Field of Art The present invention relates to novel aromatic analogs of choline, to synthesis of these analogs, preparation of radiolabeled forms thereof and the medical use thereof as imaging agents and therapeutics, especially in oncological, inflammatory, and neurological diseases.

Background Art

The treatment of tumorous diseases greatly profits from the tools of nuclear medicine, including functional imaging methods; such as planar scintigraphy, positron emission tomography (PET), and single -photon emission computed tomography (SPECT) [Zaidi H. (Ed.) (2006), Quantitative Analysis in Nuclear Medicine Imaging, Springer US, 1st Issue, ISBN 978-0-387-25444-9; Welch M. J., Redvanly C. S. (2003), Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley & Sons, Ltd. ISBN: 0-471-49560-3]. The above-mentioned functional imaging methods, in contrast to anatomical imaging methods (computer tomography - CT, magnetic resonance - MRI), allow visualization of biochemical changes in tissues, and are therefore used for diagnosis of various diseases [Pimlott S. L., Sutherland A. (2011), Chem. Soc. Rev. 40, 149-162].

Planar scintigraphy, positron emission tomography, and single -photon emission computed tomography all rely on radioactively labeled imaging agents. Unfortunately, many imaging agents suffer from drawbacks, such as a very short half -life of the used radioisotopes, complicated synthesis, or unsuitable distribution in the organism [Pimlott S. L., Sutherland A. (2011), Chem. Soc. Rev. 40, 149-162]. Only a few radiopharmaceuticals that are used for example in oncology allow a theranostic approach, which means that the same molecule can be used for both diagnostic and therapeutic purposes. Theranostic approach allows a significantly more accurate monitoring of dosage, of distribution of the radiopharmaceutical in the organism, and of the results of the treatment [Velikyan I. (2012), Theranostics 2, 424-426; Del Vecchio S. et al. (2007) /. Nucl. Med. Mol. Imaging 51, 152- 63]. Examples of medicaments which enable the theranostic approach include several radioiodinated pharmaceuticals that are commonly labeled by 123 I or 131 I for diagnostic purposes and by 131 I for therapeutic purposes [Giammarile F. et al. (2008), Eur. J. Nucl. Med. Mol. Imaging 35, 1039-1047]. Choline transporters represent a molecular structure suitable for targeting diagnostic and therapeutic radiopharmaceuticals, as they are overexpressed on the surface of certain types of tumor cells, in particular prostate carcinoma, small-cell lung carcinoma, and glioma [Hara T. et al. (2006), Mol. Imaging 5, 498-509; Inazu M. et al. (2013), Pharmacol. Res. 76, 119-131]. This fact is already exploited in the clinical practice for visualization of certain types of tumor focuses, particularly prostate carcinoma. Three radiolabeled derivatives of choline - [ ¹⁸ F]fluoromethylcholine, [ ¹⁸ F]fluoroethylcholine, and [ ⁿ C]-choline - are currently employed in tumor imaging [Umbehr M. H. et al. (2013), Eur. Urol. 64, 106-117; Evangelista L. et al. (2015), Nucl. Med. Biol. 42, 340-348]. These compounds preferentially accumulate in tumor cells which overexpress choline transporters.

[ ¹⁸ F]Fluoromethylcholine, [ ¹⁸ F]fluoroethylcholine, and [ ⁿ C]choline contain positron emitters in their molecules. The emitted positron provides - by annihilation with an electron - an antiparallel pair of photons. These antiparallel photons are captured by a detector, and the resulting data are then used for reconstruction of the three-dimensional image of the tumor focus [Pimlott S. L., Sutherland A. (2011), Chem. Soc. Rev. 40, 149-162].

Expression of choline transporters is also increased in a number of other tissues - in myocardium, digestive tract, neural tissue, in endocrine glands, in bone marrow and blood, in the skin, and in the lungs. Pathological function or expression of the choline transporters thus accompanies a number of human diseases - not only tumors, but for example also neuro-muscular diseases [Barwick K. E. S. et al. (2012), Am. J. Hum. Genet. 91, 1103-1107], atherosclerosis [Neumann S. A. et al. (2012), Int. J. Cardiovasc. Imaging 28, 243-250], alcohol addiction [Stankiewitz A. M. et al. (2015), Pharmacol. Biochem. Behav. 139 Part A, 27-38], and psychiatric disorders, such as depression [Hahn M. K. et al. (2008), Genes Brain Behav. 7, 487-495] or attention deficit disorder [English B. A. et al. (2009), /. Neurodev. Disord. 1, 252-263].

However, the above-mentioned choline derivatives used in clinical practice, suffer from serious drawbacks. Due to the very short half -life of ^U C (t _1/2 = 20 min) and ¹⁸ F (t _1/2 = 110 min) an on-site cyclotron is necessary for a successful application of such medicaments to the patient. The short half- life also imposes strict requirements on the synthesis of the radiopharmaceutical and on the production quality control. Furthermore, in the visualization of prostate carcinoma, the short half -life of [ ¹⁸ F]fluoromethylcholine, [ ¹⁸ F]fluoroethylcholine, and [ ⁿ C]-choline causes problems in combination with rapid renal excretion [Bauman G. et al. 2012, Prostate Cancer Prostatic Dis. 15, 45-55]. The image quality is significantly decreased by background activity accumulated in the bladder and the short half-life does not allow acquiring the image after most of the radioactivity has been eliminated from the bladder. Due to the characteristics of the emitted radiation [ ¹⁸ F]fluoromethylcholine, [ ¹⁸ F]fluoroethylcholine, and [ ⁿ C]choline are applicable only for diagnostic purposes, and they cannot be used in therapy.

Disclosure of the Invention

Object of the present invention are compounds of general formula I:

wherein

- Z atoms are independently selected from the group consisting of carbon, nitrogen, oxygen, sulfur;

- n is 1 or 0; hence the ring comprising the atoms Z is an aromatic or heteroaromatic five-membered or six-membered ring;

123 124 12 127 131

- R are independently selected from the group consisting of H; I; I; I; I; I; Q to C _M alkyl; C ₂ to CM alkenyl; C ₂ to C _M alkynyl; Ce to do aryl; C ₃ to Ce heteroaryl comprising at least one heteroatom selected from O, S, N; C ₃ to Ce heterocyclyl comprising one to two heteroatoms selected from O, S, N; Q to C _M alkoxy; Ce to do aryloxy; C ₃ to C heteroaryloxy comprising at least one heteroatom selected from O, S, N; C ₃ to Ce heterocyclyloxy comprising one to two heteroatoms selected from O, S, N; benzyloxy; Q to C _M alkylthio; halogen; OH; SH; NH ₂ ; Q to C _M alkylamino; Ce to Cio arylamino; Ci to C _M acylamino; di(Ci to C _M alkyl)amino, wherein the alkyls are the same or different; di(C ₆ to C ₁₀ aryl)amino, wherein the aryls are the same or different; di(Q to C ₁₄ acyl)amino, wherein the acyls are the same or different; CN; nitro and COOR _n , wherein R _n is H or Q to C ₁₄ alkyl or aryl;

- whereas at least one of the substituents R is ¹²³ I; ¹²⁴ I; ¹²⁵ I; ¹²⁷ I or ¹³¹ I,

- R ⁶ is C to C ₄ linear alkyl, whereas C ₂ -C ₄ linear alkyl can optionally be terminally substituted by OH group,

- R ⁷ and R ⁸ are independently selected from the group consisting of Q to C ₆ alkyl; C ₂ to C ₆ alkenyl; C ₂ to Ce alkynyl; C ₃ to C ₈ cycloalkyl; C ₃ to C ₈ cycloalkenyl; C ₆ to C ₁₀ aryl; C ₃ to C ₆ heteroaryl comprising at least one heteroatom selected from O, S, N; C to Ce heterocyclyl comprising one or two heteroatoms selected from O, S, N; whereas R ⁷ and R ⁸ can optionally be substituted by OH group or Q-C hydroxy alkyl group; or

- R ⁷ and R ⁸ together with the nitrogen atom form a four- to seven-membered ring, optionally comprising at least one further heteroatom selected from O, S, N, a optionally substituted by OH group or Q-C hydroxy alkyl group; or

- R ⁶ , R ⁷ and R ⁸ together with the nitrogen atom form a bicyclic group containing 5 to 12 carbon atoms and optionally at least one further heteroatom selected from O, S, N, and optionally substituted by OH group or Ci-C hydroxyalkyl group; preferably, such bicyclic group is quinuclidine;

- whereas at least one, preferably exactly one, of substituents R ⁶ , R ⁷ and R ⁸ is substituted by OH group or by Q-C ₄ hydroxyalkyl group; - Y is a linker formed by a linear or branched hydrocarbon chain having 1 to 10 carbon atoms, whereas optionally 1 to 2 carbon atoms can be replaced by oxygen and/or sulfur atoms; and

- X is a pharmaceutically acceptable anion;

with the proviso that:

123 124 12 127 131

- when exactly one of the substituents R is '"I; '"I; I; I or and is in the ortho position, and all other substituents R are hydrogen atoms, Y is -CH ₂ -, R ⁶ is 2-hydroxyethyl, then NR ⁷ R ⁸ is not dimethylamino or pyrrolidino group,

123 124 12 127 131

- when exactly one of the substituents R is '"I; '"I; I; I or and is in the ortho position, and all other substituents R are hydrogen atoms, Y is -CH ₂ -, R ⁶ is ethyl or 2-hydroxyethyl, R ⁷ is ethyl or 2-hydroxyethyl, then R ⁸ is not methyl.

The general formula I of the present invention is meant to include all isomers, enantiomers and diastereomers. Preferably, at most one Z is other than carbon.

Preferably, the ring containing Z atoms is selected from benzene ring and thiophene ring.

Preferably, R ⁶ is 2-hydroxyethyl, 3-hydroxypropyl, or 4-hydroxybutyl.

Preferably, when R ⁷ and R ⁸ together with the nitrogen atom form a four- to seven-membered ring, this ring is substituted by OH or Q-C4 hydroxyalkyl group in the position 3 or 4.

Preferably, R ⁷ and R ⁸ together with the nitrogen atom for a five- to six-membered ring.

Preferably, when R ⁶ , R ⁷ and R ⁸ together with the nitrogen atom form a bicyclic group, this group is substituted by OH or C1-C4 hydroxyalkyl group in the position 3 or 4.

Preferably, the alkyl chains comprise 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms.

Preferably, the alkenyl or alkynyl chains comprise 2 to 10 carbon atoms, or 2 to 8 carbon atoms, or 2 to 6 carbon atoms, or 2 to 4 carbon atoms.

Preferably, Y is an alkylene chain. More preferably, when Y is a branched hydrocarbon chain, then a phenyl substituted by R substituents and the nitrogen atom are bound to the same atom of the branched hydrocarbon chain, and the chain contains 2 to 8, preferably 2 to 5 carbon atoms. More preferably, when Y is a linear hydrocarbon chain, it contains 1 to 3 carbon atoms, or 2 to 3 carbon atoms.

Pharmaceutically acceptable salts (anions, cations) include inorganic or organic anions, inorganic or organic cations, addition salts with inorganic or organic acids or bases, co-crystals, inclusion compounds and other salts suitable for physiological administration [Stahl P. H., Wermuth, C. G. (Ed.) (2011), Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2. Vydani, ISBN 978-3-90639-051 -2] .

Suitable pharmaceutically acceptable anions according to the present invention preferably include anions derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, boric acid, tetrafluoroboric acid, phosphoric acid, nitric acid, carbonic acid, sulfuric acid, sulfurous acid; and from organic acids including alifatic, cycloalifatic, aromatic, arylalifatic, heterocyclic, carboxylic and sulfonic acids. Suitable organic anions are preferably selected from the group comprising acetate, isethionate, lactobionate, methanesulfonate, trifluoromethanesulfonate, 4-methylbenzenesulfonate, trifluoroacetate, formiate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartrate, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilate, stearate, salicylate, 4-hydroxybenzoate, phenylacetate, mandelate, pamoate, ethanesulfonate, benzenesulfonate, panthotenate, 2-hydroxyethanesulfonate, sulfanilate, N-cyclohexylsulfamate, 3-hydroxybutyrate, galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerolfosfate, heptanoate, hexanoate, nicotinate, 2-naphthalenesulfonate, oxalate, pectinate,

3- phenylpropionate, picrate, pivalate, thiocyanate, octanoate, decanoate, undecanoate, oxoglutarate, cinnamate, l-hydroxy-2-naphthoate, 4-acetamidobenzoate, gentisate, glucoheptanoate, hippurate, isobutyrate, undecylenate, pyroglutamate, naphthalene-l ,5-disulfonate, laurate, oleate, sebacate, pentanoate and palmitate.

