The invention concerns a compound, the compound for use in a diagnostic meth od, the compound for use in a diagnostic method for diagnosing a disorder in gen eration, degradation, distribution or function of norepinephrine transporter (NET) and a method for synthesizing the compound.

Norepinephrine (NE) is a neurotransmitter. Functions of NE comprise a general mobilization of brain and body as well as an increase of heart rate and blood pres sure. NET is responsible for the reuptake of NE released into the synaptic cleft into the presynaptic neuron. Therewith, NET regulates concentration of NE in the syn aptic cleft. For detecting NET in the human or animal body several tracers that bind to NET and that can be detected in Positron Emission Tomography (PET) have been developed.

WO 2013/036869 A2 discloses compositions, methods and systems for the syn thesis and use of imaging agents comprising ¹⁸ F. The imaging agent may be used to image an area of interest in a subject, including the heart, cardiovascular sys tem, cardiac vessels, brain and other organs. In certain embodiments the dis closed methods include a method of detecting NET.

From WO 2008/083056 compounds for use as imaging agents within nuclear med icine applications (PET imaging) for imaging of cardiac innervations and methods of synthesis for obtaining these compounds in radiolabeled form are known.

Vaidyanathan G. et al., Nucl Med Biol. 2015 August, 42(8), pages 673 to 684 dis close synthesis and evaluation of 4-[ ¹⁸ F]fluoropropoxy-3-iodobenzylguanidine.

Werner, R. A., J Nucl Med. 2015 September, 56(9), pages 1429 to 1433 disclose ¹⁸ F-N-[3-bromo-4-(3-fluoro-propoxy)-benzyl]-guanidine as PET tracer for noninva- sive assessment of sympathetic innervation of the heart. From Jung, Yong-Woon et al. , ACS Chem. Neurosci. 2017, 8, pages 1530 to 1542 radiotracers of cardiac sympathetic innervation are known. The radiotracers are 4- [ ¹⁸ F]fluoro-m-hydroxyphenethylguanidine ([ ¹⁸ F]4F-MFIPG, [ ¹⁸ F]1 ) and its structural isomer 3-[ ¹⁸ F]fluoro-p-hydroxyphenethylguanidine ([ ¹⁸ F]3F-PFIPG, [ ¹⁸ F]2) having the following formulae

According to this document fluorine-18 has a sufficiently long half-life such that the production and distribution of ¹⁸ F-labeled radiopharmaceuticals from a central pro duction facility to stand-alone PET imaging centers is feasible. In the document it is further stated that PET studies showed very long neuronal retention times for [ ¹⁸ F]1 and [ ¹⁸ F]2 in nonhuman primate myocardium consistent with rapid vesicular uptake and very little diffusion of the tracers from storage vesicles. Furthermore, the guanidine group of the side chain of [ ¹⁸ F]1 and [ ¹⁸ F]2 confers stability against neuronal enzymes.

Chen, X. et al., EJNMMI Research (2018) 8:12, pages 1 to 8 discloses ¹⁸ F-N-[3- bromo-4-(3-fluoro-propoxy)-benzyl]-guanidine ( ¹⁸ F-LMI1195) as PET tracer. This tracer is designed for assessment of sympathetic innervation of the heart. It is de scribed that for ¹⁸ F-LMI1195 exists an easy and high-yield labeling procedure that is convenient and eligible for commercial preparation and application. The problem to be solved by the present invention is to provide an alternative tracer binding to NET, the tracer for use in diagnostic methods as well as a meth od for synthesizing the tracer.

The problem is solved by the features of claims 1 , 2, 3 and 6. Embodiments are subject-matter of claims 4, 5 and 7 to 13.

According to the invention a compound according to the following formula

is provided, wherein R1 is an F or an I residue, in particular an F residue.

The invention further concerns the compound according to the invention for use in a diagnostic method practiced on the living human or animal body, i. e., in vivo. This diagnostic method may be a diagnostic method for diagnosing a disorder in generation, degradation, distribution or function of NET in the human or animal body. The inventors found that the compound according to the invention shows a particular slow uptake and a quick flushing out by the liver in vivo.

The diagnostic method may comprise Positron Emission Tomography (PET) of the human or animal body, to which body the compound has been administered be fore performing the PET. The disorder in generation, degradation, distribution or function of NET may be associated with a cardiovascular disease, a dysregulated blood pressure, a renal disorder, a neuroendocrine tumor disease or Parkinson's disease.

The invention further concerns a method for synthesizing the compound according to the invention, wherein the method comprises the following steps or consists of the following steps: a) Removing the methyl group of the following compound 14

from this compound to receive the following

compound 15

, wherein R1 is a halogen residue, b) chloroalkylation of compound 15 by use of 1 -chloro-3-iodopropane to receive the following compound 25

c) performing a Wittig reaction with compound 25 by use of a (methox- ymethyl)triphenylphosphonium halide and potassium fe/f-butoxide or sodi um hydride to receive the following compound 26

d) reduction of compound 26 in a two step one pot reaction by addition of mercury acetate to allow the formation of an intermediate followed by ad dition of alkali metal borohydride, in particular sodium borohydride or potas sium borohydride, to receive the following compound 28

e) performing a Mitsunobu reaction with compound 28 in the presence of triphenylphosphine, an azodicarboxylate and the following compound 24

CHg

BocHN N rV N°

T CH 3

NBoc

24 to receive the following compound 29

f) replacing the chlorine atom at the terminal of the alkyl chain of the compound resulting from any of steps b) to e) by iodine in a Finkelstein re action followed by a tosylation using silver p-toluenesulfonate in the dark and if any of steps a) to e) have not been performed up to the present step performing all of steps a) to e) that have not been performed up to the pre sent step to receive the following compound 30

g) radiolabeling compound 30 by a nucleophilic substitution using an al kali metal salt of ¹⁸ F, in particular K ¹⁸ F, followed by deprotection under acid ic condition at a temperature of at least 70 °C to receive the following com pound

¹⁸ F-tracer .wherein the halogen residue is an F or I residue, in particular an F residue.

If the halogen residue is an F residue the resulting compound is the compound designated as ¹⁸ F-AF78 in this application and if the halogen residue is an I resi due the resulting compound is the compound designated as ¹⁸ F-37 in this applica tion. For both of these compounds efficiency is shown in the embodiments of the invention.

The steps modifying the aldehyde group of compound 14 and groups resulting directly or indirectly from this aldehyde group can be performed partly or complete ly prior to or after all or of a part of the steps modifying the methoxy group of com pound 14 and groups resulting directly or indirectly from this methoxy group. This means that above steps a) to f) can be performed in the given order but also in any other order as long as performing the steps results in compound 30. Perfor mances in different orders are illustrated in the reaction schemes according to Figs. 1 and 2 and 4 to 6. If steps a) to f) are performed in the given order, all of steps b) to f) are performed in each case with the product of the preceding step which product is identified in the preceding step by a number.

In an embodiment the chlorine atom at the terminal of the alkyl chain of the com pound resulting from e) is replaced in step f) by iodine in a Finkelstein reaction.

An advantage of the method according to the invention illustrated in Fig. 1 is that the reaction to extend a benzaldehyde residue by one more methylene groups to achieve a phenethyl moiety which reaction usually takes four steps only takes two steps. These two steps are a Wittig reaction from benzaldehyde 25 to enol ether 26, and a one-pot reaction to phenylethanol 28 through putative intermediate phe- nylacetaldehyde 27. The intermediate 27 was first planned to be prepared in acidic condition from 26. But after having applied multiple demethylation conditions, such as hydrochloric acid or formic acid, no desired compound could be obtained.

Compound 26 decomposes very fast after the addition of acid, even at low tem perature and anhydrous condition, and formed the starting material compound 25 again. In the end, a one-pot reaction using mercury (II) acetate is applied as the mercury salt probably stabilizes the putative phenylacetaldehyde intermediate 27, which is not isolated and is directly reduced by addition of NaBFU in basic solution. As a result, the synthetic scheme is markedly shortened with successful formation of alcohol 28. Though compound 28 is even formed when the alkali metal borohy- dride is added immediately after addition of mercury acetate, the yield of com pound 28 is low in this case. The inventors found that yield is improved when the intermediate 27 has enough time to form between the addition of the mercury ace tate and the addition of the reducing agent alkali metal borohydride. If the time be tween the addition of the mercury acetate and the addition of the reducing agent is too long, hydrolysis back to the benzaldehyde 25 may occur.

