New cryptate-type (bicyclic) chelators and their use as ligands for MN(II)-based MRI contrast agents, 52MN-based pet diagnostic agents, and 64 / 67cu(II)-isotope-based pet diagnostic and therapeutic agents

Abstract

Magnetic resonance imaging (MRI) has been the most important and most advanced diagnostic imaging modality of the past decade. The advantages of the method include, among others, high resolution and good differentiation of soft tissues. Contrast agents (CAs) are used for the most of examinations. Currently, only Gd(III)-based MRI CAs are available for human clinical applications. However, the use of toxic Gd(III)-ion-based agents can cause a serious problem; Gd(III) is not completely eliminated from the body even in patients with satisfactory renal function. As an alternative, the use of Mn(II) complexes has come to the fore. However, Mn(II) ion can cause neurodegenerative changes in the case of prolonged exposure or high doses, which is associated with symptoms similar to Parkinson's disease. It is therefore advisable to use a contrast agent containing Mn(II) ions that does not dissociate or dissociates only to a very small extent during its stay in the body. The subject of the present patent application is cryptate-type ligands obtained by the fusion of two macrocyclic compound families that individually perform satisfactorily for Mn(II) complexation.

Description

[0001] New cryptate-type (bicyclic) chelators and their use as ligands for Mn(II)-based MRI contrast agents,52Mn-based PET diagnostic agents, and64 / 67Cu(II)-isotope-based PET diagnostic and therapeutic agents

[0002] The subject of the invention is the production of new cryptate-type (bicyclic) chelators and their use as ligands for production of Mn(II)-based MRI contrast agents,52Mn(II) or64Cu(II) isoptope-based PET diagnostic agents and67Cu(II)-isopote-based therapeutic agents, which are formed during the crosslinking of 3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca- 1(14), 11(15), 12-triene, 2,11-diaza[3.3](2,6)-pyridinophane and 6-oxa-3,9,15-triaza- bicyclo[9.3.1]pentadeca-1(14),11(15),12-triene 12-membered macrocycles below in 1 : 1 or 2:2 type ring closure reactions.

[0003] Magnetic resonance imaging (MRI) has been the most important and most advanced diagnostic imaging modality of the past decade. The advantages of the method include the high resolution and good differentiation of soft tissues, which is based on the difference in the relaxation times of the protons that make up the tissues. Contrast agents (CAs) are used for most examinations, which can improve imaging as well as reveal details that may be hidden or indistinguishable. Further advantage of using CAs is that the time required for the test is significantly reduced. MR imaging is based on the physical basis of the relaxation of1H nuclei in the body following excitation by radiofrequency in a strong magnetic field. The relaxation time varies depending on the chemical environment of the protons, which will result in contrast in the images between different tissues. Commonly used extracellular CAs increase signal intensity as a function of tissue water content, which is achieved by accelerating the relaxation of water protons. This can be achieved by the use of paramagnetic materials, which are most likely to be paramagnetic metal ions such as Gd(III), Mn(II) or Fe(III). However, large amounts of free metal ions are toxic to the living organism, and are therefore usually "packaged" in chelates by complexing agents. At present, only Gd(III)-based MRI CAs are available for human clinical use.

[0004] The success story of Gd(III)-based CAs has been overshadowed by several problems over the past two decades. On the one hand, the disease nephrogenic systemic fibrosis (NSF), discovered in the mid-2000s and associated with the use of Gd(III)-containing CAs in patients with severe kidney disease, highlighted that even with a strict set of requirements, serious problems can arise from the use of toxic Gd(III)-ion-based agents. These problems could have been remedied by a more careful use of the CAs and by the warnings that were placed on their packaging. However, in the mid-2010s, it was also confirmed that even in patients with satisfactory renal function, Gd(III) is not completely cleared from the body, creating hyperintense regions in certain tissues (e.g. the brain). In addition to these problems, the increase in Gd(III) content in surface waters recognised in the 1990s (which became known as the “gadolinium anomaly”) resulted in the authorities (Food and Drug Administration - FDA, European Medicines Agency - EMA, etc.) suspending the use of or withdrawing from the market several open chain MRI CA compounds. In parallel, intensive research has been launched to find alternatives to Gd(III). The use of Mn(II) complexes as MRI CAs is not new. The central paramagnetic metal ion of Mangafodipir (Teslascan), which is also used in practice, was also a Mn(II) ion, which could be used primarily in liver diagnostics due to the different uptake of Mn(II) by healthy and abnormal liver cells. In the case of the contrast agent Mangafodipir, the Mn(II) ions released after the dissociation of the complex are absorbed due to the low kinetic inertness of the complex. However, despite the endogenous nature of the Mn(II) ion, literature data indicate that prolonged exposure or high doses may cause neurodegenerative changes, which are associated with symptoms similar to Parkinson's disease. It is therefore advisable to use a contrast agent containing Mn(II) ions that does not dissociate or dissociates only to a very small extent during its stay in the body.

[0005] The renewed research is primarily focused on fine-tuning the physicochemical parameters of the complexes (stability, inertness) and parameters related to their efficiency (e.g. the effect on the relaxation rate, which can be defined as the relaxivity with respect to a 1 mM concentration of paramagnetic particles). In connection with this research, our research group produced and patented trans-CDTA-bis(amide)-type chelators (WO2016135234 - ETHYLENEDIAMINETETRAACETIC ACID BIS(AMIDE) DERIVATIVES AND THEIR RESPECTIVE COMPLEXES WITH MN(II) ION FOR USE AS MRI CONTRAST AGENT). These open-chain complexing agents have significantly improved the inertness of Mn(II) chelates (which can be best illustrated by the rate constant for acid-mediated dissociation and / or the half-lives calculated for the given conditions). The transdiaminocyclohexane platform is also used to develop the [Mn(PyC3A)]- chelate, which entered Phase I clinical trials in 2022 with the support of Reveal Pharmaceuticals (EricM. Gale, Iliyana P. Atanasova, Francesco Blasi, Ilknur Ay, and Peter Caravan, J. Am. Chem. Soc. 2015, 137, 49, 15548 15557). Significant improvements were achieved with macrocyclic chelators, including the 12-membered 3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca- 1(14), 11(15),12-triene (piclene), 2,11-diaza[3.3](2,6)-pyridinophane (bispiclene), 6-oxa-3,9,15-triaza-bicyclo[9.3.1]pentadeca- 1(14), 11(15), 12-triene (O-piclene), and the 15-membered 3,12,18-triaza-6,9- dioxabicyclo[12.3.1]octadeca-1(18),14,16-triene (15-PyN3O2) macrocycles {3,6,9,15- tetraaza-bicyclo [9.3.1]pentadeca-l(14), 11(15), 12-triene based compounds and their application as ligands of essential metal ion based MRI and 52MN based PET contrast agents Patent number: 10568976; 2,11-diaza-[3.3](2,6)pyridinophane compounds and their application as ligands of essential metal ion based MRI contrast agents and 52MN based PET contrast agents Patent number: 10968224 and NEW 6-0X4- 3, 9, 15 -TRIAZA - BICYCLO [9.3.1]PENTADECA-1(14),11(15), 12-TRIENE DERIVATIVES BASED

[0006] COMPOUNDS AND THEIR APPLICATION AS LIGANDS OF ESSENTIAL METAL ION BASED MRI AND 52MN BASED PET CONTRAST AGENTS Publication number: 20180354969 and Inorg. Chem. 2010, 49, 7, 3224-3238 https: / / doi.org / 10.1021 / ic9020756). Piclen-based Mn(II) chelates have also been patented by General Electric (WO2018115314 - TETRAAZABICYCLO-MACROCYCLE BASED MANGANESE CHELATE COMPOUNDS SUITABLE AS MRI IMAGING AGENTS).

[0007] The best parameters in terms of inertness of the complexes for the complexation of the Mn(II) ion were obtained with the so-called bispidine-type chelators (the rate constant characterizing the proton-assisted dissociation was 1.6x10-3M-1s-1, which was obtained as 2.2x10-3M-1s-1in the studies performed with the Zn(II) exchange metal ion) [Angew. Chem. Int. Ed. 2020, 13;59(29): 11958-11963. doi: 10.1002 / anie.202003685.]. However, these chelates formed significantly more slowly than usual, and it was also observed that some of the chelates formed dissociated relatively quickly (an observation that was answered in a recent publication), which depends on the conformation of the bispidine rings [Chem. Eur. J. 2023, 29(62) e202301880, https: / / doi. org / 10. 1002 chem.202301880] .

[0008] The subject of the present invention is the cryptate-type ligands obtained by the fusion of two macrocyclic compound families (the 12-membered piclene, bispiclene and O-piclene and the 15-membered 15-PyN5and 15-PyN3O2), which individually perform satisfactorily for Mn(II) complexation, and which, when "built into each other", result in cryptate-type chelators whose Mn(II) and Cu(II) complexes have unexpected kinetic inertness, surpassing the most inert Mn(II) complexes known from the literature. Two derivatives belonging to this family of compounds have been previously prepared (Helv. Chim Acta, 1988, 71(5), 1042- 1052 https: / / doi. org / 10.1002 / hlca.19880710515 and Tetrahedron Letters, 2003, 44(27), 5087- 5089 https: / / doi.org / 10.1016 / S0040-4039(03)01150-X), however, their complexes have not been studied, so their properties cannot be deduced from the properties of other complexes or the starting parent ligands.

[0009] The subject of the present invention is 1. Mn(II) or Cu(II), preferably Mn(II) complexes of the compound of general formula (I) wherein Z is absent or CH2;

[0010] R1and R1’ is H, or together with R3and R3’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring;

[0011] R2and R2’ are H, or together with R4and R4’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring; R3and R3’ are H, or combined with R1and R1’ groups as described above;

[0012] R4and R4’ are H, or combined with R2and R2’ groups as described above; or R1, R1’, R2, R2’, R3, R3’, R4and R4’ and -C-Y-C-Z-C-Y-C- moiety to which they are attached form 2,2'-bipyridyl or 1,10-phenatroline; each Y is O or NR9; R5and R5’ are H, or R6and R6’ groups and -C-X-C- moiety to which they are attached form a 6-membered heteroaryl ring;

[0013] R6and R6’ are H, or combined with R5and R5’ groups as described above;

[0014] X is O or NR10;

[0015] R7and R8together form a bond, or two compounds of formula (I) form a dimer and R7forms a bond with R8group of a second compound of formula (I), and in this case R8forms a bond with R7group of this second compound; Z, Y, X and RkR10are identical in the two compounds of formula (I);

[0016] R9is absent or H;

[0017] R11, R12and R13are independently each other H, C1-C4 alkyl, CF3, OH, OC1-C4 alkyl, -CN, NH2, NH(C1-C4 alkyl), N(C1-C4 alkyl)2, benzyl, phenyl, p-ethoxybenzyl, or pharmaceutically acceptable salts, hydrates, solvates and isomers thereof. 2. The complex according to point 1, wherein the compound of formula (I) is the compound of formula (1-1)

[0018] 5 compound of formula (1-2)

[0019]

[0020] 4. The complex according to any one of points 1 to 3, wherein

[0021] R1, R1’, R2, R2’, R3, R3’, R4and R4’ are H, or

[0022] R1and R1’ together with R3and R3’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring, and R2and R2’ together with R4and R4’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring; or

[0023] R1, R1’, R2, R2’, R3, R3’, R4and R4’ and -C-Y-C-Z-C-Y-C- moiety to which they are attached form 1,10-phenatroline.

[0024] 5. The complex according to any one of points 1 to 4, wherein R1and R1’ together with R3and R3’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring, and R2and R2’ together with R4and R4’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring; wherein the 6-membered heteroaryl ring is preferably pyridine; or

[0025] R1, R1’, R2, R2’, R3, R3’, R4and R4’ and -C-Y-C-Z-C-Y-C- moiety to which they are attached form 2,2'-bipyridyl or 1,10-phenatroline; and

[0026] Y is N; and

[0027] Z is absent.

