰ Br, ⁸² Br, ⁸⁹ Sr, ⁹⁰ Nb, ⁹⁰ Y, ¹⁰³ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹²¹ Sn, ¹²⁷ Te, ¹³¹ I, ¹⁴⁰ La, ¹⁴¹ Ce, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁴ Pr, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷² Tm, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹¹ Bi, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²¹⁴ Bi, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac and ²²⁷ Th; and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , - SO ₃ R ₃₇ , -PO ₃ (R ₃₇ )2, -PO ₄ (R ₃₇ )2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In another preferred embodiment, the Label is selected from the group consisting of -OR ₃₇ , -N(R ₃₇ )2, -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O)R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ ,

NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2, SC(=O)N(R ₃₇ )2, OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2,

NR ₃₇ C(=O)N(R ₃₇ )2, NR ₃₇ C(=S)N(R ₃₇ )2, C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ ) ₂ , C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , C ₁ -C ₁₂ alkyl groups, C ₂ -C ₁₂ alkenyl groups, C ₂ -C ₁₂ alkynyl groups, C ₆ -C ₁₂ aryl groups, C2- C ₁₂ heteroaryl groups, C ₃ -C ₁₂ cycloalkyl groups, C ₅ -C ₁₂ cycloalkenyl groups, C ₁₂ -C ₁₂ cycloalkynyl groups, C ₃ -C ₁₂ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)aryl(cyclo)alkyl, C ₄ -C ₁₂ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₁₂ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₁₂ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₁₂ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, and C ₄ -C ₁₂ cycloalkylalkyl groups; the R ₃₇ groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, (cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups, (cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups, (cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with and/or chelating at least one isotope selected from the group consisting of ³ H, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ¹⁹ F, ⁴⁴ Sc, ⁵¹ Cr, ⁵² Fe, ⁵² Mn, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Zn, ⁶² Cu, ⁶³ Zn, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁰ As, ⁷¹ As, ⁷² As, ⁷⁴ As, ⁷⁵ Se, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^8O Br, ⁸² Br, ⁸² Rb, ⁸⁶ Y, ⁸⁸ Y, ⁸⁹ Sr, ⁸⁹ Zr, ⁹⁷ Ru, ^99m Tc, ¹¹⁰ In, ¹¹¹ In, ¹¹³ In, ¹¹⁴ In, ¹¹⁷ Sn, ¹²⁰ I, ¹²² Xe, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁹³ Pt, ¹⁹⁵ Pt, ²⁰¹ Tl, ²⁰³ Pb, ²⁴ Na, ³² P, ³³ P, ⁴⁷ Sc, ⁵⁹ Fe, ⁶⁷ Cu, ⁷⁶ As, ⁷⁷ As, ⁸⁰ Br, ⁸² Br, ⁸⁹ Sr, ⁹⁰ Nb, ⁹⁰ Y, ¹⁰³ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹²¹ Sn, ¹²⁷ Te, ¹³¹ I, ¹⁴⁰ La, ¹⁴¹ Ce, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁴ Pr, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷² Tm, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹¹ Bi, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²¹⁴ Bi, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac and ²²⁷ Th; and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, - SO ₃ R ₃₇ , -PO ₃ (R ₃₇ ) _2, -PO ₄ (R ₃₇ ) _2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each Label is independently selected from the group consisting of -OR ₃₇ , -N(R ₃₇ )2, -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2,

OC(=O)R ₃₇ , SC(=O)R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)- R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ ,

NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , C ₁ -C ₆ alkyl groups, C ₂ -C ₆ alkenyl groups, C ₂ -C ₆ alkynyl groups, C ₆ aryl groups, C ₂ -C ₆ heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C8 cycloalkynyl groups, C ₃ -C ₆ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₆

(hetero)aryl(cyclo)alkyl, C ₄ -C ₆ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₆ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₆ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₆ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₆ alkylcycloalkyl groups, and C ₄ -C ₆ cycloalkylalkyl groups; the R ₃₇ groups, alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, (cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl groups, (cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups, (cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups comprise, are substituted with and/or chelating at least one isotope selected from the group consisting of ³ H, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ¹⁹ F, ⁴⁴ Sc, ⁵¹ Cr, ⁵² Fe, ⁵² Mn, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Zn, ⁶² Cu, ⁶³ Zn, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁰ As, ⁷¹ As, ⁷² As, ⁷⁴ As, ⁷⁵ Se, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^8O Br, ⁸² Br, ⁸² Rb, ⁸⁶ Y, ⁸⁸ Y, ⁸⁹ Sr, ⁸⁹ Zr, ⁹⁷ Ru, ^99m Tc, ¹¹⁰ In, ¹¹¹ In, ¹¹³ In, ¹¹⁴ In, ¹¹⁷ Sn, ¹²⁰ I, ¹²² Xe, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁹³ Pt, ¹⁹⁵ Pt, ²⁰¹ Tl, ²⁰³ Pb, ²⁴ Na, ³² P, ³³ P, ⁴⁷ Sc, ⁵⁹ Fe, ⁶⁷ Cu, ⁷⁶ As, ⁷⁷ As, ⁸⁰ Br, ⁸² Br, ⁸⁹ Sr, ⁹⁰ Nb, ⁹⁰ Y, ¹⁰³ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹²¹ Sn, ¹²⁷ Te, ¹³¹ I, ¹⁴⁰ La, ¹⁴¹ Ce, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁴ Pr, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷² Tm, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹¹ Bi, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²¹⁴ Bi, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac and ²²⁷ Th; and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , - SO ₃ R ₃₇ , -PO ₃ (R ₃₇ )2, -PO ₄ (R ₃₇ )2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In a preferred embodiment the Label is derived from a prosthetic group. The person skilled in the art will understand that a prosthetic group is a precursor that can be radiolabeled with a radionuclide like ¹³¹ I thus forming the Label.

In another preferred embodiment, the Label is selected from the group consisting of C ₁ -C ₁₂ alkyl groups, C ₂ -C ₁₂ alkenyl groups, C ₂ -C ₁₂ alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C ₁₂ cycloalkyl groups, C ₅ -C ₁₂ cycloalkenyl groups, C ₁₂ -C ₁₂ cycloalkynyl groups, C ₃ -C ₁₂ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)aryl(cyclo)alkyl, C ₄ -C ₁₂ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₁₂ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₁₂ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₁₂ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, and C ₄ -C ₁₂ cycloalkylalkyl groups; said groups comprise, are substituted with, at least one isotope selected from the group consisting of ³ H, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ¹⁹ F, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^8O Br, ⁸² Br, ¹²⁰ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ³² P, ³³ P, ¹³¹ I, ²¹¹ At, and are optionally further substituted with a moiety selected from the group consisting of -Cl, -F, -Br, - I, -OR ₃₇ , -N(R ₃₇ ) ₂ , -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ ) _2, -PO ₄ (R ₃₇ ) _2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In a preferred embodiment the Label comprises a chelating moiety. In a preferred embodiment, the Label is a chelating moiety selected from the group consisting of conjugates of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid), DOTAGA anhydride (2,2¢,2”-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)- 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), NOTA (1,4,7- triazacyclononane-N,N',N"-triacetic acid), TETA (1,4,8,11- tetraazacyclotetradecane-N,N',N",N'-tetraacetic acid), OTTA (N1-(p- isothiocyanatobenzyl)-diethylenetriamine-N ₁ ,N ₂ ,N ₃ ,N ₃ -tetraacetic acid), deferoxamine or DFO (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4- dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N- hydroxybutanediamide), and the DFO derivative called DFO*, and HYNIC (hydrazinonicotinamide); and the chelating moiety chelates a metal selected from the group consisting of ⁴⁴ Sc, ⁵¹ Cr, ⁵² Fe, ⁵² Mn, ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Zn, ⁶² Cu, ⁶³ Zn, ⁶⁴ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁰ As, ⁷¹ As, ⁷² As, ⁷⁴ As, ⁷⁵ Se, ⁸² Rb, ⁸⁶ Y, ⁸⁸ Y, ⁸⁹ Sr, ⁸⁹ Zr, ⁹⁷ Ru, ^99m Tc, ¹¹⁰ In, ¹¹¹ In, ¹¹³ In, ¹¹⁴ In, ¹¹⁷ Sn, ¹²² Xe, ¹⁶⁶ Ho, ¹⁶⁷ Tm, ¹⁶⁹ Yb, ¹⁹³ Pt, ¹⁹⁵ Pt, ²⁰¹ Tl, ²⁰³ Pb, ²⁴ Na, ⁴⁷ Sc, ⁵⁹ Fe, ⁶⁷ Cu, ⁷⁶ As, ⁷⁷ As, ⁸⁹ Sr, ⁹⁰ Nb, ⁹⁰ Y, ¹⁰³ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹¹ In, ¹²¹ Sn, ¹²⁷ Te, ¹⁴⁰ La, ¹⁴¹ Ce, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁴ Pr, ¹⁴⁹ Pm, ¹⁴⁹ Tb, ¹⁵¹ Pm, ¹⁵³ Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁷² Tm, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹¹ At, ²¹¹ Bi, ²¹² Bi, ²¹² Pb, ²¹³ Bi, ²¹⁴ Bi, ²²³ Ra, ²²⁴ Ra, ²²⁵ Ac and ²²⁷ Th.

In a preferred embodiment the metal chelate comprises an acyclic derivative of ethylenediaminotetraacetic acid (EDTA) or

diethylenediaminotetraacetic acid (DTPA):

, wherein the dashed line denotes a bond to the rest of the molecule.