The time between the addition of the mercury acetate and the reducing agent may be in the range of 5 minutes to 25 minutes, in particular 6 minutes to 20 minutes, in particular 7 minutes to 15 minutes, in particular 8 minutes to 15 minutes, in par ticular 9 minutes to 12 minutes. In particular the time between the addition of the mercury acetate and the reducing agent is 10 minutes or about 10 minutes.

Furthermore, the reaction to extend the benzaldehyde residue by one more meth ylene group should occur at a temperature in the range of 0 °C to 5 °C, in particu lar 0 °C to 4 °C, in particular 0 °C to 2 °C, in particular 0 °C to 1 °C, in particular at a temperature of 0 °C. Compound 24 is used in Mitsunobu reaction to introduce a fully protected guani dine moiety in compound 28 to result in the compound 29. The chlorine atom at the terminal of the alkyl chain is replaced by iodine in a Finkelstein reaction which iodine is subsequently replaced by a tosylate residue for the radiolabeling by re acting with silver tosylate in the dark. The total reaction scheme is illustrated in Fig. 1 .

Radiofluorination is performed by a nucleophilic method illustrated in Fig. 2. The tosylate moiety is a very good leaving group, which is replaced by [ ¹⁸ F]fluorine.

TLC using autoradiography showed the formation of the fluorinated intermediate before deprotection. The intermediate formed after the removal of both Boc groups still contained the triazinanone structure on the guanidine. This structure turned out to be more stable than expected. Therefore, 6N HCI was used for decomposi tion of this moiety. The total synthesis time of labeling is approximately 120 min. The average overall radiochemical yield was 7.9 ± 3.1 % (decay-corrected based on the starting radioactivity, calculated from 5 times of labeling records) and > 97% radiochemical purity.

The (methoxymethyl)triphenylphosphonium halide of step c) may be (methoxyme- thyl)triphenylphosphonium chloride.

In an embodiment the azodicarboxylate of step e) is diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD).

The deprotection under acidic condition of step g) may be performed at a tempera ture of at most 100 °C.

Compound 24 can be synthesized in a method comprising the following steps: h) Reacting benzyl carbamate, L/,L/'-dimethylurea and water at a tem perature of at least 80 °C to receive the following compound 23 i) reacting compound 23 with hydrogen catalyzed by palladium on acti vated charcoal to receive compound 22

j) reacting compound 22 with the following compound 13

the presence of A/,/V-diisopropylethylamine (DIPEA) to receive compound 24, wherein Boc is tert-butoxycarbonyl pro- tecting group.

In an embodiment compound 13 of step j) is synthesized by reacting 1 ,3-bis(tert- butoxycarbonyl)-2-methyl-2-thiopseudourea and 5-chlorobenzotriazole in the presence of mercury (II) chloride (HgCte).

The total reaction scheme of synthesis of compound 24 is illustrated in Fig. 3. For synthesizing compound 24 the triazinane intermediate 22 is prepared first. When using benzylamine as starting material, the hydrogenation step from resulting compound 21 , wherein Bn is benzyl, to compound 22 would re quire very high pressure, which is difficult to achieve in lab conditions and also unfeasible for a scaling-up preparation. The improved method of the invention us es benzyl carbamate instead of benzylamine for the preparation of triazinane in termediate 23, which undergoes a much milder condition in the removal of protec tion group. While forming the fully protected guanidine compound 24 from com pound 22, a reaction with pseudourea

BocHN SCH ₃

T

NBoc

did not work because the triazinane intermediate decomposed. However, the inventors found that compound 24 can be obtained when compound 13 is used instead of the pseudourea.

Embodiments of the invention:

Fig. 1 shows a reaction scheme for synthesis of compound 30 as precursor of tracer 2 (= ¹⁸ F-AF78),

Fig. 2 shows a reaction scheme for synthesis of ¹⁸ F-AF78,

Fig. 3 shows a reaction scheme for synthesis of compound 24,

Fig. 4 shows a reaction scheme for synthesis of compound 1 17,

Fig. 5 shows a reaction scheme for synthesis of compound 123 as precursor of tracer ¹⁸ F-37,

Fig. 6 shows a reaction scheme for synthesis of compound ¹⁸ F-37, Fig. 7 shows dose-response curves of ¹³¹ I-MIBG uptake in SK-N-SH cells in the presence of increasing concentrations of non-radioactive compounds,

Fig. 8 shows uptake of ¹⁸ F-AF78 in SK-N-SFI cells with and without NET inhibitor desipramine (DMI),

Fig. 9 shows dose-response curves of ¹⁸ F-AF78 uptake in SK-N-SFI cells in the presence of increasing concentrations of non-radioactive references,

Fig. 10 shows an autoradiography of slices from rats' left ventricular short axis after administration of ¹⁸ F-AF78 with and without prior NET blockade,

Fig. 1 1 shows results of a tissue distribution study after administration of ¹⁸ F-AF78 with and without prior NET blockade,

Fig. 12 shows static PET images of cardiac uptake of ¹⁸ F-AF78 in healthy rats with and without prior NET blockade,

Fig. 13 shows static PET images of cardiac uptake of ¹⁸ F-37 in healthy rats with and without prior NET blockade,

Fig. 14 shows time-activity curves of ¹⁸ F-AF78 generated from dynamic PET im ages in a control monkey with and without prior NET blockade,

Fig. 15 shows time-activity curves of ¹⁸ F-37 generated from dynamic PET images in rat (left panel) and results of biodistribution studies in rats (n = 4) show ing heart-to-blood (H/B), heart-to-liver (H/L) and heart-to-muscle ratios (FI/M) after 10 min of tracer injection (right panel),

Fig. 16 shows static PET images of cardiac uptake of ¹⁸ F-AF78 in healthy rats, rabbits, pigs and monkeys, Fig. 17 shows the results of a biodistribution study with ¹⁸ F-AF78 in monkey and

Fig. 18 shows the results of a kinetic study performed with ¹⁸ F-AF78 in monkeys’ hearts. Top panel shows the cardiac tracer uptake with (DM I blocking) and without (CONT) the pretreatment of NET blocker desipramine (DMI). Mid dle panel shows the tracer uptake with DMI chase (DMI injection after tracer administration) compared with control (CONT). Lower panel shows the tracer uptake with tyramine chase (TYR chase, tyramine, a catechola mine releasing agent, injected after tracer administration) compared with control (CONT).

Synthesis of the compound according to the invention

The conditions of the synthesis according to Fig. 1 were as follows:

(a) 1 -chloro-3-iodopropane, K2CO3, acetone, 60°C, 14 h, 87%; (b) (methoxyme- thyl)triphenylphosphonium chloride, potassium tert- butoxide (KOtBu), THF, 0°C- >r.t., 24 h, 50%; (c) Hg(OAc) ₂ , THF, H20, 0°C, 15 min; (d) NaBH ₄ , K2CO3, H2O, 0°C, 30 min, 68%; (e) compound 24, triphenylphosphine (PPh3), diisopropyl azodi- carboxylate (DIAD), THF, 0°C->r.t., 16 h, 84%; (f) Nal, acetone, 70°C, 14 h, 99%; (g) silver p-toluenesulfonate, CH3CN, 0°C->r.t., dark, 72 h, 98%.

Conditions of the synthesis according to Fig. 3 were as follows:

(a) H2O, 100°C, 14 h, 77%; (b) CHsOH, H2O, 100°C, 14 h, 35%; (c) H2, Pd/C, r.t., 14 h, 88%; (d) compound 13, DIPEA, CHsCN, 50°C, 14 h, 58%; (e) compound 23, HgCI ₂ , DMF, N2, 50°C, 14 h.

Detail of synthesis of the compounds of Figs. 1 to 3:

Solvents were dried according to published methods and freshly distilled before use. All other reagents were commercially available compounds and were used without further purification unless noted otherwise. All reactions were carried out under nitrogen atmosphere. ¹ H and ¹³ C NMR spectral data were obtained from a Bruker Avance II 400 MHz. ¹ H and ¹³ C NMR chemical shifts (d) are reported in parts per million (ppm) relative to internal standard TMS and coupling constants (J) are in Hz.

Thin layer chromatography (TLC) was performed on silica gel 60 with a 254 nm fluorescent indicator. UV light or iodine vapor were used for detection.

Purity was evaluated via LCMS performed on an LCMS-system by Shimadzu Products, containing a LC20AB liquid chromatograph, an SPD-20A UVA/is detec tor and a DGU-20A3R degassing unit. Stationary phase was a Synergi 4 pm fu- sion-RP (150 ^c 4.6 mm) column (Phenomenex, Aschaffenburg, Germany). Mass spectra were obtained by a Shimadzu LCMS-2020 (confirming purity >95%).