[0028] 6. The complex according to any one of points 1 to 4, wherein

[0029] R1, R1’, R2, R2’, R3, R3’, R4and R4’ are H, and Y is O or NH, and

[0030] Z is absent or CH2.

[0031] 7. The complex according to point 6, wherein

[0032] Y is O or NH, and Z is absent; or

[0033] Y is O, and

[0034] Z is CH2.

[0035] 8. The complex according to any one of points 1 to 7, wherein

[0036] R5, R5’, R6and R6’ are H, and X is O or NR10; or R5and R5’ together with R6and R6’ groups and -C-X-C- moiety to which they are attached form a 6-membered heteroaryl ring, and X is N; wherein the 6-membered heteroaryl ring is preferably pyridine.

[0037] 9. The complex according to any one of points 1 to 8, wherein the compound of formula (I) is selected from the following:

[0038]

[0039]

[0040] The preparation of the compounds of the invention is illustrated in the following examples.

[0041] Synthesis examples

[0042] 1,2-Bis(2-iodoethoxy)ethane (2, [36839-55-1], TCI Chemicals); 3,7-dioxa-1,9- nonanediol (16, [67439-82-1], Merck KGaA); p-toluenesulfonyl chloride (17, [98-59-9], TCI Chemicals); 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, [74065-64-8], TCI Chemicals); 4- nitrobenzenesulfonyl chloride (37, [98-74-8], TCI Chemicals); 4-

[0043] (trifluoromethyl)benzenesulfonyl chloride (39, [2991-42-6], Alfa Aesar); 4- cyanobenzenesulfonyl chloride (41, [49584-26-1], Molar Chemicals Ltd.); 2- (chloromethyl)pyridine x HC1 (43, [6959-47-3], TCI Chemicals); 1-bromododecane (45, [143-15-7], Merck KGaA); 2-hydroxy-5-nitrobenzyl bromide (47, [772-33-8], TCI Chemicals); 2-bromomethyl-3 -hydroxypyridine x HBr (49, [87440-88-8], FluoroChem); 1- (bromomethyl)-4-ethoxybenzene (51, 2606-57-7], BLDpharm); methyl 4-

[0044] (bromomethyl)benzoate (53, [2417-72-3], Merck KGaA); tert-butyl piperazin- 1-yl carbamate (60, [147081-80-9], BLDpharm); 4-bromobutyryl bromide (61, [56489-06-6], Alfa Chemistry); 2-bromoacetyl bromide (66, [598-21-0], Merck KGaA); 2-chloroethanol (71, [107-07-3], Merck KGaA) and 2-(2-(tert-butoxycarbonyl)hydrazinyl)acetic acid (75, [115262- 99-2], Ambeed) are commercially available. 3,6-ditosyl-3,6-diazaoctane-1,8-di-(toluene-4- sulfonate) (9) [1]; 2,9-bis(chloromethyl)-1,10-phenanthroline (28) [2]; N-(phthalimido)- 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11-13-triene-6-ethanamine (33) [3]; 6-(2- nitroimidazol-l-yl)-hexan- 1-amine (56) [4]; CDPA (58) [5]; 2-(4'-aminophenyl)-6- methoxybenzothiazole (6-MeO-BTA-O) (65) [5]; 2-(4'-nitrophenyl)-6-methoxybenzothiazole (70) [6] were prepared according to methods known from the literature.

[0045] General description 1 (preparation of CBMC compounds by incorporating the crosslinking element):

[0046] The starting macrocycle (1 eq.) is weighed into a two-necked flask and dissolved in acetonitrile solvent. A solution of the base used (5 eq.) in 20 mL of water is added to the solution. The mixture is heated to 85 °C and stirred. A solution of the crosslinking reagent (1.1 eq.) in acetonitrile is weighed into a dropping funnel and added dropwise to the reaction over 1 hour. The reaction is then stirred at 85 °C, and the progress of the reaction is monitored by analytical HPLC. After the starting macrocycle has completely reacted, the solvent is removed from the reaction using a rotary vacuum evaporator. The evaporation residue is purified by preparative HPLC.

[0047] General description 2 (removal of tert-butoxycarbonyl protecting group (-Boc)):

[0048] The 6-Boc-CBPC derivatives were dissolved in 15 mL of dichloromethane in a flask and the reaction was cooled to 0 °C using an ice-water bath. 7 mL of trifluoroacetic acid was slowly added dropwise to the cold solution and then the reaction was stirred at room temperature for 30 min. The solvent was evaporated from the reaction, the residue was dissolved in a 1 :1 mixture of water and acetonitrile and the sample was freeze-dried. The product was isolated by preparative HPLC. Preparation of CB8O2PC (4)

[0049] The preparation of compound 6-Boc-CB8O2PC (3) was carried out according to general description 1, in which 200 mg of 6-Boc-piclene (1, 0.653 mmol, 1 eq.) (in 200 mL of acetonitrile) was used with 66 mg of 1,2-bis(2-iodoethoxy)ethane (2, 0.718 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 444 mg of sodium acetate (3.26 mmol, 5 eq.) (in 20 mL of water). The product obtained was a slightly yellow oil (120 mg, 43.7 % yield). 1H-NMR (360 MHZ, CD3OD)-. 8.15 (t, 1H), 7.62 (d, 2H), 5.08 (d, 2H), 4.77 (d, 2H), 4.18 (m, 2H), 4.05 (m, 4H), 3.92 (m, 4H), 3.76 (m, 2H), 3.66 (m, 4H), 3.58 (m, 2H), 2.99 (m, 2H), 1.58 (s, 9H).

[0050] ESI-MS (m / z): [M+Na]+calculated: 443.26; [M+Na]+measured: 443.21.

[0051] The preparation of compound CB8O2PC (4) was carried out according to general description 2 using 300 mg of 6-Boc-CB8O2PC (3, 0.713 mmol, 1 eq.). The product was obtained as a pale yellow oil (182 mg, 79.8 % yield). 1H NMR (360 MHz, CD3OD)-. 8.13 (t, 1H), 7.59 (d, 2H), 5.03 (d, 2H), 4.76 (d, 2H), 4.20 (m, 2H), 4.07 (m, 4H), 3.90 (m, 4H), 3.72 (m, 2H), 3.64 (m, 4H), 3.59 (m, 2H), 2.93 (m, 2H).

[0052] ESI-MS (m / z) : [M+Na]+calculated: 343.21; [M+Na]+measured: 343.21.

[0053] Preparation of CB8O2QPC (6)

[0054] The preparation of compound CB8O2OPC (6) was carried out according to general description 1, using 200 mg of O-piclene (5, 0.965 mmol, 1 eq.) (in 200 mL of acetonitrile) with 393 mg of 1,2-bis(2-iodoethoxy)ethane (2, 1.06 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 667 mg of potassium carbonate (4.82 mmol, 5 eq.) (in 20 mL of water). The product obtained was a colorless oil (230 mg, 74.1 % yield).1H NMR (360 MHz, CD3OD)-. 8.12 (t, 1H), 7.59 (d, 2H), 5.05 (d, 2H), 4.82 (d, 2H), 3.99 (m, 2H), 3.91 (t, 4H), 3.87-3.74 (m, 8H), 3.73-3.49 (m, 6H).

[0055] 13C-NMR (360 MHZ, CD3OD)-. 151.1 (2 x Cquat(aromatic)), 141.7, 124.2 (2+1 C(aromatic)),

[0056] 71.1, 64.6, 64.0, 59.2, 57.2, 56.7 (6 x 2 -CH2-)

[0057] ESI-MS (m / z)-. [M+Na]+calculated: 344.1944; [M+Na]measured: 344.1942.

[0058] Preparation of CB8O2BP (8)

[0059] The preparation of compound CB8O2BP (8) was carried out according to general description 1, using 200 mg of bispiclene (7, 0.833 mmol, 1 eq.) (in 200 mL of acetonitrile) with 339 mg of 1,2-bis(2-iodoethoxy)ethane (2, 0.916 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 1.36 g of cesium carbonate (4.16 mmol, 5 eq.) (in 20 mL of water). The obtained product (8) was a colorless oil (120 mg, 40.7 % yield).1H-NMR (400 MHZ, D2O): 7.52 (t, 2H), 7.07 (d, 4H), 4.87 (d, 4H), 4.53 (d, 4H), 4.01 (m, 4H), 3.89 (m, 4H), 3.83 (m, 4H).

[0060] ESI-MS (m / z): [M+H]+calculated: 355.2129; [M+H]+measured: 355.2128.

[0061] General description 3 (removal of p-toluenesulfonyl (hereinafter tosyl) protecting groups by acid hydrolysis):

[0062] The compounds containing tosyl groups were dissolved in 2 mL of concentrated sulfuric acid, and then the hydrolysis was carried out in a microwave reactor (30 min, 20 W, 120 °C). After the reaction, the sample was cooled to room temperature and then to approx. 8- 10 °C in a refrigerator. The cooled reaction was added dropwise to 100 mL of cold diethyl ether, the diethyl ether was decanted from the precipitated material. The solid was then dissolved in a 1 :1 mixture of water and acetonitrile and the pH of the sample was neutralized by adding NaOH solution. The product was isolated from the resulting solution by preparative HPLC technique. Preparation of CB8N2PC (11)

[0063] The preparation of compound Ts2-6-Boc-CB8N2PC (10) was carried out according to the general description 1, using 200 mg of 6-Boc-piclene (1, 0.653 mmol, 1 eq.) (in 200 mL of acetonitrile) with 549 mg of 3,6-ditosyl-3,6-diazaoctane-1,8-di-(toluene-4-sulfonate) (in 50 mL of acetonitrile) (9, 0.718 mmo1, 1.1 eq.) and 444 mg of sodium acetate (3.26 mmol, 5 eq.) (in 20 mL of water). The product was a white crystalline substance (105 mg, 22.8 % yield). 1H-NMR (360 MHZ, CDCl3): 7.75 (t, 1H), 7.64 (d, 4H), 7.43 (d, 4H), 7.23 (d, 2H), 5.01 (d, 2H), 4.84 (d, 2H), 4.08 (m, 2H), 3.98 (m, 4H), 3.84 (m, 4H), 3.76 (m, 4H), 3.62 (m, 2H), 3.48 (m, 2H), 2.78 (m, 2H), 2.42 (s, 6H), 1.58 (s, 9H).

[0064] ESI-MS (m / z): [M+H]+calculated: 727.3306; [M+H]+measured: 727.3305.

[0065] Compound CB8N2PC (11) was prepared according to the general description 3 using 100 mg of compound Ts2-6-Boc-CB8N2PC (10, 0.138 mmol, 1 eq.). The final product was a yellow oil (12.4 mg, 28.3 % yield). 1H NMR (360 MHz, CD3OD)-. 8.11 (t, 1H), 7.62 (d, 2H), 5.07 (d, 2H), 4.72 (d, 2H), 4.24 (m, 2H), 4.02 (m, 4H), 3.90 (m, 4H), 3.72 (m, 4H), 3.52 (m, 2H), 3.44 (m, 2H), 2.87 (m, 2H).

[0066] ESI-MS (m / z): [M+Na]+calculated: 341.2424; [M+Na]measured: 341.2424.

[0067] Preparation of CB8N2OPC (13)

[0068] The preparation of compound Ts2-CB8N2OPC (12) was carried out according to the general description 1, using 150 mg of O-piclene (5, 0.724 mmol, 1 eq.) (in 200 mL of acetonitrile) with 609 mg of 3,6-ditosyl-3,6-diazaoctane-1,8-di-(toluene-4-sulfonate) (in 50 mL of acetonitrile) (9, 0.796 mmol, 1.1 eq.) and 500 mg of potassium carbonate (3.62 mmol, 5 eq.) (in 20 mL of water). A white powder was obtained as the product (147 mg, 32.4 % yield).1H-NMR (360 MHZ, CDCl3): 7.62 (t, 1H), 7.52 (d, 4H), 7.34 (d, 4H), 7.25 (d, 2H), 5.01 (d, 2H), 4.80 (d, 2H), 3.99 (m, 2H), 3.90 (t, 4H), 3.79-3.63 (m, 8H), 3.62-3.41 (m, 6H), 2.38 (s, 6H).