In another preferred embodiment the metal chelate comprises an acyclic chelator containing carboxy-pyridine groups:

, wherein the dashed line denotes a bond to the rest of the molecule.

In another preferred embodiment the metal chelate comprises a cyclic derivative of 1,4,7,10-tetraazadodecane (cyclen):

, wherein the dashed line denotes a bond to the rest of the molecule. In another preferred embodiment the metal chelate comprises a derivative of 1,4,7-triazacyclononane (TACN):

Ĭ wherein the dashed line denotes a bond to the rest of the molecule.

In yet another preferred embodiment the metal chelate comprises a macrocyclic chelator containing N and O heteroatoms:

, wherein the dashed line denotes a bond to the rest of the molecule.

In another preferred embodiment the metal chelate comprises a derivative of the cryptand agent sarcophagine (Sar):

, wherein the dashed line denotes a bond to the rest of the molecule.

In another preferred embodiment the metal chelate comprises a linear or cyclic chelator containing hydroxamate groups:

, wherein the dashed line denotes a bond to the rest of the molecule.

In yet another preferred embodiment the metal chelate comprises a linear or cyclic chelator containing 3-hydroxy-4-pyridinone (3,4-HOPO) groups and derivatives:

, wherein the dashed line denotes a bond to the rest of the molecule.

In another preferred embodiment the metal chelate comprises a linear or cyclic chelator containing N, S and P heteroatoms:

, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of ^99m Tc, ¹⁸⁶ Re, and ¹⁸⁸ Re.

In another preferred embodiment the metal chelate comprises glycine, serine, cysteine, lysine and alanine residues:

, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of ^99m Tc, ¹⁸⁶ Re, and ¹⁸⁸ Re.

In another preferred embodiment the metal chelate contains hydrazinonicotinic acid derivatives (HYNIC) and a co-ligand:

, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of ^99m Tc, ¹⁸⁶ Re, and ¹⁸⁸ Re.

In another preferred embodiment the chelate comprises carbonyl groups and a chelator containing N, O and S heteroatoms or a

cyclopentadienyle:

, wherein the dashed line denotes a bond to the rest of the molecule M denotes a radionuclide selected from the group consisting of ^99m Tc, ¹⁸⁶ Re, and ¹⁸⁸ Re.

In some preferred embodiments of the present invention the label contains ¹⁸ F and can be produced by the skilled person on the basis of known synthesis routes using known labeled synthons or prosthetic groups. Several non limiting examples of ¹⁸ F-containing labels are depicted below:

, wherein the dashed line denotes a bond to the rest of the molecule.

In preferred embodiments of the present invention the label contains at least one isotope selected from the group consisting of ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, and ²¹¹ At; and is synthesized by the skilled person on the basis of known synthesis routes using prosthetic groups. Several preferred embodiments of such labels are depicted below:

, wherein the dashed line denotes a bond to the rest of the molecule and X denotes ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I or ²¹¹ At.

In yet another preferred embodiment of the present invention the label contains at least one isotope selected from the group consisting of ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, and ²¹¹ At;and is synthetized by the skilled person on the basis of known synthesis routes using a closo-decaborate(2-) group:

, wherein the dashed line denotes a bond to the rest of the molecule and X denotes ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I or ²¹¹ At. Administration Agent The Administration Agent can be any construct of which it is desired to modify it with a Label for imaging or radiotherapy and of which it is desired to remove its imaging or radiotherapy label at a particular time after injection. This particularly is the case in the event of targeted imaging and radiotherapy to a site, such as a tumor, within the body of a subject, notably a human subject. The sole requirement is that it can be provided with a Trigger T ^R , which is further linker to a Label. The precise linkage of the Trigger to the Administration Agent will depend on the molecular structure of both, but it should be noted that this does not normally present a particular challenge to the person skilled in the art, as many proven conjugation methods and linkage moieties for various biomolecules exist. The linkage can, optionally, be via a spacer such as a polyethylene glycol (PEG) chain.

Typically the Administration Agent can bind to a Primary Target, as defined herein. Said Primary Target can be a target to which a Targeting Agent binds or it can be a therapeutic target upon which a drug has its effect. In a preferred embodiment the Primary Target is a therapeutic target and the Targeting Agent is a drug and binds said Primary Target.

In preferred embodiments, the Administration Agent is a Targeting Agent as defined herein.

Preferably, the Administration Agent is selected from the group consisting of proteins, peptoids and peptides. Most preferably, the

Administration Agent is an antibody.

In another preferred embodiment, the Administration Agent is selected from the group consisting of antibodies, antibody-drug conjugates, antibody derivatives, antibody fragments, proteins, polymers, polymer-drug conjugates, drug containing liposomes and polymersomes, nanoparticles, microparticles, aptamers, oligopeptides, oligonucleotides, oligosaccharides, carbohydrates, as well as peptides, peptoids, steroids, toxins, hormones, cytokines, and chemokines.

In other preferred embodiments, the Administration Agent equals a Targeting Agent, and the Targeting Agent is radiolabeled with a therapeutic radioisotope in order to target therapeutic radiation to tissues expressing a Primary Target.

In other preferred embodiments, the Administration Agent equals a Targeting Agent, and the Targeting Agent is radiolabeled with a diagnostic radioisotope in order to image tissues expressing a Primary Target.

In preferred embodiments, the Administration Agent is an antibody, more preferably an antibody that comprises an FcRn binding domain, more preferably an intact IgG antibody.

In other preferred embodiments, the Administration Agent is an antibody that comprises an albumin-binding moiety. In other preferred embodiments, the Administration Agent is a protein that comprises an albumin-binding moiety. In other preferred embodiments the

Administration Agent equals a drug. In other preferred embodiments the Administration Agent equals a drug and the drug is labeled using the presented invention for the purpose of imaging in vivo drug distribution.

Drugs that can be used in an Administration Agent relevant to this invention are pharmaceutically active compounds, such as antibodies, antibody-drug conjugates, antibody derivatives, antibody fragments, proteins, biomolecules, polymer-drug conjugates, drug containing liposomes and polymersomes, aptamers, oligopeptides, oligonucleotides,

oligosaccharides, carbohydrates, as well as peptides, peptoids, steroids, toxins, hormones, cytokines, and chemokines. Other drugs that can be used are low to medium molecular weight compounds, preferably organic compounds, (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da, more preferably about 300 to about 1000 Da).

In a preferred embodiment the pharmaceutically active compound or drug is selected from the group consisting of cytotoxins,

antiproliferative/antitumor agents, antiviral agents, antibiotics, anti- inflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti- angiogenic factors, and enzyme inhibitors.

In other preferred embodiments the drug is designed to act in the central neural system, for example in the context of Alzheimer’s disease and Parkinsons’ disease, for example antibodies against beta-amyloïd and Tau proteins.

Exemplary cytotoxic drug types, for example for use in cancer therapy, include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors.

Exemplary immunemodulators are antibodies against PD-L1, PD-1, LAG-3, OX40, TIGIT, TIM-3, B7H4, Vista, CTLA-4, APRIL, cytokines, including IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, TNF, interferon gamma, GMCSF, NDV-GMCSF, and agonists and antagonists of STING, agonists and antagonists of TLRs including TLR1/2, TLR3, TLR4 , TLR7/8, TLR9, TLR12, agonists and antagonists of GITR, CD3, CD28, CD40, CD74, CTLA4, OX40, PD1, PDL1, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A.

It will be understood that chemical modifications may also be made to the Administration Agent in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.

In a preferred embodiment, the Administration Agent before conjugation to the remainder of the compound of Formula (19) comprises at least one moiety selected from the group consisting of -OH, -NHR’, -CO ₂ H, - SH, -S-S-, -SCH ₃ - -N3, terminal alkynyl, terminal alkenyl, -C(O)R', C8-C ₁₂ (hetero)cycloalkynyl, nitrone, nitrile oxide, (imino)sydnone, isonitrille, and (oxa)norbornene, tetrazine, wherein R' equals R ₃₇ , said moiety used for conjugation to a moiety comprising the dienophile, the Label and R32 so as to form the compound satisfying Formula (19), and comprising a C ^M2 or C ^X moiety.

In preferred embodiments the Administration Agent is bound to the remainder of the compound of Formula (19) via a C ^M2 selected from the group consisting of amine, amide, thioamide, aminooxy, carbamate, thiocarbamate, urea, thiourea, sulfonamide, and sulfoncarbamate. In preferred embodiments C ^M2 equals R ₁₀ as defined herein. Preferably, the Administration Agent is coupled as described above in relation to C ^M2 and _C ^X _.

Preferably, the Administration Agent, preferably an antibody, is modified with further moieties that equal Formula (19), except that these further moieties do not comprise an Administration Agent (as the first mentioned Administration Agent is already coupled to said moiety). In a preferred embodiment, the Administration Agent, preferably an antibody, is coupled to further moieties as defined in this paragraph at 1 to 8 positions, more preferably from 1 to 6 positions, even more preferably at 1 to 4 positions. Further embodiments in relation to the compound of Formula (19) In a preferred embodiment, in the compound of the invention the Administration Agent comprises an antibody, and preferably the

Administration Agent is an antibody.

In a preferred embodiment, in the compound of the invention the Label is a radiolabel, preferably a chelating moiety that chelates a

radioisotope. Diene The combinations according to the invention comprise a compound according to Formula (19) and a diene, preferably a tetrazine. In a preferred embodiment, the combination is in the form of a kit.

