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EP-3887359-B1 - NOVEL TETRAZINE COMPOUNDS FOR IN VIVO IMAGING

EP3887359B1EP 3887359 B1EP3887359 B1EP 3887359B1EP-3887359-B1

Inventors

  • KJAER, ANDREAS
  • PETERSEN, Ida Nymann
  • HERTH, Matthias Manfred
  • KRISTENSEN, Jesper Langgard

Dates

Publication Date
20260506
Application Date
20191129

Claims (15)

  1. A tetrazine compound having the following formula I: wherein one of R 1 -R 5 is 18 F, at least two of the remaining R 1 -R 5 are H, and the other remaining R 1 -R 5 are the same or different and are selected from H, alkyl, halogen, - CF 3 , -CN, -O-alkyl, -S-alkyl, -NH-alkyl, -N(alkyl) 2 , -NH(C=O)-alkyl, -N-alkyl-(C=O)-alkyl, -SO 2 -alkyl, -SO 2 -NH 2 , -SO 2 -NHalkyl, -SO 2 -N(alkyl) 2 -C(=O)-NH 2 , -C(=O)-NH-alkyl, -C(=O)-N(alkyl) 2 , -C(=O)-OH, - C(=O)-O-alkyl, -CH 2 -NH 2, - CH 2 -NH-alkyl, -OH, CH 2 -O-alkyl, CH 2 -O-aryl, CH 2 -O-phenyl, CH 2 -O-naphthyl, wherein n is an integer from 1 to 4, and R 6 is selected from H, CH 3 , phenyl, wherein the curly bond indicates the link to the tetrazine moiety.
  2. A compound according to claim 1, wherein alkyl is selected from linear or branched C 1 -C 6 alkyl, cyclic C 3 -C 6 alkyl, optionally substituted with -OH, -NH 2 or halogen.
  3. A compound according to claim 1 or 2, wherein alkyl is selected from linear or branced methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert. butyl, pentyl, and hexyl.
  4. A compound according to any of the preceding claims, wherein halogen is selected from I, Br, or Cl.
  5. A compound according to any of the preceding claims selected from the compounds, wherein: a) R 1 is 18 F and all other R's are H; or b) R 2 is 18 F and all other R's are H; or c) R 3 is 18 F and all other R's are H.
  6. A compound according to any of claims 1-4, selected from compounds wherein: a) R 1 is 18 F and R 2 , R 3 , R 4 , and R 5 are H, and R 6 is H, or or b) R 2 is 18 F and R 1 , R 3 , R 4 , and R 5 are H, and R 6 is H, or c) R 3 is 18 F and R 1 , R 2 , R 4 , and R 5 are H, and R 6 is H or
  7. A tetrazine precursor having the formula III: wherein one of R 1 -R 5 is SnR 3 , B(OR) 2 , B(OH) 2 . R is a linear or branched C 1 -C 6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, optionally substitute with -OH, -NH 2 or halogen; and at least two of the remaining R 1 -R 5 are H, and the other remaining R 1 -R 5 are the same or different and are selected from H, alkyl, halogen, - CF 3 , -CN, -O-alkyl, - S-alkyl, -NH-alkyl, -N(alkyl) 2 , -NH(C=O)-alkyl, -N-alkyl-(C=O)-alkyl, -SO 2 -alkyl, -SO 2 -NH 2 , -SO 2 -NHalkyl, -SO 2 -N(alkyl) 2 -C(=O)-NH 2 , -C(=O)-NH-alkyl, -C(=O)-N(alkyl) 2 , -C(=O)-OH, -C(=O)-O-alkyl, -CH 2 -NH 2, - C-NH-alkyl, -OH, CH 2 -O-alkyl, CH 2 -O-aryl, CH 2 -O-phenyl, CH 2 -O-naphthyl wherein n is an integer from 1 to 4, and R 6 is selected from H, CH 3 , phenyl, wherein the curly bond indicates the link to the tetrazine moiety.
  8. A compound as defined in any of claims 1-6 for use in bioorthogonal chemistry.
  9. A compound as defined in any of claims 1-6 for use in diagnostics.
  10. A compound as defined in any of claims 1-6 for use in in vivo imagining.
  11. A compound for use as defined in any of claims 8-10, wherein the compound penetrates the blood-brain-barrier.
  12. A method for preparing a compound as defined in any of claims 1-6, the method comprises converting a suitably functionalized benzonitrile having the general formula to the corresponding tetrazine having the general formula where R represents R 1 to R 6 as defined in claim 1 by reacting it with R 1 -CN and NH 2 NH 2 H 2 O by first stirring at 60°C for 24h in the presence of Zn(OTf) 2 and then adding NaNO 2 and HCl.
  13. A method according to claim 12 comprising reacting with wherein R 1 -R 6 are as defined in claim 1, in the presence of hydrazine monohydrate to obtain a compound according to any of claims 1-6.
  14. A method according to claim 12, wherein the reaction is carried out at a temperature range of from 50 to 70 °C.
  15. A method according to claim 13, wherein water is added after cooling to room temperature followed by addition of HCl and extraction with EtOAc.