[0069] ESI-MS (m / z) : [M+H]+calculated: 628.2622; [M+H]+measured: 628.2621.

[0070] Synthesis of compound CB8N2OPC (13) was carried out according to the general description 3 using 200 mg of compound Ts2-CB8N2OPC (12, 0.319 mmol, 1 eq.). The obtained product was a colorless oil (40.4 mg, 39.7 % yield).1H NMR (360 MHz, CD3OD): 8.17 (t, 1H), 7.62 (d, 2H), 5.10 (d, 2H), 4.87 (d, 2H), 4.03 (m, 2H), 3.96 (t, 4H), 3.89-3.72 (m, 8H), 3.71-3.47 (m, 6H).

[0071] ESI-MS (m / z)-. [M+Na]+calculated: 342.2264; [M+Na]measured: 342.2261.

[0072] Preparation of CB8N2BP (15)

[0073] Preparation of compound Ts2-CB8N2BP (14) was carried out according to general description 1, using 250 mg of bispiclene (7, 1.04 mmol, 1 eq.) (in 200 mL of acetonitrile) with 875 mg of 3,6-ditosyl-3,6-diazaoctane-1,8-di-(toluene-4-sulfonate) (in 50 mL of acetonitrile) (9, 1.14 mmol, 1.1 eq.) and 384 mg of lithium carbonate (5.20 mmol, 5 eq.) (in 20 mL of water). A slightly yellow powder was obtained as the product (285 mg, 41.4 % yield).1H NMR (360 MHz, CDCl3): 7.71 (t, 2H), 7.64 (d, 4H), 7.44 (d, 4H), 7.21 (d, 4H), 4.21 (d, 4H), 3.98 (d, 4H), 3.31-3.22 (m, 4H), 3.20-3.08 (m, 4H), 2.58-2.44 (m, 4H), 2.42 (s, 6H).

[0074] ESI-MS (m / z): [M+H]+calculated: 661.2625; [M+H]+measured: 661.2622.

[0075] Synthesis of compound CB8N2BP (15) was carried out according to the general description 3 using 200 mg of compound Ts2-CB8N2BP (14, 0.303 mmol, 1 eq.). The product was obtained as a yellow oil (33.5 mg, 31.4 % yield). 'H-NMR (360 MHz, CD3OD)-. 8.14 (t, 2H), 7.60 (d, 4H), 5.00 (d, 4H), 4.68 (d, 4H), 4.21-4.08 (m, 4H), 4.01-3.92 (m, 4H), 3.72-3.61 (m, 4H).

[0076] ESI-MS (m / z) -. [M+Na]+calculated: 375.2268; [M+Na]measured: 375.2266.

[0077] Preparation of l,9-ditosyl-3.,7-dioxa-l.,9-nonanediol (18)

[0078] (16) (17) (18)

[0079] 853 mg of solid NaOH (21.3 mmol, 3.5 eq.) was dissolved in 5 mL of water, to which solution was added 1.00 g of 3,7-dioxa-l,9-nonanediol (16, 6.09 mmol, 1 eq.). The reaction was cooled to 0 °C in an ice-salt bath, and a solution of 2.55 g of p-toluenesulfonyl chloride (17, 13.4 mmol, 2.2 eq.) in 10 mL of THF was slowly added with stirring. The reaction was stirred at room temperature for 1 day, and then 10 mL of water was added to the mixture. The reaction mixture was extracted with 3 x 25 mL of di chloromethane, and the collected organic phase was dried over Na2SO4. After filtering off the drying agent, the solvent was removed by rotary vacuum evaporator to give a colorless oil (2.20 g, 76.5% yield).1H-NMR (500 MHZ, CDCl3): 7.76 (d, 4H), 7.31 (d, 4H), 4.11 (t, 4H), 3.57 (t, 4H), 3.40 (t, 4H), 2.42 (s, 6H), 1.68 (quin., 2H).

[0080] ESI-MS (m / z) -. [M+H]+calculated: 473.1298; [M+H]+measured: 473.1296.

[0081] The preparation of compound 6-Boc-CB9O2PC (19) was carried out according to the general description 1, using 100 mg of 6-Boc-piclene (1, 0.326 mmol, 1 eq.) (in 300 mL of acetonitrile) with 170 mg of l,9-ditosyl-3,7-dioxa-1,9-nonanediol (18, 0.359 mmol, 1.1 eq.) (in 150 mL of acetonitrile) and 226 mg of potassium carbonate (1.63 mmol, 5 eq.). A colorless oil was obtained as the product (36 mg, 25.4 % yield).1H NMR (360 MHz, DMSO-d6): 7.81 (t, 1H), 7.22 (d, 2H), 4.05 (d, 2H), 3.91 (d, 2H), 3.47 (t, 4H), 3.34 (t, 4H), 3.05 (t, 4H), 2.61-2,54 (m, 8H), 1.62 (quin., 2H), 1.42 (s, 9H).

[0082] ESI-MS (m / z): [M+H]+calculated: 435.30; [M+H]+measured: 435.30.

[0083] The preparation of compound CB9O2PC (20) was carried out according to general description 2 using 35 mg of compound 6-Boc-CB9O2PC (19, 0.0805 mmol, 1 eq.). The product was a pale yellow oil (19 mg, 70.6 % yield).1H NMR (360 MHz, CD3OD): 8.17 (t, 1H), 7.60 (d, 2H), 5.14 (d, 2H), 4.92 (d, 2H), 3.88 (m, 4H), 3.70 (m, 4H), 3.61 (m, 4H), 3.57 (m, 4H), 2.98 (m, 4H), 1.92 (quan., 2H).

[0084] ESI-MS (m / z): [M+Na]+calculated: 357.23; [M+Na]+measured: 357.21.

[0085] Preparation of CB9O2QPC (21)

[0086] Compound CB9O2OPC (21) was prepared according to the general description 1. The reaction was carried out using 150 mg of O-piclene (5, 0.724 mmol, 1 eq.) (in 300 mL of acetonitrile), 376 mg of l,9-ditosyl-3,7-dioxa-l,9-nonanediol (18, 0.796 mmol, 1.1 eq.) (in 150 mL of acetonitrile), and 500 mg of potassium carbonate (3.62 mmol, 5 eq.). The product was a colorless oil (97 mg, 39.8 % yield).1H-NMR (360 MHZ, DMSO-d6): 1.11 (t, 1H), 7.28 (d, 2H), 4.01 (d, 2H), 3.84 (d, 2H), 3.41 (t, 4H), 3.28 (t, 4H), 3.01 (t, 4H), 2.64 (m, 4H), 2.51 (m, 4H), 1.69 (quin., 2H).

[0087] ESI-MS (m / z) : [M+Na]+calculated: 358.21; [M+Na]+measured: 358.20.

[0088] Preparation of CB9O2BP (22)

[0089] The synthesis of compound CB9O2BP (22) was carried out according to the general description 1. The reaction was carried out with 100 mg of bispiclene (7, 0.416 mmol, 1 eq.) (in 300 mL of acetonitrile), 216 mg of l,9-ditosyl-3,7-dioxa-l,9-nonanediol (18, 0.458 mmol, 1.1 eq.) (in 150 mL of acetonitrile) and 154 mg of lithium carbonate (2.08 mmol, 5 eq.). The product was a slightly yellow oil (78 mg, 51.2 % yield).1H-NMR (360 MHZ, CDCl3): 7.71 (t, 2H), 7.23 (d, 4H), 4.03 (d, 4H), 3.91 (d, 4H), 3.46 (t, 4H), 3.33 (t, 4H), 2.54 (t, 4H), 1.64 (quin., 2H).

[0090] ESI-MS (m / z): [M+H]+calculated: 369.23; [M+H]+measured: 369.21.

[0091] Preparation of CBBPPC (25)

[0092] The preparation of compound 6-BOC-CBBPPC (24) was carried out according to general description 1, using 200 mg of 6-Boc-piclene (1, 0.653 mmol, 1 eq.) (in 200 mL of acetonitrile) with 182 mg of 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, 0.718 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 241 mg of lithium carbonate (3.26 mmol, 5 eq.). A slightly yellow oil was obtained as the product (67 mg, 21.0 % yield).1H NMR (360 MHz, CDCl3): 9.11 (d, 2H), 7.74 (t, 1H), 7.55 (t, 2H) 7.25 (d, 2H), 7.08 (d, 2H) 4.02 (d, 4H), 3.88 (d, 4H), 3.11 (t, 4H), 2.65 (t, 4H), 1.48 (s, 9H).

[0093] ESI-MS (m / z): [M+H]+calculated: 487.28; [M+H]+measured: 487.45.

[0094] The synthesis of compound CBBPPC (25) was carried out according to the general description 2, using 50 mg of compound 6-BOC-CBBPPC (24, 0.103 mmol, 1 eq.). The product was a pale yellow oil (34 mg, 85.6 % yield).1H NMR (360 MHz, CDCl3): 9.13 (d, 2H), 7.69 (t, 1H), 7.51 (t, 2H) 7.19 (d, 2H), 7.03 (d, 2H) 4.05 (d, 4H), 3.93 (d, 4H), 3.17 (t, 4H), 2.71 (t, 4H).

[0095] ESI-MS (m / z)-. [M+Na]+calculated: 409.21; [M+Na]+measured: 409.22.

[0096] Preparation of CBBPOPC (26)

[0097] The preparation of compound CBBPOPC (26) was carried out according to general description 1, using 200 mg of O-piclene (5, 0.965 mmol, 1 eq.) (in 200 mL of acetonitrile) with 269 mg of 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, 1.06 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 356 mg of lithium carbonate (4.82 mmol, 5 eq.). The product was a colorless oil (125 mg, 33.4 % yield).1H NMR (360 MHz, CDCl3): 8.91 (d, 2H), 7.64 (t, 1H), 7.41 (t, 2H) 7.19 (d, 2H), 7.01 (d, 2H) 4.34 (d, 4H), 4.21 (d, 4H), 3.61 (t, 4H), 3.21 (t, 4H).

[0098] ESI-MS (m / z) [:M+H]+calculated: 388.21; [M+H]+measured: 388.19.

[0099] Preparation of CBBPBP (27)

[0100] The preparation of compound CBBPBP (27) was carried out according to general description 1, using 200 mg of bispiclene (7, 0.832 mmol, 1 eq.) (in 200 mL of acetonitrile) with 232 mg of 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, 0.915 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 307 mg of lithium carbonate (4.16 mmol, 5 eq.). The product was a colorless oil (154 mg, 44.1 % yield).1H-NMR (360 MHZ, CDCl3): 8.95 (d, 2H), 7.61 (t, 2H), 7.42 (t, 2H) 7.17 (d, 4H), 7.03 (d, 2H) 4.32 (d, 4H), 4.28 (d, 2H), 3.99 (d, 2H), 3.92 (d, 4H).

[0101] ESI-MS (m / z) [:M+Na]+calculated: 443.20; [M+Na]+measured: 443.21.

[0102] Preparation of CBTPPC (30)

[0103] The preparation of compound 6-Boc-CBTPPC (29) was carried out according to general description 1, using 150 mg of 6-Boc-piclene (1, 0.490 mmol, 1 eq.) (in 250 mL of acetonitrile) with 149 mg of 2,9-bis(chloromethyl)-l,10-phenanthroline (28, 0.539 mmol, 1.1 eq.) (in 150 mL of acetonitrile) and 181 mg of lithium carbonate (2.45 mmol, 5 eq.). A yellow oil was obtained as the product (138 mg, 55.2 % yield).1H-NMR (360 MHZ, CDCl3): 8.04 (d, 2H), 7.74 (t, 1H), 7.57 (d, 2H), 7.32 (d, 2H), 7.24 (d, 2H), 4.06 (d, 4H), 3.94 (d, 4H), 3.14 (t, 4H), 2.87 (t, 4H), 1.49 (s, 9H).