The tetrazine compound according to the invention may herein be referred to as“Activator”. The tetrazine typically reacts with the other Bio- orthogonal Reactive Group, that is a dienophile (vide supra). The diene of the Activator is selected so as to be capable of reacting with the dienophile of the TCO by undergoing a Diels-Alder cycloaddition followed by a retro Diels-Alder reaction, giving the IEDDA adduct. This intermediate adduct then releases the Construct-A, where this release can be caused by various circumstances or conditions that relate to the specific molecular structure of the IEDDA adduct.

The compound used to release one or more moieties R ₄₈ from the structure of Formula (19) is herein referred to as Activator.

In a preferred embodiment, the Activator is a tetrazine. Tetrazines are dienes and are highly reactive towards dienophiles, especially the TCO constructs (vide supra). The diene of the Activator is selected so as to be capable of reacting with the dienophile, e.g. the TCO, by undergoing a Diels- Alder cycloaddition followed by a retro Diels-Alder reaction, giving the IEDDA adduct. This intermediate adduct then releases the Construct-A. Synthesis routes to tetrazines in general are readily available to the skilled person, based on standard knowledge in the art. References to tetrazine synthesis routes include for example Lions et al, J. Org. Chem., 1965, 30, 318-319; Horwitz et al, J. Am. Chem. Soc., 1958, 80, 3155-3159; Hapiot et al, New J. Chem., 2004, 28, 387-392, Kaim et al, Z. Naturforsch., 1995, 50b, 123-127; Yang et al., Angew. Chem.2012, 124, 5312 -5315; Mao et al., Angew. Chem. Int. Ed.2019, 58, 1106-1109; Qu et al. Angew. Chem. Int. Ed. 2018, 57, 12057 -12061; Selvaraj et al., Tetrahedron Lett.2014, 55, 4795- 4797; Fan et al., Angew. Chem. Int. Ed.2016, 55, 14046-14050.

Preferably, the Activator is a tetrazine satisfying Formula (4) and preferably including pharmaceutically accepted salts thereof:

; wherein each moiety Q1 and Q2 is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , -SO ₃ , - PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O)R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O- R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2, NR ₃₇ C(=O)N(R ₃₇ )2, NR ₃₇ C(=S)N(R ₃₇ )2, C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ ) ₂ , C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, - NR ₃₇ OR ₃₇ , alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, (cyclo)alkyl(hetero)aryl groups, (hetero)aryl(cyclo)alkyl,

(cyclo)alkenyl(hetero)aryl groups, (hetero)aryl(cyclo)alkenyl groups,

(cyclo)alkynyl(hetero)aryl groups, (hetero)aryl(cyclo)alkynyl groups, alkylcycloalkyl groups, and cycloalkylalkyl groups. In Formula (4), the Q ₁ and Q ₂ groups not being H, -F, -Cl, -Br, -I, -OH, -NH ₂ , -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , are optionally substituted, preferably with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ R ₃₇ , -PO ₃ (R ₃₇ ) _2, -PO ₄ (R ₃₇ ) _2, -NO ₂ , -CF ₃ , =O, =NR ₃₇ , and -SR ₃₇ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₇ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. Preferably, each individual Q1 and Q2 group comprises at most 4 substituents, more preferably at most 3 substituents, even more preferably at most 2 substituents, and most preferably at most 1 substituent.

In Formula (4), the Q1 and Q2 groups are optionally bound to a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, an aptamer, a carbohydrate, an oligonucleotide, an oligosaccharide, a lipid, a steroid, a liposome, a Targeting Agent T ^T , R ⁸⁷ , an albumin-binding moiety, and a chelating moiety, a moiety comprising a radionuclide, or a Drug D ^D .

In Formula (4), preferably at least one of moieties Q1 and Q2 is not hydrogen.

In Formula (4), preferably each moiety Q ₁ and Q ₂ is independently selected from the group consisting of hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , - N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2,

SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ ,

NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ ) ₂ , C(=O)O- R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , - ON(R ₃₇ ) _{2, -} NR ₃₇ OR ₃₇ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ (cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups.

In preferred embodiments, the Q1 and Q2 groups not being hydrogen are not substituted.

In a preferred embodiment, Q1 and Q2 in Formula (4) are selected from the group of hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O) ₂ N(R ₃₇ ) ₂ , OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ ,

NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ ,

OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O) ₂ R ₃₇ , NR ₃₇ S(O) ₂ R ₃₇ , -ON(R ₃₇ ) _{2, -} NR ₃₇ OR ₃₇ , C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂

(hetero)arylalkyl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂

cycloalkylalkyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups.

In a preferred embodiment, Q1 and Q2 in Formula (4) are selected from the group of hydrogen, -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O) ₂ N(R ₃₇ ) ₂ , OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ ,

NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ ,

OC(=S)N(R ₃₇ ) ₂ , SC(=S)N(R ₃₇ ) ₂ , NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R ₃₇ , C(=S)R ₃₇ , C(=O)N(R ₃₇ )2, C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O) ₂ R ₃₇ , NR ₃₇ S(O) ₂ R ₃₇ , -ON(R ₃₇ ) _{2, -} NR ₃₇ OR ₃₇ , C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C2-C ₄ alkynyl groups, C ₆ -C8 aryl groups, C2-C8 heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁₀ alkyl(hetero)aryl groups, C ₃ -C ₁₀

(hetero)arylalkyl groups, C ₄ -C ₁ 0 alkylcycloalkyl groups, C ₄ -C ₁ 0 cycloalkylalkyl groups, C ₅ -C ₁₀ cycloalkyl(hetero)aryl groups and C ₅ -C ₁₀ (hetero)arylcycloalkyl groups.

In a preferred embodiment, Q1 and Q2 in Formula (4) are selected from the group of hydrogen, C ₁ -C ₈ alkyl, phenyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, 2,6-pyrimidyl, 2,5-pyrimidyl, 3,5-pyrmidyl, and 2,4-pyrimidyl; and Q1 and Q2 not being hydrogen are optionally substituted with a moiety selected from the group consisting of -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ ) ₂ , -SO ₃ , - PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O- R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ ) ₂ , SC(=O)N(R ₃₇ ) ₂ , OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2, NR ₃₇ C(=O)N(R ₃₇ )2, NR ₃₇ C(=S)N(R ₃₇ )2,

C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ ) ₂ , C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , -S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, - NR ₃₇ OR ₃₇ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ - C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄

(cyclo)alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)aryl(cyclo)alkyl, C ₄ -C ₂₄ (cyclo)alkenyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkenyl groups, C ₄ -C ₂₄ (cyclo)alkynyl(hetero)aryl groups, C ₄ -C ₂₄ (hetero)aryl(cyclo)alkynyl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, and C ₄ -C ₂₄ cycloalkylalkyl groups; preferably with a moiety selected from the group consisting of -F, -Cl, -Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2, OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)-R ₃₇ ,

NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2, SC(=O)N(R ₃₇ )2, OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2,

NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ )2, C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , - S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, -NR ₃₇ OR ₃₇ , C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, C ₂ -C ₈ alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ -C ₁₂

alkylcycloalkyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₅ -C ₁₂

cycloalkyl(hetero)aryl groups, and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups; more preferably with a moiety selected from the group consisting of -F, -Cl, - Br, -I, -OR ₃₇ , -N(R ₃₇ )2, -SO ₃ , -PO ₃ -, -NO ₂ , -CF ₃ , -SR ₃₇ , S(=O)2N(R ₃₇ )2,

OC(=O)R ₃₇ , SC(=O) R ₃₇ , OC(=S)R ₃₇ , SC(=S)R ₃₇ , NR ₃₇ C(=O)-R ₃₇ , NR ₃₇ C(=S)- R ₃₇ , NR ₃₇ C(=O)O-R ₃₇ , NR ₃₇ C(=S)O-R ₃₇ , NR ₃₇ C(=O)S-R ₃₇ , NR ₃₇ C(=S)S-R ₃₇ , OC(=O)N(R ₃₇ )2, SC(=O)N(R ₃₇ )2, OC(=S)N(R ₃₇ )2, SC(=S)N(R ₃₇ )2,

NR ₃₇ C(=O)N(R ₃₇ ) ₂ , NR ₃₇ C(=S)N(R ₃₇ ) ₂ , C(=O)R _37, C(=S)R _37, C(=O)N(R ₃₇ ) ₂ , C(=S)N(R ₃₇ ) ₂ , C(=O)O-R ₃₇ , C(=O)S-R ₃₇ , C(=S)O-R ₃₇ , C(=S)S-R ₃₇ , S(O)R ₃₇ , - S(O)2R ₃₇ , NR ₃₇ S(O)2R ₃₇ , -ON(R ₃₇ )2, -NR ₃₇ OR ₃₇ , C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C ₂ -C ₄ alkynyl groups, C ₆ -C ₈ aryl groups, C ₂ -C ₈ heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁ 0

alkyl(hetero)aryl groups, C ₃ -C ₁ 0 (hetero)arylalkyl groups, C ₄ -C ₁ 0

alkylcycloalkyl groups, C ₄ -C ₁₀ cycloalkylalkyl groups, C ₅ -C ₁₀

cycloalkyl(hetero)aryl groups, and C ₅ -C ₁ 0 (hetero)arylcycloalkyl groups.