For column chromatography, silica gel 60, 230-400 mesh by Merck was used. For preparative thin layer chromatography, silica gel 60 PF254 by Merck was used. ferf-Butyl (E)-(((ferf-butoxycarbonyl)imino)(5-chloro-1 H- benzo[c/][1 ,2,3]triazol-1 -yl)methyl)carbamate (13)

1 ,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea and 5-chlorobenzotriazole were dissolved in dimethylformamide and triethyl amine (0.35 g, 3.45 mol). Mercu- ry(ll) chloride (HgCte) was added and the reaction mixture was stirred at 50 °C for 24 h. The mixture was washed with sodium hydrocarbonate and water and ex tracted with ethyl acetate (8x50 ml). The combined organic layers were dried over sodium sulfate. The crude product was dissolved in dichloromethane and purified by column (5: 1-1 :1 petroleum ether: ethyl acetate) to give the target compound 13 as a white solid (0.74 g, 18.7 mmol. 57% yield) which was used in the next step without further purification. ESI-MS (m/z): 396.05 [M+H] ⁺ .

3-Fluoro-4-hydroxybenzaldehyde (15)

3-Fluoro-4-methoxybenzaldehyde 14 (5.00 g, 32.5 mmol) was mixed with 48%

HBr (30 ml_), heated to 140 °C and stirred under argon atmosphere for 3 h. The mixture was diluted with water (150 ml_) and extracted with dichloromethane (2x100 mL). The combined organic layers were washed with brine solution and dried over sodium sulfate. The solvent was removed in vacuo to give compound 15 as a brown solid (4.36 g, 30.2 mmol, 97% yield). NMRs are in accordance to literature. (Jiang 2014) ESI-MS (m/z): 141.00 [M+H] ⁺ .

5-Benzyl-1 ,3-dimethyl-1 ,3,5-triazinan-2-one (21 )

Benzylamine (9.81 g, 91.7 mmol) was dissolved in formaldehyde (14.7 g, 183 mmol, 40% aq. solution) and heated to 100 °C under argon atmosphere. N,N’~ dimethylurea (8.07 g, 91.7 mmol) was added and the mixture was stirred for 14 h. The mixture was washed with water and extracted with dichloromethane (2x200 mL). The combined organic layers were washed with brine solution and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was pu rified by column chromatography (1 :1 petroleum ether: ethyl acetate) to give the title compound 21 as yellow crystals (15.5 g, 70.9 mmol, 77% yield). ¹ H NMR

(CDCL, 400 MHz) d = 2.85 (s, 6 H), 3.90 (s, 2 H), 4.16 (s, 4 H), 7.28 (m, 5 H) ppm. ¹³ C NMR (CDCL, 101 MHz) d = 32.47, 55.36, 67.72, 127.70, 128.60, 129.11 , 137.49, 156.04 ppm. ESI-MS (m/z): 220.10 [M+H] ⁺ .

Benzyl 3,5-dimethyl-4-oxo-1,3,5-triazinane-1-carboxylate (23)

Benzyl carbamate (1.00 g, 6.62 mmol) was dissolved in formaldehyde (1.18 g,

14.6 mmol, 40% aq. solution) and methanol. The mixture was heated to 100 °C under argon atmosphere, N,N’-dimethylurea (0.58 g, 6.62 mmol) was added and the mixture was stirred for 14 h. The solvent was removed in vacuo and crude product was washed with water and extracted with dichloromethane (2x200 mL). The combined organic layers were washed with brine solution and dried over so dium sulfate. The solvent was removed in vacuo and the crude product was puri fied by column chromatography (1 :1 petroleum ethenethyl acetate, then 25:1 di- chloromethane:methanol) to give the title compound 23 as a colourless liquid (0.60 g, 2.29 mmol, 35% yield). ¹ H NMR (CDCL, 400 MHz) d = 2.79 (s, 6H), 4.62 (s, 4H), 5.09 (s, 2H), 7.26-7.27 (5H) ppm. ¹³ C NMR (CDCL, 101 MHz) d = 32.66, 60.34, 68.15, 128.04, 128.40, 128.58, 135.56, 154.39, 156.07 ppm. ESI-MS (m/z): 264.00 [M+H] ⁺ . 1,3-Dimethyl-1,3,5-triazinan-2-one (22)

Compound 23 (603 mg, 2.29 mmol) was dissolved in methanol, palladium on acti vated charcoal (30 mg) was added and the reaction mixture was stirred at r.t. for 14 h under hydrogen atmosphere. The mixture was filtered and the filtrate was concentrated in vacuo to give the title compound 22 as white solid (259 mg, 2.01 mmol, 88% yield). ¹ H NMR (CDCIs, 400 MHz) d = 2.77 (s, 1 H), 2.85 (s, 5H), 4.16 (s, 4H) ppm. tert-Butyl (Z)-(((tert-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-t riazinan- 1-yl)methyl)carbamate (24)

To a solution of compound 22 (137 mg, 1.06 mmol) and compound 13 (420 mg, 1.06 mmol) in acetonitrile, DIPEA (274 mg, 2.13 mmol) was added and the mixture was stirred at 50 °C for 14 h. The solvent was removed in vacuo. The residue was diluted with water (50 ml_) and extracted with ethyl acetate (2x30 ml) and di- chloromethane (2x30 ml). The combined organic phases were washed with brine, dried over sodium sulfate and the solvent was removed in vacuo. The crude prod uct was purified by column chromatography (6:1 then 2:1 petroleum ether: ethyl acetate, 1 :1 :0.05 petroleum ethenethyl acetate:triethylamine) to give the target compound 24 as white crystals (226 mg, 0.61 mmol, 58% yield). ¹ H NMR (CDCh, 400 MHz) d = 1.47-1.48 (m, 18H), 2.01 -2.02 (m, 1 H), 2.90-2.91 (m, 6H), 4.69 (s, 6H) ppm. ¹³ C NMR (CDCIs, 101 MHz) d = 28.16, 33.18, 60.47, 62.31 , 81.82, 154.01 , 157.30 ppm. ESI-MS (m/z): 372.20 [M+H] ⁺ .

4-(3-Chloropropoxy)-3-fluorobenzaldehyde (25)

To a solution of compound 15 (2.50 g, 17.9 mmol) and potassium carbonate (2.71 g, 19.6 mmol) in acetone, 1 -chloro-3-iodpropane (3.64 g, 17.9 mmol) was added and the reaction mixture was stirred at 60 °C for 14 h. The solvent was evaporated and the residue was diluted with water (100 ml_) and extracted with ethyl acetate (2x50 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was pu rified by column chromatography (5:1 petroleum ether: ethyl acetate). The solvent was removed in vacuo to give compound 25 as a pale-yellow liquid (3.36 g, 15.6 mmol, 87% yield). ¹ H NMR (CDCIs, 400 MHz) d = 2.23-2.32 (m, 2 H), 3.74-3.78 (m, 2 H), 4.22-4.28 (m, 2 H), 7.58-7.80 (m, 3 H), 9,85 (s, 1 H) ppm. ¹³ C NMR (CDCIs, 101 MHz) d = 31 .98, 41 .14, 65.69, 1 13.62, 1 13.81 , 1 15.94, 1 16.15,

123.30, 123.37, 130.20, 130.30, 147.54, 148.66, 150.66-153.12 (J(C-F) = 254.4 Hz), 189.96 ppm. ESI-MS (m/z): 217.10 [M+H] ⁺ .

(E)-1-(3-Chloropropoxy)-2-fluoro-4-(2-methoxyvinyl)benzen e (26)

(Methoxymethyl)triphenylphosphonium chloride (538 mg, 1 .57 mmol) was dis solved in dry tetrahydrofuran and cooled to 0°C. Potassium fe/f-butoxide (228 mg, 2.04 mmol) was added portionwise and the mixture was stirred for 1 h. Compound

25 (200 mg, 1 .00 mmol) in dry tetrahydrofuran was added dropwise under cooling and the reaction was warmed to room temperature over night. After mixture was quenched with water, the solvent was removed in vacuo and the residue was di luted with water (100 ml_) and extracted with ethyl acetate (2x50 ml). The com bined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (15: 1 petroleum ether: ethyl acetate) to give the target compound

26 as a colorless oil (192 mg, 0.78 mmol, 50% yield). ¹ H NMR (CDCIs, 400 MHz) d = 2.25 (m, 2 H), 3.37 (s, 2 H), 3.76-3.77 (m, 3 H), 4.16 (m, 2H), 5.13-5.14 (d, J = 6.9 Hz, 1 H), 5.70-5.73 (d, J = 13.0 Hz, 0.5 H), 6.08-6.10 (d, J = 7.0 Hz, 0.5 H), 6.87-6.89 (m, 3 H) ppm. ¹³ C NMR (CDCIs, 101 MHz) d = 32.47, 41 .52, 41 .55, 56.70, 60.78, 66.15, 66.29, 1 12.69, 1 12.88, 1 15.08, 1 15.10, 1 15.84, 1 15.86, 1 16.02, 1 16.21 , 121 .13, 121 .17, 147.57, 148.78, 151 .91 -154.35 (J(C-F) = 245.1 Hz), 153.80 ppm.