[0104] ESI-MS (m / z) [:M+H]+calculated: 511.28; [M+H]+measured: 511.26.

[0105] The synthesis of compound CBTPPC (30) was carried out according to general description 2 using 100 mg of compound 6-Boc-CBTPPC (29, 0.196 mmol, 1 eq.). The product was a yellow oil (54 mg, 67.5 % yield).1H NMR (360 MHz, CDCl3): 8.01 (d, 2H), 7.64 (t, 1H), 7.51 (d, 2H), 7.28 (d, 2H), 7.19 (d, 2H), 4.17 (d, 4H), 3.99 (d, 4H), 3.44 (t, 4H), 3.12 (t, 4H).

[0106] ESI-MS (m / z) [:M+Na]+calculated: 411.23; [M+Na]+measured: 411.21.

[0107] Preparation of CBTPOPC (31)

[0108] The preparation of compound CBTPOPC (31) was carried out according to general description 1, using 150 mg of O-piclene (5, 0.724 mmol, 1 eq.) (in 250 mL of acetonitrile) with 221 mg of 2,9-bis(chloromethyl)-l,10-phenanthroline (28, 0.796 mmol, 1.1 eq.) (in 150 mL of acetonitrile) and 267 mg of lithium carbonate (3.62 mmol, 5 eq.). A slightly yellow oil was obtained as the product (104 mg, 34.9 % yield).1H NMR (360 MHz, CDCl3): 8.11 (d, 2H), 7.74 (t, 1H), 7.63 (d, 2H), 7.34 (d, 2H), 7.21 (d, 2H), 4.21 (d, 4H), 4.04 (d, 4H), 3.51 (t, 4H), 3.29 (t, 4H).

[0109] ESI-MS (m / z) [:M+Na]+calculated: 434.20; [M+Na]+measured: 434.21.

[0110] Preparation of CBTPBP (32)

[0111] The preparation of compound CBTPBP (32) was carried out according to general description 1, using 150 mg of bispiclene (7, 0.624 mmol, 1 eq.) (in 300 mL of acetonitrile) with 190 mg of 2,9-bis(chloromethyl)-l,10-phenanthroline (28, 0.687 mmol, 1.1 eq.) (in 150 mL of acetonitrile) and 231 mg of lithium carbonate (3.12 mmol, 5 eq.). The product was a yellow oil (69 mg, 24.1 % yield).1H NMR (360 MHz, CDCl3): 8.13 (d, 2H), 7.71 (t, 1H), 7.61 (d, 2H), 7.29 (d, 2H), 7.17 (d, 2H), 4.27 (d, 4H), 4.25 (d, 2H), 4.01 (d, 4H), 3.98 (d, 2H).

[0112] ESI-MS (m / z)-. [M+Na]+calculated: 467.20; [M+Na]+measured: 467.21.

[0113] Preparation of CB8O2PC-EA (35)

[0114] (33) (34)

[0115] The preparation of compound (34) was carried out according to the general description 1, using 150 mg of N-(phthalimido)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),l 1,13- triene-6-ethanamine (33, 0.395 mmol, 1 eq.) (in 200 mL of acetonitrile) with 161 mg of 1,2- bis(2-iodoethoxy)ethane (2, 0.435 mmol, 1.1 eq.) (in 50 mL of acetonitrile) and 269 mg of sodium acetate (1.98 mmol, 5 eq.) (in 20 mL of water). A white crystalline substance was obtained as the product (76 mg, 38.7 % yield).1H-NMR (360 MHZ, CDCl3): 7.82 (dd, 2H), 7.71 (dd, 2H), 7.51 (t, 1H), 7.08 (d, 2H), 4.08 (d, 2H), 3.92 (d, 2H), 3.77 (t, 2H), 3.74 (m, 2H), 3.71 (m, 2H), 3.62 (m, 4H), 3.34 (m, 4H), 3.27 (m, 4H), 3.17 (m, 2H), 2.93 (t, 2H), 2.41 (m, 2H).

[0116] ESI-MS (m / z) -. [M+H]+calculated: 494.2762; [M+H]+measured: 494.2762.

[0117] To prepare the compound CB8O2PC-EA (35), 100 mg of starting compound (34, 0.203 mmol, 1 eq.) was dissolved in 10 mL of ethanol, to which 92 pL of 80% hydrazine hydrate (p= 1.03 g / mL, 1.52 mmol, 7.5 eq.) was dissolved. The reaction was carried out in a microwave reactor at 80 °C (120 W, 40 min). The by-product precipitated from the cooled reaction was separated by filtration, which was washed with additional ethanol. The solvent was removed from the filtrate using a rotary vacuum evaporator. After evaporation, a slightly yellow oil was recovered as the product (40 mg, 54.1 % yield).1H NMR (360 MHz, CDCl3): 7.58 (t, 1H), 7.14 (d, 2H), 4.03 (d, 2H), 3.90 (d, 2H), 3.81 (t, 2H), 3.71 (m, 2H), 3.69 (m, 2H), 3.60 (m, 4H), 3.31 (m, 4H), 3.22 (m, 4H), 3.12 (m, 2H), 3.01 (t, 2H), 2.38 (m, 2H).

[0118] ESI-MS (m / z)-. [M+Na]+calculated: 386.25; [M+Na]+measured: 386.21.

[0119] General description 4 (preparation of compounds CB8O2PC-SA):

[0120] 100 mg of compound CB8O2PC-EA (35, 0.275 mmol, 1 eq.) dissolved in 20 mL of anhydrous THF was weighed into a two-necked round-bottom flask. 76 mg of anhydrous K2CO3(0.550 mmol, 2 eq.) was added to the solution. The flask was mounted on a laboratory ball condenser and the system was filled with argon gas. An aqueous solution of the corresponding sulfonyl chloride (1.1 eq.) in 30 mL of THF was weighed into a dropping funnel attached to the flask. The flask was cooled in an ice-water cooling mixture and the reagent solution was added dropwise to the stirred, cooled reaction. After the dropwise addition, the reaction was heated to 60 °C and stirred for 1 day. After the reaction, the inorganic salt was filtered through a G3 glass filter, the solvent was removed from the filtrate using a rotary vacuum evaporator. The product was purified from the evaporation residue by preparative HPLC. Preparation of CB8O2PC-SAMe(36)

[0121] The preparation of compound CB8O2PC-SAMe(36) was carried out according to the general description 4, 100 mg of CB8O2PC-EA starting material (35, 0.275 mmol, 1 eq.) was reacted with 58 mg of p-toluenesulfonyl chloride (17, 0.303 mmol, 1.1 equiv.). The product was a yellow oil (103 mg, 72.5 % yield).1H-NMR (360 MHZ, CD3CN): 7.82 (t, 1H), 7.62 (d, 2H), 7.37 (d, 2H), 7.18 (d, 2H), 4.87 (d, 2H), 4.69 (d, 2H), 4.51 (t, 4H), 4.29 (t, 4H), 3.98 (t, 2H), 3.51 (t, 4H), 3.31 (t, 4H), 3.27 (t, 2H), 3.17 (t, 4H), 3.01 (s, 3H).

[0122] ESI-MS (m / z) -. [M+Na]+calculated: 540.26; [M+Na]+measured: 540.26.

[0123] Preparation of CB8O2PC-SANO2(38)

[0124] The preparation of compound CB8O2PC-SANO2(38) was carried out according to the general description 4, 100 mg of CB8O2PC-EA starting material (35, 0.275 mmol, 1 eq.) was reacted with 67 mg of 4-nitrobenzenesulfonyl chloride (37, 0.303 mmol, 1.1 eq.). The product was a yellow oil (111 mg, 78.2 % yield).1H NMR (360 MHz, CD3CN): 8.11 (d, 2H), 7.97 (d, 2H), 7.81 (t, 1H), 7.15 (d, 2H), 4.83 (d, 2H), 4.66 (d, 2H), 4.42 (t, 4H), 4.26 (t, 4H), 3.71 (t, 2H), 3.50 (t, 4H), 3.27 (t, 4H), 3.20 (t, 2H), 3.12 (t, 4H).

[0125] ESI-MS (m / z) [:M+Na]+calculated: 571.23; [M+Na]+measured: 571.21. Preparation of CB8O2PC-SAcr3(40)

[0126] The preparation of compound CB8O2PC-SAcr3(40) was carried out according to the general description 4, 100 mg of CB8O2PC-EA starting material (35, 0.275 mmol, 1 eq.) was reacted with 74 mg of 4-(trifluoromethyl)benzenesulfonyl chloride (39, 0.303 mmol, 1.1 eq.). After purification, a slightly orange oil was obtained (85 mg, 54.3 % yield).1H-NMR (360 MHZ, CD3CN): 7.80 (t, 1H), 7.69 (d, 2H), 7.63 (d, 2H), 7.13 (d, 2H), 4.81 (d, 2H), 4.65 (d, 2H), 4.40 (t, 4H), 4.23 (t, 4H), 3.62 (t, 2H), 3.51 (t, 4H), 3.29 (t, 4H), 3.25 (t, 2H), 3.10 (t, 4H).

[0127] ESI-MS (m / z) [:M+Na]+calculated: 594.23; [M+Na]+measured: 594.22.

[0128] Preparation of CB8O2PC-SACN(42)

[0129] The preparation of compound CB8O2PC-SACN(42) was carried out according to the general description 4, 100 mg of CB8O2PC-EA starting material (35, 0.275 mmol, 1 eq.) was reacted with 61 mg of 4-cyanobenzenesulfonyl chloride (41, 0.303 mmol, 1.1 eq.). After purification, a slightly orange oil was obtained (98 mg, 67.1 % yield).1H NMR (360 MHz, CD3CN): 7.94 (d, 2H), 7.83 (d, 2H), 7.81 (t, 1H), 7.20 (d, 2H), 4.83 (d, 2H), 4.68 (d, 2H), 4.42 (t, 4H), 4.20 (t, 4H), 3.60 (t, 2H), 3.47 (t, 4H), 3.26 (t, 4H), 3.21 (t, 2H), 3.17 (t, 4H).

[0130] ESI-MS (m / z) [:M+Na]+calculated: 551.24; [M+Na]+measured: 551.24. Preparation of CB8O2PC-DPA (44)

[0131] 120 mg of compound CB8O2PC-EA (35, 0.330 mmol, 1 eq.) was dissolved in 40 mL of anhydrous acetonitrile, to which solution 228 mg of K2CO3(1.65 mmol, 5 eq.) and 27 mg of KI (0.165 mmol, 0.5 eq.) solid powders were added. A solution of 135 mg of 2- (chloromethyl)pyridine x HCI compound (43, 0.825 mmol, 2.5 eq.) in 10 mL of anhydrous acetonitrile was weighed into a dropping funnel mounted on the flask and the system was bubbled with argon. The reagent was slowly added dropwise to the stirred reaction mixture and after addition, the reaction was further stirred at room temperature, the progress of which was monitored by analytical HPLC measurements. After the starting bicycle was completely reacted, the inorganic salt mixture was filtered off through a G3 glass filter, and the solvent was removed from the filtrate using a rotary vacuum evaporator. After purification by preparative HPLC, the product was isolated as an orange oil (118 mg, 65.8 % yield).1H-NMR (360 MHZ, CDCl3): 8.50 (d, 2H), 7.73 (t, 2H), 7.70 (t, 1H), 7.30, (d, 2H), 7.24 (d, 2H), 7.22 (d, 2H), 4.03 (d, 2H), 3.93 (d, 2H), 3.61 (t, 4H), 3.48 (t, 4H), 3.93 (s, 4H), 2.74 (t, 4H), 2.34 (t, 4H), 2.31 (t, 2H), 2.23 (t, 4H), 2.19 (t, 2H).

[0132] ESI-MS (m / z): [M+H]+calculated: 546.36; [M+H]+measured: 546.34.