In a preferred embodiment, in Formula (4):

(a) Q ₁ and Q ₂ are independently selected from the group consisting of 2- pyridyl, 3-pyridyl, and 4-pyridyl;

(b) Q1 is selected from the group consisting of 2,6-pyrimidyl, 2,5-pyrimidyl, 3,5-pyrmidyl, and 2,4-pyrimidyl; and Q ₂ is (hetero)alkyl; or

(c) Q1 is phenyl and Q2 is hydrogen;

(d) Q ₁ is phenyl and Q ₂ is phenyl;

(e) Q ₁ is phenyl and Q ₂ is C ₁ -C ₈ alkyl;

(f) Q1 and Q2 are C ₁ -C8 alkyl;

and in (a)-(f) all Q ₁ and Q ₂ not being hydrogen are optionally substituted as defined in the previous paragraph.

In preferred embodiments, the Activator can be a multimeric compound, comprising a plurality of dienes as defined herein. These multimeric compounds include but are not limited to biomolecules, proteins, peptides, peptoids, polymers, dendrimers, liposomes, micelles, particles, polymer particles, or other polymeric constructs. Preferred tetrazines Formula (4a) Preferred tetrazines are in accordance with Formula (4a), and preferably include pharmaceutically accepted salts thereof:

Formula (4a), wherein each moiety Q ₁ and Q ₂ is independently selected from the group consisting of hydrogen and moieties according to Formula (5):

, wherein the dashed line indicates a bond to the remainder of the molecule, and wherein R10, R11, and R12 are as defined herein.

In a preferred embodiment, each f in Formula (5) is an integer independently selected from a range of from 0 to 24, preferably in a range of from 1 to 12, more preferably in a range of from 2 to 6, even more preferably from 1 to 3. In a preferred embodiment, f is 1. In other preferred

embodiments f is an integer in the range from 12 to 24. In a preferred embodiment, in Formula (5) g is an integer in a range of from 0 to 12, preferably in a range of from 1 to 6, more preferably in a range of from 2 to 4. In a preferred embodiment, in Formula (5) each h is independently 0 or 1. In a preferred embodiment, g is 0, and f is 1. In a preferred embodiment, g is 1, and f is 1. In case the compound according to the invention comprises more than one moiety satisfying Formula (5), each g, h, and f is independently selected.

In a preferred embodiment, the moiety according to Formula (5) is optionally substituted with another independently selected moiety according to Formula (5). In a preferred embodiment, the moiety according to Formula (5) is not substituted with another independently selected moiety according to Formula (5).

In a preferred embodiment, the moiety according to Formula (5) is R ⁸⁷ , as defined further below.

In a preferred embodiment, the moiety according to Formula (5) satisfies molecules from Group R ^M shown further below.

It is preferred that at least one of moieties Q ₁ and Q ₂ in Formula (4a) is not hydrogen.

In preferred embodiments, Q1 in Formula (4a) is selected from the group consisting of C ₆ -C ₂₄ aryl, and C ₂ -C ₂₄ heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5).

In preferred embodiments, Q1 in Formula (4a) is selected from the group consisting of C ₆ -C ₂₄ aryl, and C ₂ -C ₂₄ heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q ₂ in Formula (4a) is selected from the group consisting of C ₆ -C ₂₄ aryl, and C ₂ -C ₂₄ heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5).

In preferred embodiments, Q1 in Formula (4a) is selected from the group consisting of C ₆ aryl, and C ₃ -C ₅ heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5). Herein, preferred heteroaryls are 2-pyridyl, 3-pyridyl, 4- pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl, phenyl, 2,3-pyrazyl, 3,4-pyrazyl, oxazol, isoxazol, thiazol, oxazoline, 2-pyrryl, 3-pyrryl, 2-thiophene, and 3-thiophene.

In preferred embodiments, Q1 in Formula (4a) is C ₃ -C ₅ heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q2 is C ₃ -C ₅ heteroaryl, and is optionally further substituted with a moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5). Herein, preferred heteroaryls are 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl, phenyl, 2,3-pyrazyl, 3,4-pyrazyl, oxazol, isoxazol, thiazol, oxazoline, 2-pyrryl, 3-pyrryl, 2-thiophene, and 3-thiophene.

In preferred embodiments, Q ₁ in Formula (4a) is C ₃ -C ₅ heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q ₂ is H.

In preferred embodiments, Q1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q2 is -H.

In preferred embodiments, Q ₁ in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q ₂ is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5).

In preferred embodiments, Q1 in Formula (4a) is a phenyl ring, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q2 is selected from the group consisting of C ₆ aryl, and C _3-5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5).

In preferred embodiments, Q ₁ in Formula (4a) is C ₁ -C ₁₂ alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q ₂ selected from the group consisting of C ₆ aryl, and C ₃ -5 heteroaryl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5).

In preferred embodiments, Q1 in Formula (4a) is C ₁ -C ₁₂ alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5), and Q2 in Formula (4a) is C ₁ -C ₁₂ alkyl, and is optionally further substituted with at least one moiety according to Formula (5), preferably not more than two, more preferably not more than one moiety according to Formula (5).

In preferred embodiments Q ₂ equals Q ₁ . R10 In preferred embodiments, each R ₁₀ is independently selected from the group consisting of -O-, -S-, -SS-, -NR ₄ -, -N=N-, -C(O)-, -C(O)NR ₄ -, -OC(O)-, - C(O)O-, -OC(O)O-, -OC(O)NR4-, -NR4C(O)-, -NR4C(O)O-, -NR4C(O)NR4-, - SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR ₄ -, -NR ₄ C(O)S-, -S(O)-, - S(O)2-, -OS(O)2-, -S(O ₂ )O-, -OS(O)2O-, -OS(O)2NR4-, -NR4S(O)2O-, - C(O)NR4S(O)2NR4-, -OC(O)NR4S(O)2NR4-, -OS(O)-, -OS(O)O-, -OS(O)NR4-, - ONR ₄ C(O)-, -ONR ₄ C(O)O-, -ONR ₄ C(O)NR ₄ -, -NR ₄ OC(O)-, -NR ₄ OC(O)O-, - NR ₄ OC(O)NR ₄ -, -ONR ₄ C(S)-, -ONR ₄ C(S)O-, -ONR ₄ C(S)NR ₄ -, -NR ₄ OC(S)-, - NR4OC(S)O-, -NR4OC(S)NR4-, -OC(S)-, -C(S)O-,-OC(S)O-, -OC(S)NR4-, - NR4C(S)-, -NR4C(S)O-, -SS(O)2-, -S(O)2S-, -OS(O ₂ )S-, -SS(O)2O-, -NR4OS(O)-, -NR ₄ OS(O)O-, -NR ₄ OS(O)NR ₄ -, -NR ₄ OS(O) ₂ -, -NR ₄ OS(O) ₂ O-,

-NR4OS(O)2NR4-, -ONR4S(O)-, -ONR4S(O)O-, -ONR4S(O)NR4-,

-ONR4S(O)2O-, -ONR4S(O)2NR4-, -ONR4S(O)2-, -OP(O)(R4)2-, -SP(O)(R4)2-, -NR ₄ P(O)(R ₄ ) ₂ -, and combinations thereof, wherein R ₄ is defined as described herein. In preferred embodiments, each R10 is independently selected from the group consisting of -O-, -S-, -SS-, -NR ₄ -, -N=N-, -C(O)-, - C(O)NR ₄ -, -OC(O)-, -C(O)O-, -OC(O)NR ₄ -, -NR ₄ C(O)-, -NR ₄ C(O)O-, - NR4C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR4-, - NR ₄ C(O)S-, -S(O)-, -S(O) ₂ -, -C(O)NR ₄ S(O) ₂ NR ₄ -, -OC(O)NR ₄ S(O) ₂ NR ₄ -, - OC(S)-, -C(S)O-, -OC(S)O-, -OC(S)NR4-, -NR4C(S)-, -NR4C(S)O-, -SS(O)2-.

Preferably, each R4 in relation to R10 is individually selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl. R11 In preferred embodiments, each R11 is independently selected from the group consisting of C ₁ -C ₂₄ alkylene groups, C ₂ -C ₂₄ alkenylene groups, C ₂ -C ₂₄ alkynylene groups, C ₆ -C ₂₄ arylene, C ₂ -C ₂₄ heteroarylene, C ₃ -C ₂₄

cycloalkylene groups, C ₅ -C ₂₄ cycloalkenylene groups, and C ₁₂ -C ₂₄

cycloalkynylene groups, and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,

cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₆ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each R11 is independently selected from the group consisting of C ₁ -C ₁₂ alkylene groups, C ₂ -C ₁₂ alkenylene groups, C2- C ₁₂ alkynylene groups, C ₆ -C ₁₂ arylene, C ₂ -C ₁₂ heteroarylene, C ₃ -C ₁₂ cycloalkylene groups, C ₅ -C ₁₂ cycloalkenylene groups, and C ₁₂

cycloalkynylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups,

cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, each R11 is independently selected from the group consisting of C ₁ -C ₆ alkylene groups, C ₂ -C ₆ alkenylene groups, C ₂ - C ₆ alkynylene groups, C ₆ -C ₆ arylene, C ₂ -C ₆ heteroarylene, C ₃ -C ₆

cycloalkylene groups, and C ₅ -C ₆ cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₆ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, the R11 groups are optionally further substituted with one or more substituents selected from the group

consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR36, -SR36, C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)arylalkyl groups, C ₄ -C ₂₄