In a preferred embodiment, the invention relates to the following derivatives of general formula I: (2-hydroxyethyl)-[2-(3-iodo-4-methoxyphenyl)ethyl]-dimethyla mmonium

4- methylbenzenesulfonate (1),

3-hydroxy-l -[2-(3-iodo-4-methoxyphenyl)ethyl]quinuclidin-l-ium 4-methylbenzenesulfonate (2), (2-hydroxyethyl)-[2-(3,5-diiodo-4-methoxyphenyl)ethyl]-dimet hylammonium

4-methylbenzenesulfonate (3),

(2-hydroxy ethyl)- [3 -(3-iodo-4-methoxyphenyl)propyl] -dimethylammonium

4-methylbenzenesulfonate (4),

(2-hydroxyethyl)-[2-(4-iodophenyl)ethyl] -dimethylammonium 4-methylbenzenesulfonate (5),

3-hydroxy-l -[2-(4-iodophenyl)ethyl]-l-methylpiperidin-l -ium 4-methylbenzenesulfonate (6),

1 -(2-hydroxy ethyl)- 1 - [2-(4-iodophenyl)ethyl]pyrrolidin- 1 -ium 4-methylbenzenesulfonate (7),

3-hydroxy-l -[2-(4-iodophenyl)ethyl]quinuclidin-l -ium 4-methylbenzenseulfonate (8),

(2-hydroxyethyl)-(3-iodobenzyl)-dimethylammonium methanesulfonate (9),

l-(2-hydroxyethyl)-l-(3-iodobenzyl)pyrrolidin-l -ium methanesulfonate (10),

(2-hydroxyethyl)-(4-iodobenzyl)-dimethylammonium bromide (11), diethyl-(2-hydroxyethyl)-(4-iodobenzyl)ammonium bromide (12),

l-(2-hydroxy ethyl)- l-(4-iodobenzyl)pyrrolidin-l-ium bromide (13),

1 -(2-hydroxy ethyl)- 1 -(2-iodobenzyl)piperidin- 1 -ium 4-methylbenzenesulfonate (14) ,

diethyl-(2-hydroxyethyl)-(2-iodobenzyl)ammonium 4-methylbenzenesulfonate (15),

3 -hydroxy- l-(2-iodobenzyl)-l-methylpiperidin-l -ium 4-methylbenzenesulfonate (16),

1 ,4-bis(2-hydroxy ethyl)- l-(2-iodobenzyl)piperazin-l -ium 4-methylbenzenesulfonate (17),

3- hydroxy-l -(2-iodobenzyl)quinuclidin-l -ium 4-methylbenzenesulfonate (18),

[3-(4-hexyloxy-3-iodophenyl)propyl]-(2-hydroxyethyl)-dime thylammonium

4- methylbenzensulfonate (21),

[3-(4-decyloxy-3-iodophenyl)propyl]-(2-hydroxyethyl)-dimethy lammonium methanesulfonate (22), (2-hydroxyethyl)-[2-(2-iodophenoxy)ethyl]-dimethylammonium bromide (23),

1 -(2-hydroxy ethyl)- 1 - [2-(2-iodophenoxy)ethyl]pyrrolidin- 1 -ium bromide (24),

(2-hydroxyethyl)-[2-(4-iodophenoxy)ethyl]-dimethylammoniu m bromide (25),

1 -(2-hydroxy ethyl)- 1 - [2-(4-iodophenoxy)ethyl]pyrrolidin- 1 -ium bromide (26),

(2-hydroxyethyl)-[2-(3-iodophenoxy)ethyl]-dimethylammoniu m bromide (27),

1 -(2-hydroxyethyl)- 1 -[2-(3-iodophenoxy)ethyl]pyrrolidin- 1 -ium bromide (28),

(2-hydroxyethyl)-[6-(3-iodophenoxy)hexyl]-dimethylammoniu m bromide (29),

1 -(2-hydroxyethyl)- 1 - [6-(3-iodophenoxy)hexyl]pyrrolidin- 1 -ium bromide (30) ,

(2-hydroxyethyl)- [4-(2-iodophenoxy)butyl]-dimethylammonium bromide (31),

1 -(2-hydroxyethyl)- 1 -[4-(2-iodophenoxy)butyl]pyrrolidin- 1 -ium bromide (32),

(2-hydroxyethyl)-[2-(5-iodothiophen-2-yl)ethyl]-dimethyla mmonium 4-methylbenzenesulfonate

(33) ,

1 -(2-hydroxyethyl)- 1 -[2-(5-iodothiophen-2-yl)ethyl]pyrrolidin- 1 -ium 4-methylbenzenesulfonate

(34) ,

4-(2-hydroxyethyl)-4-(4-iodobenzyl)morpholin-4-ium bromide (35),

4-(2-hydroxyethyl)-4-(2-iodobenzyl)morpholin-4-ium bromide (36),

(2-hydroxyethyl)-{6-[(2-iodopyridin-3-yl)oxy]hexyl}-dimet hylammonium bromide (37),

(2-hydroxyethyl)- { 4- [(2-iodo-6-methylpyridin-3-yl)oxy] butyl } -dimethylammonium bromide (38) , (2-hydroxyethyl)- { 4-[(3-iodo-[ 1 , 1 '-biphenyl] -4-yl)oxy]butyl } -dimethylammonium bromide (39), (2-hydroxyethyl)- [6 -(4-iodo-3 -nitrophenoxy)hexyl] -dimethylammonium bromide (40) .

In another aspect, object of the present invention are compounds of general formula I for use as medicaments and/or diagnostic agents, preferably for use as theranostic agents.

Object of the present invention is also a medical preparation comprising at least one compound of general formula I according to the present invention, optionally in the form of a pharmaceutically acceptable salt or solvate, and at least one pharmaceutically acceptable carrier, filler and/or diluent. A further object of the present invention are compounds of general formula la

wherein

- Z atoms are independently selected from the group consisting of carbon, nitrogen, oxygen, sulfur;

- n is 1 or 0; hence the ring comprising the atoms Z is an aromatic or heteroaromatic five-membered or six-membered ring;

123 124 12 127 131

- R are independently selected from the group consisting of H; I; I; I; I; I; Q to C _M alkyl; C ₂ to CM alkenyl; C ₂ to C _M alkynyl; Ce to do aryl; C ₃ to Ce heteroaryl comprising at least one heteroatom selected from O, S, N; C ₃ to Ce heterocyclyl comprising one to two heteroatoms selected from O, S, N; Q to C _M alkoxy; Ce to do aryloxy; C ₃ to C heteroaryloxy comprising at least one heteroatom selected from O, S, N; C ₃ to Ce heterocyclyloxy comprising one to two heteroatoms selected from O, S, N; benzyloxy; Q to C ₁₄ alkylthio; halogen; OH; SH; NH ₂ ; Q to C ₁₄ alkylamino; Ce to Cio arylamino; Q to C ₁₄ acylamino; di(Q to C ₁₄ alkyl)amino, wherein the alkyls are the same or different; di(C ₆ to C ₁₀ aryl)amino, wherein the aryls are the same or different; di(Q to C ₁₄ acyl)amino, wherein the acyls are the same or different; CN; nitro and COOR _n , wherein R _n is H or Q to C ₁₄ alkyl or aryl;

- whereas at least one of the substituents R is ¹²³ I; ¹²⁴ I; ¹²⁵ I; ¹²⁷ I or ¹³¹ I,

- R ⁶ is C to C ₄ linear alkyl, whereas C ₂ -C ₄ linear alkyl can optionally be terminally substituted by OH group,

- R ⁷ and R ⁸ are independently selected from the group consisting of Q to Ce alkyl; C ₂ to Ce alkenyl; C ₂ to Ce alkynyl; C to C ₈ cycloalkyl; C to C ₈ cycloalkenyl; Ce to Cio aryl; C to Ce heteroaryl comprising at least one heteroatom selected from O, S, N; C to Ce heterocyclyl comprising one or two heteroatoms selected from O, S, N; whereas R ⁷ and R ⁸ can optionally be substituted by OH group or Q-C hydroxyalkyl group; or

- R ⁷ and R ⁸ together with the nitrogen atom form a four- to seven-membered ring, optionally comprising at least one further heteroatom selected from O, S, N, and optionally substituted by OH group or Q-C hydroxyalkyl group; or

- R ⁶ , R ⁷ and R ⁸ together with the nitrogen atom form a bicyclic group containing 5 to 12 carbon atoms and optionally at least one further heteroatom selected from O, S, N, and optionally substituted by OH group or Q-C ₄ hydroxyalkyl group; preferably, such bicyclic group is quinuclidine; - whereas at least one, preferably exactly one, of substituents R ⁶ , R ⁷ and R ⁸ is substituted by OH group or by Q-C4 hydroxy alkyl group;

- Y is a linker formed by a linear or branched hydrocarbon chain having 1 to 10 carbon atoms, whereas optionally 1 to 2 carbon atoms can be replaced by oxygen and/or sulfur atoms; and

- X is a pharmaceutically acceptable anion;

for use in a method of treatment and/or diagnostics of disorders involving pathological function or expression of choline transporters.

The general formula la is meant to include all isomers, enantiomers and diastereomers. The preferred embodiments listed for formula I are applicable also for formula la.

Disorders involving pathological function or expression of choline transporters include in particular oncologic diseases, such as prostate carcinoma, lung carcinoma, glioma; inflammatory, neurological and neuromuscular disorders; atherosclerosis; alcohol addiction; psychiatric disorders, such as depression and attention deficit disorders.

Another object of the present invention is use of the compounds of general formula la according to the present invention for in vitro diagnostics. The compounds of the present invention may be prepared by alkylation of a tertiary amine using a corresponding alkylation agent.

The present invention provides a novel group of aromatic choline analogs which may be radiolabeled by various iodine isotopes. The novel choline analogs overcome the drawbacks of [ ¹⁸ F]fluoromethylcholine, [ ¹⁸ F]fluoroethylcholine, and [ ⁿ C]choline as described above, because they use pharmaceutically relevant iodine radioisotopes with significantly longer half-lives. By an appropriate selection of the iodine isotope [Coenen H. H. et al. (2006), Radioiodination Reactions for Pharmaceuticals: Compendium for Effective Synthesis Strategies, Springer Netherlands, 1st Ed.], radiopharmaceuticals applicable not only for positron emission tomography ( ¹²⁴ I) but also for single -photon computed emission tomography ( 123 I, 125 I, 131 I), planar scintigraphy ( 123 I, 12 I, 131 I), as well as for therapy ( ¹³¹ I) can be obtained.

The compounds of general formula I or la comprising iodine radioisotopes ( 123 I, 124 I, 12 I, 131 I) are particularly suitable for use in radiodiagnostics and radiotherapy. The compounds of general formula I or la comprising stable iodine isotope ¹²⁷ I are particularly suitable for use as precursors of these radiopharmaceuticals and radiodiagnostics, or as medicaments. The preparation of a radiopharmaceutical according to this invention is substantially facilitated by the fact that a non-radioactive pharmaceutical comprising ¹²⁷ I can be converted to radiolabeled pharmaceutical by a simple palladium-catalysed isotopic exchange. This isotopic exchange can take place also in aqueous media under mild conditions, and with short reaction times. Thus, the preparation of a radiopharmaceutical can be performed also directly at the clinical department in an easy and advantageous manner with employment of a radiopharmaceutical kit.

The novel iodinated choline derivatives according to the present invention exhibit a higher affinity to choline transporters than the currently medically utilized fluoromethylcholine (or [ ¹⁸ F]fluoromethylcholine, respectively), and some of them even possess a higher affinity than the natural ligand of the transporters - choline (or [ ⁿ C]choline, respectively). The derivatives of the present invention are more advantageous than the currently used imaging agents. Moreover, they can also be applied in the therapy of disorders involving an increased expression of choline transporters in the target tissue.

Brief Description of Drawings

Fig. 1 represents a radiochromatogram of compound 4 labeled with the radioactive isotope ¹²⁵ I prepared by the procedure described in example 23.

Fig. 2 represents an example of an inhibition curve of choline as described in Biological Assays for Examples 1-40 (inhibition of the uptake of [ ³ H]choline by non-radioactive choline).

Examples of carrying out the Invention

The invention is further illustrated by the following examples, which should not be construed as limiting the claimed scope.

Chemical shifts in the NMR spectra are given in ppm.

The notation in the NMR spectra: s (singlet), d (doublet), t (triplet), m (multiplet), br (broad signal). Abbreviations in the infrared spectra (IR): sh (shoulder in the absorption band), br (broadened band). The following abbreviations describe the intensity of the bands in the IR spectra: vs (very strong), s (strong), m (medium), w (weak), vw (very weak).

NMR spectra were recorded on Bruker DPX 300 (300 MHz for Ή, 75 MHz for ¹³ C) and Bruker Avance-III 600 (600 MHz for Ή, 150 MHz for ¹³ C) NMR spectrometers (Bruker Daltonik, Germany). Elemental analyses for C, H, N were performed on the instrument PE 2400 Series II Analyzer (Perkin Elmer, USA). Elemental analyses for sulfur and iodine were performed on the X-ray fluorescence spectrometer SPECTRO iQ II (Spectro Analytical Instruments, Germany). For chromatographic analyses, HPLC chromatograph Dionex Ultimate 3000 (Dionex, USA) with in-line measurement of radioactivity (FlowStar LB 513 Radio Flow Detector, Berthold Technologies, Germany) and a reverse-phase column (Chromolith Performance RP-18e 100 X 4.6 mm, Merck, Germany) was used. As elution mixture acetonitrile and water with 0.1 % of trifluoroacetic acid was used at a flow rate of 4 mL min ^"1 . Infrared spectra were obtained on the spectrometer Specac MKII Golden Gate Single Reflection ATR System, with a diamond crystal and ray incident angle of 45°.