[0133] General description 5 (alkylation of compound CB8O2PC (4) with various halide derivatives):

[0134] 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) was weighed into a two-necked flask and 100 mL of anhydrous acetonitrile was added. 194 mg of K2CO3(1.40 mmol, 3 eq.) and 39 mg of KI (0.234 mmol, 0.5 eq.) powder were weighed into the solution. The flask was connected to a laboratory ball condenser and a dropping funnel was mounted on the free end. The system was bubbled with argon. A solution of the halide derivative (1.1 eq.) in 20 mL of acetonitrile was weighed into the dropping funnel and slowly added to the stirred reaction. After the dropwise addition, the reaction was stirred at 70 °C under an argon atmosphere and its completion was monitored by analytical HPLC. After the reaction was complete, the solid was filtered off on a G3 glass filter and the precipitate was washed with warm acetonitrile. The solvent was evaporated from the collected filtrate, and the product was purified from the evaporation residue using a preparative HPLC system.

[0135] Preparation of CB8O2PC-C12 (46)

[0136] The preparation of compound CB8O2PC-C12 (46) was carried out according to the general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 128 mg of 1- bromo-dodecane (45, 0.515 mmol, 1.1 eq.). The product 46 was a slightly yellow oil (112 mg, 48.9 % yield).

[0137] ESI-MS (m / z) : [M+Na]+calculated: 511.40; [M+Na]+measured: 511.41.

[0138] Preparation of CB8O2PCmNO2°OHBn(48)

[0139] The preparation of compound CB8O2PCmNO2°OHB" (48) was carried out according to the general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 119 mg of 2- hydroxy-5-nitrobenzyl bromide (47, 0.515 mmol, 1.1 eq.). After purification, an orangecolored syrup was obtained (141 mg, 64.1 % yield).

[0140] ESI-MS (m / z) [:M+Na]+calculated: 494.24; [M+Na]+measured: 494.23. Preparation of CB8O2PC°OHpic(50)

[0141] The preparation of compound CB8O2PC°OHp,c(50) was carried out according to the general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 139 mg of 2- bromomethyl-3-hydroxypyridine x HBr (49, 0.515 mmol, 1.1 eq.). A yellow oil was obtained as the product (126 mg, 62.8 % yield).

[0142] ESI-MS (m / z) [:M+H]+calculated: 428.27; [M+H]+measured: 428.27. Preparation of CB8O2PC-EQB (52)

[0143] The preparation of compound CB8O2PC-EOB (52) was carried out according to the general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 111 mg of 1- (bromomethyl)-4-ethoxybenzene (51, 0.515 mmol, 1.1 eq.). The product was a colorless syrup (175 mg, 82.4 % yield).

[0144] ESI-MS (m / z) [:M+Na]+calculated: 477.28; [M+Na]+measured: 477.27. Preparation of CB8O2PCpCOOHBn(55)

[0145] The preparation of compound CB8O2PCpCOOMeB" (54) was carried out according to the general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 118 mg of methyl 4-(bromomethyl)benzoate (53, 0.515 mmol, 1.1 eq.). The product was a slightly yellow oil (188 mg, 85.8 % yield).

[0146] ESI-MS (m / z) -. [M+Na]+calculated: 491.26; [M+Na]+measured: 491.26.

[0147] The methyl group on compound CB8O2PCpCOOMeBn(54) was removed by alkaline hydrolysis, for which 150 mg of compound CB8O2PCpCOOMeBn(54, 0.320 mmol, 1 eq.) was dissolved in 30 mL of anhydrous ethanol and 26 mg of solid NaOH (0.640 mmol, 2 eq.) powder was added to the solution. After the reaction, the solvent was removed using a rotary vacuum evaporator to obtain the product CB8O2PCpCOOHBn (55) as a slightly yellow oil (127 mg, 87.2 % yield).

[0148] ESI-MS (m / z) [:M+Na]+calculated: 477.24; [M+Na]+measured: 477.24.

[0149] Preparation of CB8O2PCpAMBn-C6 N1(57)

[0150] To prepare the compound CB8O2PCpAMBn'C6'NI(57), 150 mg of the starting macrocycle (55, 0.330 mmol, 1 eq.), 131 mg of HBTU (0.347 mmol, 1.05 eq.) and 54 mg of HOBt (0.396 mmol, 1.20 eq.) coupling agents were dissolved in 10 mL of dichloromethane, to which 200 pL of N,N-diisopropylethylamine (0.742 g / mL, 1.16 mmol, 3.5 eq.) was added. The reaction mixture was stirred at 25 °C for 30 min, then a solution of 74 mg of 6-(2- nitroimidazol-l-yl)-hexan-l-amine (56, 0.347 mmol, 1.05 eq.) in 5 mL of dichloromethane was added. The reaction was further stirred and the completion of the reaction was monitored by analytical HPLC. After the starting macrocycle had completely reacted, the solvent was removed from the reaction using a rotary vacuum evaporator. The evaporation residue was purified by preparative HPLC. After evaporation, a slightly yellow solid powder was recovered as the product (95 mg, 44.4 % yield).

[0151] ESI-MS (m / z) [:M+H]+calculated: 649.38; [M+H]+measured: 649.40.

[0152] Preparation of CB8O2PCpyrDPA(59)

[0153] The preparation of compound CB8O2PCpyrDPA(59) was carried out according to the general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 175 mg of CDPA (58, 0.515 mmol, 1.1 eq.). The product was a brown syrup (209 mg, 71.7 % yield).

[0154] ESI-MS (m / z) [:M+H]+calculated: 623.38; [M+H]+measured: 623.36.

[0155] Preparation of CB8O2PC-lCHyd (64)

[0156] To prepare the compound CB8O2PC-lCHyd (64), it is first necessary to prepare the bromide derivative (62) used as the alkylating agent.

[0157] 100 mg of tert-butyl piperazin- 1-yl carbamate (60, 0.497 mmol, 1 eq.) and 369 mg of solid K3PO4 (1.74 mmol, 3.5 eq.) powder are weighed into a multi-necked round-bottom flask. To the flask, 40 mL of anhydrous acetonitrile and a few grains of heated molecular sieve are added. A dropping funnel is placed on the flask and the system is bubbled with argon. A solution of 4-bromobutyryl bromide (61, 0.745 mmol, 1.5 eq.) in 20 mL of anhydrous acetonitrile is weighed into the dropping funnel. While cooling with ice and stirring, the solution of the bromine derivative is slowly added dropwise to the reaction. The reaction is then stirred for 2 hours at room temperature and under an argon atmosphere. After the reaction, the mixture was heated to 70 °C and the solid was filtered off on a G3 glass filter. The solvent was evaporated from the filtrate, and the evaporation residue was redissolved in 25 mL of dichloromethane. The solution was first extracted with 25 mL of water, then with 25 mL of 5% citric acid solution, and finally with 15 mL of water. The organic phase was dried over MgSO4, and after filtering off the drying agent on a pleated filter, the solvent was removed from the filtrate on a rotary vacuum evaporator. The product (62) was obtained as a colorless oil (94 mg, 53.8 % yield). 1H-NMR (360 MHZ, DMSO-d6): 9.21 (s, 1H), 3.50 (t, 2H), 3.44 (t, 4H), 2.80 (t, 4H), 2.32 (t, 2H), 2.08 (quin., 2H), 1.45 (s, 9H).

[0158] ESI-MS (m / z): [M+H]+calculated: 350.10; [M+H]+measured: 350.10. Compound 63 was prepared according to the general description in 5 using 150 mg of compound CB8O2PC (4, 0.468 mmol, 1 eq.) and 180 mg of the bromide derivative (62, 0.515 mmol, 1.1 eq.). A yellow syrup was obtained as the product (134 mg, 48.6 % yield).

[0159] ESI-MS (m / z) [:M+H]+calculated: 590.40; [M+H]+measured: 590.41.

[0160] For preparation of CB8O2PC-lCHyd (64), general description 2 was used, using 100 mg of starting compound (63, 0.170 mmol, 1 eq.). The product was obtained as a slightly yellow oil (64 mg, 83.7 %).

[0161] ESI-MS (m / z)-. [M+H]+calculated: 490.35; [M+H]+measured: 490.35.

[0162] Preparation of CB8O2PC-PIB1 (69)

[0163] Compound CB8O2PC-PIB1 (69) can be prepared by reacting the bicyclic compound CB8O2PC (4) with the bromide derivative 67.

[0164] 121 mg of heated K2CO3(0.878 mmol, 1.5 eq.), 150 mg of 6-MeO-BTA-O (65, 0.585 mmol, 1 eq.) and 80 mL of dichloromethane are weighed into a three-necked flask. A dropping funnel is attached to the flask and the system is filled with argon. A solution of 130 mg of 2-bromoacetyl bromide (66, 0.644 mmol, 1.1 eq.) in 20 mL of dichloromethane is weighed into the dropping funnel. The flask is cooled with ice-water cooling. Argon gas is passed through one neck of the flask, while the outlet is led to the exhauter through the other neck. After the dropwise addition, the mixture is further stirred in the cold for one hour, then the cooling is removed and the reaction is stirred at room temperature. The completion of the reaction is checked by analytical HPLC. The solid precipitate is filtered off on a G3 glass filter and washed with dichloromethane. The organic phase is extracted with 50 mL of water, 50 mL of 5% citric acid solution and finally 50 mL of water. The collected organic phase is dried over MgSO4, and after filtering off the +ing agent, the organic phase is evaporated. The product in the evaporation residue is purified by preparative HPLC. The product (67) is obtained as an orange oil (92 mg, 41.5% yield).

[0165] ESI-MS (m / z) -. [M+H]+calculated: 377.00; [M+H]+measured: 376.99.

[0166]

[0167] Compound 68 was prepared according to general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 194 mg of bromide derivative 67 (0.515 mmol, 1.1 eq.). The product was an orange oil (82 mg, 28.5 % yield).

[0168] ESI-MS (m / z) -. [M+Na]+calculated: 639.27; [M+Na]+measured: 639.27.

[0169] The methyl group of compound 68 was cleaved with BBn reagent [5], To a solution of 50 mg of derivative 68 (0.0811 mmol, 1 eq.) in 2 mL of di chloromethane, 0.325 mL of BBn (1.0 M, 0.325 mmol, 4 eq.) in dichloromethane was added and the reaction was stirred at room temperature. After the reaction was complete, the solvent was removed by rotary vacuum evaporator and the product was isolated after purification by preparative HPLC. Compound CB8O2PC-PIB1 (69) was a yellow powder (31 mg, 63.4 % yield).

[0170] ESI-MS (m / z)-. [M+Na]+calculated: 625.26; [M+Na]+measured: 625.21.

[0171] Preparation of CB8O2PC-PIB2 (68)

[0172] For preparing compound CB8O2PC-PIB2 (68), also in this case, the preparation of the alkylating agent was required.

[0173] 1.0 mL of 2-chloroethanol (71) was added to 200 mg of 2-(4'-nitrophenyl)-6- methoxybenzothiazole (70, 0.735 mmol, 1 eq.) and the reaction was heated at 100 °C for 4 h. The reaction was then cooled to room temperature and further cooled in an ice-water mixture. 5.0 mL of thionyl chloride was slowly added dropwise to the cooled reaction. After the dropwise addition, the reaction was heated at 80 °C for an additional 4 h. After the reaction was complete, the reaction cooled to room temperature was poured into 100 mL of cold diethyl ether. The ether phase was decanted from the precipitated product and the solid product was further washed with fresh diethyl ether and then air-dried. The product obtained was an orange powder (148 mg, 54.3% yield).

[0174] ESI-MS (m / z): [M+H]+calculated: 335.03; [M+H]+measured: 335.01.

[0175] Compound 73 was prepared according to general description 5 using 150 mg of CB8O2PC (4, 0.468 mmol, 1 eq.) and 191 mg of chloride derivative 72 (0.515 mmol, 1.1 eq.). The product 73 was an orange oil (53 mg, 18.4 % yield).

[0176] ESI-MS (m / z) -. [M+Na]+calculated: 642.26; [M+Na]+measured: 642.26.