(hetero)arylalkenyl groups, C ₄ -C ₂₄ (hetero)arylalkynyl groups, C ₄ -C ₂₄ alkenyl(hetero)aryl groups, C ₄ -C ₂₄ alkynyl(hetero)aryl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, C ₆ -C ₂₄ alkylcycloalkenyl groups, C ₁₃ -C ₂₄

alkylcycloalkynyl groups, C ₄ -C ₂₄ cycloalkylalkyl groups, C ₆ -C ₂₄

cycloalkenylalkyl groups, C ₁ 3-C ₂₄ cycloalkynylalkyl groups, C ₅ -C ₂₄

alkenylcycloalkyl groups, C ₇ -C ₂₄ alkenylcycloalkenyl groups, C ₁₄ -C ₂₄ alkenylcycloalkynyl groups, C ₅ -C ₂₄ cycloalkylalkenyl groups, C ₇ -C ₂₄ cycloalkenylalkenyl groups, C ₁₄ -C ₂₄ cycloalkynylalkenyl groups, C ₅ -C ₂₄ alkynylcycloalkyl groups, C ₇ -C ₂₄ alkynylcycloalkenyl groups, C ₁ 4-C ₂₄ alkynylcycloalkynyl groups, C ₅ -C ₂₄ cycloalkylalkynyl groups, C ₇ -C ₂₄ cycloalkenylalkynyl groups, C ₁₄ -C ₂₄ cycloalkynylalkynyl groups, C ₅ -C ₂₄ cycloalkyl(hetero)aryl groups, C ₇ -C ₂₄ cycloalkenyl(hetero)aryl groups, C ₁ 4- C ₂₄ cycloalkynyl(hetero)aryl groups, C ₅ -C ₂₄ (hetero)arylcycloalkyl groups, C ₇ -C ₂₄ (hetero)arylcycloalkenyl groups, and C ₁₄ -C ₂₄ (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more

heteroatoms selected from the group consisting of O, S, NR ₃₆ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, the R ₁₁ groups are optionally further substituted with one or more substituents selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR ₃₆ , -SR ₃₆ , C ₁ -C ₁₂ alkyl groups, C ₂ -C ₁₂ alkenyl groups, C ₂ -C ₁₂ alkynyl groups, C ₆ -C ₁₂ aryl groups, C ₂ -C ₁₂ heteroaryl groups, C ₃ -C ₁₂ cycloalkyl groups, C ₅ -C ₁₂ cycloalkenyl groups, C ₁₂ cycloalkynyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ -C ₁₂

(hetero)arylalkenyl groups, C ₄ -C ₁₂ (hetero)arylalkynyl groups, C ₄ -C ₁₂ alkenyl(hetero)aryl groups, C ₄ -C ₁₂ alkynyl(hetero)aryl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₆ -C ₁₂ alkylcycloalkenyl groups, C ₁₃ -C ₁₈

alkylcycloalkynyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₆ -C ₁₂

cycloalkenylalkyl groups, C ₁₃ -C ₁₈ cycloalkynylalkyl groups, C ₅ -C ₁₂ alkenylcycloalkyl groups, C ₇ -C ₁₂ alkenylcycloalkenyl groups, C ₁₄ -C ₁₆ alkenylcycloalkynyl groups, C ₅ -C ₁₂ cycloalkylalkenyl groups, C ₇ -C ₁₂ cycloalkenylalkenyl groups, C ₁₄ -C ₁₆ cycloalkynylalkenyl groups, C ₅ -C ₁₂ alkynylcycloalkyl groups, C ₇ -C ₁₂ alkynylcycloalkenyl groups, C ₁₄ -C ₁₆ alkynylcycloalkynyl groups, C ₅ -C ₁₂ cycloalkylalkynyl groups, C ₇ -C ₁₂ cycloalkenylalkynyl groups, C ₁₄ -C ₁₆ cycloalkynylalkynyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups, C ₇ -C ₁₂ cycloalkenyl(hetero)aryl groups, C ₁ 4- C ₁₆ cycloalkynyl(hetero)aryl groups, C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, C ₇ -C ₁₂ (hetero)arylcycloalkenyl groups, and C ₁₄ -C ₁₆ (hetero)arylcycloalkynyl groups, wherein the substituents optionally contain one or more

heteroatoms selected from the group consisting of O, S, NR ₃₆ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, the R ₁₁ groups are optionally further substituted with one or more substituents selected from the group

consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H _, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR ₃₆ , -SR ₃₆ , C ₁ -C ₆ alkyl groups, C ₂ -C ₆ alkenyl groups, C ₂ -C ₆ alkynyl groups, C ₆ aryl groups, C2-C ₆ heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ - C ₆ cycloalkenyl groups, C ₃ -C ₆ alkyl(hetero)aryl groups, C ₃ -C ₆

(hetero)arylalkyl groups, C ₄ -C ₆ (hetero)arylalkenyl groups, C ₄ -C ₆

(hetero)arylalkynyl groups, C ₄ -C ₆ alkenyl(hetero)aryl groups, C ₄ -C ₆ alkynyl(hetero)aryl groups, C ₄ -C ₆ alkylcycloalkyl groups, C ₆

alkylcycloalkenyl groups, C ₄ -C ₆ cycloalkylalkyl groups, C ₆ cycloalkenylalkyl groups, C ₅ -C ₆ alkenylcycloalkyl groups, C ₇ alkenylcycloalkenyl groups, C ₅ -C ₆ cycloalkylalkenyl groups, C ₇ cycloalkenylalkenyl groups, C ₅ -C ₆

alkynylcycloalkyl groups, C ₇ alkynylcycloalkenyl groups, C ₅ -C ₆

cycloalkylalkynyl groups, C ₅ -C ₆ cycloalkyl(hetero)aryl groups, and C ₅ -C ₆ (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, the R11 groups are optionally further substituted with one or more substituents selected from the group

consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR36, -SR36, C ₁ -C ₆ alkyl groups, C2-C ₆ alkenyl groups, C2-C ₆ alkynyl groups, C ₆ aryl groups, C ₂ -C ₆ heteroaryl groups, C ₃ -C ₆ cycloalkyl groups, C ₅ - C ₆ cycloalkenyl groups, C ₃ -C ₇ alkyl(hetero)aryl groups, C ₃ -C ₇ (hetero)arylalkyl groups, C ₄ -C ₈ (hetero)arylalkenyl groups, C ₄ -C ₈

(hetero)arylalkynyl groups, C ₄ -C8 alkenyl(hetero)aryl groups, C ₄ -C8 alkynyl(hetero)aryl groups, C ₄ -C ₆ alkylcycloalkyl groups, C ₆ -C ₇

alkylcycloalkenyl groups, C ₄ -C ₆ cycloalkylalkyl groups, C ₆ -C ₇

cycloalkenylalkyl groups, C ₅ -C ₆ alkenylcycloalkyl groups, C ₇ -C8

alkenylcycloalkenyl groups, C ₅ -C ₆ cycloalkylalkenyl groups, C ₇ -C8

cycloalkenylalkenyl groups, C ₅ -C ₆ alkynylcycloalkyl groups, C ₇ -C ₈

alkynylcycloalkenyl groups, C ₅ -C ₆ cycloalkylalkynyl groups, C ₅ -C9

cycloalkyl(hetero)aryl groups, and C ₅ -C ₆ (hetero)arylcycloalkyl groups, wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR36, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

It is preferred that when f > 2, that R11 is independently selected from the group consisting of C ₁ -C ₆ alkylene groups, C ₂ -C ₆ alkenylene groups, C ₂ - C ₆ alkynylene groups, C ₆ -C ₆ arylene, C2-C ₆ heteroarylene, C ₃ -C ₆

cycloalkylene groups, and C ₅ -C ₆ cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₆ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In a preferred embodiment, the R ₁₁ substituents do not contain heteroatoms. In a preferred embodiment, the R ₁₁ groups are not substituted. In another preferred embodiment, the R11 groups do not contain

heteroatoms. R12 R12 is selected from the group consisting of -H, -OH, -NH ₂ , -N3, -Cl, -Br, -F, - I, a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, a biomolecule, an aptamer, a carbohydrate, an oligonucleotide, an

oligosaccharide, a lipid, a steroid, a liposome, a micelle, a Targeting Agent T ^T , R ⁸⁷ , a Drug D ^D , an imaging moiety, an albumin-binding moiety, and a chelating moiety.

Non-limiting examples of chelating moieties for use in R12 are

DTPA (diethylenetriaminepentaacetic acid),

DOTA (1,4,7,10- tetraazacyclododecane-N,N',N",N"-tetraacetic acid),

NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid),

TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N'-tetraacetic acid), OTTA (N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N ₁ ,N ₂ ,N ₃ ,N ₃ - tetraacetic acid), deferoxamine or DFO (N'-[5-[[4-[[5- (acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamin o]pentyl]-N- (5-aminopentyl)-N-hydroxybutanediamide) or HYNIC

(hydrazinonicotinamide).

In a preferred embodiment, when R ¹² is a polymer, a particle, a peptide, a peptoid, a dendrimer, a protein, a biomolecule, an oligonucleotide, an oligosaccharide, a lipid, a liposome, a micelle, a Targeting Agent T ^T , or a R ⁸⁷ , then f is at most 2, preferably at most 1. Formulae (6), (7), (8), (9), (10), (11), (12), and (13) In preferred embodiments of the invention the tetrazine is in accordance with any one of the Formulae (6), (7), (8), (9), (10), (11), (12), or (13):

wherein each moiety Q, Q1, Q2, Q3, and Q4 is independently selected from the group consisting of hydrogen and moieties according to Formula (5) as defined herein; and wherein R ₁ , R ₂ , and R ₃ are as defined herein.