2-(4-(3-Chloropropoxy)-3-fluorophenyl)ethan-1 -ol (28)

Compound 26 (90 mg, 0.37 mmol) was dissolved in tetrahydrofuran and a solution of mercury(ll) acetate (129 mg, 0.41 mmol) in water was added under cooling in an ice/water bath. The reaction mixture was stirred for 15 mins and sodium boro- hydride (56 mg, 1 .48 mmol) in a saturated potassium carbonate solution was add ed dropwise. The reaction mixture was stirred for 30 min, diluted with water and extracted with ethyl acetate (4x10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (4: 1 petrole um ether: ethyl acetate) to give the target compound 28 as colorless oil (58.5 mg, 0.25 mmol, 68% yield). ¹ H NMR (CDCIs, 400 MHz) d = 2.22-2.28 (m, 2 H), 2.77- 2.80 (t, J = 6.5 Hz, 2 H), 3.75-3.83 (m, 4 H), 4.15-4.18 (t, J = 5.8 Hz, 2H), 6.91 - 6.98 (m, 3 H) ppm. ¹³ C NMR (CDCIs, 101 MHz) d = 31 .27, 38.82, 41.32, 41 .43, 62.64, 65.98, 1 13.39, 1 13.46, 1 16.63, 1 16.84, 125.48, 125.56, 133.36, 133.39, 144.45, 144.85, 153.22-156.17 (J(C-F) = 251 .5 Hz) ppm. ferf-butyl (E)-(((ferf-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-t riazinan- 1-yl)methyl)(4-(3-chloropropoxy)-3-fluorophenethyl)carbamate (29)

Compound 28 (72 mg, 0.31 mmol), compound 24 (172 mg, 0.46 mmol) and tri phenyl phosphine (122 mg, 0.46 mmol) were dissolved in dry tetrahydrofuran. A solution of DIAD (94 mg, 0.46 mmol) in dry tetrahydrofuran was added dropwise at 0 °C and the reaction mixture was slow warmed up to room temperature and stirred for 16 h. The mixture was diluted with water (40 ml_) and extracted with di ethyl ether (4x10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude prod uct was purified by column chromatography (2.5: 15:0.5 petroleum

ether:acetone:ethyl acetate) to give the target compound 29 as a colorless liquid (152 mg, 0.26 mmol, 84% yield). ¹ H NMR (CDCIs, 400 MHz) d = 1 .47 (s, 18 H), 2.22-2.25 (m, 2 H), 2.84 (m, 6 H), 3.57 (bs, 4 H), 3.74-3.77 (m, 2 H), 4.13-4.16 (m, 2 H), 4.52 (bs, 4 H), 6.89-6.95 (m, 3 H) ppm. ¹³ C NMR (CDCIs, 101 MHz) d = 28.14, 28.25, 41 .43, 48.87, 60.49, 61 .02, 66.02, 80.84, 83.00, 1 15.48, 1 16.55, 1 16.73, 124.40, 124.43, 131 .50, 131 .56, 145.60, 145.71 , 150.98, 151 .52-153.97 (J(C-F) = 246.8 Hz), 152.31 , 156.98, 158.39 ppm. ESI-MS (m/z): 586.25, 588.25 [M+H]l ferf-butyl (E)-(((ferf-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-t riazinan- 1-yl)methyl)(3-fluoro-4-(3-iodopropoxy)phenethyl)carbamate (30a)

Compound 29 (72 mg, 0.12 mmol) and sodium iodine (37 mg, 0.25 mmol) were dissolved in acetone and the reaction mixture was stirred at 70 °C for 14 h. The mixture diluted with water (40 ml_) and extracted with diethyl ether (4 x 10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give the target compound 30a as a yellow liquid (83 mg, 0.12 mmol, 99% yield) and was used directly in the next step without further purification. ESI-MS (m/z): 678.20 [M+H] ⁺ , 700.20 [M+Na] ⁺ .

(E)-3-(4-(2-(/V,/V'-bis(ferf-butoxycarbonyl)-3,5-dimethyl -4-oxo-1,3,5-triazinane- 1-carboximidamido)ethyl)-2-fluorophenoxy)propyl 4-methylbenzenesulfonate (30)

Compound 30a (72 mg, 0.1 1 mmol) was dissolved in acetonitrile and silver p- toluenesulfonate (148 mg, 0.53 mmol) in darkness at 0 °C. The reaction mixture was stirred at room temperature for 72 h. The crude product was diluted with water (40 ml_) and extracted with ethyl acetate (4x10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give the target compound 30 as a yellow liquid (75 mg, 0.10 mmol,

98% yield). ¹ H NMR (CDCIs, 400 MHz) d = 1 .48 (s, 18 H), 2.1 1 -2.14 (m, 2 H), 2.14 (s, 3H), 2.85 (s, 6H), 3.85 (bs, 4H), 3.89-4.01 (m, 2H), 4.22-4.25 (m, 2H), 4.55 (bs, 4H), 6.77-6.93 (m, 3H), 7.26-7.28 (m, 2H), 7.75-7.77 (d, J = 8.3 Hz) ppm. ¹³ C NMR (CDCIs, 101 MHz) d = 28.15, 28.26, 29.05, 29.80, 33.22, 33.66, 60.50, 61 .20, 64.87, 66.98, 80.93, 83.06, 1 15.32, 1 16.49, 1 16.67, 124.42, 127.98, 129.95, 131 .55, 132.92, 144.92, 145.36, 145.47, 151.37, 152.28, 153.82, 156.95 ppm. ESI-MS (m/z): 722.35 [M+H] ⁺ , 744.30 [M+Na] ⁺ .

Radiochemistry

¹⁸ F-Fluoride trapped on a Sep-Pak QMA cartridge was first washed with distilled water (3 ml_) to remove ¹⁸ 0-water. ¹⁸ F-Fluoride was then eluted from the cartridge with K2CO3 solution (0.3 ml_, 23 pmol/mL) into a vial that contains Kryptofix222 (22.5 mg, 59.7 pmol). Acetonitrile (0.5 ml_x4) was added to the vial, and the solu- tion was dried azeotropically under argon at 120°C. Labeling is carried out by add ing the solution of the precursor (4.8 mg) in dry acetonitrile (0.3 mL), followed by heating and stirring at 110°C for 10 min. Hydrochloric acid (6N, 0.3 mL) was added to the reaction mixture, and the reaction continued for 20 min at 100°C. The mix ture was cooled, diluted with 1 mL of a mixture solution of water and acetonitrile (1 :1 ), and applied to the semi-preparative HPLC for purification. HPLC condition: Column: 9.4x250 mm 5-micron ZORBAX Eclipse XDB-C18 (P.N.: 990967-202). Mobile phase: Phase A: Millipore water with 0.1 % formic acid; Phase B: Methanol with 0.1 % formic acid. Condition: 0-20 min, 30%-60% B, 20-22 min 60-100% B, 22-50 min 100% B; Flow rate 3 mL/min. Condition of autoradiography: TLC with normal phase silica gel 60. Solvent system: 3 mL methanol + 20 pL formic acid,

Rf.: 0.6. ¹³¹ I-MIBG was purchase from GE Healthcare (Freiburg im Breigau, Ger many) and used within 2 h after calibration time. ¹³¹ I-MIBG was chosen instead of ¹²³ I-MIBG due to its relative longer half-life, which is convenient for research pur poses and financial reasons.