[0177] The preparation of compound CB8O2PC-PIB2 (74) requires the reduction of the nitro group in derivative 73. 50 mg of compound 73 (0.0807 mmol, 1 eq.) was dissolved in 15 mL of anhydrous ethanol. 122 mg of anhydrous tin(II) chloride (0.645 mmol, 8 eq.) was added to this solution. The reaction was heated under argon at 70 °C. After two hours, the ethanol was evaporated from the reaction and the evaporation residue was suspended in 10 mL of ethyl acetate. The organic phase was extracted with 5 mL of 1 M NaOH solution, then 10 mL of water, and then dried over MgSO4. After filtration of the drying agent and removal of the solvent by a rotary vacuum evaporator, the product was purified by preparative HPLC. The product (CB8O2PC-PIB2 (74)) was obtained as an orange powder (16 mg, 33.6% yield). ESI-MS (m / z)-. [M+Na]+calculated: 612.29; [M+Na]+measured: 612.24. Preparation of CB8O2PC-EAAMHyd(77)

[0178] 200 mg of compound CB8O2PC-EA (35, 0.550 mmol, 1 eq.) was dissolved in 30 mL of anhydrous acetonitrile. 126 mg of 2-(2-(tert-butoxycarbonyl)hydrazinyl)acetic acid (75, 0.66 mmol, 1.2 eq.) and 143 mg of N,N-diisopropyl ethylamine (1.10 mmol, 2 eq.) were added to the solution. A solution of 251 mg of HATU coupling agent (0.66 mmol, 1.2 eq.) in 20 mL of anhydrous acetonitrile was slowly added dropwise to the stirred reaction. The reaction was stirred for an additional hour at room temperature, and the completion of reaction of starting bicycle was monitored by analytical HPLC injection. The solvent was evaporated from the reaction and the residue was dissolved in 50 mL of ethyl acetate. The organic phase was extracted with 25 mL of 5% citric acid and then with 25 mL of water. After drying the collected organic phase over MgSO4, the solvent was removed under reduced pressure. The product (76) was isolated as a yellow oil (85 mg, 84.9 % yield) after purification by preparative HPLC.

[0179] ESI-MS (m / z) -. [M+Na]+calculated: 558.34; [M+Na]+measured: 558.31.

[0180] After removal of the tert-butoxycarbonyl protecting groups of compound 76, the ligand CB8O2PC-EAAMHyd(77) can be isolated, which was carried out according to the general description 2. 50 mg of compound 76 (0.0933 mmol, 1 eq.) was reacted with trifluoroacetic acid, and a yellow oil was obtained as the product (30 mg, 73.8 % yield). ESI-MS (m / z)-. [M+Na]+calculated: 458.29; [M+Na]+measured: 458.28.

[0181] Preparation of the products of 2:2 ring closure reactions

[0182] It is a well-known fact among researchers studying on the preparation of macrocyclic compounds that in addition to the expected 1 : 1 ring closure, the formation of 2:2, 3:3, etc. products must be taken into account. In general, we can say that the formation of these compounds was observed during the previously presented cross-coupling reactions in addition to the 1 : 1 cryptate-type products. Based on our studies, it can be said that using a base containing a larger cation (e.g. base containing a Cs+ion) results in a higher proportion of the 2:2 ring closure products, in which two macrocycles are connected by two cross-linking “elements”. We were able to produce these derivatives in acceptable according to the general description below.

[0183] General description 6 (production of 2:2 ring closure products):

[0184] 100 mg of the starting macrocycle (1 eq.) was weighed into a two-necked flask and dissolved in 300 mL of anhydrous acetonitrile. CS2CO3base (10 eq.) was added to the solution, and then the mixture was heated to 85 °C and stirred at this temperature. A solution of the reagent (1.0 eq.) in 150 mL of anhydrous acetonitrile was weighed into a dropping funnel and added dropwise to the reaction mixture over 2 hours. Samples were taken from the reaction mixture stirred at 85 °C at 2-hour intervals and analyzed by analytical HPLC. After the complete conversion of the starting macrocycle, the solid was filtered from the still warm reaction mixture on a G3 glass filter, which was washed with hot acetonitrile (3 x30 mL). The solvent was evaporated from the filtrate under reduced pressure and the product was recovered from the evaporation residue by preparative HPLC technique.

[0185] Preparation of CB28O2PC2 (79)

[0186] According to the description 6, 1.06 g of CS2CO3(3.26 mmol, 10 eq.) and a solution prepared with 121 mg of 1,2-bis(2-iodoethoxy)ethane (2, 0.326 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of 6-Boc-piclene (1, 0.326 mmol, 1 eq.). The product (78) was a slightly yellow oil (43 mg, 31.4 % yield).1H-NMR (360 MHZ, CDCl3): 7.80 (t, 2H), 7.22 (d, 4H), 3.97 (s, 8H), 3.52 (t, 8H), 3.48 (t, 8H), 3.04 (t, 8H), 2.65 (t, 8H), 2.51 (t, 8H), 1.44 (s, 18H).

[0187] ESI-MS (m / z): [M+2H]2+calculated: 421.78; [M+2H]2+measured: 421.76.

[0188] The removal of the tert-butoxycarbonyl protecting groups from compound 78 was carried out according to description 2. 40 mg of the protected derivative 78 (0.0476 mmol, 1 eq.) was used for the reaction The product CB28O2PC2 (79) was a slightly yellow oil (14 mg, 45.2 % yield).1H-NMR (360 MHZ, CDCl3): 7.78 (t, 2H), 7.24 (d, 4H), 3.92 (s, 8H), 3.50 (t, 8H), 3.32 (t, 8H), 3.02 (t, 8H), 2.46 (t, 8H), 2.32 (t, 8H).

[0189] ESI-MS (m / z) [:M+2H]2+calculated: 321.73; [M+2H]2+measured: 321.72.

[0190] Preparation of CB28O2QPC2 (80)

[0191] According to the description 6, 1.57 g of CS2CO3(4.83 mmol, 10 eq.) and a solution prepared with 179 mg of 1,2-bis(2-iodoethoxy)ethane (2, 0.483 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of O-piclene (5, 0.483 mmol, 1 eq.). The product (80) was a slightly yellow oil (50 mg, 32.1 % yield).1H NMR (360 MHz, CDCl3): 7.79 (t, 2H), 7.26 (d, 4H), 3.90 (s, 8H), 3.51 (t, 8H), 3.46 (t, 8H), 3.00 (t, 8H), 2.62 (t, 8H), 2.31 (t, 8H).

[0192] ESI-MS (m / z) [:M+2H]2+calculated: 322.72; [M+2H]2+measured: 322.71.

[0193] Preparation of CB28O2BP2 (81)

[0194] According to the description 6, 1.36 g of CS2CO3(4.16 mmol, 10 eq.) and a solution prepared by dissolving 154 mg of 1,2-bis(2-iodoethoxy)ethane (2, 0.416 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of bispiclene (7, 0.416 mmol, 1 eq.). The product (81) was a slightly yellow oil (53 mg, 36.1 % yield).1H NMR (360 MHz, CDCl3): 7.76 (t, 4H), 7.19 (d, 8H), 3.92 (s, 16H), 3.50 (t, 8H), 3.05 (t, 8H), 2.65 (t, 8H).

[0195] ESI-MS (m / z) [:M+2H]2+calculated: 355.72; [M+2H]2+measured: 355.71.

[0196] Production of CB28N2PC2 (83)

[0197] According to the description 6, 1.06 g of CS2CO3(3.26 mmol, 10 eq.) and a solution prepared with 249 mg of 3,6-ditosyl-3,6-diazaoctane-l,8-di-(toluene-4-sulfonate) (9, 0.326 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of 6-Boc-piclene (1, 0.326 mmol, 1 eq.). The product (82) was a yellow oil (101 mg, 42.7 % yield).

[0198] ESI-MS (m / z) [:M+2H]2+calculated: 727.83; [M+2H]2+measured: 727.81.

[0199] The protecting groups were removed from compound 82 according to description 3. Using 100 mg of the protected derivative 82 (0.0688 mmol, 1 eq.), 23 mg (51.7 % yield) of CB28N2PC2 (83) was obtained as a brown oil.

[0200] ESI-MS (m / z): [M+2H]2+calculated: 319.76; [M+2H]2+measured: 319.74.

[0201] Production of CB28N2OPC2 (85)

[0202] According to the description 6, 1.57 g of CS2CO3(3.26 mmol, 10 eq.) and a solution prepared with 369 mg of 3,6-ditosyl-3,6-diazaoctane-l,8-di-(toluene-4-sulfonate) (9, 0.483 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of O-piclene (5, 0.483 mmol, 1 eq.). The product (84) was a slightly yellow oil (89 mg, 29.3 % yield).

[0203] ESI-MS (m / z) -. [M+2H]2+calculated: 628.77; [M+2H]2+measured: 628.77.

[0204] The protecting groups were removed from compound 84 according to description 3. 50 mg of the protected derivative 84 (0.0398 mmol, 1 eq.) was used. The product CB28N2OPC2 (85) was a brown oil (16 mg, 64.2 % yield).

[0205] ESI-MS (m / z) [:M+2H]2+calculated: 320.75; [M+2H]2+measured: 320.71.

[0206] Production of CB28N2BP (87)

[0207] According to the description 6, 1.36 g of CS2CO3(4.16 mmol, 10 eq.) and a solution obtained by dissolving 318 mg of 3,6-ditosyl-3,6-diazaoctane-l,8-di-(toluene-4-sulfonate) (9, 0.416 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of bispiclene (7, 0.416 mmol, 1 eq.). The product (86) was an orange oil (132 mg, 47.9 % yield).

[0208] ESI-MS (m / z)-. [M+2H]2+calculated: 661.77; [M+2H]2+measured: 661.76.

[0209] The protecting groups were removed from compound 86 according to description 3. 100 mg of the protected derivative 86 (0.0757 mmol, 1 eq.) was used. The product CB28N2BP2 (87) was a dark brown oil (34 mg, 63.8 % yield).

[0210] ESI-MS (m / z): [M+2H]2+calculated: 353.75; [M+2H]2+measured: 353.71.

[0211] Prep According to the description 6, 1.06 g of CS2CO3(3.26 mmol, 10 eq.) and a solution prepared with 154 mg of l,9-ditosyl-3,7-dioxa-l,9-nonanediol (18, 0.326 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of 6-Boc-piclene (1, 0.326 mmol, 1 eq.). The product (88) was a slightly yellow oil (86 mg, 60.4 % yield).

[0212] ESI-MS (m / z) -. [M+2H]2+calculated: 435.80; [M+2H]2+measured: 435.80.

[0213] The tert-butoxycarbonyl protecting groups of compound 88 were removed according to the description 2, starting from 50 mg of protected derivative 88 (0.0575 mmol, 1 eq.), which results in 29 mg (74.3 % yield) of product CB29O2PC2 (89) as a yellow oil.

[0214] ESI-MS (m / z): [M+2H]2+calculated: 335.75; [M+2H]2+measured: 335.76.

[0215] Preparation of CB29O2QPC2 (90)

[0216] According to the description 6, 1.57 g of CS2CO3(4.83 mmol, 10 eq.) and a solution obtained by dissolving 228 mg of l,9-ditosyl-3,7-dioxa-l,9-nonanediol (18, 0.483 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of O-piclene (5, 0.483 mmol, 1 eq.). The product (90) was a yellow oil (91 mg, 56.0 % yield).

[0217] ESI-MS (m / z) [:M+2H]2+calculated: 336.73; [M+2H]2+measured: 336.71.

[0218] Preparation of CB29O2BP2 (91) According to the description 6, 1.36 g of CS2CO3(4.16 mmol, 10 eq.) and a solution prepared with 197 mg of l,9-ditosyl-3,7-dioxa-l,9-nonanediol (18, 0.416 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of bispiclene (7, 0.416 mmol, 1 eq.). The product (91) was an orange oil (72 mg, 46.7 % yield).

[0219] ESI-MS (m / z) [:M+2H]2+calculated: 369.73; [M+2H]2+measured: 369.73.