In preferred embodiments, in the tetrazines according to any one of Formulae (6), (7), (8), (9), (10), (11), (12), and (13), at most one moiety selected from the group consisting of Q, Q ₁ , Q ₂ , Q ₃ , and Q ₄ is hydrogen.

In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), at most two moieties selected from the group consisting of Q, Q ₁ , Q ₂ , Q ₃ , and Q ₄ are hydrogen.

In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), at most three moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 are hydrogen.

In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), all moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 are hydrogen.

In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), at most one moiety selected from the group consisting of Q, Q1, Q2, Q3, and Q4 is not hydrogen. In preferred embodiments, in the tetrazines according to any one of Formulae (7), (8), (9), (10), (11), (12), and (13), at most two moieties selected from the group consisting of Q, Q1, Q2, Q3, and Q4 is not hydrogen. Molecular weight Preferably, for all compounds disclosed herein comprising a group Q, Q1, Q2, Q3, Q4 or -(CH ₂ )y-((R1)p-R2)n-(R1)p-R3, at least one of these groups has a molecular weight in a range of from 100 Da to 3000 Da. Preferably, at least one of these groups has a molecular weight in a range of from 100 Da to 2000 Da. More preferably, at least one of these groups has a molecular weight in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, at least one of these groups has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.

Preferably, for all compounds disclosed herein comprising a group Q, Q1, Q2, Q3, Q4 or -(CH ₂ ) _y -((R ₁ ) _p -R ₂ ) _n -(R ₁ ) _p -R ₃ , none of these groups has a molecular weight of more than 3000 Da, in particular in the case the

Activator needs to efficiently extravasate into tissues. Group -(CH ₂ ) _y -((R ₁ ) _p -R ₂ ) _n -(R ₁ ) _p -R ₃ In preferred embodiments, y is an integer in a range of from 1 to 12, preferably from 1 to 10, more preferably from 1 to 8, even more preferably from 2 to 6, most preferably from 2 to 4. In preferred embodiments, y is at least 2, preferably y is at least 3. In preferred embodiments, p is 0 or 1, wherein each p is independently selected. In preferred embodiments, each n is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3, most preferably n is 0 or 1. In preferred embodiments n is preferably an integer from 12 to 24. In preferred embodiments, n is 1. In preferred embodiments, the entire group -((R ₁ ) _p -R ₂ ) _n -(R ₁ ) _p -R ₃ is R ⁸⁷ . In preferred embodiments, the entire group -((R1)p-R2)n-(R1)p-R3 has a molecular weight in a range of from 100 Da to 3000 Da. Preferably, the entire group -((R ₁ ) _p -R ₂ ) _n -(R ₁ ) _p -R ₃ has a molecular weight in a range of from 100 Da to 2000 Da. More preferably, the entire group -((R1)p-R2)n-(R1)p-R3 has a molecular weight in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, the entire group -((R1)p-R2)n-(R1)p-R3 has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.

It is preferred that when n > 2, that R2 is independently selected from the group consisting of C ₁ -C ₆ alkylene groups, C ₂ -C ₆ alkenylene groups, C ₂ - C ₆ alkynylene groups, C ₆ arylene, C2-C ₆ heteroarylene, C ₃ -C ₆ cycloalkylene groups, and C ₅ -C ₆ cycloalkenylene groups; and wherein preferably the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, and cycloalkynylene groups optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₃₆ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, R ⁸⁷ or the entire group -((R1)p-R2)n-(R1)p-R3 satisfies molecules from Group R ^M shown below: R ^M :

, wherein the wiggly line denotes a bond to a tetrazine group as disclosed herein or to a group R1 or R2.

In preferred embodiments, the group -((R1)p-R2)n-(R1)p-R3 satisfies molecules from Group R ^M , wherein it is understood that when n is more than 1, -((R1)p-R2)n-(R1)p-R3 may be preceded by a group -((R1)p-R2)- so as to form a group -((R1)p-R2)-((R1)p-R2)n-1-(R1)p-R3. It is understood that this follows from the definition of how to write out the repeating units, i.e. - ((R1)p-R2)2- would first be written as -(R1)p-R2-(R1)p-R2- before R1, p, and R2 are independently selected. R ₁ , R ₂ , R ₃

R1 is as defined for R10. R2 is as defined for R11.R3 is as defined for R12.

R4

Preferably, each R ₄ is independently selected from the group consisting of hydrogen, C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl, C ₂ -C ₂₄ heteroaryl, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, and C ₁₂ -C ₂₄ cycloalkynyl groups.

In a preferred embodiment, each R4 is independently selected from the group consisting of hydrogen, C ₁ -C ₁₂ alkyl groups, C ₂ -C ₁₂ alkenyl groups, C ₂ -C ₁₂ alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C ₁₂ cycloalkyl groups, C ₅ -C ₁₂ cycloalkenyl groups, and C ₁₂ cycloalkynyl groups.

In a preferred embodiment, each R ₄ is independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C2-C ₄ alkynyl groups, C ₆ aryl, C2-C ₆ heteroaryl, C ₃ -C8 cycloalkyl groups, C ₅ - C ₈ cycloalkenyl groups, and C ₈ cycloalkynyl groups.

Preferably, the R4 groups not being hydrogen, optionally contain one or more heteroatoms selected from the group consisting of O, S, NR5, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized. Preferably, the R ₄ groups not being hydrogen, are optionally further substituted with one or more substituents selected from the group

consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR ₅ , -SR ₅ , C ₁ -C ₂₄ alkyl groups, C ₂ -C ₂₄ alkenyl groups, C ₂ -C ₂₄ alkynyl groups, C ₆ -C ₂₄ aryl groups, C ₂ -C ₂₄ heteroaryl groups, C ₃ -C ₂₄ cycloalkyl groups, C ₅ -C ₂₄ cycloalkenyl groups, C ₁₂ -C ₂₄ cycloalkynyl groups, C ₃ -C ₂₄ alkyl(hetero)aryl groups, C ₃ -C ₂₄ (hetero)arylalkyl groups, C ₄ -C ₂₄

(hetero)arylalkenyl groups, C ₄ -C ₂₄ (hetero)arylalkynyl groups, C ₄ -C ₂₄ alkenyl(hetero)aryl groups, C ₄ -C ₂₄ alkynyl(hetero)aryl groups, C ₄ -C ₂₄ alkylcycloalkyl groups, C ₆ -C ₂₄ alkylcycloalkenyl groups, C ₁₃ -C ₂₄

alkylcycloalkynyl groups, C ₄ -C ₂₄ cycloalkylalkyl groups, C ₆ -C ₂₄

cycloalkenylalkyl groups, C ₁₃ -C ₂₄ cycloalkynylalkyl groups, C ₅ -C ₂₄

alkenylcycloalkyl groups, C ₇ -C ₂₄ alkenylcycloalkenyl groups, C ₁ 4-C ₂₄ alkenylcycloalkynyl groups, C ₅ -C ₂₄ cycloalkylalkenyl groups, C ₇ -C ₂₄ cycloalkenylalkenyl groups, C ₁₄ -C ₂₄ cycloalkynylalkenyl groups, C ₅ -C ₂₄ alkynylcycloalkyl groups, C ₇ -C ₂₄ alkynylcycloalkenyl groups, C ₁ 4-C ₂₄ alkynylcycloalkynyl groups, C ₅ -C ₂₄ cycloalkylalkynyl groups, C ₇ -C ₂₄ cycloalkenylalkynyl groups, C ₁₄ -C ₂₄ cycloalkynylalkynyl groups, C ₅ -C ₂₄ cycloalkyl(hetero)aryl groups, C ₇ -C ₂₄ cycloalkenyl(hetero)aryl groups, C ₁ 4- C ₂₄ cycloalkynyl(hetero)aryl groups, C ₅ -C ₂₄ (hetero)arylcycloalkyl groups, C ₇ -C ₂₄ (hetero)arylcycloalkenyl groups, and C ₁₄ -C ₂₄ (hetero)arylcycloalkynyl groups; wherein the substituents optionally contain one or more

heteroatoms selected from the group consisting of O, S, NR ₅ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

Preferably, the R ₄ groups not being hydrogen, are optionally further substituted with one or more substituents selected from the group

consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , -SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NR ₅ , -SR ₅ , C ₁ -C ₄ alkyl groups, C ₂ -C ₄ alkenyl groups, C ₂ -C ₄ alkynyl groups, C ₆ aryl groups, C2-C ₄ heteroaryl groups, C ₃ -C ₄ cycloalkyl groups, C ₅ - C ₄ cycloalkenyl groups, C ₁₂ cycloalkynyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ -C ₁₂ (hetero)arylalkenyl groups, C ₄ -C ₁₂ (hetero)arylalkynyl groups, C ₄ -C ₁₂ alkenyl(hetero)aryl groups, C ₄ -C ₁₂ alkynyl(hetero)aryl groups, C ₄ -C ₁₂ alkylcycloalkyl groups, C ₆ -C ₁₂

alkylcycloalkenyl groups, C ₁ 3-C ₁₂ alkylcycloalkynyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₆ -C ₁₂ cycloalkenylalkyl groups, C ₁ 3

cycloalkynylalkyl groups, C ₅ -C ₁₂ alkenylcycloalkyl groups, C ₇ -C ₁₂

alkenylcycloalkenyl groups, C ₁ 4 alkenylcycloalkynyl groups, C ₅ -C ₁₂ cycloalkylalkenyl groups, C ₇ -C ₁₂ cycloalkenylalkenyl groups, C ₁₄