Detail of synthesis of the compounds of Figs. 4 to 6: (E/Z)-2-lodo-1-methoxy-4-(2-methoxyvinyl)benzene C10H11O2I (107)

(Methoxymethyl)triphenylphosphonium chloride (3.94 g, 11.50 mmol, 1.50 equiv.) was dissolved in dry tetrahydrofuran (60 mL) under inert gas atmosphere and po tassium fe/f-butoxide (1.47 g, 13.13 mmol, 1.71 equiv.) was added at 0 °C. The reaction mixture was stirred for 2 min at 0 °C before commercially available

3-iodo-4-methoxybenzaldehyde 106 (2.01 g, 7.66 mmol, 1.00 equiv.) was added. The solution was stirred at room temperature for 18 h and evaporated to dryness. The residue was partitioned between in ethyl acetate (50 mL) and water (50 mL) followed by layer separation. The aqueous layer was extracted with ethyl acetate (2 x 50 mL) and the combined organic layers were washed with brine (1 ^c 100 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude material was purified by column chromatography (S1O2, petroleum ether/ethyl acetate = 12: 1 , Rf = 0.37) affording the desired product 107 as a pale yellow oil (1 .79 g, 81 % yield). ¹ H-NMR (CDC , 400 MHz) d = 8.02 (1 H, d, J = 2.13 Hz, H ^h ), 7.66 (1 H, d, J = 2.18 Hz, H ^h ), 7.50 (1 H, dd, J = 2.13 Hz, 8.54 Hz, H ^d ), 7.15 (1 H, dd, J = 2.25 Hz, 8.46 Hz, H ^d ), 6.92 (1 H, d, J = 12.92 Hz, H ^a ), 6.78 - 6.71 (2H, m, H ^e/e ), 6.07 (1 H, d, J = 6.94 Hz, H ^a’ ), 5.70 (1 H, d, J = 13.00 Hz, H ^b ), 5.09 (1 H, d, J = 6.98 Hz, H ^b ), 3.86/3.85 (6H, s, H ^i/r ), 3.77 (3H, s, H ^j ), 3.66 (3H, s, H ) ppm. ¹³ C-NMR (CDCh, 101 MHz) d = 156.4 (C ^f ), 156.1 (C ^f ), 148.5 (C ^a ), 147.3 (C ^a’ ), 139.1 (C ^h’ ), 136.1 (C ^h ), 131 .3 (C ^c ), 131 .0 (C ^c ), 129.4 (C ^d’ ), 126.3 (C ^d ), 1 1 1 .2/1 10.7 (C ^e/e’ ), 103.9 (C ^b’ ), 103.4 (C ^b ), 86.5 ( ), 86.0 ( ^' ), 60.8 (CO,

56.7/56.6/56.5 (C^) ppm. ESI-MS (m/z): 290.80 [M+H] ⁺ .

2-(3-lodo-4-methoxyphenyl)ethan-1 -ol C9H11 O2I (108)

(E/Z)-2-lodo-1 -methoxy-4-(2-methoxyvinyl)benzene 107 (1 .79 g, 6.17 mmol,

1 .00 equiv.) was suspended in tetrahydrofuran (46 ml_) and water (74 ml_) before mercury acetate (2.18 g, 6.84 mmol, 1 .10 equiv.) was added at 0 °C. The reaction mixture was stirred at 0 °C for 30 min followed by the addition of a 50% aqueous potassium carbonate solution (30 ml_) and sodium borohydride (942 mg,

24.90 mmol, 4.00 equiv.). After the reaction mixture was stirred at 0 °C for 2.5 h, the precipitate was filtered off and the filtrate was extracted with ethyl acetate (3 x 80 ml_). The combined organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The crude material was purified by column chromatography (S1O2, petroleum ether/ethyl acetate = 1 : 1 , Rf = 0.45) to obtain alcohol 108 as a yellow oil (1 .36 g, 79% yield). ¹ H-NMR (CDCh, 400 MHz) d = 7.65 (1 H, d, J = 2.13 Hz, H ^h ), 7.16 (1 H, dd, J = 2.14 Hz, 8.35 Hz, H ^d ), 6.76 (1 H, d, J = 8.35 Hz, H ^e ), 3.85 (3H, s, H'), 3.81 (2H, t, J = 6.55 Hz, H ^a ), 2.76 (2H, t,

J = 6.53 Hz, H ^b ), 1 .69 (1 H, s, Hi) ppm. ¹³ C-NMR (CDCh, 101 MHz) d = 157.0 (C ^f ), 139.9 (C ^h ), 132.9 (C ^c ), 130.2 (C ^d ), 1 1 1 .1 (C ^e ), 86.2 (C^), 63.7 (C ^a ), 56.5 (C ¹ ), 37.8 (C ^b ) ppm. ESI-MS (m/z): 278.80 [M+H] ⁺ . 3-lodo-4-methoxyphenethyl acetate C11 H13O3I (113)

2-(3-lodo-4-methoxyphenyl)ethan-1 -ol 108 (1 .04 g, 3.74 mmol, 1 .00 equiv.) was dissolved in acetone (20 ml_) and acetyl chloride (800 mI_, 1 1 .21 mmol,

3.00 equiv.) was added at 0 °C. The reaction mixture was stirred at room tempera ture for 18 h before the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (20 ml_) and washed with saturated aqueous sodi um bicarbonate solution (1 ^c 20 ml_) and brine (1 ^c 20 ml_). The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The crude material was purified by column chromatography (S1O2, petroleum ether/ethyl acetate = 3:1 , Rf = 0.54) to obtain acetate 113 as a yellow oil (1 .07 g, 90% yield). ¹ H-NMR (CDCIs, 400 MHz) d = 7.63 (1 H, d, J = 2.15 Hz, H ^h ), 7.15 (1 H, dd, J = 2.16 Hz, 8.39 Hz, H ^d ), 6.75 (1 H, d, J = 8.38 Hz, H ^e ), 4.22 (2H, t, J = 7.01 Hz, H ^a ), 3.85 (3H, s, H'), 2.83 (2H, t, J = 7.01 Hz, H ^b ), 2.03 (3H, s, H ^k ) ppm. ¹³ C-

3-lodo-4-methoxyphenethyl acetate 113 (588 mg, 1 .87 mmol, 1.00 equiv.) was dissolved in dry dichloromethane (12 ml_) under inert gas atmosphere and a solu tion of boron tribromide (445 mI_, 4.69 mmol, 2.50 equiv.) in dry dichloromethane (12 mL) was added at -20 °C. The reaction mixture was stirred for 2 h and parti tioned between ethyl acetate (60 mL) and a 50% aqueous sodium bicarbonate solution (60 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2 ^c 60 mL). The combined organic layers were washed with brine (1 ^c 180 mL) and dried over sodium sulfate. Evaporation of the solvent under reduced pressure and purification by column chromatography (S1O2, petroleum ether/ethyl acetate = 3:1 , Rf = 0.36) afforded phenol 114 as a yellow oil (602 mg, 92% yield). ¹ H-NMR (CDCIs, 400 MHz) d = 7.51 (1 H, d, J = 2.08 Hz, H ^h ), 7.08 (1 H, dd, J = 2.05 Hz, 8.28 Hz, H ^d ), 6.91 (1 H, d, J = 8.29 Hz, H ^e ), 5.47 (1 H, bs, H'), 4.22 (2H, t, J = 7.02 Hz, H ^a ), 2.83 (2H, t, J = 6.99 Hz, H ^b ), 2.04 (3H, s, H ^k ) ppm. ¹³ C- NMR (CDCIs, 101 MHz) d = 171 .2 ( ), 153.8 (C ^f ), 138.5 (C ^h ), 132.0 (C ^c ), 130.8 (C ^d ), 1 15.1 (C ^e ), 85.6 (C ⁹ ), 65.0 (C ^a ), 33.8 (C ^b ), 21 .1 (C ^k ) ppm. ESI-MS (m/z):

328.85 [M+Na] ⁺ .