List of abbreviations

BCA Bicinchoninic acid assay

IR Infrared spectrum

NMR Nuclear magnetic resonance

eq. Equivalent

meq. Milliequivalent (amount of substance multiplied by the ion charge)

DMF N,N-dimethylformamide

DMSO Dimethyl sulfoxide

v/v Volume to volume ratio

MeOH Methanol

Et ₂ 0 Diethyl ether

EtOAc Ethyl acetate

Ms Methanesulfonyl

MsO Methanesulfonate anion

M mol . L ¹

Tris 2- Amino-2-hydroxymethyl-propane- 1 ,3 -diol

Ts 4-Methylbenzenesulfonyl

TsO 4-Methylbenzenesulfonate anion

m.p. Melting point

ATR Attenuated total reflectance

ESI Electrospray ionization

HRMS High resolution mass spectrometry

PBS Phosphate buffered saline

cps Counts per second (number of registered radionuclide decays per second)

HPLC High-performance liquid chromatography

TLC Thin-layer chromatography I. Synthesis of compounds

General procedure for the preparation of compounds 1 to 40 using alkylating agents A to T and the corresponding amines:

Magnetic stirrer, a corresponding alkylating agent, and a corresponding amine were put into a flask. Unless otherwise indicated, DMF was used as a solvent. The flask was flushed with argon, sealed with a rubber septum, immersed into a heated oil bath, and the reaction mixture was stirred. The course of the reaction was monitored by TLC. The solvent was subsequently removed in vacuo by co-evaporation with toluene. Unless otherwise indicated, the product was purified by re -precipitation, which was done as follows: the evaporated residue was dissolved in a minimal quantity of MeOH, transferred into a centrifugation tube, precipitated with a mixture of Et ₂ 0 and hexane, or with pure Et ₂ 0, and the resulting suspension was centrifuged and decanted. The product was then dissolved in MeOH, transferred to a 25 mL pear-shaped flask using a Pasteur pipette, and the solvent was subsequently removed on a rotary evaporator by co-evaporation with toluene. The residual volatiles were finally removed under high vacuum.



Example 1

Preparation of (2-hydroxyethyl)-[2-(3-iodo-4-methoxyphenyl)ethyl]-dimethyla mmonium

4-methylbenzenesulfonate (1)

The alkylating agent A (200 mg, 0.463 mmol, 1 eq.), DMF (2 mL), and 2-(dimethylamino)ethanol (140 μL·, 1.39 mmol, 3 eq.) were used for the synthesis of compound 1. The reaction time was 3 h at the temperature of 80 °C. The precipitation was done twice using a mixture of Et ₂ 0 and hexane (1 :1 v/v, 45 mL). The product was obtained as a white solid (137 mg, 0.263 mmol, 57 %).

H NMR (600 MHz, DMSO-<¾) δ 7.75 (d, / = 1.9 Hz, 1H), 7.48 (d, / = 8.0 Hz, 2H), 7.30 (dd, / = 8.4, 1.8 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 6.97 (d, / = 8.4 Hz, 1H), 5.34 (t, / = 5.0 Hz, 1H), 3.88 - 3.83 (m, 2H), 3.80 (s, 3H), 3.52 - 3.47 (m, 2H), 3.48 - 3.44 (m, 2H), 3.13 (s, 6H), 2.98 - 2.94 (m, 2H), 2.28 (s, 3H). ¹³ C NMR (151 MHz, DMSO-<¾) δ 156.76, 145.69, 139.27, 137.65, 130.47, 130.43, 128.09, 125.49, 111.62, 86.24, 64.82, 64.68, 56.42, 55.04, 50.87, 26.73, 20.82. IR (diamond- ATR): v (cm ¹ ) 3300m,br, 3081vw, 3036vw, 3006vw, 2946w, 2918vw, 2867vw, 2840vw, 1981vw, 1912vw, 1598w, 1566vw, 1491m, 1461m, 1439m, 1407w, 1394w, 1356w, 1280m, 1254m, 1217s, 1194s, 1178s, 1170s, 1119s, 1102m, 1052m, 1032s, 1018m, 1008s, 939w, 923m, 895w, 882w, 856w, 821s, 798w, 780w, 748w, 713w, 680s, 662m, 564s, 551s, 516w, 496w. HRMS (ESI) m/z: [M ⁺ ] (C ₁₃ H ₂₁ IN0 ₂ ⁺ ) calculated: 350.061148, found: 350.06124. Elem. anal. C ₂ iH ₂₈ IN0 ₄ S, calculated: C (46.07), H (5.41), N (2.69), S (6.15), found: C (46.02), H (5.48), N (2.58), S (6.24).

Example 2

Preparation of 3-hydroxy- 1- [2-(3-iodo-4-methoxyphenyl)ethyl]quinuclidin- 1-ium

4-methylbenzenesulfonate (2)

The alkylating agent A (200 mg, 0.463 mmol, 1 eq.), DMF (2 mL), and quinuclidin-3-ol (178 mg, 1.400 mmol, 3 eq.) were used for the synthesis of compound 2. The reaction time was 3 h at the temperature of 80 °C. The precipitation was done first using a mixture of Et ₂ 0 and hexane (1 :1 v/v, 45 mL), and subsequently with pure Et ₂ 0 (45 mL). The product was obtained as an off-white solid (238 mg, 0.425 mmol, 92 %).

M.p. 173-176 °C. *H NMR (600 MHz, DMSO-<¾) δ 7.75 (d, / = 2.2 Hz, 1H), 7.48 (d, / = 8.0 Hz, 2H), 7.29 (dd, / = 8.4, 2.2 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 6.97 (d, / = 8.5 Hz, 1H), 5.60 (d, / = 3.5 Hz, 1H), 4.11 - 4.06 (m, 1H), 3.80 (s, 3H), 3.68 (ddd, / = 12.7, 8.2, 2.9 Hz, 1H), 3.47 - 3.41 (m, 1H), 3.40 - 3.27 (m, 5H), 3.09 (dt, / = 12.8, 2.8 Hz, 1H), 2.96 - 2.86 (m, 2H), 2.28 (s, 3H), 2.16 - 2.10 (m, 1H), 2.06 - 2.02 (m, 1H), 1.96 - 1.89 (m, 1H), 1.81 - 1.71 (m, 2H). ¹³ C NMR (151 MHz, DMSO-<¾) δ 156.73, 145.73, 139.24, 137.62, 130.61, 130.43, 128.08, 125.49, 111.58, 86.22, 63.44, 62.65, 56.42, 54.03, 52.71, 26.36, 26.05, 20.90, 20.81, 17.39. IR (diamond- ATR): v (cm ¹ ) 3274m,br, 3014w, 2938w, 2918w, 2885w, 2840w, 1662vw, 1600w, 1570vw, 1491m, 1461w, 1421w, 1392w, 1363w, 1338w, 1324w, 1284w, 1254m, 1212s, 1180s, 1168s, 1119s, 1098m.l049m, 1032m, 1008s, 975w, 946w, 923w, 906w, 884w, 820m, 761w, 710w, 682s, 664m, 638w, 563s, 492w, 468w. HRMS (ESI) m/z: [M ⁺ ] (C ₁₆ H ₂₃ IN0 ₂ ⁺ ) calculated: 388.076798, found: 388.07672. Elem. anal. C ₂ 3H3 ₀ INO ₅ S, calculated: C (49.38), H (5.40), N (2.50), I (22.68), S (5.73), found: C (49.51) H (5.35), N (2.24), I (22.40), S (5.76).

Example 3

Preparation of (2-hydroxyethyl)-[2-(3,5-diiodo-4-methoxyphenyl)ethyl]-dimet hylammonium 4-methylbenzenesulfonate (3)

The alkylating agent B (350 mg, 0.627 mmol, 1 eq.), DMF (3 mL), and 2-(dimethylamino)ethanol (190 μL·, 1.888 mmol, 3 eq.) were used for the synthesis of compound 3. The reaction time was 4 h at the temperature of 85 °C. The precipitation was done once using a mixture of Et ₂ 0 and hexane (1 :1 v/v, 45 mL), and subsequently with pure Et ₂ 0 (40 mL). The product was obtained as a white solid (258 mg, 0.399 mmol, 64 %).

M.p. 178-180 °C, *H NMR (600 MHz, DMSO-<¾) δ 7.82 (s, 2H), 7.48 (d, / = 8.1 Hz, 2H), 7.11 (d, / = 7.9 Hz, 2H), 5.34 (t, / = 5.0 Hz, 1H), 3.88 - 3.83 (m, 2H), 3.73 (s, 3H), 3.52 - 3.48 (m, 2H), 3.46 - 3.43 (m, 2H), 3.11 (s, 6H), 3.00 - 2.93 (m, 2H), 2.28 (s, 3H). ¹³ C NMR (151 MHz, DMSO-<¾) δ 157.38, 145.67, 140.12, 137.64, 136.74, 128.08, 125.48, 91.52, 64.94, 64.21, 60.29, 55.00, 50.90, 26.26, 20.82. IR (diamond- ATR): v (cm ¹ ) 3322m,br, 3032w, 2999vw, 2948vw, 2934vw, 2915vw, 2870vw, 2850vw, 2817vw, 1991vw, 1980vw, 1930vw, 191 lvw, 1893vw, 1803vw, 1752vw, 1599vw, 1581w, 1536w, 1493w, 1480w, 1461m, 1414m, 1396w, 1377w, 1360w, 1337w, 1326w, 1306w, 1248m, 1207m, 1196s, 1173s, 1150m, 1122s, 1102m, 1094m, 1054w, 1032s, 1009s, 996s, 978w, 961w, 938m, 91 lw, 889w, 869w, 854w, 840w, 812m, 799w, 772w, 742w, 706m, 682s, 622w, 610w, 564s, 552s, 517w, 493w, 463w. HRMS (ESI) m z: [M ⁺ ] (C ₁₃ H ₂₀ I ₂ NO ₂ ⁺ ) calculated: 475.957791, found: 475.95731, Elem. anal. C ₂₀ H ₂₇ I ₂ NO ₅ S, calculated: C (37.11), H (4.20), N (2.16), I (39.21), S (4.95), found: C (37.14), H (4.27), N (1.98), 1 (39.23), S (5.17).

Example 4

Preparation of (2-hydroxyethyl)-[3-(3-iodo-4-methoxyphenyl)propyl]-dimethyl ammonium 4-methylbenzenesulfonate (4)

The alkylating agent C was used (342 mg, 0.766 mmol, 1 eq.), DMF (3 mL), and 2-(dimethylamino)ethanol (190 μL·, 1.888 mmol, 3 eq.) were used for the synthesis of compound 4. The reaction time was 2 h at the temperature of 80 °C. The product was purified by chromatography on Cis-silica gel (eluent 4:6 MeOH: water v/v). The product was obtained as a white solid (354 mg, 0.661 mmol, 86 %).

M.p. 120-122 °C. *H NMR (600 MHz, DMSO-<¾) δ 7.67 (d, / = 2.2 Hz, 1H), 7.49

(d, / = 8.0 Hz, 2H), 7.23 (dd, / = 8.4, 2.2 Hz, 1H), 7.12 (d, / = 7.9 Hz, 2H), 6.93 (d, / = 8.5 Hz, 1H), 5.28 (br s, 1H), 3.82 - 3.78 (m, 2H), 3.79 (s, 3H), 3.40 - 3.36 (m, 2H), 3.34 - 3.30 (m, 2H), 3.05 (s, 6H), 2.49 (t, / = 8.0 Hz, 2H), 2.28 (s, 3H), 1.98 - 1.92 (m, 2H). ¹³ C NMR (151 MHz, DMSO-<¾ δ 156.22, 145.70, 138.53, 137.66, 134.67, 129.62, 128.10, 125.49, 111.44, 86.14, 64.63, 63.60, 56.37, 54.97, 50.91, 30.23, 23.86, 20.82. IR (diamond- ATR): v (cm ^_1 ) 3324m,br, 3035vw, 3021vw, 3000vw, 2972vw, 2958vw, 2937w, 2862vw, 2840vw, 1981vw, 1915vw, 1655vw,

1598w, 1562vw, 1486m, 1474w, 1459w, 1441w, 1418w, 1400w, 1362w, 1290w, 1278w, 1253m, 1227m, 1214m, 1173s, 1150m, 1118s, 1096m, 1073m, 1048m, 1030s, 1020w, 1008s, 968w, 944w, 893w, 846w, 814s, 800w, 756w, 712w, 680s, 663m, 598w, 562s, 540m, 512w, 494w. HRMS (ESI) m z: [M) ⁺ ] (Ci ₄ H ₂₃ IN0 ₂ ⁺ ) calculated: 364.076798, found: 364.07690. Elem. anal. C ₂₁ H ₃₀ INO ₅ S, calculated: C (47.11), H (5.65), N (2.62), S (5.99), found: C (47.18), H (5.63), N, (2.47), S (6.04).