[0220] Preparation of CB2BPPC2 (93)

[0221] According to the description 6, 1.06 g of CS2CO3(3.26 mmol, 10 eq.) and a solution obtained by dissolving 83 mg of 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, 0.326 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of 6-Boc-piclene (1, 0.326 mmol, 1 eq.). The product (92) was an orange oil (65 mg, 40.9 % yield). 1H-NMR (360 MHZ, CDCl3): 9.07 (d, 4H), 7.81 (t, 2H), 7.62 (t, 4H), 7.24 (d, 4H), 7.08 (d, 4H), 4.01 (s, 8H), 3.98 (s, 8H), 3.11 (t, 8H), 2.74 (t, 8H), 1.63 (s, 18H).

[0222] ESI-MS (m / z) -. [M+2H]2+calculated: 487.79; [M+2H]2+measured: 487.66.

[0223] The tert-butoxycarbonyl protecting groups of compound 92 were removed according to description 2. 50 mg of the protected derivative 92 (0.0514 mmol, 1 eq.) was used to obtain 25 mg (63.3 % yield) of the product CB2BPPC2 (93) as a brown powder. 1H NMR (360 MHz, CDCl3): 9.05 (d, 4H), 7.83 (t, 2H), 7.60 (t, 4H), 7.22 (d, 4H), 7.11 (d, 4H), 4.05 (s, 8H), 3.96 (s, 8H), 3.34 (t, 8H), 2.92 (t, 8H).

[0224] ESI-MS (m / z): [M+2H]2+calculated: 387.73; [M+2H]2+measured: 387.71. Preparation of CB2BPOPC2 (94)

[0225] According to the description 6, 1.57 g of CS2CO3(4.83 mmol, 10 eq.) and a solution obtained by dissolving 122 mg of 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, 0.483 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of O-piclene (5, 0.483 mmol, 1 eq.). The product (94) was an orange oil (70 mg, 37.5 % yield).1H-NMR (360 MHZ, CDCl3): 9.03 (d, 4H), 7.82 (t, 2H), 7.65 (t, 4H), 7.20 (d, 4H), 7.13 (d, 4H), 4.04 (s, 8H), 3.99 (s, 8H), 3.24 (t, 8H), 2.81 (t, 8H).

[0226] ESI-MS (m / z) -. [M+2H]2+calculated: 388.72; [M+2H]2+measured: 388.71.

[0227] Preparation of CB2BPBP2 (95)

[0228] According to the description 6, 1.36 g of CS2CO3(4.16 mmol, 10 eq.) and a solution obtained by dissolving 105 mg of 6,6'-bis(chloromethyl)-2,2'-bipyridyl (23, 0.416 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of bispiclene (7, 0.416 mmol, 1 eq.). The product (95) was a brown oil (112 mg, 63.8 % yield).1H NMR (360 MHz, CDCl3): 9.00 (d, 4H), 7.85 (t, 4H), 7.61 (t, 4H), 7.25 (d, 8H), 7.08 (d, 4H), 4.01 (s, 16H), 3.89 (s, 8H).

[0229] ESI-MS (m / z) [:M+2H]2+calculated: 421.72 [M+2H]2+measured: 421.65. Preparation of CB2TPPC2 (97)

[0230] According to the description 6, 1.06 g of CS2CO3(3.26 mmol, 10 eq.) and a solution obtained by dissolving 90 mg of 2,9-bis(chloromethyl)-l,10-phenanthroline (28, 0.326 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of 6-Boc-piclene (1, 0.326 mmol, 1 eq.). The product (96) was a brown powder (80 mg, 48.1 % yield).

[0231] ESI-MS (m / z) [:M+2H]2+calculated: 511.79; [M+2H]2+measured: 511.77.

[0232] The tert-butoxycarbonyl protecting groups of compound 96 were removed according to the description 2. Using 80 mg of protected derivative 96 (0.0784 mmol, 1 eq.), 45 mg (69.9 % yield) of CB2TPPC2 (97) was obtained as a brown powder.

[0233] ESI-MS (m / z) [:M+2H]2+calculated: 411.73; [M+2H]2+measured: 411.71.

[0234] Preparation of CB2TPOPC2 (98)

[0235] According to the description 6, 1.57 g of CS2CO3(4.83 mmol, 10 eq.) and a solution obtained by dissolving 134 mg of 2,9-bis(chloromethyl)-l,10-phenanthroline (28, 0.483 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of O-piclene (5, 0.483 mmol, 1 eq.). The product (98) was a dark brown powder (61 mg, 30.7 % yield).

[0236] ESI-MS (m / z) [:M+2H]2+calculated: 412.72; [M+2H]2+measured: 412.72.

[0237] Preparation of CB2TPBP2 (99)

[0238] According to the description 6, 1.36 g of CS2CO3(4.16 mmol, 10 eq.) and a solution obtained by dissolving 115 mg of 2,9-bis(chloromethyl)-l,10-phenanthroline (28, 0.416 mmol, 1 eq.) in anhydrous acetonitrile were added to 100 mg of bispiclene (7, 0.416 mmol, 1 eq.). The product (99) was a dark brown powder (120 mg, 64.7 % yield).

[0239] ESI-MS (m / z) [:M+2H]2+calculated: 445.72; [M+2H]2+measured: 445.71.

[0240] Experimental data

[0241] The first screen for potential Mn(II)-based contrast agent candidates is the physicochemical parameters of the chelates (stability, rate of formation, inertness, relaxation effect, etc.). Therefore, we studied the protonation constants of the prepared compounds, examined the formation of Mn(II) complexes, the rate constants characteristic of their acid- assisted dissociation, and we determined the relaxivity values characteristic of the complex at 25 and 37 °C at field strengths of 0.49 and 1.41 T, at a pH close to physiological. Our studies were performed in all cases in the presence of 0.15 M NaCl, which is the same as the electrolyte concentration found under physiological conditions. Based on the formation kinetic data, we found that for most of the investigated systems, quantitative complex formation (confirmed by HPLC and relaxometry methods) can be achieved in aqueous solution at room temperature, albeit in a slow reaction. Based on these, using the protonation constants of the chelators, it is possible to give a numerical value for the lower limit of the stability constant (or the pMn value, which can be considered as an apparent constant), with which the compounds can be placed in the range of chelators studied so far, since the determination of stability constants in such systems is very complicated and time consuming.

[0242] Table 1 Protonation constants of the studied ligands, the lower limit of stability and pMn constants of their Mn(II) complexes (25 °C, 0.15 M NaCl). a pMn was estimated at pH=7.4 with CMn=CL=10-5M concentrations; b formation of the complex in aqueous medium was not observed (at pH=7.40); c complex formation took several months at pH=7.40;ca 5th protonation constant can also be calculated 2.04(5);din the given system a dinuclear Mn(II)-chelate is also formed logAMnLxMn(min)=9.5.

[0243] It is now accepted that kinetic data is of much greater importance among the thermodynamic parameters for complexes designed for application. A complex intended for application can dissociate in several ways, so we distinguish spontaneous dissociation (k0), proton-assisted dissociation reaction pathways (kH kHH), where the process occurs via a protonated intermediate. Essential metal ions can also catalyze the dissociation when we can talk about a reaction that occurs with the attack of the exchanging metal ion (kCu), during which a dinuclear intermediate is formed. In this intermediate, the donor atoms are transferred one by one from the Mn(II) ion to the attacking metal ion. In a further possibility, the attack of the exchanging Cu(II) ion occurs on the protonated complex (kCuH) . In addition, hydroxide- catalyzed dissociation has also been observed in the case of certain metal ion complexes (forming stable hydroxide complexes), although this pathway was not observed in the case of Mn(II) complexes. Similarly, chelators with different protonation can also induce dissociation of the complexes (kL, kLH), if there is a possibility of formation of mixed ligand intermediates. The equilibrium constants K1H, K2H, KM, KMH, KLand KLHshown in the figure below are the protonation and stability constants of the protonated, dinuclear intermediate and the mixed ligand complexes.

[0244] Figure 1 Possible dissociation pathways of Mn(II) complexes (where M’=Cu or Zn; L’=DTPA or CDTA). The one-way arrows indicate the dissociation steps, while the back- and-forth arrows indicate the (pre)equilibrium.

[0245] Based on the scheme presented above, the kobspseudo-first-order rate constants obtained for each reaction can generally be given by the following equation, taking into account the individual reaction pathways and the stability constants of the intermediates formed: wherein

[0246] In the case of metal complexes formed with macrocyclic ligands, the mechanism outlined above is simplified to proton-assisted dissociation pathways (although in case of some complexes the role of spontaneous dissociation has also been documented). In our case, preliminary experiments already showed that complexes with excellent inertness were formed, so we investigated acid-assisted dissociation in the case of our Mn(II) complexes. For some complexes, we performed measurements in a wide range of acid concentrations (e.g. [Mn(CB2O8BP)]2+, [Mn(CB2O8PC)]2+, Mn(CB2O9PC)]2+), while for others (for the most inert complexes, e.g. [Mn(CB2BP2PC2)]2+), we determined the pseudo-first-order rate constant only at 1 M acid concentration, which is comparable to the rate constant characteristic of acid- assisted dissociation under such conditions. A comparison of our results with the data of the commercial available complexes [Gd(D03A-BT)] and [Gd(HP-D03A)], as well as with the most inert Mn(II) complex (bispidine-based ligand) published so far is shown below (Table 2).

[0247] Based on the data, it can be seen that the inertness of the Mn(II) complexes of the ligands we prepared exceeds the parameters published for the commercial contrast agent [Gd(HP- D03A)] and the most inert Mn(II) chelate published so far, and approaches the data characteristic of [Gd(DO3A-BT)].

[0248] Figure 2 H+-ion concentration dependence of the pseudo-first-order rate constants observed for [Mn(CB2O8BP)]2+(green), [Mn(CB2O8PC)]2+(blue) and [Mn(CB2O9PC)]2+(red) complexes.

[0249] Table 2 Rate constants characteristic of the acid-assisted dissociation of [Mn(CB2O8BP)]2+, [Mn(CB2O8PC)]2+, [Mn(CB2O9PC)]2+and [Mn(CB2BP2PC2)]4+complexes, with data for some commercially available Gd(III) complexes (25 °C, 0.15 M NaCl). a: for these complexes the dependence of the pseudo-first-order rate constants on H+ion concentration is quadratic.

[0250] In addition to being of the appropriate kinetic inertness, the complex must also have the appropriate relaxivity for practical use (relaxivity (mM-1s-1): the relaxation rate increase resulted by a 1 mM solution of the paramagnetic material compared to that measured in a diamagnetic medium). The higher the relaxivity of a complex, the greater its contrast- enhancing effect, which also means that less of the complex having higher relaxivity needs to be injected into the body to achieve the same image quality. We determined the relaxivity values of the Mn(II) complexes at pH=7.4, 25 and 37 °C at field strengths of 0.49 and 1.41 T.

[0251] The relaxivity values of the complexes are presented in Table 3 for the practically used DOTAREM ([Gd(DOTA)]“ complex, r1=3.83 mM-1s-1), and the relaxivity values of the already withdrawn MAGNEVIST ([Gd(DTPA)]2-complex, r1=4.02 mM-1s-1) contrast agents under the same conditions are also presented. Although the parameters of our complexes only approximate the values typical of commercial Gd(III)-based contrast agents, it is worth noting that fine-tuning of this parameter can be easily achieved with various tricks (e.g. by coupling the complex to macromolecules). Table 3 Relaxivity values of the [Mn(CB2O8BP)]2+, [Mn(CB2O8PC)]2+, [Mn(CB2O9PC)]2+and [Mn(CB2BP2PC2)]4+complexes (pH=7.4)

[0252] In vivo experiments

[0253] In the case of a potential contrast agent, in addition to the initial physicochemical characterization, a key question is how the complex behaves during injection. Therefore, our best performing compound based on kinetic parameters, the52Mn isotope labeled chelate of the [Mn(CB2BP2PC2)]4+complex, was also investigated in vivo (dynamic in vivo PET / MRI studies), and then ex vivo organ distribution studies were performed. However, this required optimization of the labeling of the CB2BP2PC2 ligand (chelator concentration, pH, time, temperature, etc.). A radiochemical purity (RCP %) of 83.9-95.4% was achieved with the optimized parameters.