cycloalkynylalkenyl groups, C ₅ -C ₁₂ alkynylcycloalkyl groups, C ₇ -C ₁₂ alkynylcycloalkenyl groups, C ₁ 4-C ₁₂ alkynylcycloalkynyl groups, C ₅ -C ₁₂ cycloalkylalkynyl groups, C ₇ -C ₁₂ cycloalkenylalkynyl groups, C ₁₄

cycloalkynylalkynyl groups, C ₅ -C ₁₂ cycloalkyl(hetero)aryl groups, C ₇ -C ₁₂ cycloalkenyl(hetero)aryl groups, C ₁ 4 cycloalkynyl(hetero)aryl groups, C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, C ₇ -C ₁₂ (hetero)arylcycloalkenyl groups, and C ₁ 4 (hetero)arylcycloalkynyl groups; wherein the substituents optionally contain one or more heteroatoms selected from the group consisting of O, S, NR ₅ , P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In a preferred embodiment, the R4 substituents do not contain heteroatoms. In a preferred embodiment, the R ₄ groups are not substituted. In another preferred embodiment, the R4 groups do not contain

heteroatoms. R5

Preferably, each R5 is independently selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl groups, C ₂ -C ₈ alkenyl groups, C ₂ -C ₈ alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ - C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₅ -C ₁₂

cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein the R ₅ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , - SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In a preferred embodiment, the R ₅ groups are not substituted. In another preferred embodiment, the R5 groups do not contain heteroatoms. Moieties Q, Q ₁ , Q ₂ , Q ₃ , Q ₄

In preferred embodiments, g is an integer in a range of from 0 to 12, preferably from 0 to 10, more preferably from 0 to 8, even more preferably from 1 to 6, most preferably from 2 to 4. In other preferred embodiments g is 0. In case more than one moiety selected from the group consisting of Q, Q ₁ , Q2, Q3, and Q4 within one compound satisfies Formula (5), each g is independently selected. In preferred embodiments, h is 0 or 1. In case more than one moiety selected from the group consisting of Q, Q1, Q2, Q3, and Q4 within one compound satisfies Formula (5), each h is independently selected. In preferred embodiments, each f belonging to a moiety Q, Q ₁ , Q ₂ , Q3, or Q4 is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3, most preferably f is 0 or 1. In preferred embodiments f is preferably an integer from 12 to 24. In other preferred embodiments, f is 1.

In preferred embodiments, the group -((R ₁₀ ) _h -R ₁₁ ) _f -(R ₁₀ ) _h -R ₁₂ satisfies molecules from Group R ^M shown above.

In preferred embodiments, the group -((R10)h-R11)f-(R10)h-R12 satisfies molecules from Group R ^M , wherein it is understood that when f is more than 1, e.g. -((R10)h-R11)f-1-(R10)h-R12 may be preceded by a group -(R10)h-R11- so as to form a group -(R10)h-R11-((R10)h-R11)f-1-(R10)h-R12. It is understood that this follows from the definition of how to write out the repeating units, i.e. - ((R ₁₀ ) _h -R ₁₁ ) ₂ - would first be written as -(R ₁₀ ) _h -R ₁₁ -(R ₁₀ ) _h -R ₁₁ - before R ₁₀ , h, and R11 are independently selected. Formula (14)

In a preferred embodiment, the Activator is a tetrazine satisfying Formula (14):

Formula (14), and preferably including pharmaceutically acceptable salts thereof, wherein, Ya is selected from the group consisting of Y1, Y2, Y3, Y4, Y5, Y6, and Y ₇ :

Y7 wherein, Y _b is selected from the group consisting of Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , hydrogen, X ₄₇ , and–(S ^P ) _D –R ⁸⁷ ; wherein D is 0 or 1;wherein when Y _a is Y ₆ , then Yb is hydrogen, wherein each Q1 and Q5, are individually selected from the group consisting of X _45, hydrogen, X ₄₇ and–(S ^P ) _D –R ⁸⁷ ; wherein each Q ₂ and Q4, are individually selected from the group consisting of X46, hydrogen, X ₄₇ , and–(S ^P ) _D –R ⁸⁷ ; wherein each Q ₃ is individually selected from the group consisting of hydrogen, X47, and–(S ^P )D–R ⁸⁷ ; wherein preferably the compound of Formula (14) comprises at least one X45 or X46 group, wherein each X ₄₅ individually is selected from the group consisting of N(X ₅₀ ) ₂ ,

C(X51)2N(X50)2, NX50C(O)X51, NX50C(S)X51, OH, SH, C(O)OH, C(S)OH, C(O)SH, C(S)SH, NX50C(O)OX51, NX50C(S)OX51, NX50C(O)SX51,

NX ₅₀ C(S)SX _51, NX ₅₀ C(O)N(X ₅₁ ) ₂ , NX ₅₀ C(S)N(X ₅₁ ) ₂ , NX ₅₀ SO ₂ X _51, NX ₅₀ SO ₃ X _51, NX50OX51, SO ₃ H, and PO ₃ H ₂ ; wherein each X46 individually is selected from the group consisting of N(X ₅₀ ) ₂ , C(X ₅₁ ) ₂ N(X ₅₀ ) _2, NX ₅₀ C(O)X ₅₁ , NX ₅₀ C(S)X _51, , OH, SH, C(O)OH, C(S)OH, C(O)SH, C(S)SH, NX ₅₀ C(O)OX _51, NX ₅₀ C(S)OX _51, NX50C(O)SX51, NX50C(S)SX51, NX50C(O)N(X51)2, NX50C(S)N(X51)2,

NX ₅₀ SO ₂ X _51, NX ₅₀ SO ₃ X _51, NX ₅₀ OX ₅₁ , SO ₃ H, and PO ₃ H ₂ ; wherein each X ₅₀ and X51 individually is selected from the group consisting of hydrogen, X48, and–(S ^P )D–R ⁸⁷ ; wherein each X48 is preferably independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C ₂ -C ₄ alkenyl groups, and C ₄ -6 (hetero)aryl groups; wherein for X48 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, -SH, - SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , and -NO ₂ ; and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si- , wherein the N, S, and P atoms are optionally oxidized, wherein each X ₄₇ is selected from the group consisting of -F, -Cl, -Br, -I, -OX49, -N(X49)2, -SO ₃ , - PO ₃ - _, -NO ₂ , -CF ₃ , -SX ₄₉ , S(=O) ₂ N(X ₄₉ ) _2, OC(=O)X ₄₉ , SC(=O) X ₄₉ , OC(=S)X ₄₉ , SC(=S)X _49, NX ₄₉ C(=O)-X ₄₉ , NX ₄₉ C(=S)-X ₄₉ , NX ₄₉ C(=O)O-X ₄₉ , NX ₄₉ C(=S)O- X49, NX49C(=O)S-X49, NX49C(=S)S-X49, OC(=O)N(X49)2, SC(=O)N(X49)2, OC(=S)N(X ₄₉ ) ₂ , SC(=S)N(X ₄₉ ) ₂ , NX ₄₉ C(=O)N(X ₄₉ ) ₂ , NX ₄₉ C(=S)N(X ₄₉ ) _2,

C(=O)X49, C(=S)X49, C(=O)N(X49)2, C(=S)N(X49)2, C(=O)O-X49, C(=O)S-X49, C(=S)O-X49, C(=S)S-X49, -S(O)X49, -S(O)2X49, NX49S(O)2X49, -ON(X49)2, - NX ₄₉ OX ₄₉ , C ₁ -C ₈ alkyl groups, C ₂ -C ₈ alkenyl groups, C ₂ -C ₈ alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ - C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₅ -C ₁₂

cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, aryl, heteroaryl, cycloalkyl groups, cycloalkenyl groups, alkyl(hetero)aryl groups,

(hetero)arylalkyl groups, alkylcycloalkyl groups, cycloalkylalkyl groups, cycloalkyl(hetero)aryl groups and (hetero)arylcycloalkyl groups are optionally substituted with a moiety selected from the group consisting of - Cl, -F, -Br, -I, -OX ₄₉ , -N(X ₄₉ ) ₂ , -SO ₃ X ₄₉ , -PO ₃ (X ₄₉ ) _2, -PO ₄ (X ₄₉ ) _2, -NO ₂ , -CF ₃ , =O, =NX ₄₉ , and -SX ₄₉ , and optionally contain one or more heteroatoms selected from the group consisting of O, S, NX49, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally

quaternized; wherein X49 is selected from the group consisting of hydrogen, C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C ₈ cycloalkyl groups, C ₅ -C ₈ cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ -C ₁₂

alkylcycloalkyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₅ -C ₁₂

cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein the X49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , - SO ₃ H, -PO ₃ H _, -PO ₄ H _2, -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized,

wherein preferably at most two, more preferably at most one of Q1, Q2, Q3, Q _4, and Q ₅ are R ⁸⁷ ; wherein the compound according to Formula (14) preferably comprises for each individual Ya and Yb at most four R ⁸⁷ groups, more preferably at most two R ⁸⁷ groups, most preferably at most one R ⁸⁷ ; wherein the compound according to Formula (14) preferably comprises at least one R ⁸⁷ ; wherein preferably for each individual Ya and Yb at most three, more preferably at most two of Q ₁ , Q ₂ , Q ₃ , Q _4, and Q ₅ are not hydrogen; wherein preferably for each individual Ya and Yb at most two of Q1, Q2, Q3, Q4, and Q5 are X45 or X46, wherein preferably for each individual Y _a and Y _b one of Q ₁ , Q ₂ , Q _4, and Q ₅ is X ₄₅ or X ₄₆ , wherein preferably both Y _a and Yb comprise at least one X45 or X46,wherein preferably both Ya and Yb comprise one X45 or X46, wherein preferably both Ya and Yb comprise one X45 or X ₄₆ , whereby the X ₄₅ comprised in Y _a is the same as the X ₄₅ comprised in Yb, and/or the X46 comprised in Ya is the same as the X46 comprised in Yb, wherein preferably Y _a and Y _b are both independently selected Y ₁ , or both independently selected Y ₂ , or both independently selected Y ₃ , or both independently selected Y4, or both independently selected Y5; or both independently selected Y ₇ .