Example 5

Preparation of (2-hydroxyethyl)-[2-(4-iodophenyl)ethyl]-dimethylammonium

4-methylbenzenesulfonate (5)

The alkylating agent D was used (400 mg, 0.99 mmol, 1 eq.), DMF (5 mL), and 2-(dimethylamino)ethanol (300 μL·, 2.98 mmol, 3 eq.) were used for the synthesis of compound 5. The reaction time was 2 h at the temperature of 60 °C and 2 h at the temperature of 80 °C. The precipitation was done once using the mixture of Et ₂ 0 and hexane (1 : 1 v/v, 45 mL) and subsequently twice with pure Et ₂ 0 (40 mL). The product was obtained as a white solid (270 mg, 0.55 mmol,

56 %).

M.p. 204-206 °C. *H NMR (600 MHz, DMSO-<¾) δ 7.70 (d, / = 8.2 Hz, 2H), 7.48

(d, / = 8.0 Hz, 2H), 7.13 (d, / = 8.2 Hz, 2H), 7.11 (d, / = 8.0 Hz, 2H), 5.35 (t, / = 5.0 Hz, 1H), 3.88 - 3.83 (m, 2H), 3.55 - 3.50 (m, 2H), 3.49 - 3.44 (m, 2H), 3.13 (s, 6H), 3.03 - 2.97 (m, 2H), 2.28 (s, 3H). ¹³ C NMR (151 MHz, DMSO-<¾ δ 145.66, 137.67, 137.38, 136.25, 131.48, 128.10, 125.48, 92.88, 64.78, 64.27, 55.04, 50.90, 27.73, 20.82. IR (diamond- ATR): v (cm ^_1 ) 3347m,br, 3041w, 3026w, 3003w, 2968w, 2947w, 2915w, 2864w, 1981vw, 1936vw, 1902vw, 1596w, 1484m, 1468w,

1442w, 1424w, 1406w, 1400w, 1354w, 1322w, 1306w, 1287w, 1218s, 1181s, 1168s, 1154m, 1118s, 1102m, 1084w, 1063m, 1053w, 1030s, 1007s, 975w, 946w, 923m, 907w, 883w, 858w, 832m, 824m, 802m, 730w, 713w, 682s, 628w, 564s, 552s, 512m, 453w. HRMS (ESI) m z: [M ⁺ ] (C ₁₂ H ₁₉ INO ⁺ ) calculated: 320.050584, found: 320.05048. Elem. anal. Ci ₉ H ₂₆ lN0 ₄ S, calculated: C (46.44), H (5.33), N (2.85), I (25.83), S (6.53), found: C (46.65), H (5.32), N (2.64), I (25.56), S (6.55).

Example 6

Preparation of 3-hydroxy-l-[2-(4-iodophenyl)ethyl]-l-methylpiperidin-l-ium

4-methylbenzenesulfonate (6)

The alkylating agent D (400 mg, 0.99 mmol, 1 eq.), DMF (5 mL), and l-methylpiperidine-3-ol (344 μΕ, 2.98 mmol, 3 eq.) were used for the synthesis of compound 6. The reaction time was 2 h at the temperature of 60 °C, 2 h at the temperature of 80 °C, and 2 h at the temperature of 90 °C.

The precipitation was done three times using a mixture of Et ₂ 0 and hexane (1 : 1 v/v, 45 mL). The product was obtained as a mixture of two diastereomers (1 : 1 ratio according to the NMR analysis) as a pale-yellow solid(269 mg, 0.52 mmol, 53 %).

lU NMR (600 MHz, DMSO-<¾) δ 7.71 (d, / = 8.3 Hz, 1H, diastereomer 1), 7.70 (d, / = 8.3 Hz, 1H, diastereomer 2), 7.48 (d, / = 8.2 Hz, 2H, 4-methylbenzenesulfonate), 7.16 (d, / = 8.2 Hz, 1H), 7.13 (d, / = 8.2 Hz, 1H), 7.11 (d, / = 8.2 Hz, 2H, 4-methylbenzenesulfonate), 5.52 (d, / = 4.5 Hz, 1H), 5.44 (d, / = 4.2 Hz, 1H), 4.06 - 3.99 (m, 2H), 3.72 - 3.66 (m, 1H), 3.58 - 3.49 (m, 2H), 3.48 - 3.30 (m, 6H), 3.21 (s, 3H), 3.16 (d, / = 7.0 Hz, 1H), 3.14 (d, / = 6.9 Hz, 1H), 3.10 (s, 3H), 3.07 - 2.94 (m,

4H), 2.28 (s, 6H), 1.98 - 1.87 (m, 2H), 1.87 - 1.75 (m, 4H), 1.52 - 1.40 (m, 2H). ^lj C NMR (151 MHz, DMSO-<¾ δ 145.70, 137.64, 137.41, 137.34, 136.37, 136.19, 131.58, 131.50, 128.09, 125.49, 92.89, 92.88, 65.02, 63.55, 63.03, 62.78, 61.45, 61.31, 60.02, 59.84, 49.93, 47.83, 30.00, 29.38, 27.24, 26.93, 20.82, 17.06, 16.70. IR (diamond- ATR): v (cm ^"1 ) 3320m,br, 3056vw, 3021vw, 2998vw, 2949w, 2922vw, 2869vw, 1979vw, 1968vw, 1954vw, 1933vw, 1915vw, 1906vw, 1598w, 1585w, 1562w, 1493w, 1457m, 1383w, 1354w, 1278w, 1206m, 1176s, 1119s, 1080m, 1052w, 1032s, 1008s, 914w, 876m, 816m, 765m, 745w, 734w, 71 lw, 679s, 648m, 563s, 518m, 494m. HRMS (ESI) m z: [M ⁺ ] (Ci ₄ H ₂ iINO ⁺ ) calculated: 346.066234, found: 346.06600, Elem. anal. C ₂ iH ₂₈ IN0 ₄ S, calculated: C (48.75), H (5.45), N (2.71), I (24.53), S (6.20), found: C (48.91), H (5.48), N (2.59), I (24.25), S (6.25). Example 7

Preparation of l-(2-hydroxyethyl)-l-[2-(4-iodophenyl)ethyl]pyrrolidin-l-ium

4-methylbenzenesulfonate (7)

The alkylating agent D (400 mg, 0.99 mmol, 1 eq.), DMF (5 mL), and 2-(pyrrolidin-l-yl)ethanol (350 μL·, 2.99 mmol, 3 eq.) were used for the synthesis of compound 7. The reaction time was 2 h at the temperature of 60 °C, 2 h at the temperature of 80 °C, and 2 h at the temperature of 90 °C. The precipitation was done three-times using a mixture of Et ₂ 0 and hexane (1 :2 v/v, 45 mL). The product was obtained in as a pale-yellow solid (165 mg, 0.32 mmol, 32 %).

M.p. 152-154 °C. *H NMR (600 MHz, DMSO-<¾) δ 7.69 (d, / = 7.9 Hz, 2H), 7.48 (d, / = 7.8 Hz, 2H), 7.15 (d, / = 7.9 Hz, 2H), 7.11 (d, / = 7.8 Hz, 2H), 5.40 (t, / = 5.0 Hz, 1H), 3.88 - 3.83 (m, 2H), 3.66 - 3.54 (m, 4H), 3.52 - 3.46 (m, 4H), 3.04 - 2.99 (m, 2H), 2.28 (s, 3H), 2.10 - 2.02 (m, 4H). ¹³ C NMR (151 MHz, DMSO-<¾ δ 145.67, 137.66, 137.32, 136.36, 131.51, 128.09, 125.48, 92.84, 62.78, 60.27, 60.08, 55.73, 28.36, 21.21, 20.82. IR (diamond- ATR): v (cm ^_1 ) 3314m,br, 3064vw, 3040vw, 3021vw, 3007vw, 2978vw, 2965vw, 2916vw, 2890vw, 2862vw, 1995vw, 1981vw, 1935vw, 1912vw, 1823vw, 1795vw, 1670vw, 1598w, 1484m, 1468w, 1443w, 1418w, 1402w, 1389w, 1379w, 1359w, 1346w, 1321w, 1307w, 1212s, 1200m, 1184s, 1168s, 1120s, 1104m, 1086m, 1060w, 1032s, 1007s, 973w, 941w, 908w, 884w, 835m, 822m, 800m, 724w, 712w, 682m, 655m, 641m, 562s, 553s,sh, 524w, 508m, 496w. HRMS (ESI) m z: [M) ⁺ ] (Ci ₄ H ₂ iINO ⁺ ) calculated: 346.066234, found: 346.06629, Elem. anal. C ₂ iH ₂₈ IN0 ₄ S, calculated: C (48.75), H (5.45), N (2.71), I (24.53), S (6.20), found: C (48.93), H (5.46), N (2.53), I (24.07), S (6.30).

Example 8

Preparation of 3-hydroxy-l-[2-(4-iodophenyl)ethyl]quinuclidin-l-ium 4-methylbenzenesulfonate

(8)

The alkylating agent D (400 mg, 0.99 mmol, 1 eq.), DMF (5 mL), and quinuclidin-3-ol (380 mg, 2.99 mmol, 3 eq.) were used for the synthesis of compound 8. The reaction time was 3 h at the temperature of 60 °C. The precipitation was done once using a mixture of Et ₂ 0 and hexane (1 : 1 v/v, 45 mL), and twice with pure Et ₂ 0 (40 mL). The product was obtained as an off-white solid (425 mg, 0.80 mmol, 80 %).

M.p. 217-218 °C. H NMR (600 MHz, DMSO-<¾) δ 7.70 (d, / = 8.2 Hz, 2H), 7.48 (d, / = 8.0 Hz, 2H), 7.14 - 7.10 (m, 4H), 5.60 (d, / = 3.5 Hz, 1H), 4.10 - 4.06 (m, 1H), 3.69 (ddd, / = 12.7, 8.3, 3.0 Hz, 1H), 3.48 - 3.42 (m, 1H), 3.41 - 3.28 (m, 5H), 3.09 (dt, / = 12.9, 2.8 Hz, 1H), 2.99 - 2.90 (m, 2H), 2.28 (s, 3H), 2.17 - 2.09 (m, 1H), 2.06 - 2.01 (m, 1H), 1.95 - 1.88 (m, 1H), 1.81 - 1.70 (m, 2H). ¹³ C NMR (151 MHz, DMSO- ₆ ) δ 145.69, 137.65, 137.35, 136.43, 131.46, 128.09, 125.49, 92.83, 63.44, 63.02, 62.67, 54.04, 52.74, 27.06, 26.36, 20.90, 20.82, 17.39. IR (diamond- ATR): v (cm ¹ ) 3259m,br, 3061vw, 3036vw, 3020vw, 301 lvw, 2956w, 2920vw, 2879vw, 1991 v , 1980vw, 1920vw, 1899vw, 1802vw, 1655vw, 1596vw, 1486m, 1456w, 1386w, 1360w, 1338w, 1323w, 1306w, 1217s, 1184s, 1137w, 1120s, 1096m, 1060m, 1031s, 1006s, 969w, 954w, 944w, 922w, 906w, 884w, 841w, 816m, 806m,sh, 720w, 711w, 680s, 639w, 564s, 551m,sh, 508m, 485w. HRMS (ESI) m z: [M ⁺ ] (d ₅ H ₂₁ INO ⁺ ) calculated: 358.066234, found: 358.06608, Elem. anal. C ₂ 2H ₂₈ IN0 ₄ S, calculated: C (49.91), H (5.33), N (2.65), I (23.97), S (6.06), found: C (50.12), H (5.37), N (2.46), I (23.73), S (6.21).

Example 9

Preparation of (2-hydroxyethyl)-(3-iodobenzyl)-dimethylammonium methanesulfonate (9)

The alkylating agent E (200 mg, 0.67 mmol, 1 eq.), DMF (1.5 mL), and 2-(dimethylamino)ethanol (134 μL·, 1.33 mmol, 2 eq.) were used for the synthesis of compound 9. The reaction time was 1 h at the temperature of 80 °C. The the product was precipitated once from a dichloromethane solution with a mixture of Et ₂ 0 and hexane (2:1 v/v, 45 mL) and once from MeOH solution by a mixture of Et ₂ 0 and hexane (2:1 v/v, 45 mL). The product was obtained in as a colorless viscous liquid (259 mg, 0.67 mmol, 100 %).

δ 7.99 (dd, / = 1.8, 1.7 Hz, 1H), 7.91 (ddd, / = 7.9, 7.7, 1.7, 1.1 Hz, 1H), 7.32 (dd, / = 7.9, 7.7 Hz, 1H), 2H), 3.95 - 3.87 (m, 2H), 3.41 - 3.34 (m, 2H), 3.01

Example 10

Preparation of l-(2-hydroxyethyl)-l-(3-iodobenzyl)pyrrolidin-l-ium methanesulfonate (10)

The alkylating agent E (400 mg, 1.27 mmol, 1 eq.), DMF (2.0 mL), and 2-(pyrrolidin-l-yl)ethanol

(223 μL·, 1.91 mmol, 1.5 eq.) were used for the synthesis of compound 10. The reaction time was 1 h at the temperature of 75 °C. The product was precipitated from a MeOH solution once with a mixture of Et ₂ 0 and hexane (2: 1 v/v, 45 mL) and once with pure Et ₂ 0 (45 mL). The product was obtained as a yellowish viscous liquid (500 mg, 1.16 mmol, 91 %).