4-(3-Chloropropoxy)-3-iodophenethyl acetate C13H16O3ICI

4-Hydroxy-3-iodophenethyl acetate 114 (330 mg, 1 .08 mmol, 1 .00 equiv.) was dis solved in acetone (17 mL) followed by the addition of 3-chloro-1 -iodopropane (177 pL, 1 .65 mmol, 1 .53 equiv.) and cesium carbonate (702 mg, 2.15 mmol,

2.00 equiv.). The reaction mixture was stirred at 70 °C for 18 h before water (85 mL) was added. After an extraction with ethyl acetate (3 ^c 85 mL), the com bined organic layers were washed with brine (1 ^c 215 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (S1O2, petroleum ether/ethyl acetate =

5: 1 , Rf = 0.42) to give the title compound as a yellow oil (372 mg, 90% yield). ¹ H- NMR (CDCIs, 400 MHz) d = 7.63 (1 H, d, J = 2.16 Hz, H ^h ), 7.13 (1 H, dd, J = 2.15 Hz, 8.33 Hz, H ^d ), 6.76 (1 H, d, J = 8.35 Hz, H ^e ), 4.22 (2H, t, J = 6.99 Hz, H ^a ), 4.14 (2H, t, J = 5.68 Hz, H ^k ), 3.84 (2H, t, J = 6.33 Hz, H ^m ), 2.83 (2H, t, J = 6.99 Hz, H ^b ), 2.23 (2H, quint, J = 6.00 Hz, H'), 2.03 (3H, s, W) ppm. ¹³ C-NMR (CDCIs, 101 MHz) d = 171.1 (C ¹ ), 156.1 (C ^f ), 139.8 (C ^h ), 132.5 (C ^c ), 130.0 (C ^d ), 1 12.2 (C ^e ), 86.8 (C°), 65.6 (C ^k ), 64.9 (C ^a ), 41 .8 (C ^m ), 33.8 (C ^b ), 32.3 (C), 21 .1 (C ppm. ESI-MS (m/z): 404.80 [M+Na] ⁺ . 2-(4-(3-Chloropropoxy)-3-iodophenyl)ethan-1 -ol Cn H14O2ICI (117)

4-(3-Chloropropoxy)-3-iodophenethyl acetate (189 mg, 0.49 mmol, 1.00 equiv.) was dissolved in methanol (9 ml_) before potassium carbonate (650 mg,

4.70 mmol, 9.60 equiv.) was added. The reaction mixture was stirred at room tem perature for 3 h and was partitioned between water (60 ml_) and dichloromethane (60 ml_) followed by layer separation. The aqueous layer was extracted with di chloromethane (2 x 60 ml_) and the combined organic layers were washed with brine (1 ^c 180 ml_) and dried over sodium sulfate. The solvent was removed under reduced pressure to obtain alcohol 117 as a yellow oil (175 mg, quant.) which was used directly in the next step. ¹ H-NMR (CDCh, 400 MHz) d = 7.65 (1 H, d, J = 2.15 Hz, H ^h ), 7.16 (1 H, dd, J = 2.12 Hz, 8.32 Hz, H ^d ), 6.77 (1 H, d, J = 8.36 Hz, H ^e ), 4.14

(2H, t, J = 5.72 Hz, H'), 3.87 - 3.78 (4H, m, H ^{a k} ), 2.77 (2H, t, J = 6.48 Hz, H ^b ), 2.27

(2H, quint, J = 6.00 Hz, Hi) ppm. ¹³ C-NMR (CDCh, 101 MHz) d = 156.1 (C ^f ), 139.9 (C ^h ), 133.3 (C ^c ), 130.2 (C ^d ), 1 12.3 (C ^e ), 87.0 ( ), 65.7 (C ¹ ), 63.7 (C ^a ), 41 .8 (C ^k ),

37.9 (C ^b ), 32.3 (Ci) ppm. ESI-MS (m/z): 362.80 [M+Na] ⁺ . ferf-Butyl (((ferf-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1 ,3,5-triazinan-1 - yl)methyl)(4-(3-chloropropoxy)-3-iodophenethyl)carbamate C27H41 N5O6ICI (121 )

2-(4-(3-Chloropropoxy)-3-iodophenyl)ethan-1 -ol 117 (160 mg, 0.47 mmol,

1 .00 equiv.), triphenylphosphine (185 mg, 0.71 mmol, 1 .51 equiv.) and 24

(175 mg, 0.47 mmol, 1 .00 equiv.) were dissolved in dry tetrahydrofuran (23 ml_) under inert gas atmosphere. DIAD (140 pl_, 0.71 mmol, 1 .51 equiv.) was added at 0 °C and the reaction mixture was stirred at room temperature for 18 h. The sol vent was removed under reduced pressure and the residue was partitioned be tween water (20 ml_) and ethyl acetate (20 ml_) followed by layer separation. The aqueous layer was extracted with ethyl acetate (2 ^c 20 mL) and the combined or ganic layers were washed with brine (1 ^c 60 mL) and dried over sodium sulfate. Evaporation of the solvent under reduced pressure and purification by column chromatography (S1O2, petroleum ether/acetone/ethyl acetate = 5:3: 1 , Rf = 0.38) gave the title compound 121 as a pale green oil (272 mg, 83% yield). ¹ H-NMR (CDCh, 400 MHz) d = 7.61 (1 H, d, J = 1 .81 Hz, H ^h ), 7.12 (1 H, dd, J = 1 .71 Hz,

8.25 Hz, H ^d ), 6.74 (1 H, d, J = 8.31 Hz, H ^e ), 4.50 (4H, bs, H ^P ), 4.16 - 4.07 (3H, m, H'), 3.82 (2H, t, J = 6.25 Hz, H ^k ), 3.57 (2H, bs, H ^a ), 2.88 - 2.80 (8H, m, H ^b ), 2.26 (2H, quint, J = 5.96 Hz, Hi), 1 .48 (9H, s, H°), 1 .47 (9H, s, H°) ppm. ¹³ C-NMR (CDCh, 101 MHz) d = 156.3 (C ^{f r} ), 152.4/151 .3 (C ^{l m} ), 139.6 (C ^h ), 132.5 (C ^c ), 129.9 (C ^d ), 1 12.3 (C ^e ), 87.0 (C ⁹ ), 83.1/80.9 (C ⁿ ), 65.7 (C ¹ ), 61 .2 (C^), 49.0 (C ^a ), 41 .7 (C ^k ), 33.2 (C ), 33.1 (C ^b ), 32.2 ( ), 28.3/28.2 (C°) ppm. ESI-MS (m/z): 694.20 [M+H] ⁺ , 716.05 [M+Na] ⁺ . ferf-Butyl (((ferf-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1 ,3,5-triazinan-1 - yl)methyl)(3-iodo-4-(3-iodopropoxy)phenethyl)carbamate C27H41 N5O6I2 (122)

121 (236 mg, 0.34 mmol, 1 .00 equiv.) was dissolved in acetone (30 mL) and sodi um iodide (510 mg, 3.40 mmol, 10.00 equiv.) was added. The reaction mixture was stirred at 70 °C for 18 h and the solvent was removed under reduced pres sure. Since LC-MS indicated incomplete conversion of the starting material, an additional amount of sodium iodide (816 mg, 5.44 mmol, 16.00 equiv.) was added. The reaction mixture was stirred at 70 °C for another day. After adding another amount of sodium iodide (816 mg, 5.44 mmol, 16.00 equiv.), the reaction mixture was stirred at 70 °C for additional 18 h before partitioning between water (15 ml_) and diethyl ether (15 ml_) followed by layer separation. The aqueous layer was extracted with diethyl ether (2 x 15 ml_) and the combined organic layers were washed with brine (1 ^c 45 ml_) and dried over sodium sulfate. The solvent was removed under reduced pressure to obtain iodide 122 as a yellow oil (221 mg, 83% yield) which was used directly in the next step. ¹ H-NMR (CDCh, 400 MHz) d

= 7.62 (1 H, d, J = 2.16 Hz, H ^h ), 7.13 (1 H, dd, J = 2.22 Hz, 8.31 Hz, H ^d ), 6.75 (1 H, d, J = 8.33 Hz, H ^e ), 4.51 (4H, bs, H ^P ), 4.05 (3H, t, J = 5.69 Hz, H'), 3.58 (2H, bs,

H ^a ), 3.46 (3H, t, J = 6.60 Hz, H ^k ), 2.90 - 2.75 (8H, m, H^), 2.32 - 2.24 (2H, m, Hi),

1.48 (9H, s, H°), 1 .47 (10H, s, H°) ppm. ¹³ C-NMR (CDCh, 101 MHz) d = 157.0 (C ^r ), 156.2 (C ^f ), 152.3/151 .1 (C ^{l m} ), 139.6 (C ^h ), 132.6 (C ^c ), 129.8 (C ^d ), 1 12.4 (C ^e ), 87.0 (C ⁹ ), 83.1/81 .0 (C ⁿ ), 68.7 (C ¹ ), 61 .2 (C ^P ), 49.0 (C ^a ), 33.3 (C^), 33.1 (C ^b ), 32.9 (C*), 28.3/28.2 (C°), 3.1 (C ^k ) ppm. ESI-MS (m/z): 786.00 [M+H] ⁺ , 807.90 [M+Na] ⁺ .

