Synthesis of Novel Fluorinated Xanthine Derivatives with High Adenosine A2B Receptor Binding Affinity

The G protein-coupled adenosine A2B receptor is suggested to be involved in various pathological processes accompanied by increased levels of adenosine as found in inflammation, hypoxia, and cancer. Therefore, the adenosine A2B receptor is currently in focus as a novel target for cancer therapy as well as for noninvasive molecular imaging via positron emission tomography (PET). Aiming at the development of a radiotracer labeled with the PET radionuclide fluorine-18 for imaging the adenosine A2B receptor in brain tumors, one of the most potent and selective antagonists, the xanthine derivative PSB-603, was selected as a lead compound. As initial biodistribution studies in mice revealed a negligible brain uptake of [3H]PSB-603 (SUV3min: 0.2), structural modifications were performed to optimize the physicochemical properties regarding blood–brain barrier penetration. Two novel fluorinated derivatives bearing a 2-fluoropyridine (5) moiety and a 4-fluoro-piperidine (6) moiety were synthesized, and their affinity towards the four adenosine receptor subtypes was determined in competition binding assays. Both compounds showed high affinity towards the adenosine A2B receptor (Ki (5) = 9.97 ± 0.86 nM; Ki (6) = 12.3 ± 3.6 nM) with moderate selectivity versus the other adenosine receptor subtypes.


Introduction
Adenosine is an important signaling molecule that is released from cells or generated extracellularly after the cleavage of adenosine 5'-triphosphate (ATP) by ectonucleotidases. Extracellular adenosine activates G protein-coupled receptors, which are classified into four subtypes: adenosine A 1 , A 2A , A 2B , and A 3 receptors [1][2][3][4]. Compared to the other subtypes, the adenosine A 2B receptor (A 2B receptor) has a low expression under physiological conditions and is only activated at higher adenosine concentrations [1,5]. Such high levels can be reached under pathophysiological conditions (e.g., inflammation, cancer, infection, and hypoxia) [6].
Due to its involvement in tumor growth, angiogenesis, metastasis, and immunomodulation, the A 2B receptor is an interesting target for the imaging and treatment of cancer [7,8]. It shows an increased expression in various solid tumors and tumor cell lines relative to normal tissues. These findings can be an outcome of the hypoxic conditions and is associated with increased adenosine concentrations in the tumor microenvironment [9][10][11].
To date, numerous different antagonists targeting this adenosine receptor subtype have been developed [8,12]. One of the most specific and potent antagonists is the xanthine derivative PSB-603 [13] (see Table 1), which was recently shown to suppress the γ-irradiation-induced translocation of the epidermal growth factor receptor (EGFR) triggering DNA damage repair and radioresistance of tumors. These studies showed a significant involvement of the adenosine A 2B receptor by suppression of EGFR translocation and reduction of tumor growth in mice due to pretreatment with PSB-603 [14,15].
One important noninvasive in vivo imaging tool to diagnose cancer and to follow up on therapy responses is positron emission tomography (PET). For this technique, a specific radiotracer with high affinity is needed, which is labeled with a positron-emitting radionuclide, such as carbon-11 (t 1/2 = 20.4 min) or fluorine-18 (t 1/2 = 109.8 min). As the main objective of our work was to characterize the expression of the A 2B receptor using PET, the development of a specific radioligand labeled with fluorine-18 would support the investigation of this promising target in vitro and in vivo. The examination of the contribution of this receptor subtype in brain tumors was of particular interest to us.
The first PET radiotracer for imaging of the adenosine A 2B receptor was published by Petroni et al. [16] (see Figure 1). The carbon-11-labeled triazinobenzimidazole compound 1 showed only a moderate affinity (IC 50 (A 2B ) = 210.2 ± 12.3 nM [16]). Its pharmacokinetic properties were investigated in rats. The authors concluded that the radiotracer provides potential for the development of more potent and selective A 2B receptor PET imaging tools [16]. The first fluorine-18-labeled radiotracer 2 (see Figure 1) was published in 2018 by our group and showed good affinity towards the adenosine A 2B receptor with moderate selectivity towards the other adenosine receptor subtypes [17]. However, metabolism studies in mice revealed a rapid metabolic degradation of 2. One of the radiometabolites was found to be the cleavage product of the amide bond, resulting in an amino derivative that was able to penetrate the blood-brain barrier (BBB). Recently, we developed the bicyclic radiotracer 3 with a pyridopyrimidine-2,4-dione core ( Figure 1) [18]. This compound showed good affinity and selectivity towards the A 2B receptor with considerably improved in vivo stability; however, its brain uptake was rather low.