*H NMR (300 MHz, DMSO-<¾) δ 8.02 (t, / = 1.7 Hz, 1H), 7.89 (dt, / = 7.9, 1.1

8 - δ 4,

20.84.

Example 11

Preparation of (2-hydroxyethyl)-(4-iodobenzyl)-dimethylammonium bromide (11)

The alkylating agent F (400 mg, 1.35 mmol, 1 eq.), DMF (2 mL), and 2-(dimethylamino)ethanol (303 μL·, 3.01 mmol, 2.2 eq.) were used for the synthesis of compound 11. The reaction time was 30 min at the temperature of 75 °C. The from the product was precipitated from a MeOH solution once with a mixture of Et ₂ 0 and hexane (3: 1 v/v, 40 mL) and once with pure Et ₂ 0 (40 mL). The product was obtained as a white solid (472 mg, 1.22 mmol, 91 %).

-<¾ δ 7.88 (d, / = 8.2 Hz, 2H), 7.39 (d, / = 8.2 Hz, 2H), (s, 2H), 3.94 - 3.86 (m, 2H), 3.43 - 3.37 (m, 2H), 3.03 (s, MSO-<¾ δ 137.67, 135.10, 127.75, 98.01 , 66.51 , 64i

10

Example 12

Preparation of diethyl-(2-hydroxyethyl)-(4-iodobenzyl)ammonium bromide (12)

The alkylating agent F (400 mg, 1.35 mmol, 1 eq.), DMF (2 mL), and 2-(diethylamino)ethanol (268 μL·, 2.02 mmol, 1.5 eq.) were used for the synthesis of compound 12. The reaction time was 30 min at the temperature of 75 °C. The precipitation was performed from a MeOH solution once with the mixture of Et ₂ 0 and hexane (3: 1 v/v, 40 mL) and once with pure Et ₂ 0 (40 mL). The product was obtained as a white solid (459 mg, 1.11 mmol, 82 %).

1 ^{h nmr} (300 MHz, DMSO-<¾ δ 7.87 (d, / = 8.3 Hz, 2H), 7.36 (d, / = 8.3 Hz, 2H), 5.38 (t, / = 5.1 Hz, 1H), 4.58 (s, 2H), 3.88 (dt, / = 5.2, 5.1 Hz, 1H), 3.30 - 3.20 (m, 6H), 1.31 (t, / = 7.1 Hz, 6H). ¹³ C NMR (75 MHz, DMSO-<¾ δ 137.76, 134.87, 127.48, 97.87, 60.30, 58.06, 54.45, 53.25, 7.79. Example 13

Preparation of l-(2-hydroxyethyl)-l-(4-iodobenzyl)pyrrolidin-l-ium bromide (13)

The alkylating agent F (400 mg, 1.35 mmol, 1 eq.), DMF (2 mL), and 2-(pyrrolidin-l -yl)ethanol (236 μL·, 2.02 mmol, 1.5 eq.) were used for the synthesis of compound 13. The reaction time was 30 min at the temperature of 75 °C. The product was precipitated from a MeOH solution once with a mixture of Et ₂ 0 and hexane (3: 1 v/v, 40 mL) and once with pure Et ₂ 0 (40 mL). The product was obtained as a white solid(484 mg, 1.17 mmol, 87 %).

*H NMR (300 MHz, DMSO-<¾ δ 7.87 (d, / = 8.3 Hz, 2H), 7.42 (d, / = 8.3 Hz, 2H), 5.43 (t, / = 4.9 Hz, 1H), 4.63 (s, 2H), 3.93 (dt, / = 4.9, 4.9 Hz, 2H), 3.64 - 3.45 (m, 4H), 3.31 - 3.23 (m, 2H), 2.14 - 1.99 (m, 4H). ¹³ C NMR (75 MHz, DMSO- ₆ ) δ 137.75, 134.86, 128.42, 97.83, 61.71 , 60.90, 59.75, 55.31 , 20.78. Example 14

Preparation of l-(2-hydroxyethyl)-l-(2-iodobenzyl)piperidin-l-ium 4-methylbenzenesulfonate

(14)

The alkylating agent G (600 mg, 2.38 mmol, 1 eq.) and l-(2-hydroxyethyl)piperidine (4 mL, 30 mmol, 12.5 eq.) without any additional solvent were used for the synthesis of compound 14. The reaction was carried out for 18 h at the temperature of 80 °C. The reaction mixture was diluted with water (50 mL) and the residual amine was removed by extraction with ethyl acetate (4 X 40 mL). The aqueous phase was concentrated, the oily residue was dissolved in MeOH and was slowly passed through a column containing a strong anion exchange resin (30 mL, particle size 650 - 820 μπι, 1 mekv. mL ¹ ) in TsO cycle. The solvent from the collected solution was removed in vacuo.The product was obtained in the form of brown viscous liquid, which subsequently crystallized upon cooling (938 mg, 1.81 mmol, 76 %).

*H NMR (300 MHz, DMSO-ifc) δ 8.08 (dd, / = 8.0, 1.3 Hz, 1H), 7.68 (dd, /

1.6 Hz, 1H), 7.54 (ddd, / = 7.5, 7.5, 1.3 Hz, 1H), 7.49 (d, / = 8.1 Hz, 2H), 7.26

(ddd, / = 7.8, 7.8, 1.5 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 5.48 (t, / = 4.7 Hz, 1H),

4.80 (s, 2H), 4.08 - 3.87 (m, 2H), 3.73 - 3.58 (m, 4H), 3.19 - 3.07 (m, 2H), 2.28 (s, 3H), 2.05 - 1.83 (m, 2H), 1.82 - 1.67 (m, 2H), 1.65 - 1.47 (m, 1H), 1.40 - 1.17 (m, 1H). ¹³ C NMR (75 MHz, DMSO-<¾ δ 145.66, 140.99, 137.65, 135.25, 132.16, 130.87, 128.79, 128.07, 125.48, 105.67, 69.34, 58.65, 57.77, 54.94, 20.80, 20.09, 19.29. IR (diamond- ATR): v (cm ¹ ) 3343m,br, 3086vw, 3063vw, 3023w, 2960w, 2918vw, 2884w, 1982vw, 1967vw, 1953vw, 1916vw, 1660vw, 1598vw, 1586vw, 1492w, 1465w, 1455m, 1436w, 1400w, 1346w, 1315w, 1290w, 1213s, 1178s, 1120s, 1103m, 1052m, 1034s, 1010s, 990w, 946w, 932w, 908w, 868w, 818m, 798w, 763s, 726w, 712w, 679s, 650m, 623w, 561s, 464w. HRMS (ESI) m/z: [M ⁺ ] (Ci ₄ H ₂ iINO ⁺ ) calculated: 346.066234, found: 346.06628, Elem. anal. C ₂ iH ₂₈ IN0 ₄ S, calculated: C (48.75), H (5.45), N (2.71), S (6.20), found: C (49.07), H (5.58), N (2.62), S (6.28).

General procedure for preparation of compounds 15 to 18 with the alkylating agent H:

A magnetic stir bar, alkylating agent H (400 mg, 1.16 mmol, 1 eq.), DMF (5 mL), and an appropriate amine (3.49 mmol, 3 eq.) were put into a pear-shaped flask. The flask was flushed with argon, closed by a rubber septum, immersed into an oil bath heated to 50 °C, and the reaction mixture was stirred for 2 h. The course of the reaction was monitored by TLC. The solvent was removed on a rotary evaporator by co-evaporation with toluene. The residue was then dissolved in a minimum amount of MeOH, transferred into a centrifugation tube, precipitated with a mixture of Et ₂ 0 and hexane (1 :1 v/v, 45 mL), centrifuged and decanted. The resulting pellet was then re -dissolved in a minimal volume of MeOH, precipitated with pure Et ₂ 0, centrifuged and decanted (this procedure was repeated two times). The obtained product was dissolved in MeOH and was passed slowly through a column containing a strong anion exchange resin (15 mL, particle size 650 - 820 μπι, 1 meq. mL ¹ ) in TsO cycle. The solvent was removed from the solution on a rotary evaporator. In the case of liquid samples, solvent were removed by co-evaporation with toluene. Residual solvents were then removed under high vacuum.

Example 15

Preparation of Diethyl-(2-hydroxyethyl)-(2-iodobenzyl)ammonium 4-methylbenzenesulfonate

(15)

A colorless viscous liquid, which crystallized upon cooling (477 mg, 0.94 mmol, 81 %).

TsO" M.p. 103-105°C. *H NMR (600 MHz, DMSO-<¾) δ 8.07 (dd, / = 7.9, 1.3 Hz, 1H),

7.67 (dd, / = 7.8, 1.4 Hz, 1H), 7.55 (ddd, / = 7.6, 7.6, 1.3 Hz, 1H), 7.48 (d, / = 8.0 Hz, 2H), 7.26 (ddd, / = 7.7, 7.7, 1.6 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 5.44 (br s, 1H), 4.72 (s, 2H), 3.87 (br s, 2H), 3.47 - 3.37 (m, 6H), 2.28 (s, 3H), 1.28 (t, / = 7.1 Hz, 6H). ¹³ C NMR (151 MHz, DMSO-<¾) δ 145.71, 140.92, 137.63, 134.11, 132.17,

131.26, 129.07, 128.08, 125.49, 105.51, 65.31, 59.38, 54.76, 54.32, 20.81, 8.18. IR (diamond- ATR): v (cm ¹ ) 3270m,br, 3010w, 2990vw, 2972w, 2916vw, 2889vw, 2870vw, 1990vw, 1980vw, 1646vw, 1596vw, 1581vw, 1567vw, 1494w, 1468w, 1457w, 1443w, 1424w, 1402w, 1382w, 1357w, 1304w, 1274w, 1215m, 1186s, 1160m, 1116m, 1090m, 1052w, 1033m, 1010s, 982w, 963w, 934w, 890w, 858w, 820m, 802w, 785w, 760m, 730w, 721m, 713m, 678s, 648m, 624w, 562s, 526w, 496w, 458w. HRMS (ESI) m/z: [M ⁺ ] (C ₁₃ H ₂₁ INO ⁺ ) calculated: 334.066234, found: 334.06648. Elem. anal. C ₂₀ H ₂₈ INO ₄ S, calculated: C (47.53), H (5.58), N (2.77), I (25.11), S (6.34), found: C (47.42), H (5.60), N (2.74), I (25.01), S (6.51).

Example 16

Preparation of 3-hydroxy-l-(2-iodobenzyl)-l-methylpiperidin-l-ium 4-methylbenzenesulfonate

(16)

Mixture of diastereomers (2: 1 ratio according to the NMR analysis) in the form of an off-white hygroscopic solid (424 mg, 0.84 mmol, 73 %).

_Ts0 - 30 *H NMR (300 MHz, DMSO-<¾) δ 8.08 (dd, / = 8.0, 1.3 Hz, 1H), 7.72 (dd, / =

7.8, 1.7 Hz, 0.67H), 7.65 (dd, / = 7.7, 1.7 Hz, 0.33H), 7.58 - 7.52 (m, 1H), 7.49 (d, / = 8.0 Hz, 2H), 7.27 (ddd, / = 7.7, 7.7, 1.7 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 5.50 (s, 1H), 4.84 - 4.58 (m, 2H), 4.22 - 4.14 (m, 0.33H), 4.05 - 3.93 (m, 0.67H), 3.57 - 3.01 (m, 4H), 3.14 (s, 1H), 3.04 (s, 2H), 2.29 (s, 3H), 2.11 - 1.69 (m, 3H), 1.68 - 1.52 (m, 0.33H), 1.41 - 1.22 (m, 0.67H). ¹³ C NMR (75 MHz, DMSO-<¾) δ 145.70, 145.69, 141.01, 137.61, 135.16, 134.84, 132.25, 132.23, 130.76, 130.71, 128.78, 128.72, 128.06, 125.48, 105.15, 105.10, 72.17, 63.77, 63.54, 61.94, 60.99, 60.45, 59.76, 49.23, 46.65, 30.51, 28.50, 20.79, 17.76, 17.75, 16.31. IR (diamond-ATR): v (cm ¹ ) 3320m,br, 3056vw, 3021vw, 2998vw, 2949w, 2922vw, 2869vw, 1979vw, 1968vw, 1954vw, 1933vw, 1915vw, 1906vw, 1598w, 1585w, 1562w, 1493w, 1457m, 1383w, 1354w, 1278w, 1206m, 1176s, 1119s, 1080m, 1052w, 1032s, 1008s, 914w, 876m, 816m, 765m, 745w, 734w, 71 lw, 679s, 648m, 563s, 518m, 494m. HRMS (ESI) m/z: [M ⁺ ] (Ci ₃ H ₁₉ INO ⁺ ) calculated: 332.050584, found: 332.05079. Elem. anal. C ₂₀ H ₂₆ INO ₄ S O.5H ₂ O, calculated: C (46.88), H (5.31), N (2.73), I (24.77), S (6.26), found: C (47.13), H (5.36), N (2.78), I (24.67). Example 17

Preparation of 1 ,4-bis(2-hydroxyethyl) - 1 -(2-iodobenzyl)piperazin- 1 -ium

4-methylbenzenesulfonate (17)

White amorphous highly hygroscopic solid (502 mg, 0.89 mmol, 77 %).