3-(4-(2-(/V,AP-Bis(ferf-butoxycarbonyl)-3,5-dimethyl-4-ox o-1,3,5-triazinane-1 - carboximidamido)ethyl)-2-iodophenoxy)propyl 4-methylbenzenesulfonate C34H48N5O9SI (123)

122 (191 mg, 0.24 mmol, 1 .00 equiv.) was dissolved in acetonitrile (10 ml_) and silver p-toluenesulfonate (340 mg, 1 .22 mmol, 5.00 equiv.) was added. The reac tion mixture was stirred in darkness at room temperature for 72 h before the sol vent was removed under reduced pressure. The residue was partitioned between water (15 ml_) and ethyl acetate (15 ml_) followed by layer separation. The aque ous layer was extracted with ethyl acetate (2 x 15 ml_) and the combined organic layers were washed with brine (1 ^c 40 ml_) and dried over sodium sulfate. Evapo ration of the solvent under reduced pressure and purification by column chroma- tography by column chromatography (S1O2, petroleum ether/acetone/ethyl acetate = 6:3: 1 , Rf = 0.33) afforded the target compound 123 as a colourless foam

(1 13 mg, 56% yield). ¹ H-NMR (CDCIs, 400 MHz) d = 7.75 (2H, d, J = 8.33 Hz, H‘), 7.58 (1 H, d, J = 2.06 Hz, H ^h ), 7.23 (2H, d, J = 8.32 Hz, H ^u ), 7.10 (1 H, dd, J = 2.04 Hz, 8.33 Hz, H ^d ), 6.62 (1 H, d, J = 8.22 Hz, H ^e ), 4.52 (3H, bs, H ^P ), 4.31 (2H, t, J = 6.02 Hz, H ^k ), 3.96 (2H, t, J = 5.78 Hz, H'), 3.56 (2H, bs, H ^a ), 2.88 - 2.79 (8H, m, H ^{b q} ), 2.36 (3H, s, H ^w ), 2.19 - 2.10 (2H, m, Hi), 1 .48 (9H, s, H°), 1 .47 (9H, s, H°) ppm. ¹³ C-NMR (CDCIs, 101 MHz) d = 157.0 (C ^r ), 156.0 (C ^f ), 152.3/151 .0 (C ^{l m} ), 144.9 (C ^v ), 139.6 (C ^h ), 132.9 (C ^s ), 132.5 (C ^c ), 130.0 (C ^u ), 129.7 (C ^d ), 127.9 (C‘), 1 12.1 (C ^e ), 86.8 (C ⁹ ), 83.0/81 .0 (C ⁿ ), 67.2 (C ^k ), 64.4 (C ¹ ), 61 .2 (C ^P ), 49.1 (C ^a ), 33.3 (C ^q ), 33.2 (C ^b ), 29.0 (Ci), 28.3/28.2 (C°), 21 .8 (C ^w ) ppm. ESI-MS (m/z): 830.10 [M+H] ⁺ , 852.05 [M+Na] ⁺ .

Radiochemistry

Radiofluorination was performed using K ¹⁸ F to replace the tosylate group of pre cursor 123 with ¹⁸ F. By the addition of 6N HCI, cleavage of the boc groups and the triazinanone moiety was achieved (Fig. 6). The desired radioactive labelled com pound ¹⁸ F-37 was obtained with an overall radiochemical yield of 22% (decay cor rected) and >99% radiochemical purity.

Competitive binding/Cell uptake studies

SK-N-SH cells expressing NET were cultivated according to the instructions from the supplier (Sigma-Aldrich Chemie GmbH, Munich, Germany). The cells were transferred to a 12 well plate and incubated in 1 ml_ EMEM while reaching 2x10 ⁵ cells/well density the day for testing. The medium was removed, and the cells were washed with 1 ml_ EMEM. A solution of ¹⁸ F-AF78 (3.7 kBq) in EMEM (700 pL) was added to each well with or without inhibitors (NE, MHPG, cold AF78 each with a final concentration 100 nM, 1 pM, 10 pM and 100 pM, respectively;

Desipramine 10 pM). The plate was incubated for 60 min at 37°C. The cells were washed with ice-cold PBS buffer (1 ml_x2) in order to remove unbound tracer. NaOH solution (0.1 N, 500pL) was added to each well followed by the collection of cell pellet and the measurement in g-counter (FH412, Frieseke & Flopfner, Erlan gen, Germany) using differential energy windows (±20%) for ¹⁸ F and ¹³¹ 1.

Cold, i. e. non-radioactive, reference compound AF78 of the compound according to the invention and the following compound 1

rvri-i-\_F i

were synthesized. The compounds were tested in SK-N- SH cells expressing NET in order to evaluate their competitive binding affinity against ¹³¹ I-MIBG. Fig. 7 shows the dose-response curves of ¹³¹ I-MIBG uptake in SK-N-SFI cells in the presence of increasing concentrations of the following non radioactive compounds: NE (·), MFIPG (■), cold reference 1 (= compound 1 ) (¨), cold reference 2 (= compound AF78) ( A). Results are expressed as percent of control MIBG uptake. IC50 of NE, MFIPG, and cold reference 2 AF78 are 1 .38 ± 0.25 mM, 6.80 ± 0.73 pM, and 2.57 ± 1 .37 pM, respectively. (IC50 of AF78 vs. MHPG p value < 0.01 )

NE and cold MIBG were used as references. As a result, cold reference 2 (AF78) could inhibit the cell uptake of ¹³¹ I-MIBG in a concentration-dependent manner. In contrast, cold reference 1 was not able to block the uptake of ¹³¹ I-MIBG even at the highest concentration tested. Inhibition was only 40% at 100 pM. This result provides a hint for a structure-activity relationship. The introduction of long alkyl chain at 3-position on the benzene ring seems not to be tolerated, whereas intro duction at the 4-position seems to be well tolerated.

After successful radiolabeling, ¹⁸ F-AF78 was also tested in SK-N-SFI cells in order to check cell uptake. Antidepressant desipramine (DMI) was used as the inhibitor of NET. Results are shown in Fig. 8. The addition of DMI blocks cell uptake of tracers specifically transported by NET. The results show that 94% of the uptake of ¹⁸ F-AF78 was inhibited by the addition of DMI compared to 87% of ¹³¹ l-MIBG, as a reference. To further confirm the tracer’s binding affinity, ¹⁸ F-labeled AF78 was used in com petitive inhibition assay, to which different concentrations of NE, MFIPG, and cold reference 2 (AF78) were added. Fig. 9 shows dose-response curves of ¹⁸ F-AF78 uptake in SK-N-SFI cells in the presence of increasing concentrations of the follow ing non-radioactive compounds: NE (·), MFIPG (■), and cold AF78 ( A). Results are expressed as percentage of control AF78 uptake. NE, MFIPG, and cold AF78 could inhibit the uptake of ¹⁸ F-AF78 at an IC50 value of 0.60 ± 0.27, 0.85 ± 0.63, and 2.68 ± 0.83 mM, respectively. (IC50 of MFIPG vs. AF78, p value < 0.05). These results are in accordance with the outcomes achieved by using ¹³¹ I-MIBG as a ref erence. Therefore, it further confirmed that the structural modification retained the property of being specifically taken up by NET as compared to the original refer ence and the clinically used tracer ¹³¹ I-MIBG.

Ex vivo autoradiography and tissue counting studies

Standard protocols and data analysis methods for non-invasive PET imaging of small animals have been established in the working group of the inventors

(Rischpler C. et al. , Eur J Nucl Med Mol Imaging. 2013; 40(7): 1077-83). Healthy male Wistar rats (Japan SLC, Inc., Japan) each weighing 200-250 g were used. The rats are anaesthetized with 2% isoflurane. 10-20 MBq of the tracer was ad ministered via tail vein injection. Ten minutes after the tracer administration, the animals were immediately euthanized. Hearts, livers and blood were obtained for ex vivo analysis with autoradiography (Typhoon FLA 7000) and tissue counts with y-counter (1480 WIZARD™ 3”). Following weight and decay correction of tissue counts, the heart to blood (H/B) and heart to liver (H/L) count ratio were calculated, respectively. For autoradiography, the rats were first injected via tail vein either with or without (control) NET blocker phenoxynezamine 50 mg/kg after anaesthe sia. After 10 min, the tracer (10-20 MBq) was administered. The hearts were har vested 10 min later, frozen, and cut into 20 pm short axis slices using a cryostat (Cryotome FSE, Thermo Fisher Scientific). To obtain the distribution of the tracer, autoradiography plates (Fuji SR-type image plate, Fujifilm Corporation, Tokyo, Ja- pan) were exposed to the short axis slices immediately for 18 h and thereafter im aged using a digital autoradiographic system (Typhoon FLA 7000).