To date, numerous different antagonists targeting this adenosine receptor subtype have been developed [8,12]. One of the most specific and potent antagonists is the xan thine derivative PSB-603 [13] (see Table 1), which was recently shown to suppress the γ irradiation-induced translocation of the epidermal growth factor receptor (EGFR) trigger ing DNA damage repair and radioresistance of tumors. These studies showed a significan involvement of the adenosine A2B receptor by suppression of EGFR translocation and re duction of tumor growth in mice due to pretreatment with PSB-603 [14,15].
One important noninvasive in vivo imaging tool to diagnose cancer and to follow up on therapy responses is positron emission tomography (PET). For this technique, a specific radiotracer with high affinity is needed, which is labeled with a positron-emitting radio nuclide, such as carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.8 min). As the main objective of our work was to characterize the expression of the A2B receptor using PET, the development of a specific radioligand labeled with fluorine-18 would support the inves tigation of this promising target in vitro and in vivo. The examination of the contribution of this receptor subtype in brain tumors was of particular interest to us.
The first PET radiotracer for imaging of the adenosine A2B receptor was published by Petroni et al. [16] (see Figure 1). The carbon-11-labeled triazinobenzimidazole compound 1 showed only a moderate affinity (IC50 (A2B) = 210.2 ± 12.3 nM [16]). Its pharmacokinetic properties were investigated in rats. The authors concluded that the radiotracer provide potential for the development of more potent and selective A2B receptor PET imaging tools [16]. The first fluorine-18-labeled radiotracer 2 (see Figure 1) was published in 2018 by ou group and showed good affinity towards the adenosine A2B receptor with moderate selec tivity towards the other adenosine receptor subtypes [17]. However, metabolism studies in mice revealed a rapid metabolic degradation of 2. One of the radiometabolites was found to be the cleavage product of the amide bond, resulting in an amino derivative tha was able to penetrate the blood-brain barrier (BBB). Recently, we developed the bicyclic radiotracer 3 with a pyridopyrimidine-2,4-dione core ( Figure 1) [18]. This compound showed good affinity and selectivity towards the A2B receptor with considerably im proved in vivo stability; however, its brain uptake was rather low. In recent years, high affinity and selectivity of xanthine-based compounds have been achieved by several groups, resulting in a wide range of structural diversity [8,13,[19][20][21][22][23] One important structural modification of xanthines is N-alkylation at the nitrogen atoms N1 and N3 (see Table 1). The most promising results were obtained with ethyl or propy substitutions at position N1 [23]. Moreover, various substitutions in position 8 of the xan thine core also resulted in compounds with high affinity and selectivity, such as CVT-6975 [20], MRS-1754 [19], PSB-603 [13], and PSB-1901 [23] (see Table 1). All of the substituents contain phenyl or heteroaromatic moieties, of which the phenylsulfonamide-based deriv atives show the highest affinity towards the adenosine A2B receptor [23]. The most poten and selective xanthine-based compounds are PSB-603 and PSB-1901, which were pub lished by the Müller group in 2009 [13] and 2019 [23]. They are characterized by propy substitution on position N1 and the phenylsulfonamide, substituted with a 4-chloro phenyl (PSB-603) or 4-bromophenyl (PSB-1901) piperazine moiety, respectively (Table 1) In recent years, high affinity and selectivity of xanthine-based compounds have been achieved by several groups, resulting in a wide range of structural diversity [8,13,[19][20][21][22][23]. One important structural modification of xanthines is N-alkylation at the nitrogen atoms N1 and N3 (see Table 1). The most promising results were obtained with ethyl or propyl substitutions at position N1 [23]. Moreover, various substitutions in position 8 of the xanthine core also resulted in compounds with high affinity and selectivity, such as CVT-6975 [20], MRS-1754 [19], PSB-603 [13], and PSB-1901 [23] (see Table 1). All of the substituents contain phenyl or heteroaromatic moieties, of which the phenylsulfonamide-based derivatives show the highest affinity towards the adenosine A 2B receptor [23]. The most potent and selective xanthine-based compounds are PSB-603 and PSB-1901, which were published by the Müller group in 2009 [13] and 2019 [23]. They are characterized by propyl substitution on position N1 and the phenylsulfonamide, substituted with a 4-chlorophenyl (PSB-603) or 4-bromophenyl (PSB-1901) piperazine moiety, respectively (Table 1).