*H NMR (600 MHz, DMSO-<¾) δ 8.09 (dd, / = 8.0, 1.2 Hz, 1H), 7.71

(dd, / = 7.7, 1.6 Hz, 1H), 7.56 (ddd, / = 7.6, 7.6, 1.2 Hz, 1H), 7.49 (d, / = 8.0 Hz, 2H), 7.28 (ddd, / = 7.7, 7.7, 1.6 Hz, 1H), 7.12 (d, / = 7.9 Hz,

2H), 5.52 (t, / = 4.8 Hz, 1H), 4.86 (s, 2H), 4.49 (br s, 1H), 4.03 - 3.99 (m, 2H), 3.77 - 3.65 (m, 4H), 3.44 (q, / = 5.7 Hz, 2H), 3.24 (t, / = 10.7 Hz, 2H), 2.96 - 2.86 (m, 2H), 2.82 - 2.65 (m, 2H), 2.46 - 2.41 (m, 2H), 2.28 (s, 3H). ¹³ C NMR (151 MHz, DMSO-<¾ δ 145.64, 141.06, 137.70, 135.55, 132.33, 130.64, 128.85, 128.11, 125.50, 105.87, 69.36, 58.71, 58.47, 58.27, 56.81, 54.94, 46.05, 20.82. IR (diamond-ATR): v (cm ¹ ) 3352m,br, 3082vw, 3056vw, 3018vw, 2937w, 2920w, 2869vw, 2834w, 1993vw, 1966vw, 1925 vw, 1904vw, 1654vw, 1598w, 1584w, 1562w, 1494w, 1453m, 1360w, 1283w, 1207s, 1173s, 1120s, 1081w, 1053w, 1031s, 1008s, 988w, 944w, 932w, 888w, 855w, 816m, 800w, 764m, 728w, 71 lw, 680s, 648w, 633w, 601w, 563s, 493w, 450w. HRMS (ESI) m/z: [M ⁺ ] (C ₁₅ H ₂₄ IN ₂ 0 ₂ ⁺ ) calculated: 391.087697, found: 391.08771.

Example 18

Preparation of 3-hydroxy-l-(2-iodobenzyl)quinuclidin-l-ium 4-methylbenzenesulfonate (18)

White solid (496 mg, 0.96 mmol, 83 %).

M.p. 154-156 °C. ^! H NMR (600 MHz, DMSO-<¾) δ 8.07 (dd, / = 8.0, 1.2 Hz, 1H), 7.66 (dd, / = 7.7, 1.6 Hz, 1H), 7.54 (ddd, / = 7.5, 7.5, 1.3 Hz, 1H), 7.48 (d, / = 8.1 Hz, 2H), 7.25 (ddd, / = 7.7, 7.7, 1.6 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 5.60 (s, 1H), 4.59 (d, / = 13.5 Hz, 1H), 4.54 (d, / = 13.5 Hz, 1H), 4.08 - 4.02 (m, 1H), 3.74 (ddd, / = 12.7, 8.3, 3.0 Hz, 1H), 3.51 (tt, / = 11.2, 3.8 Hz, 1H), 3.47 - 3.32 (m, 3H), 3.14 (dt, / = 12.7, 2.8 Hz, 1H), 2.28 (s, 3H), 2.15 - 2.07 (m, 1H), 2.03 - 1.99 (m, 1H), 1.94 - 1.87 (m, 1H), 1.80 - 1.70 (m, 2H). ¹³ C NMR (151 MHz, DMSO-<¾ δ 145.73, 140.90, 137.63, 135.04, 132.17, 130.73, 128.78, 128.08, 125.50, 105.03, 69.40, 63.31, 63.21, 54.68, 53.51, 26.39, 20.97, 20.82, 17.47. IR (diamond-ATR): v (cm- ¹ ) 3185m,br, 3088w, 3060w, 3052w, 3013w, 2982w, 2946w, 2888w, 1931vw, 1855vw, 1826vw, 1664vw, 1598w, 1586w, 1560w, 1490w, 1460m, 1436w, 1425w, 1412w, 1385w, 1347w, 1318w, 1279w, 1217m, 1171s, 1133m,sh, 1119s, 1092m, 1070w, 1048w, 1032s, 1010s, 997m, 966w, 940w, 910w, 889w, 852w, 827m, 802w, 774m, 733w, 714w, 678s, 648w, 619w, 601w, 560s, 550m,sh, 484w, 469w. HRMS (ESI) m/z: [M ⁺ ] (Ci ₄ H ₁₉ INO ⁺ ) calculated: 344.050584, found: 344.05065. Elem. anal. C ₂₁ H ₂₆ INO ₄ S, calculated: C (48.94), H (5.08), N (2.72), I (24.62), S (6.22), found: C (48.64), H (5.15), N (2.59), 1 (24.21), S (6.11).

Example 19

Preparation of (2-hydroxyethyl)-(2-iodobenzyl)-dimethylammonium 4-methylbenzenesulfonate

(19)

Colorless viscous liquid (500 mg, 1.05 mmol, 90%).

*H NMR (600 MHz, DMSO-<¾) δ 8.08 (dd, / = 8.0, 1.2 Hz, 1H), 7.72 (dd, / = 7.7, 1.6 Hz, 1H), 7.55 (ddd, / = 7.5, 7.5, 1.3 Hz, 1H), 7.48 (d, / = 8.0 Hz, 2H), 7.27 (ddd, / = 7.7, 7.7, 1.6 Hz, 1H), 7.11 (d, / = 7.8 Hz, 2H), 5.41 (br s, 1H), 4.72 (s, 2H), 3.97 - 3.91 (m, 2H), 3.56 - 3.53 (m, 2H), 3.09 (s, 6H), 2.28 (s, 3H). ¹³ C NMR (151 MHz, DMSO- d ₆ ) δ 145.71, 140.97, 137.64, 134.94, 132.28, 131.16, 128.82, 128.09, 125.50, 105.11,

70.48, 66.54, 55.15, 50.31, 20.82. IR (diamond-ATR): v (cm ¹ ) 3321m,br, 3082w, 3046w, 3024w, 2965w, 2919w, 2868w, 1970vw, 1965vw, 1941vw, 1912vw, 1641w, 1598w, 1584w, 1563w, 1467m, 1434m, 1382w, 1354w, 1278w, 1206s, 1174s, 1119s, 1106m, 1093m, 1052w, 1032s, 1009s, 978w, 953w, 912w, 860w, 816m, 767m, 741m, 71 lw, 680s, 648m, 562s, 494w, 466w. HRMS (ESI) m/z: [M ⁺ ] (C„H ₁₇ INO ⁺ ) calculated: 306.034934, found: 306.03521.

Example 20

Preparation of l-(2-hydroxyethyl)-l-(4-iodobenzyl)pyrrolidin-l-ium 4-methylbenzenesulfonate

(20)

Pale-yellow viscous liquid which crystallized upon cooling (453 mg, 0.90 mmol, 78 %).

M.p. 97-99 °C. ^l U NMR (300 MHz, DMSO-<¾) δ 8.07 (dd, / = 8.0, 1.2 Hz, 1H), 7.74

(dd, / = 7.8, 1.6 Hz, 1H), 7.54 (ddd, / = 7.5, 7.5, 1.3 Hz, 1H), 7.48 (d, / = 7.9 Hz, 2H), 7.26 (ddd, / = 7.7, 7.7, 1.7 Hz, 1H), 7.11 (d, / = 7.8 Hz, 2H), 5.47 (t, / = 4.4 Hz, 1H), 4.80 (s, 2H), 3.99 - 3.93 (m, 2H), 3.83 - 3.63 (m, 2H), 3.54 - 3.45 (m, 2H), 3.44 - 3.29 (m, 2H), 2.28 (s, 3H), 2.14 - 1.87 (m, 4H). ¹³ C NMR (75 MHz, DMSO-

d ₆ ) δ 145.69, 140.81, 137.63, 134.80, 132.16, 131.57, 128.94, 128.07, 125.48, 105.48, 66.43, 62.17, 60.26, 55.70, 22.41, 20.80. IR (diamond- ATR): v (cm ¹ ) 3356m,br, 3038vw, 3021vw, 2982vw, 2965vw, 2916vw, 2902vw, 2874vw, 2849vw, 1978vw, 1965vw, 1954vw, 1923vw, 1900vw, 1668vw, 1646vw, 1598vw, 1585vw, 1598vw, 1585vw, 1562vw, 1496w, 1470w, 1457m, 1438w, 1356w, 1306w, 1277w, 1256vw, 1214m, 1186s, 1120s, 1106m, 1075m, 1051w, 1036s, 1013s, 993m, 959m, 908w, 892w, 875w, 822m, 799w, 765m, 730w, 713w, 696w, 681s, 649w, 626w, 590w, 563s, 547m, 521w, 450w. HRMS (ESI) m/z: [M ⁺ ] (C ₁₃ H ₁₉ INO ⁺ ) calculated: 332.050584, found: 332.05093. Elem. anal. C ₂₀ H ₂₆ INO ₄ S, calculated: C (47.72), H (5.21), N (2.78), S (6.37), found: C, (47.68), H (5.21), N (2.78), S (6.31).

Example 21

Preparation of [3-(4-hexyloxy-3-iodophenyl)propyl]-(2-hydroxyethyl)-dimethy lammonium 4-methylbenzenesulfonate (21)

The alkylating agent I (338 mg, 0.655 mmol, 1 eq.), DMF (0.8 mL), and 2-(dimethylamino)ethanol (350 μL·, 3.48 mmol, 5.3 eq.) were used for the synthesis of compound 21. The reaction time was 2 h at the temperature of 75 °C. Precipitation was performed from a dichloromethane solution with hexane (45 mL), the product was then re -dissolved in a mixture of dichloromethane and MeOH (2 mL, 9: 1 v/v) and precipitated with a mixture of Et ₂ 0 and hexane. The product was then once more re-dissolved in pure dichloromethane and precipitated with pure hexane (45 mL). The product was obtained as a white solid (391 mg, 0.65 mmol, 99 %).

*H NMR (300 MHz, DMSO-<¾) δ 7.66 (d, / = 2.1 Hz, 1H), 7.49 (d, / = 8.0 Hz, 2H), 7.21 (dd, / = 8.4, 2.1 Hz, 1H), 7.11 (d, / = 7.9 Hz, 2H), 6.91 (d, / = 8.4 Hz, 1H), 5.30 (t, / = 5.0 Hz, 1H), 3.98 (t, / = 6.2 Hz, 2H), 3.81 (br s, 2H), 3.42 - 3.28 (m, 4H), 3.06 (s, 6H), 2.49 (t, / = 7.4 Hz, 2H), 2.28 (s, 3H), 2.02 - 1.88 (m, 2H), 1.77 - 1.65 (m, 2H), 1.53 - 1.40 (m, 2H), 1.37 - 1.24 (m, 4H), 0.93 - 0.84 (m, 3H), ¹³ C NMR (75 MHz, DMSO- ¾>) δ 155.62, 145.74, 138.43, 137.61, 134.57, 129.56, 128.06, 125.48, 112.43, 86.81, 68.63, 64.63, 63.63, 54.95, 50.90, 30.88, ₃ 30.25, 28.53, 25.22, 23.84, 22.08, 20.78, 13.91.

Example 22

Preparation of [3-(4-decyloxy-3-iodophenyl)propyl]-(2-hydroxyethyl)-dimethy lammonium methanesulfonate (22)

The alkylating agent J (250 mg, 0.504 mmol, 1 eq.), DMF (0.7 mL), and 2-(dimethylamino)ethanol (200 μL·, 1.99 mmol, 3.9 eq.) were used for the synthesis of compound 22. The reaction was run 1 h at the temperature of 65 °C, the temperature was then for increased to 75 °C for 45 min. However, as the conversion was still not complete, the temperature was increased to 82 °C for additional 30 min. The product was purified by precipitation from a MeOH solution using a mixture of Et ₂ 0 and hexane (1:4 v/v). The product was obtained as a white waxy solid (186 mg, 0.318 mmol, 63 %).

'H NMR (600 MHz, DMSO-<¾) δ 7.67 (s, 1H), 7.21 (d, / = 8.4 Hz, 1H), 6.92 (d, / = 8.4 Hz, 1H), 5.28 (t, / = 4.9 Hz, 1H), 3.98 (t, / = 6.1 Hz, 2H), 3.81 (br s, 2H), 3.40 - 3.37 (m, 2H), 3.35 - 3.30 (m, 2H), 3.06 (s, 6H), 2.49 (t, / = 7.5 Hz, 2H), 2.30 (s, 3H), 2.00 - 1.92 (m, 2H), 1.75 - 1.67 (m, 2H), 1.49 - 1.42 (m, 2H), 1.36 - 1.20 (m, 12H), 0.86 (t, / = 6.6 Hz, 3H). ¹³ C NMR (151 MHz, CDC1 ₃ ) δ 156.57, 139.05, 133.57, 129.55, 112.29, 87.02, 69.49, 65.78, 65.27, 56.24, 51.95, 51.94, 39.77, 32.02, 30.87, 29.69, 29.67, 29.45, 29.45, 29.22, 26.20, 24.80, 22.81, 14.26.