Cardiac imaging studies in rats and monkeys

In an autoradiography study of left ventricular short axis (LVSA) slices from rats, ¹⁸ F-AF78 showed homogeneous tracer uptake throughout the left ventricular walls in the healthy myocardium. The result is shown in Fig. 10. ¹⁸ F-AF78 demonstrated an even distribution throughout the myocardium (control), which could be inhibited by a pretreatment with phenoxybenzamine (PhB, 50 mg/kg i.v. injection) 10 min before tracer injection. A lack of delineation of the right ventricular wall might be due to its thinness and the slicing position.

An ex vivo tissue counting study showed that the tracer has a reasonable uptake in the heart as demonstrated in both Heart-to-Blood (H/B) and Heart-to-Liver (H/L) ratio counting 12.54 and 6.14, respectively. This uptake could be specifically blocked by PhB (50 mg/kg) and led to approximately 6 fold decline in the H/B ratio and 5 fold decline in the H/L ratio, counting 2.41 and 1 .30, respectively. Results of the tissue distribution study are shown in Fig. 1 1 . The columns are the mean value of 2 rats. Data were determined 10 min post injection. Y-axis represents the heart/tissue distribution ratio. Both the Heart-to-Blood ratio (H/B) and the Heart-to- Liver (H/L) ratio decreased significantly after pretreatment with PhB (50 mg/kg, NET blockade) 10 min before tracer injection. In comparison, according to Raffel, D. et al. , J. Nucl. Med. 2017, 58(Suppl 1 ): 96 MHPG and PHPG show tissue con centration ratios at a time frame of 50-60 min imaging as follows: MHPG with H/B 3.6 and H/L 2.3; PHPG with 5.4 and 1 .1 , respectively. Therefore, the low liver up take of ¹⁸ F-AF78 facilitates the possibility of evaluating the inferior wall activity of the heart. This highly specific cardiac uptake shows that ¹⁸ F-AF78 is an almost ideal PET imaging of the heart.

Cardiac PET imaging in animals

Rats were maintained anaesthetized during the whole experiment by 2% isoflu- rane. All scans were obtained using a dedicated small-animal PET system (mi- croPET FOCUS 120, SIEMENS, Germany). The PET imaging protocol was de signed to assess the systemic and myocardial tracer distribution of ¹⁸ F-AF78. Shortly before the injection of 10-20 MBq of ¹⁸ F-AF78 via the tail vein, a dynamic PET scan was initiated. In order to evaluate the cardiac uptake mechanism of the tracer, rats were pretreated with phenoxybenzamine (50 mg/kg intravenously, Sigma-Aldrich, Tokyo, Japan). Ten minutes after the pretreatment, 10-20 MBq of ¹⁸ F-AF78 was administered via the tail vein. A 10 min dynamic PET session was started shortly before the tracer injection. PET image was acquired in list-mode format. The data were sorted into 3-demensional sonograms, which were then rebind with a Fourier algorithm to reconstruct dynamic images using a 2- dimensinal ordered-subset expectation maximization (OSEM) algorithm. All imag es were corrected for ¹⁸ F decay, random, and dead time; correction for attenuation was not performed (Higuchi JNM 2013). The obtained PET images were analyzed with the public domain tool AMIDE imaging software (A Medical Imaging Data Ex aminer, version 1 .01 ).

Systemic distribution of ¹⁸ F-AF78 ¹⁸ F-37 in the healthy rat myocardium by in vivo PET with (NET Blockade) or without (No pretreatment) pretreatment with NET blocker PhB (50 mg/kg iv injection) 10 min before the tracer injection is shown in Figs. 12 and 13. The static PET images show homogeneous and clear tracer up take throughout the left ventricular wall. Pretreatment with NET blocker PhB (50 mg/kg, intravenously via tail vein) significantly decreased the myocardial tracer uptake (Figs. 12 and 13, NET Blockade).

For control, Cynomolgus monkey (male, 5.3 kg) was maintained anaesthesized during the imaging study using 3.3% seboflurane. Shortly before receiving an in travenous injection of the tracer (44 MBq), a 120 min dynamic PET session was started. In addition, to investigate the inhibition of cardiac neuronal uptake-1 , DMI (1 mg/kg, iv.) was injected to Cynomolgus monkey (female, 5.6 kg) 10 min before the tracer (18.2 MBq) injection and PET assessment started. PET was conducted using a PCA-2000A positron scanner (Toshiba Medical Systems Corporation, Otawara, Japan). Sinograms were collected with the following pattern: 10 sec*12, 30 sec*6, 300 sec *23. The tissue uptake was determined as arbitrary unit (A.U.), from which a time-activity curves of different organs were generated.

A tissue bio-distribution consistent with that observed in rat could be observed with clear long term and stable cardiac uptake during the total 120 min scan whereas uptake in adjacent liver and lungs was low. Representative time activity curves derived from ¹⁸ F-AF78 dynamic images in control and sympathetic nerve blocked monkey were generated from the scanning data. The time activity curves ex pressed in arbitrary unit, A.U. versus time in minutes are shown Fig. 14. ¹⁸ F-AF78 cardiac uptake showed a high tracer activity after the first washout and maintained activity throughout the entire 120 min scan time. The pretreatment with uptake-1 inhibitor DMI (1 mg/kg, iv) 10 min before the tracer injection markedly reduced the heart uptake level, which dropped almost instantly to the same level as in blood. In the control, activity in the liver reached a peak a little while after the tracer injec tion, then decreased slowly to blood level after 60 min. The results show a stable long-term cardiac specific uptake with favorable Fleart/Blood and Fleart/Liver rati os. Tracer uptake in the liver was not affected by pretreatment with DMI.

Further biodistribution studies were performed in rats (n = 4) 10 min after injection of the tracer ¹⁸ F-37 demonstrating favourable heart-to-blood (H/B, approx. 6: 1 ) and heart-to-liver (H/L, approx. 2: 1 ) ratios (Fig. 15, right panel). Heart-to-muscle ratios (H/M) were very high with a value of approx. 12:1 . Time activity curves indicated stable long term cardiac uptake (expressed in arbitrary unit, A.U.) which is dis played in Fig. 15 (left panel). 16 MBq of tracer activity was injected.

Static PET images of cardiac uptake of ¹⁸ F-AF78 in healthy rats, rabbits, pigs and monkeys (Fig. 16) show that ¹⁸ F-AF78 works in all of these species such that it can be assumed that it works in all mammals including humans.

A further biodistribution study with ¹⁸ F-AF78 in monkey has been conducted. Re sults calculated as SUV (standardized uptake value) are shown in Fig. 17. The results show a stable cardiac uptake along with fast blood pool washout and rela- tive fast liver uptake washout. The liver uptake is decreasing and is lower than cardiac uptake 35 min after tracer administration.

A kinetic study has been performed with ¹⁸ F-AF78 in monkeys’ hearts. The results confirmed the specific uptake of ¹⁸ F-AF78 via NET and stable vesicular storage mechanism in the sympathetic nerve terminals. When injected 10 min before the tracer administration, NET blocker desipramine (DMI) specifically blocked the tracer uptake with significant difference (p<0.0001 ) compared to control (Fig. 18, upper panel). In contrast, when performing DMI chase (injected 10 min after the tracer administration), no difference can be seen compared to control group (Fig. 18, middle panel). Furthermore, a continuous tyramine (TYR) chase was applied after tracer administration. Tyramine is known as agent causing a catecholamine release thus enhancing tracer washout. The cardiac uptake of ¹⁸ F-AF78 de creased considerable. Decrease was statistically significant (p< 0.005) compared to control group (Fig. 18, lower panel).

Statistical analysis

All results are displayed as mean ± SD. The two-tailed paired Student t-test was used to compare differences between two dependent groups, and the two-tailed independent Student t-test for differences between independent groups. Multiple group comparisons were performed using analysis of variance (ANOVA). A p val ue of less than 0.05 was assumed to be statistically significant. Statistical analysis was performed with StatMate III (ATMS Co., Ltd.).

The meaning of abbreviations used in this specification is as follows:

DIAD, diisopropyl azodicarboxylate; DIPEA, A/,/V-diisopropylethylamine; DMF, /V,/V-dimethylformamide; DMI, desipramine; ¹¹ C-HED, ¹¹ C-(-)-/7?- hydroxyephedrine; iv, intravenously; ¹⁸ F-MHPG, ¹⁸ F-4-fluoro-3- hydroxyphenethylguanidine; ^123/131 l-MIBG, ^123/131 l-mefa-iodobenzylguanidine; NE, norepinephrine; NET, norepinephrine transporter; PET, positron emission tomog raphy; PhB, phenoxybenzamine; ¹⁸ F-PHPG, ¹⁸ F-3-fluoro-4- hydroxyphenethylguanidine; SPECT, single photon emission computed tomogra phy; THF, tetrahydrofuran