Based on these structure-activity relationship results, PSB-603 was used in this study as lead compound for the development of a fluoro-containing PET radiotracer. Initial biodistribution studies in mice were performed to investigate whether [ 3 H]PSB-603 would be able to penetrate the BBB. The measured standardized uptake value (SUV) of 0.2 at 3 min p.i. (results are in Section 2.1) revealed that the brain uptake was insufficient for a suitable PET radiotracer for brain imaging [24,25]. Therefore, structural modifications of the PSB-603 lead compound (M = 529 g/mol, clogP = 4.9) were envisaged to improve the brain uptake. Accordingly, the molar mass was reduced by shortening the phenylpiperazinyl moiety, which additionally contributed to a reduction in the high lipophilicity of PSB-603. Furthermore, in order to allow labeling with fluorine-18, fluoropyridine and 4-fluoropiperidine rings were selected as suitable moieties as we have previously shown [17,18,26]. Based on this strategy, the two novel xanthine derivatives 5 and 6 (see Scheme 1) demonstrated, with a molar mass of 444 and 435 g/mol and clogP values of 2.7 and 3.0, respectively, improved physicochemical properties regarding the potential to sufficiently penetrate the BBB for PET imaging. In particular, the clogP values were in the range of 1 to 4, which has been reported to be beneficial for a passive diffusion of molecules through the BBB [27][28][29]. Based on these structure-activity relationship results, PSB-603 was used in this study as lead compound for the development of a fluoro-containing PET radiotracer. Initial biodistribution studies in mice were performed to investigate whether [ 3 H]PSB-603 would be able to penetrate the BBB. The measured standardized uptake value (SUV) of 0.2 at 3 min p.i. (results are in Section 2.1) revealed that the brain uptake was insufficient for a suitable PET radiotracer for brain imaging [24,25]. Therefore, structural modifications of the PSB-603 lead compound (M = 529 g/mol, clogP = 4.9) were envisaged to improve the brain uptake. Accordingly, the molar mass was reduced by shortening the phenylpiperazinyl moiety, which additionally contributed to a reduction in the high lipophilicity of PSB-603. Furthermore, in order to allow labeling with fluorine-18, fluoropyridine and 4-fluoropiperidine rings were selected as suitable moieties as we have previously shown [17,18,26]. Based on this strategy, the two novel xanthine derivatives 5 and 6 (see Scheme 1) demonstrated, with a molar mass of 444 and 435 g/mol and clogP values of 2.7 and 3.0, respectively, improved physicochemical properties regarding the potential to sufficiently penetrate the BBB for PET imaging. In particular, the clogP values were in the range of 1 to 4, which has been reported to be beneficial for a passive diffusion of molecules through the BBB [27][28][29].

Organ Distribution of [ 3 H]PSB-603 in Mice
To estimate the ability of [ 3 H]PSB-603 to penetrate the BBB in vivo, an initial biodistribution study in healthy CD1 mice was performed.
For mouse brain, an SUV of 0.21 ± 0.07 (n = 3) at 3 min p.i. of [ 3 H]PSB-603 was determined, indicating a low uptake of the radioligand.