Example 23

Preparation of (2-hydroxyethyl)-[2-(2-iodophenoxy)ethyl]-dimethylammonium bromide (23)

The alkylating agent K (350 mg, 1.07 mmol, 1 eq.), DMF (1 mL), and 2-(dimethylamino)ethanol (646 μL·, 6.42 mmol, 6.0 eq.) were used for the synthesis of compound 23. The reaction time was 1 h obtained as a white solid (398 mg, = 7.7, 1.5 Hz, 1H), 7.42 - 7.38 (m, / = 7.7, 7.7, 1.1 Hz, 1H), 5.35 (t, / 8 (m, 4H), 3.64 - 3.60 (m, 2H),

Example 24

Preparation of l-(2-hydroxyethyl)-l-[2-(2-iodophenoxy)ethyl]pyrrolidin-l-iu m bromide (24)

The alkylating agent K (350 mg, 1.07 mmol, 1 eq.), DMF (1 mL), and 2-(pyrrolidin-l-yl)ethanol (376 μL·, 3.21 mmol, 3.0 eq.) were used for the synthesis of compound 24. The reaction time was 90 min at the temperature of 77 °C. The product was obtained as a pale -yellow viscous liquid (331 mg, 0.75 mmol, 70 %).

H NMR (300 MHz, DMSO-<¾) δ 7.81 (dd, / = 7.7, 1.6 Hz, 1H), 7.41 (ddd, / =

8.3, 7.4, 1.6 Hz, 1H), 7.06 (dd, / = 8.3, 1.2 Hz, 1H), 6.81 (ddd, / = 7.7, 7.4, 1.2

Hz, 1H), 5.34 (t, / = 4.8 Hz, 1H), 4.54 - 4.46 (m, 2H), 3.95 - 3.85 (m, 4H), 3.83

3.65 (m, 4H), 3.63 - 3.55 (m, 2H), 2.23 - 2.06 (m, 4H). Example 25

Preparation of (2-hydroxyethyl)-[2-(4-iodophenoxy)ethyl]-dimethylammonium bromide (25)

The alkylating agent L (350 mg, 1.07 mmol, 1 eq.), DMF (1 mL), and 2-(dimethylamino)ethanol ynthesis of compound 25. The The product was obtained as a 2H), 6.90 - 6.83 (m, 2H), 5.31 0 (m, 4H), 3.58 - 3.50 (m, 2H),

Example 26

Preparation of l-(2-hydroxyethyl)-l-[2-(4-iodophenoxy)ethyl]pyrrolidin-l-iu m bromide (26)

Example 27

Preparation of (2-hydroxyethyl)-[2-(3-iodophenoxy)ethyl]-dimethylammonium bromide (27)

The alkylating agent M (300 mg, 0.918 mmol, 1 eq.), DMF (1 mL), and 2-(dimethylamino)ethanol

(554 μΕ, 5.51 mmol, 6.0 eq.) were used for the synthesis of compound 27. The reaction time was 2 h at the temperature of 70 °C. The product was obtained as a white solid (368 mg, 0.88 mmol, 96 %).

*H NMR (300 MHz, DMSO-<¾) δ 7.40 - 7.34 (m, 2H), 7.15 - 7.08 (m, 1H), 7.05 - 7.00 (m, 1H), 5.32 (t, / = 5.0 Hz, 1H), 4.51 - 4.42 (m, 2H), 3.92 - 3.81 (m, 4H),

3.58 - 3.51 (m, 2H), 3.20 (s, 6H).

Example 28

Preparation of l-(2-hydroxyethyl)-l-[2-(3-iodophenoxy)ethyl]pyrrolidin-l-iu m bromide (28)

The alkylating agent M (300 mg, 0.918 mmol, 1 eq.), DMF (1 mL), and 2-(pyrrolidin-l-yl)ethanol (322 μΕ, 2.75 mmol, 3.0 eq.) were used for the synthesis of compound 28. The reaction time was 2 h at the temperature of 70 °C. The product was obtained as a yellow viscous liquid which crystallized upon cooling (380 mg, 0.86 mmol, 80 %).

*H NMR (300 MHz, DMSO-<¾) δ 7.40 - 7.33 (m, 2H), 7.15 - 7.07 (m, 1H), 7.05 - 6.98 (m, 1H), 5.32 (t, / = 5.0 Hz, 1H), 4.45 (t, / = 4.7 Hz, 2H), 3.92 - 3.79 (m, 4H), 3.78 - 3.58 (m, 4H), 3.56 - 3.47 (m, 2H), 2.17 - 2.02 (m, 4H).

Example 29

Preparation of (2-hydroxyethyl)-[6-(3-iodophenoxy)hexyl]-dimethylammonium bromide (29)

The alkylating agent N (350 mg, 0.914 mmol, 1 eq.), DMF (1 mL), and 2-(dimethylamino)ethanol

(552 μL·, 5.48 mmol, 6.0 eq.) were used for the synthesis of compound 29.

The reaction time was 2 h at the temperature of 70 °C. The product was obtained as a white solid (395 mg, 0.84 mmol, 92 %).

*H NMR (300 MHz, DMSO-<¾) δ 7.30 - 7.24 (m, 2H), 7.10 - 7.03 (m, 1H), 6.98 - 6.91 (m, 1H), 5.27 (t, / = 5.0 Hz, 1H), 3.96 (t, / = 6.4 Hz, 2H), 3.87 - 3.76 (m, 2H), 3.46 - 3.28 (m, 4H), 3.07 (s, 6H), 1.78 - 1.62 (m, 4H), 1.51 - 1.39 (m, 2H), 1.39 - 1.24 (m, 2H).

Example 30

Preparation of l-(2-hydroxyethyl)-l-[6-(3-iodophenoxy)hexyl]pyrrolidin-l-iu m bromide (30)

The alkylating agent N (350 mg, 0.914 mmol, 1 eq.), DMF (1 mL), and 2-(pyrrolidin-l-yl)ethanol

Example 31

Preparation of (2-hydroxyethyl)-[4-(2-iodophenoxy)butyl]-dimethylammonium bromide (31)

The alkylating agent O (355 mg, 1.00 mmol, 1 eq.), DMF (1 mL), and 2-(dimethylamino)ethanol (604 μΕ, 6.00 mmol, 6.0 eq.) were used for the synthesis of compound 31. The reaction time was 2.5 h at the temperature of 70 °C. The product was obtained as a colorless viscous liquid (418 mg, 0.94 mmol, 94 %). *H NMR (300 MHz, DMSO-<¾) δ 7.77 (dd, / = 7.7, 1.6 Hz, 1H), 7.36 (ddd, / = 8.2, 7.4, 1.6 Hz, 1H), 7.01 (dd, / = 8.3, 1.3 Hz, 1H), 6.79 - 6.72 (m, 1H), 5.28 (t, / = 5.0 Hz, 1H) 4.08 (t, / = 6.4 Hz, 2H), 3.90 - 3.79 (m, 2H), 3.52 - 3.37 (m, 4H), 3.10 (s, 6H), 2.01 - 1.86 (m, 2H), 1.86 - 1.71 (m, 2H).

Example 32

Preparation of l-(2-hydroxyethyl)-l-[4-(2-iodophenoxy)butyl]pyrrolidin-l-iu m bromide (32)

The alkylating agent O (355 mg, 1.00 mmol, 1 eq.), DMF (1 mL), and 2-(pyrrolidin-l-yl)ethanol (468 μL·, 4.00 mmol, 4.0 eq.) were used for the synthesis of compound 32. The reaction time was was obtained as a yellow 1.6 Hz, 1H), 7.35 (ddd, / = 6.78 - 6.72 (m, 1H), 5.28 .77 (m, 2H), 3.72 - 3.59 .99 - 1.85 (m, 2H) 1.85 - Example 33

Preparation of (2-hydroxyethyl)-[2-(5-iodothiophen-2-yl)ethyl]-dimethylammo nium

4-methylbenzenesulfonate (33)

The alkylating agent P (250 mg, 0.612 mmol, 1 eq.), DMF (1 mL), and 2-(dimethylamino)ethanol (370 μL·, 3.67 mmol, 6.0 eq.) were used for the synthesis of compound 33. The reaction time was 2 h at the temperature of 65 °C. The product was obtained as a white solid (273 mg, 0.55 mmol, 90 %).

H NMR (300 MHz, DMSO-<¾) δ 7.48 (d, / = 8.0 Hz, 2H), 7.22 (d, /

= 3.6 Hz, 1H), 7.11 (d, / = 8.0 Hz, 2H), 6.74 (d, / = 3.6 Hz, 1H), 5.33

(t, / = 4.9 Hz, 1H), 3.89 - 3.80 (m, 2H), 3.63 - 3.53 (m, 2H), 3.50

3.43 (m, 2H), 3.35 - 3.26 (m, 2H), 3.12 (s, 3H), 2.29 (s, 3H).

Example 34

Preparation of l-(2-hydroxyethyl)-l-[2-(5-iodothiophen-2-yl)ethyl]pyrrolidi n-l-ium

4-methylbenzenesulfonate (34) The alkylating agent P (250 mg, 0.612 mmol, 1 eq.), DMF (1 mL), and 2-(pyrrolidin-l-yl)ethanol

(358 μL·, 3.06 mmol, 5.0 eq.) were used for the synthesis of compound 34. The reaction time was 2 h at the temperature of 65 °C. The product was obtained as brown solid (120 mg, 0.23 mmol, 37 %).

*H NMR (300 MHz, DMSO) δ 7.47 (d, / = 8.0 Hz, 2H), 7.22 (d, / = 3.6 Hz, 1H), 7.11 (d, / = 8.0 Hz, 1H), 6.75 (d, / = 3.6 Hz, 1H), 5.36 (t, / =

4.8 Hz, 2H), 3.90 - 3.76 (m, 2H), 3.70 - 3.51 (m, 6H), 3.51 - 3.43 (m, 2H), 2.29 (s, 3H), 2.13 - 2.00 (m, 4H).

Example 35

Preparation of 4-(2-hydroxyethyl)-4-(4-iodobenzyl)morpholin-4-ium bromide (35)

The alkylating agent F (300 mg, 1.01 mmol, 1 eq.) and 4-(2-hydroxyethyl)mo holine (4.00 mL, 33,0 mmol, 32.7 eq.) were used for the synthesis of compound 35. The reaction time was 18 h at the pink solid / = 8.3 .6 Hz,

Example 36

Preparation of 4-(2-hydroxyethyl)-4-(2-iodobenzyl)morpholin-4-ium chloride (36)

The alkylating agent G (300 mg, 1.19 mmol, 1 eq.) and 4-(2-hydroxyethyl)morpholine (4.00 mL, 33,0 mmol, 27.7 eq.) were used for the synthesis of compound 36. The reaction time was 18 h at the temperature of 72 °C. The product was obtained as an amorphous brown solid (364 mg, 0.948 mmol, 80 %).

DMSO-<¾) δ 8.12 - 8.06 (m, 1H), 7.81 - 7.74 (m, 1H), 7.32 - 7.24 (m, 1H), 5.86 (t, / = 5.0 Hz, 1H), 4.96 (s, 4H), 3.95 - 3.82 (m, 4H), 3.77 - 3.68 (m, 2H), 3.41 -

Example 37

Preparation of (2-hydroxyethyl)-{6-[(2-iodopyridin-3-yl)oxy]hexyl}-dimethyl ammonium bromide (37)

The alkylating agent Q (200 mg, 0.521 mmol, 1 eq.) and 2-(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 38.2 eq.) were used for the synthesis of compound 37. The reaction time was 40 min at the temperature of 75 °C. The product was obtained as a colorless viscous liquid (240 mg, 0.51 mmol, 97 %).

*H NMR (300 MHz, DMSO-<¾) δ 7.95 (dd, / = 3.0, 3.0 Hz, 1H), 7.37 - 7.32 (m, 2H), 5.27 (t, / = 4.9 Hz, 1H), 4.08 (t, / = 6.2 Hz, 2H), 3.87 - 3.77 (m, 2H), 3.44 - 3.29 (m, 4H), 3.08 (s, 6H), 1.83 - 1.65 (m, 4H), 1.59 - 1.46 (m, 2H), 1.42 - 1.28 (m, 2H).

Example 38

Preparation of (2-hydroxyethyl)-{4-[(2-iodo-6-methylpyridin-3-yl)oxy]butyl} - dimethylammonium bromide (38)

The alkylating agent R (200 mg, 0.540 mmol, 1 eq.) and 2-(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 36.9 eq.) were used for the synthesis of compound 38. The reaction time was 40 min at the temperature of 75 °C. The product was obtained as a colorless viscous liquid (246 mg, 0.54 mmol, 99 %).