[0254] Dynamic in vivo PET / MRI studies

[0255] The results of the dynamic studies show (Figure 2) that a significant part (75%) of [52Mn]Mn(CB2BP2PC) is excreted into the urine via the kidneys and liver after approximately 10 minutes. In addition, a small increase in accumulation can be observed in the small intestine after 30 minutes, which, however, does not reach the uptake value of either the kidneys or the liver. After 20 minutes from injection, the accumulation in the organs / tissues showing higher activity stabilizes, which does not change significantly within the 90-minute study period. Static in vivo PET / MRI studies

[0256] Considering the average SUV values of the different organs and tissues (Figure 3), it can be said that the accumulation in the bladder is high in the first 2 hours, then after 1 day it decreases below SUV = 1, which can also be said in the case of the colon. At all time points, the accumulation in the liver and kidney shows an outstanding value, while at later time points the spleen, pancreas, salivary gland and stomach should also be mentioned. The above suggests rapid excretion and clearance, which can also be seen in the figure showing the percentage SUV ratio of the different organs and tissues (Figure 4). It can be observed here that in the first 2 hours, nearly two-thirds (60.91%) of the injected [52Mn]Mn(CB2BP2PC) activity is excreted into the bladder with the urine. 6 hours after injection, only 29.3% of the labeled substance can be found in the animals compared to the 2-hour imaging. This value is only 17.5% after one day, which then decreases continuously to 6.6% only after 14 days. Considering the uptake rate of the organs, it can be said that 2 hours after injection, in addition to the bladder, the accumulation is significantly in the kidney, liver and colon, of which the kidney and liver showed high values until the end of the study. Since the radiochemical purity of the injected product was 92.4 %, the question may arise whether the non-specifically bound so-called “free” [52Mn]Mn(II) in the sample influences our data. Based on the results found in the literature, after the injection of isotonic saline containing52Mn(II), the radioisotope was not excreted into the urine via the kidneys, but was excreted into the feces via the liver. (Wooten AL et al. Biodistribution and PET Imaging of pharmacokinetics of manganese in mice using Manganese-52. PLoS One. 2017; 12(3); Hernandez R et al. Radiomanganese PET Detects Changes in Functional β-Cell Mass in Mouse Models of Diabetes. Diabetes. 2017 ;66(8):2163-74; Graves SA et al. Novel Preparation Methods of (52)Mn for ImmunoPET Imaging. Bioconjug Chem. 2015;26(10):2118-24). This can also be seen in Figure 3, where the intestinal uptake is comparable to the kidney and liver uptake values after 2 hours (SUVcolon= 3.02 ± 2.27; SUVsmaii intestine = 0.49 ± 0.33; SUViiver = 3.71 ± 0.25; SUVkidney= 2.98 ± 0.50). Knowing the structure of the compound, the liver uptake is not surprising, as the ligand has a common motif (aromatic moieties, e.g. ethoxybenzyl in EOB-DTPA) with most liver-specific contrast agents (candidates). Furthermore, it can be observed in Figure 4 that the percentage SUV of colon at the same time point is approximately what would be expected based on the radiochemical purity (-10%). At 6-hour data, both absolute and proportional accumulation decrease, consistent with the excretion of “free”52Mn(II) via the feces, which is also supported by that the accumulation in heart and lung, which is typical of this chemical form of the radioisotope, cannot be observed at early measurement points.

[0257]

[0258] Figure 4 Percentage SUV ratios of organs examined by in vivo static PET / MRI imaging at a given measurement time point after injection of [52Mn]Mn(CB2BP2PC) chelate Ex vivo organ distribution studies

[0259] The ex vivo distributions show a similar pattern and trend (Figure 5) as the in vivo static imagings.

[0260] Figure 5 ID% / g values of ex vivo organ distribution measurements at given measurement times, with urine (A) and without urine (B)

[0261] Significant activity can be seen in the urine (ID% / g = 253.04; SD = 128.50), where, if we also considere the percentage of uptake values (Figure 6), it can be observed that 77.18% of the total activity of organs and tissues is excreted. During ex vivo measurements, urine showed a high accumulation until the fifth day (day 5th: ID% / g = 7.096; SD = 9.482), which at first glance contradicts the in vivo results, where after 6 hours we see SUV values lower in the bladder than in the liver and kidney (which is explained by the fact that the mice's urination could not be controlled at later times, so the bladders had different saturations during imaging and dissection). In the case of the other organs and tissues, a similar trend can be observed in the figures showing %ID / g and %ID / g percentage ratio (Figures 5 and 6), as well as in the figures presenting SUV and percentage SUV ratio. An outstanding result can be seen in the kidney at 6 hours in Figure 6. In this case, an exceptionally high value was measured in one of the 3 mice in the group, which is also presented by the standard deviation, in this case, it was probably a difference in the accumulation characteristic of the individual.

[0262] Figure 6 Percentage %ID / g ratios of ex vivo organ distributions at a given measurement time

[0263] In summary, after 2 hours, 70 - 80% of the injected [52Mn]Mn(CB2BP2PC) is rapidly excreted via the urine, but approximately 20 - 30% has accumulated in the kidneys and liver. Accumulation in these organs has steadily decreased, although it is still visible after 14 days in both ex vivo and in vivo measurements (less than 1% of the initial injected activity). The agent also accumulated in the spleen, pancreas and salivary glands, which, although typically do not reach the accumulation in the kidneys and liver, produced visible uptake. References

[0264] 1. Csupasz, T.; Lihi, N.; Fekete, Z.; Nagy, A.; Botar, R.; Forgacs, V.; Szikra, D.; May, N.V.; Tircso, G.; Kalman, F.K. Exceptionally fast formation of stable rigidified cross-bridged complexes formed with Cu(II) isotopes for molecular imaging. Inorg. Chem. Front. 2022, 9, 1217-1223, doi: 10.1039 / dlqi01526e.

[0265] 2. Casula, A.; Nairi, V.; Fernandez -Moreira, V.; Laguna, A.; Lippolis, V.; Garau, A.; Gimeno, M.C. Re(i) derivatives functionalised with thioether crowns containing the 1,10- phenanthroline subunit as a new class of chemosensors. Dalt. Trans. 2015, 44, 18506-18517, doi: 10.1039 / c5dt02723c.

[0266] 3. Botar, R.; Molnar, E.; Garda, Z.; Madarasi, E.; Trencsenyi, G.; Kiss, J.; Kalman, F.K.; Tircso, G. Synthesis and characterization of a stable and inert Mn(II)-based Zn(II) responsive MRI probe for molecular imaging of glucose stimulated zinc secretion (GSZS). Inorg. Chem. Front. 2022, 9, 577-583, doi: 10.1039 / DlQI00501D.

[0267] 4. Hoigebazar, L.; Min Jeong, J.; Kyung Hong, M.; Ju Kim Y.; Youn Lee, J.; Shetty, D.; Lee Y.-S.; Soo Lee D.; Chung J.-K.; Chui Lee, M. Synthesis of 68Ga-labeled DOTA- nitroimidazole derivatives and their feasibilities as hypoxia imaging PET tracers Bioorg. Med. Chem. 2011, 19(7), 2176-2181, doi: 10.1016 / j.bmc.2011.02.041

[0268] 5. Jia, J.; Wu, L.; Ding, Y.; Huang, C.; Zhu, W.; Xua, Y.; Qiana, X. A DPA-based highly selective and sensitive fluorescent probe for mercuric ions and its imaging in living cells. Dalt. Trans. 2016, 45, 9402-9406, doi: 10.1039 / C6DT01258B.

[0269] 6. Mathis, C.A.; Wang, Y.; Holt, D.P.; Huang, G.; Debnath, M.L.; Klunk, W.E. Synthesis and evaluation of HC-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents. J. Med. Chem. 2003, 46, 2740-2754, doi: 10.1021 / jm030026b.

[0270] Acknowledgement

[0271] The patent application was prepared with the professional support of the National Research, Development and Innovation Office (NRDI Office), grant no. 134694, the University of Debrecen LTD-IMPACT Discovery subprogram, grant no. DISC / 2022 / 1 / 021, the Cooperative Doctoral Programme for Doctoral Scholarships (KDP-2020) of Ministry of Innovation and Technology and the New National Excellence Program, code number UNKP-23-2, of the Ministry of Culture and Innovation financed from the National Research Development and Innovation Fund. The scientific results presented were prepared with the support of the Richter Gedeon Talentum Foundation established by Richter Gedeon Pic., within the framework of the “Richter Talentum PhD. Scholarship”.

Claims

Claims1. Mn(II) or Cu(II) complexes of the compound of general formula (I)whereinZ is absent or CH2;R1and R1’ are H, or together with R3and R3’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring;R2and R2’ are H, or together with R4and R4’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring;R3and R3’ are H, or combined with R1and R1’ groups as above;R4and R4’ are H, or combined with R2and R2’ groups as above; or R1, R1’, R2, R2’, R3, R3’, R4and R4’ and -C-Y-C-Z-C-Y-C- moiety to which they are attached form 2,2'-bipyridyl or 1,10-phenatroline; each Y is O or NR9;R5and R5’ are H, or together with R6and R6’ groups and -C-X-C- moiety to which they are attached form a 6-membered heteroaryl ring;R6and R6’ are H, or combined with R5and R5’ groups as above;X is O or NR10;R7and R8together form a bond, or two compounds of formula (I) form a dimer and R7forms a bond with R8group of the second compound of formula (I), and in this case R8forms a bond with R7group of this second compound; Z, Y, X and RCR10are identical in the two compounds of formula (I);R9is absent or H;R11, R12and R13are independently each other H, C1-C4 alkyl, CF3, OH, OC1-C4 alkyl, -CN, NH2, NH(C1-C4 alkyl), N(C1-C4 alkyl)2, benzyl, phenyl, p-ethoxybenzyl, or pharmaceutically acceptable salts, hydrates, solvates and isomers thereof.

2. The complex according to claim 1, wherein the compound of formula (I) is the compound of formula (1-1)3. The complex according to claim 1, wherein the compound of formula (I) is the compound of formula (1-2)4. The complex according to any one of claims 1 to 3, whereinR1, R1’, R2, R2’, R3, R3’, R4and R4’ are H, orR1and R1’ together with R3and R3’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring, and R2and R2’ together with R4and R4’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring; orR1, R1’, R2, R2’, R3, R3’, R4and R4’ together with the -C-Y-C-Z-C-Y-C- moiety to which they are attached form 1,10-phenatroline.

5. The complex according to any one of claims 1 to 4, whereinR1and R1’ together with R3and R3’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring, and R2and R2’ together with R4and R4’ groups and -C-Y-C- moiety to which they are attached form a 6-membered heteroaryl ring; wherein the 6-membered heteroaryl ring is preferably pyridine; orR1, R1’, R2, R2’, R3, R3’, R4and R4’ and -C-Y-C-Z-C-Y-C- moiety to which they are attached form 2,2'-bipyridyl or 1,10-phenatroline; andY is N; andZ is absent.

6. The complex according to any one of claims 1 to 4, whereinR1, R1’, R2, R2’, R3, R3’, R4and R4’ are H, andY is O or NH, andZ is absent or CH2.

7. The complex according to claim 6, whereinY is O or NH, andZ is absent; orY is O, andZ is CH2.

8. The complex according to any one of claims 1 to 7, whereinR5, R5’, R6and R6’ are H, and X is O or NR10; orR5and R5’ together with R6and R6’ groups and -C-X-C- moiety to which they are attached form a 6-membered heteroaryl ring, and X is N; wherein the 6-membered heteroaryl ring is preferably pyridine.

9. The complex according to any one of claims 1 to 8, wherein the compound of formula (I) is selected from:

10. The complex according to any one of claims 1 to 9 for use in PET / MRI diagnostic procedures.

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