The moderate uptake in the pituitary gland corresponds to the higher expression pattern of adenosine A2B receptors, as shown by Rees et al. [30]. However, it may also be explained by the fact that the pituitary gland lacks a BBB.
Although, RT-PCR studies suggest widespread distribution of the A2B receptor with the highest gene expression in proximal colon, lung, uterus, eye, and bladder [31], the uptake of [ 3 H]PSB-603 observed in the bladder, appendix, and lung was low to moderate ( Figure 2). The high uptake in the liver is likely related to the metabolic degradation of [ 3 H]PSB-603 in this organ. Based on these structure-activity relationship results, PSB-603 was used in this study as lead compound for the development of a fluoro-containing PET radiotracer. Initial biodistribution studies in mice were performed to investigate whether [ 3 H]PSB-603 would be able to penetrate the BBB. The measured standardized uptake value (SUV) of 0.2 at 3 min p.i. (results are in Section 2.1) revealed that the brain uptake was insufficient for a suitable PET radiotracer for brain imaging [24,25]. Therefore, structural modifications of the PSB-603 lead compound (M = 529 g/mol, clogP = 4.9) were envisaged to improve the brain uptake. Accordingly, the molar mass was reduced by shortening the phenylpiperazinyl moiety, which additionally contributed to a reduction in the high lipophilicity of PSB-603. Furthermore, in order to allow labeling with fluorine-18, fluoropyridine and 4-fluoropiperidine rings were selected as suitable moieties as we have previously shown [17,18,26]. Based on this strategy, the two novel xanthine derivatives 5 and 6 (see Scheme 1) demonstrated, with a molar mass of 444 and 435 g/mol and clogP values of 2.7 and 3.0, respectively, improved physicochemical properties regarding the potential to sufficiently penetrate the BBB for PET imaging. In particular, the clogP values were in the range of 1 to 4, which has been reported to be beneficial for a passive diffusion of molecules through the BBB [27][28][29].

Organ Distribution of [ 3 H]PSB-603 in Mice
To estimate the ability of [ 3 H]PSB-603 to penetrate the BBB in vivo, an initial biodistribution study in healthy CD1 mice was performed.
For mouse brain, an SUV of 0.21 ± 0.07 (n = 3) at 3 min p.i. of [ 3 H]PSB-603 was determined, indicating a low uptake of the radioligand.
The moderate uptake in the pituitary gland corresponds to the higher expression pattern of adenosine A2B receptors, as shown by Rees et al. [30]. However, it may also be explained by the fact that the pituitary gland lacks a BBB.
Although, RT-PCR studies suggest widespread distribution of the A2B receptor with the highest gene expression in proximal colon, lung, uterus, eye, and bladder [31], the uptake of [ 3 H]PSB-603 observed in the bladder, appendix, and lung was low to moderate ( Figure 2). The high uptake in the liver is likely related to the metabolic degradation of Based on these structure-activity relationship results, PSB-603 was used in this study as lead compound for the development of a fluoro-containing PET radiotracer. Initial biodistribution studies in mice were performed to investigate whether [ 3 H]PSB-603 would be able to penetrate the BBB. The measured standardized uptake value (SUV) of 0.2 at 3 min p.i. (results are in Section 2.1) revealed that the brain uptake was insufficient for a suitable PET radiotracer for brain imaging [24,25]. Therefore, structural modifications of the PSB-603 lead compound (M = 529 g/mol, clogP = 4.9) were envisaged to improve the brain uptake. Accordingly, the molar mass was reduced by shortening the phenylpiperazinyl moiety, which additionally contributed to a reduction in the high lipophilicity of PSB-603. Furthermore, in order to allow labeling with fluorine-18, fluoropyridine and 4-fluoropiperidine rings were selected as suitable moieties as we have previously shown [17,18,26]. Based on this strategy, the two novel xanthine derivatives 5 and 6 (see Scheme 1) demonstrated, with a molar mass of 444 and 435 g/mol and clogP values of 2.7 and 3.0, respectively, improved physicochemical properties regarding the potential to sufficiently penetrate the BBB for PET imaging. In particular, the clogP values were in the range of 1 to 4, which has been reported to be beneficial for a passive diffusion of molecules through the BBB [27][28][29].