*H NMR (300 MHz, DMSO-<¾) δ 7.26 (d, / = 8.2 Hz, 1H), 7.19 (d, / = 8.2 Hz, 1H), 5.29 (t, / = 4.9 Hz, 1H), 4.08 (t, / = 6.3 Hz, 2H), 3.90 - 3.76 (m, 2H), 3.52 - 3.32 (m, 4H), 3.10 (s, 6H), 2.36 (s, 3H), 1.97 - 1.84 (m, 2H), 1.82 - 1.69 (m, 2H).

Example 39

Preparation of (2-hydroxyethyl)-{4-[(3-iodo-[l,l'-biphenyl]-4-yl)oxy]butyl} -dimethylammonium bromide (39)

The alkylating agent S (200 mg, 0.464 mmol, 1 eq.) and 2-(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 42.9 eq.) were used for the synthesis of compound 39. The reaction time was 40 min at the temperature of 75 °C. The product was obtained as a colorless viscous liquid which crystallized upon cooling (238 mg, 0.46 mmol, 99 %).

*H NMR (300 MHz, DMSO-<¾) δ 8.04 (d, / = 2.3 Hz, 1H), 7.66 (dd, / = 8.5, 2.3 Hz, 1H), 7.64 - 7.57 (m, / = 7.3, 1.3 Hz, 2H), 7.47 - 7.39 (m, 2H), 7.36 - 7.30 (m, 1H), 7.09 (d, / = 8.5 Hz, 1H), 5.30 (t, / = 5.0 Hz, 1H), 4.13 (t, / = 5.9 Hz, 2H), 3.90 - 3.81 (m, 2H), 3.55 - 3.39 (m, 4H), 3.12 (s, 6H), 2.03 - 1.88 (m, 2H), 1.87 - 1.70 (m, 2H).

Example 40

Preparation of (2-hydroxyethyl)-[6-(4-iodo-3-nitrophenoxy)hexyl]-dimethylam monium bromide (40)

II. Synthesis of radiolabeled substances

Example 23

Solution A: PdCl ₂ (1 mg) and acetonitrile (1 mL) were put into 2 mL micro-centrifugation tube, the resulting suspension was agitated in an ultrasonic bath for 15 min. The obtained suspension was then centrifuged and decanted from the non-dissolved solid PdCl ₂ .

Solution B: Ascorbic acid (30 mg) in deionized water (1 mL)

Solution C: Compound 4 (1 mg) in deionized water (100 μί)

The solutions A (4 μΕ), B (2 μΕ), and C (1 μί) were put into a 0.5 mL microcentrifuge tube. The tube was gently shaken, the mixture was left for 15 min at the room temperature and was subsequently centrifuged. Then, an aqueous solution of Na ¹²⁵ I (8 μL·, 74.0 MBq) was added, and the reaction mixture was gently shaken. The tube was sealed by a plastic film, centrifuged, and subsequently it was immersed into a water bath preheated to 75 °C. After 1 h, the tube was removed from the bath, centrifuged, and immersed into the bath again so that the solvent vaporized at the top of the tube was returned back into the remaining solution. After another 1 h, the tube was removed, cooled to room temperature, and the reaction mixture was diluted with deionized water (100 μί). Strong anion exchange resin in the TsO cycle (5 grains, particle size 650 - 820 μπι, 1 meq. mL ¹ ) was inserted to the tube to remove most of the free ¹²⁵ Γ. The grains of anion exchange resin were subsequently decanted, the solution was transferred into a clean centrifugation tube, and the grains of the anion exchange resin were rinsed with deionized water (2 X 50 μί), which was then added to the first solution. The resulting solution was filtered through a 0.20 μηι syringe filter. The radio-labeled substance 4 (55.8 MBq, 75 %) was obtained in a good radiochemical purity according to the radio-HPLC analysis (Figure 1). III. Biological tests

In vitro determination of IC ₅₀ values of the choline analogs

IC ₅₀ value corresponds to a concentration of the given analog of choline which reduces the accumulation of choline chloride, the natural ligand of choline transporterss, to 50 % of the maximum value. For the assay, the tritium labeled choline chloride, i.e. [ ³ H]choline chloride, was used. The assay was performed in vitro on the cell line PC-3 derived from a human prostate carcinoma, which is known to overexpress choline transporters. For the determination of IC ₅₀ , the PC-3 cells were cultivated in 24-well cultivation plates to confluence of 90 %. As the negative control, a cell line of human ovarian carcinoma SKOV3 prepared to the confluence of 90 % in 24-well plates was used.

The cultivation medium was removed from the thus prepared cells in cultivation plates and the cells were rinsed with phosphate buffered saline (PBS). Subsequently, 0.3 mL of "incubation buffer" (25 mM Tris/HCl, 5.4 mM KC1, 1.8 mM CaCl ₂ , 0.8 mM MgS0 ₄ , 5 mM glucose, 140 mM NaCl, pH adjusted to 7.4) was added to the cells. Then 0.1 mL of the solution of the tested substance (analog of choline) in the incubation buffer was added to the PC-3 cells in such concentrations, that after adding [ ³ H]choline chloride the resulting concentrations of the tested analog would be following: 0 M, 10 ^s M, 10 ⁷ M, 10 ⁶ M, 5x10 ⁶ M, 10 ^s M, 10 ⁴ M, 5x10 ⁴ M, 10 ³ M, 5x10 ³ M. Each concentration was tested in triplicate.

To the SKOV3 cells, 0.1 mL of the tested compound (analog of choline) solution in the incubation buffer was added in such concentrations, that after the addition of [ ³ H]choline chloride the concentrations of the analog of choline in the medium with cells were 0 M and 10 ⁴ M. Each concentration was tested in triplicate.

Subsequently, all cells prepared in this manner (PC-3 and SKOV3) were incubated with the analogs of choline for 15 minutes at the temperature of 37 °C.

Then 0.1 mL of the solution of the [ ³ H]choline chloride in the incubation buffer was added to all cell-containing wells, so that 0.1 mL of the solution had the activity of 15 kBq and that the final concentration of the [ ³ H] choline chloride in the medium with the cells was 0.81 x 10 ⁶ M. The cells were then incubated at 37 °C for a period of 60 min. After the incubation, all buffer solution was removed from the cells. The cells were rinsed with PBS, which was subsequently removed, and 0.2 mL of aqueous NaOH solution (0.1 M) was added to each well. The cells were incubated with sodium hydroxide for 30 min at 37 °C. The lysate was then homogenized with a pipette and 10 μL· of the lysate were used for the determination of protein concentration by bicinchonic acid assay (BCA). The remaining lysate (180 μί) was quantitatively transferred into scintillation vials containing a previously prepared scintillation solution (2 mL of the solution in each vial). Each well was then additionally rinsed with 0.2 mL of pure water and this solution was added to the corresponding scintillation vials. Scintillation vials were closed and their content was stirred on a shaker.

To another three scintillation vials containing scintillation solution (2 mL of the solution in each vial) the standard samples of [ ³ H]choline chloride were added (i.e. 10 μΐ of the [ ³ H]choline chloride solution together with 180 μΐ of aqueous NaOH solution (0.1 M) and 200 μΐ of water). The vials were closed and their content was stirred on the shaker.

Subsequently, radioactivity of each scintillation vial was measured using a liquid scintillation counter (Tri-Carb 2910TR Liquid Scintillation Counter, PerkinElmer). The recorded counts per second (cps) were then used to calculate the accumulation of [ ³ H]choline chloride in the individual cell samples. These results were corrected to the applied amount of radioactivity ([ ³ H]choline chloride standards) and the quantity of proteins in each cell samples. These values were used to construct a sigmoid curve of dependence between the rate of [ ³ H]choline chloride accumulation and the competing ligand (analog of choline) concentration(inhibition curve, Figure 2). The IC ₅₀ values were calculated from these inhibition curves. Assessment of the IC ₅₀ values obtained in vitro for the analogs of choline

Using the above-described method, the IC ₅₀ values were determined for 40 analogs of choline according to the present invention, and also for hemicholinium-3, fluoromethylcholine, and choline (refer to Table 1). Choline was chosen as it is a natural ligand of choline transporters, hemicholinium-3 was chosen as a classic representative of the inhibitors of choline transporters, and the radioactive fluorine ( ¹⁸ F) labeled fluoromethylcholine is routinely used in nuclear medicine to diagnose prostate carcinoma. The IC ₅₀ values determined for hemicholinium-3 and choline were in accordance with the published data (7.3 x 10 ⁶ mol . L- ¹ for choline and 1.7 x 10 ⁵ mol . L- ¹ for hemicholinium-3 [Muller S. A. et al. (2009), Eur. J. Nucl. Med. Mol. Imaging 36, 1434-1442]). Twenty-six derivatives of choline according to the the present invention possessed the same or higher IC ₅₀ values than hemicholinium-3, and eighteen derivatives of choline according to the present invention exhibited higherIC ₅₀ values than fluoromethylcholine, which is routinely used in nuclear medicine to diagnose prostate carcinoma. In the case of eight derivatives according to the present invention, the IC ₅₀ values were even higher than the IC ₅₀ value determined for choline (natural ligand).

Determination of basic pharmacological properties of analogs of choline For twenty of the tested analogs of choline, stability in the plasma, binding to plasmatic proteins, microsomal stability and the permeability through artificial membrane (PAMPA) (Table 2) were determined. Most of the analogs of choline under study have demonstrated sufficient stability in plasma after two-hour incubation (> 70 to 80 % of the original quantity of the substance). The binding to plasmatic proteins influences mainly the distribution of the drug in the organism. Plasma protein binding has shown relatively higher values (> 20 % bound to plasmatic proteins) for six analogs of choline under study. More than a half of the tested derivatives of choline was quite quickly (internal clearance > 50 μΐ. . min ¹ . mg ^_1 ) metabolized by hepatic microsomes. All studied compounds under exhibited a low ability to pass across the artificial membrane by passive diffusion. This is a desirable property, which enables a specific distribution of these substances and further supports their planned diagnostic or therapeutic use (choline transporters are localized on the plasmatic membrane of the cells). In general, the substances have shown suitable pharmacological properties for both diagnostic and therapeutic applications. Table 1. IC ₅₀ of the novel derivatives

Choline analog ICso (mol . L ^"1 )

Example 1 4.7x10 ^s

Example 2 9.2x10 ^s

Example 3 2.5x10 ^s

Example 4 1.9x10 ^s

Example 5 7.4xl0 ⁶

Example 6 8.3x10 ^s

Example 7 1.6x10 ^s

Example 8 1.2x10 ⁴

Example 9 9.9x10 ^s

Example 10 1.3x10 ^s

Example 11 1.5xl0 ⁴

Example 12 8.0x10 ^s

Example 13 2.9x10 ^s

Example 14 1.9x10 ⁴

Example 15 8.4x10 ⁶

Example 16 2.4x10 ⁴

Example 17 7.4xl0 ⁴

Example 18 1.8xl0 ⁴

Example 19 1.6xl0 ⁴ Example 20 6.3xl0 ⁶

Example 21 4.3xl0 ⁷

Example 22 1.9xl0 ⁶

Example 23 2.5x10 ^s

Example 24 4.7xl0 ⁶

Example 25 2.3x10 ^s

Example 26 1.8x10 ^s

Example 27 2.2x10 ^s

Example 28 1.5x10 ^s

Example 29 4.4x10 ^s

Example 30 9.1xl0 ⁷

Example 31 1.1x10 ^s

Example 32 5.1xl0 ⁶

Example 33 4.5xl0 ⁷

Example 34 1.0x10 ^s

Example 35 2.7xl0 ⁴

Example 36 4.3x10 ^s

Example 37 1.4x10 ⁶

Example 38 1.8x10 ^s

Example 39 1.3xl0 ⁶

Example 40 1.4x10 ⁷

Hemicholinium-3 2.9x10 ^s

Fluoromethylcholine 1.6x10 ^s

Choline 3.7xl0 ⁶

Table 2. In vitro pharmacokinetic properties of derivatives 1-8 and 14-20

„Parallel Artificial

Plasma Plasmatic

Membrane

Choline stability protein Microsomal stability

Permeability analog of (120 min) binding

Assay" Example

(PAMPA) tl Internal clearance

[%] [%] log Pe

[min] [μΐ _^ . min- . mg ^" ]

1 28.4 19.5 165.0 8.4 -9.88 2 59.0 25.2 97.6 14.2 -9.98

3 76.4 48.8 1.3 1035.8 -10.20

4 63.9 28.5 1.2 1123.6 -10.50

5 43.0 34.7 15.6 88.6 -10.26

6 81.3 26.5 1.1 1232.0 -8.48

7 97.6 16.7 15.3 90.6 -8.49

8 89.9 28.3 34.8 39.8 -9.04

14 80.7 16.9 61.3 22.6 -9.49

15 84.9 18.6 18.8 73.6 -7.74

16 79.0 4.4 119.5 11.6 -8.30

17 58.4 5.7 1732.5 0.8 -7.75

18 73.4 9.7 22.0 63.0 -7.55

19 91.3 33.9 8.3 166.4 -10.12

20 81.3 7.88 4.5 310 -8.44

Industrial Applicability

The invention can be used in medicine for localization and treatment of several types of tumorous diseases and other disorders characterized by pathological function or expression of choline transporters.