In a preferred embodiment, in Formula (14), when Q1 is a X47 or– (S ^P )D–R ⁸⁷ , then for Q1 the X47 and–(S ^P )D–R ⁸⁷ are not a group in accordance with the definition of X ₄₅ . In a preferred embodiment, in Formula (14), when Q5 is a X47 or–(S ^P )D–R ⁸⁷ , then for Q5 the X47 and–(S ^P )D–R ⁸⁷ are not a group in accordance with the definition of X45. In a preferred embodiment, in Formula (14), when Q ₂ is a X ₄₇ or–(S ^P ) _D –R ⁸⁷ , then for Q ₂ the X ₄₇ and–(S ^P ) _D – R ⁸⁷ are not a group in accordance with the definition of X46. In a preferred embodiment, in Formula (14), when Q4 is a X47 or–(S ^P )D–R ⁸⁷ , then for Q4 the X ₄₇ and–(S ^P ) _D –R ⁸⁷ are not a group in accordance with the definition of X ₄₆ .

In preferred embodiments Ya equals Yb.

In preferred embodiments Y _a is selected from Y ₁ , Y ₂ , Y ₃ , Y ₄ or Y ₅ and Y _b is hydrogen, X ₄₇ or–(S ^P ) _D –R ⁸⁷ . In preferred embodiments Y _a is selected from Y1, Y2, Y3, Y4 or Y5 and Yb is hydrogen. In preferred embodiments the compound according to Formula (14) does not comprise–(S ^P ) _D –R ⁸⁷ .

In preferred embodiments, X50 is hydrogen.

In preferred embodiments when X45 or X46 is N(X50)2, then one X50 is hydrogen and one X ₅₀ is X ₄₈ or–(S ^P ) _D –R ⁸⁷ .

In preferred embodiments Formula (14) does not comprise X46. In preferred embodiments, both Q ₁ in Formula (14) are X ₄₅ . In preferred embodiments, both Q2 in Formula (14) are X46. In preferred embodiments, both Q5 in Formula (14) are X45. In preferred embodiments, both Q4 in Formula (14) are X ₄₆ . X45

In a preferred embodiment, each X45 individually is selected from the group consisting of N(X ₅₀ ) ₂ , NX ₅₀ C(O)X ₅₁ , NX ₅₀ C(S)X _51, OH, SH, NX ₅₀ C(O)OX _51, NX50C(S)OX51, NX50C(O)SX51, NX50C(S)SX51, NX50C(O)N(X51)2,

NX ₅₀ C(S)N(X ₅₁ ) ₂ , NX ₅₀ SO ₂ X _51, NX ₅₀ SO ₃ X _51, NX ₅₀ OX ₅₁ .

In a preferred embodiment, each X ₄₅ individually is selected from the group consisting of N(X50)2, NX50C(O)X51, NX50C(S)X51, OH and SH.

In a preferred embodiment, each X ₄₅ individually is selected from the group consisting of NX ₅₀ C(O)OX _51, NX ₅₀ C(S)OX _51, NX ₅₀ C(O)SX _51,

NX50C(S)SX51, NX50C(O)N(X51)2, NX50C(S)N(X51)2, NX50SO ₂ X51, NX50SO ₃ X51, NX ₅₀ OX ₅₁ .

In a preferred embodiment, X45 is selected from the group consisting of NHX50, C(X51)2NH ₂ , CHX51NH ₂ , CH ₂ N(X50)2, CH ₂ NHX50, NHC(O)X51, NHC(S)X ₅₁ , OH, and SH.

In a preferred embodiment, X45 is NHX50. In a preferred embodiment, X45 is C(X ₅₁ ) ₂ NH ₂ . In a preferred embodiment, X ₄₅ is CHX ₅₁ NH ₂ . In a preferred embodiment, X ₄₅ is CH ₂ N(X ₅₀ ) ₂ . In a preferred embodiment, X ₄₅ is

CH ₂ NHX50. In a preferred embodiment, X45 is NH ₂ . In a preferred

embodiment, X ₄₅ is CH ₂ NH ₂ . In a preferred embodiment, X ₄₅ is NHC(O)X ₅₁ . In a preferred embodiment, X45 is NHC(S)X51. In a preferred embodiment, X45 is OH. In a preferred embodiment, X45 is SH. X ₄₆

In a preferred embodiment, X46 is individually selected from the group consisting of N(X50)2, NX50C(O)X51, NX50C(O)OX51, andNX50C(O)N(X51)2,. In a preferred embodiment, X ₄₆ is selected from the group consisting of N(X ₅₀ ) ₂ , and NX ₅₀ C(O)X _51, . In a preferred embodiment, X ₄₆ is selected from the group consisting of NHX50 and NHC(O)X51. In a preferred embodiment, X46 is NHX50. In a preferred embodiment, X46 is NH ₂ .In a preferred embodiment, X ₄₆ is NHC(O)X _51. X47

In a preferred embodiment, each X ₄₇ is individually selected from the group consisting of F, -OH, -NH ₂ , -SO ₃ -, -NO ₂ , -CF ₃ , -SH, C ₁ -C ₆ alkyl groups, C ₆ aryl groups, C ₄ -C ₅ heteroaryl groups, C ₅ -C ₈ alkyl(hetero)aryl groups, C ₅ -C ₈ (hetero)arylalkyl groups, C ₄ -C ₈ alkylcycloalkyl groups, and C ₄ -C ₈

cycloalkylalkyl groups. In a more preferred embodiment, each X47 is individually selected from the group consisting of F, -SO ₃ - _, -NO ₂ , -CF ₃ , C ₁ -C ₆ alkyl groups, C ₆ aryl groups, C ₄ -C ₅ heteroaryl groups, C ₅ -C8

alkyl(hetero)aryl groups, C ₅ -C8 (hetero)arylalkyl groups, C ₄ -C8

alkylcycloalkyl groups, and C ₄ -C ₈ cycloalkylalkyl groups. In a preferred embodiment, the X47 substituents do not contain heteroatoms. In a preferred embodiment, the X47 groups are not substituted. In another preferred embodiment, the X ₄₇ groups do not contain heteroatoms. X48

In a preferred embodiment, each X ₄₈ is independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C ₂ -C ₄ alkenyl groups, and C ₄ -6 (hetero)aryl groups. For X48 the alkyl groups, alkenyl groups, and (hetero)aryl groups are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , =O, -SH, -SO ₃ H, -PO ₃ H, - PO ₄ H ₂ and -NO ₂ ; and optionally contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized.

In a preferred embodiment, X48 is C ₁ -C ₄ alkyl. In a preferred embodiment, the X ₄₈ substituents do not contain heteroatoms. In a preferred embodiment, the X ₄₈ groups are not substituted. In another preferred embodiment, the X48 groups do not contain heteroatoms. X49

In preferred embodiments, X ₄₉ is selected from the group consisting of hydrogen, C ₁ -C8 alkyl groups, C2-C8 alkenyl groups, C2-C8 alkynyl groups, C ₆ -C ₁₂ aryl, C ₂ -C ₁₂ heteroaryl, C ₃ -C8 cycloalkyl groups, C ₅ -C8 cycloalkenyl groups, C ₃ -C ₁₂ alkyl(hetero)aryl groups, C ₃ -C ₁₂ (hetero)arylalkyl groups, C ₄ - C ₁₂ alkylcycloalkyl groups, C ₄ -C ₁₂ cycloalkylalkyl groups, C ₅ -C ₁₂

cycloalkyl(hetero)aryl groups and C ₅ -C ₁₂ (hetero)arylcycloalkyl groups, wherein the X ₄₉ groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , - SO ₃ H, -PO ₃ H _, -PO ₄ H _2, -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.

In preferred embodiments, X49 is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl groups, C2-C ₄ alkenyl groups, C2-C ₄ alkynyl groups, C ₆ -C ₈ aryl, C ₂ -C ₈ heteroaryl, C ₃ -C ₆ cycloalkyl groups, C ₅ -C ₆ cycloalkenyl groups, C ₃ -C ₁ 0 alkyl(hetero)aryl groups, C ₃ -C ₁ 0 (hetero)arylalkyl groups, C ₄ - C ₈ alkylcycloalkyl groups, C ₄ -C ₈ cycloalkylalkyl groups, C ₅ -C ₁₀

cycloalkyl(hetero)aryl groups and C ₅ -C ₁₀ (hetero)arylcycloalkyl groups, wherein the X49 groups not being hydrogen are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH ₂ , - SO ₃ H, -PO ₃ H, -PO ₄ H ₂ , -NO ₂ , -CF ₃ , =O, =NH, and -SH, and optionally contain one or more heteroatoms selected from the group consisting of O, S, NH, P, and Si, wherein the N, S, and P atoms are optionally oxidized, wherein the N atoms are optionally quaternized.