(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/004338 Al 04 January 2018 (04.01.2018) W !P O PCT (51) International Patent Classification: o Vondellaan 11, 561 1 NX Eindhoven (NL). HOEBEN, A61K 47/54 (2017.01) B82Y 5/00 (201 1.01) Freek Johannes Maria; c/o Vondellaan 11, 561 1NX Ein d A61K 47/69 (2017.01) C07D 257/08 (2006.01) hoven (NL). VERSTEEGEN, Ronny Mathieu; c/o Von dellaan 11, 561 1NX Eindhoven (NL). JANSSEN, Hendri- (21) International Application Number: cus Marie; c/o Vondellaan 11, 561 1 NX Eindhoven (NL). PCT/NL20 17/050427 VAN ONZEN, Arthur Henry Antoon Marie; c/o Vondel (22) International Filing Date: laan 11, 561 1NX Eindhoven (NL). ROSSIN, Raffaella; c/ 27 June 2017 (27.06.2017) o Vondellaan 11, 561 1 NX Eindhoven (NL). (25) Filing Language: English (74) Agent: JANSEN, CM.; V.O., Carnegieplein 5, 2517 KJ Den Haag (NL). (26) Publication Language: English (81) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of national protection available): AE, AG, AL, AM, 16176416.2 27 June 2016 (27.06.2016) EP AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (71) Applicant: TAGWORKS PHARMACEUTICALS B.V. CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, [NL/NL]; Vondellaan 11, 561 1 NX Eindhoven (NL). DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (72) Inventors: ROBILLARD, Marc Stefan; c/o Vondellaan KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 11, 561 1 NX Eindhoven (NL). TEN HOEVE, Wolter; c/ MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (54) Title: CLEAVABLE TETRAZINE USED IN BIO-ORTHOGONAL DRUG ACTIVATION Fig. 1 o (57) Abstract: Disclosed is an advancement in provoked chemical cleavage. Thereby the invention provides the use of a diene as a chemically cleavable group attached to a Construct, and the use of a dienophile to provoke the release of the Construct by allowing the diene to react with a dienophile capable of undergoing an inverse electron demand Diels Alder reaction with the diene. The invention o includes a kit for releasing a Construct C bound to a Trigger T , the kit comprising a tetrazine and a dienophile, wherein the Trigger © is the tetrazine. The invention also includes the use of the formation of a pyridazine by reacting a tetrazine comprising a Construct 00 C bound thereto and a dienophile, as a chemical tool for the release, in a chemical, biological or physiological environment, of said o Construct. o [Continued on nextpage] WO 2018/004338 Al llll II II 11III II I II II i II 11III II I II OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: CLEAVABLE TETRAZINE USED IN BIO-ORTHOGONAL DRUG ACTIVATION FIELD OF THE INVENTION The invention pertains to the field of provoked chemical cleavage. Particularly, the invention pertains to providing chemical substances with a functional group capable of acting as a Trigger for release of an entity attached to said chemical group. This plays a role, e.g., in drug delivery, prodrug activation, modification of molecules under physiological conditions (e.g., in a cellular environment), in solid phase synthesis, biomolecule interactions, modification of surfaces (including cell surfaces). BACKGROUND The current state of the art in organic chemistry enables the preparation of highly complex molecular structures, by application of a wide toolbox of synthetic transformations. Unfortunately, the vast majority of chemical techniques is executed under strictly defined conditions, requiring toxic solvents, stoichiometric reagents, extreme temperatures, exclusion of moisture or oxygen, and with carefully designed protective group protocols. One of the most difficult challenges in synthetic chemistry remains the ability to have precise control over chemical reactivity and selectivity. These demands are amplified when it is necessary to perform selective reactions in chemically complex environments, such as those found in biology. Unfortunately, only a few chemical transformations are so mild and precise that they can be used to selectively modify biochemical structures, such as proteins or nucleic acids, which typically proceed in water and at near-ambient temperature. Moreover, such biomolecular modification must be highly chemospecific in the sense that only a single functionality of interest is selectively modified in the presence of a plethora of other functional groups. Even more challenging is to apply a chemical reaction on or in living cells or whole organisms. 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. Truly bio-orthogonal reactions are not yet available as each reaction so far is orthogonal to a subset of the functionalities found in biological systems. Thus, extremely selective and high-yielding bio-orthogonal coupling chemistry reactions continue to receive interest. Bio-orthogonal reactions are broadly useful tools with applications that span synthesis, materials science, chemical biology, diagnostics, and medicine. They are generally used in coupling reactions of small molecules, peptides, proteins, oligonucleotides, other types of polymers, glycans, nanoparticles, and on surfaces (e.g., glass slides, gold, resins). Further examples include: compound library synthesis, protein engineering, functional proteomics, activity-based protein profiling, target guided synthesis of enzyme inhibitors, chemical remodeling of cell surfaces, tracking of metabolite analogues, and imaging tagged biomolecules in live cells and animals. Analogous to selective coupling reactions, chemoselective cleavable linkers and protecting groups have widespread application in the manipulation of conjugates and derivatives of: small molecules, polymers, and biomolecules such as peptides, proteins and DNA within the context of chemical synthesis, materials science, chemical biology, diagnostics, and medicine. [G.C. Rudolf, W. Heydenreuter, S.A. Sieber, Curr. Opin. Chem. Biol. 2013, 17, 110-117] For example, linker systems that allow mild cleavage under conditions ideally orthogonal to functionalities present in the biological system at hand have found application in activity- based protein profiling. Protease substrates linked to a biotin tag through a cleavable linker have been used to capture and isolate specific enzymes by the binding of the biotin tag to avidin coated resin or beads. Subsequently the captured complex can then be released under mild conditions by cleavage of the linker instead of having to break the biotin-avidin interaction. Also, chemically cleavable linkers have emerged as powerful tools in solid-phase organic synthesis, especially for the rapid production of highly diverse organic compound libraries created through combinatorial or parallel chemistry methods. Especially prominent application areas for cleavable linkers 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. However, chemoselective cleavable linkers and protecting groups have a limited orthogonality compared to the relatively highly bio-orthogonal coupling reactions listed above and with respect to the wide range of functionalities present in the complex environments encountered e.g. in vivo, in biological media, biomolecules, or complex materials. A wider and more uniform application scope may be achieved for chemoselective linkers and protecting groups if the aforementioned bio-orthogonal coupling reactions could be adapted to effect selective release instead of selective conjugation. We have demonstrated that the fastest click reaction, the inverse-electron-demand Diels Alder (IEDDA) reaction, can be used for pretargeted radioimmunoimaging, treating tumor-bearing mice with irafts-cyclooctene(TCO)-tagged antibody or antibody fragments, followed 1-3 days later by administration and selective conjugation of a radiolabeled tetrazine probe to the TCO tag of the tumor-bound antibody [Rossin, M. S. Robillard, Curr. Opin. Chem. Biol. 2014, 21, 161-169]. Based on the IEDDA conjugation we have recently developed a new release reaction, which we 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. We 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 C0 2 and Dox via a novel 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. Aforementioned technology has also been disclosed in WO2012/156919, WO2012156918A1, WO 2014/081303, and US20 150297741. Herein a dienophile is used as a chemically cleavable group.