Description

"The project leading to this patent application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668532." Field of the invention The present invention is within the field of bioorthogonal chemistry and relates to novel tetrazine compounds for use in pretargeted in vivo imaging. The compounds are suitable for use in click chemistry, i.e. reactions that join a targeting molecule and a reporter molecule. Background of the invention Click chemistry has emerged as a versatile tool for pretargeted imaging, radiotherapy and recently also for specific drug release in vivo. Click chemistry is of particular interest in bioorthogonal chemistry. Bioorthogonal chemistry refers to any chemical reaction that can occur inside living systems without interfering with native biochemical processes. A pretargeting strategy makes use of bioorthogonal chemistry and proceeds in two steps. A first step is where a substrate is modified with a bioorthogonal functional group (denoted chemical reporter or target vector) and introduced to the patient. Normally, a substrate can be a metabolite, an enzyme inhibitor, monoclonal antibody, nanomedicine, polymer, nanoparticle, etc. The second step is where a probe, that contains the complementary functional group, is introduced and reacts and labels the substrate. The probe is a small effector molecule carrying the label (payload). Figure 1 displays the principle. Especially, the tetrazine ligation based on highly reactive tetrazines (Tzs) and trans-cy-clooctenes (TCO's)) has been investigated as candidates for pretargeted strategies due to their extremely rapid reaction kinetics sans catalyst. WO2012012612 describes the Tetrazine-trans-cyclooctene system and the synthesis of three 18F tetrazines. However, their low yield led to the conclusion that the 18F labelling should be on the trans cyclooctene. The present invention has overcome the yield challenges in WO2012012612 and various new compounds have been invented. As mentioned above, a pretargeted strategy involves a vector tagged with a reactive moiety. The vector accumulates at the target site after administration. Subsequently, a small effector molecule carrying a payload of interest (e.g. a radiolabelled probe) is administered. A ligation reaction (click chemistry) between the targeting vector and the small effector molecule carrying the payload takes place in vivo, whereby the payload is coupled to the target of interest. When target vectors such as monoclonal antibodies (mAbs), polymers or nanoparticles (NPs) are applied, advantages in respect to nuclear medicine applications can be obtained. The advantages include improved imaging contrast (up to 100-fold when additional blood circulating targeting vector is deactivated or removed), lower radiation burden to healthy tissue, and maximized therapeutic doses within the target region compared to more traditional approaches. The advantages are mainly a result of a two-step process, where the first step is the slow targeting process of the vector and the second step is a rapid targeting process of the payload. Positron-Emission-Tomography (PET) is a powerful and routinely used diagnostic imaging tool in precision medicine. This is because it is highly sensitive, it offers isotropism, and it is quantifiable, i.e. it can be used to quantify the amount of nanomedicine delivered to the target region. Fluorine-18 (18F) is considered as the "gold standard" PET radionuclide for clinical applications. It is ideal because of its relatively short positron range (2.4 mm max. range in water), good branching ratio (96.7 % positron decay) and its half-life (t1/2 = 110 min), resulting in good resolution, relatively low radiation burden and ability to distribute within a several hundred kilometres range. Consequently, in recent years there have been several efforts to develop 18F-imaging agents for tetrazine (Tz) ligation-based pretargeted imaging. Initially, TCO's have been explored for 18F-labeling strategies. However, it seems as if TCOs are better suited to be attached to the targeting vector itself rather than used as a small effector molecule. Tzs with relatively low reactivity are suitable for 18F-aliphatic substitution (SN2), whereas access to higher reactive and more clinically relevant structures was not reachable. To address this problem, multi-step synthon-based 18F-labeling procedures have been developed. However, none of these procedures appears to be optimal suited for clinical applications since multi-step procedures are usually challenging to set up for clinical routine. Recently, chelator approaches to label Tz's with Al[18F]F have been successfully explored. However, these strategies exclude most likely targets beyond cell membranes or the blood-brain-barrier since passive diffusion is limited due to the polar chelator character of these structures. Furthermore, targets that are located extracellular could be difficu