Organ Distribution of [ 3 H]PSB-603 in Mice
To estimate the ability of [ 3 H]PSB-603 to penetrate the BBB in vivo, an initial biodistribution study in healthy CD1 mice was performed.
For mouse brain, an SUV of 0.21 ± 0.07 (n = 3) at 3 min p.i. of [ 3 H]PSB-603 was determined, indicating a low uptake of the radioligand.
The moderate uptake in the pituitary gland corresponds to the higher expression pattern of adenosine A2B receptors, as shown by Rees et al. [30]. However, it may also be explained by the fact that the pituitary gland lacks a BBB.
Although, RT-PCR studies suggest widespread distribution of the A2B receptor with the highest gene expression in proximal colon, lung, uterus, eye, and bladder [31], the uptake of [ 3 H]PSB-603 observed in the bladder, appendix, and lung was low to moderate ( Figure 2). The high uptake in the liver is likely related to the metabolic degradation of [ 3 H]PSB-603 in this organ. Based on these structure-activity relationship results, PSB-603 was used in this study as lead compound for the development of a fluoro-containing PET radiotracer. Initial biodistribution studies in mice were performed to investigate whether [ 3 H]PSB-603 would be able to penetrate the BBB. The measured standardized uptake value (SUV) of 0.2 at 3 min p.i. (results are in Section 2.1) revealed that the brain uptake was insufficient for a suitable PET radiotracer for brain imaging [24,25]. Therefore, structural modifications of the PSB-603 lead compound (M = 529 g/mol, clogP = 4.9) were envisaged to improve the brain uptake. Accordingly, the molar mass was reduced by shortening the phenylpiperazinyl moiety, which additionally contributed to a reduction in the high lipophilicity of PSB-603. Furthermore, in order to allow labeling with fluorine-18, fluoropyridine and 4-fluoropiperidine rings were selected as suitable moieties as we have previously shown [17,18,26]. Based on this strategy, the two novel xanthine derivatives 5 and 6 (see Scheme 1) demonstrated, with a molar mass of 444 and 435 g/mol and clogP values of 2.7 and 3.0, respectively, improved physicochemical properties regarding the potential to sufficiently penetrate the BBB for PET imaging. In particular, the clogP values were in the range of 1 to 4, which has been reported to be beneficial for a passive diffusion of molecules through the BBB [27][28][29].

Organ Distribution of [ 3 H]PSB-603 in Mice
To estimate the ability of [ 3 H]PSB-603 to penetrate the BBB in vivo, an initial biodistribution study in healthy CD1 mice was performed.
For mouse brain, an SUV of 0.21 ± 0.07 (n = 3) at 3 min p.i. of [ 3 H]PSB-603 was determined, indicating a low uptake of the radioligand.
The moderate uptake in the pituitary gland corresponds to the higher expression pattern of adenosine A2B receptors, as shown by Rees et al. [30]. However, it may also be explained by the fact that the pituitary gland lacks a BBB.
Although, RT-PCR studies suggest widespread distribution of the A2B receptor with the highest gene expression in proximal colon, lung, uterus, eye, and bladder [31], the uptake of [ 3 H]PSB-603 observed in the bladder, appendix, and lung was low to moderate ( Figure 2). The high uptake in the liver is likely related to the metabolic degradation of [ 3 H]PSB-603 in this organ.

Organ Distribution of [ 3 H]PSB-603 in Mice
To estimate the ability of [ 3 H]PSB-603 to penetrate the BBB in vivo, an initial biodistribution study in healthy CD1 mice was performed.
For mouse brain, an SUV of 0.21 ± 0.07 (n = 3) at 3 min p.i. of [ 3 H]PSB-603 was determined, indicating a low uptake of the radioligand.
The moderate uptake in the pituitary gland corresponds to the higher expression pattern of adenosine A 2B receptors, as shown by Rees et al. [30]. However, it may also be explained by the fact that the pituitary gland lacks a BBB.
Although, RT-PCR studies suggest widespread distribution of the A 2B receptor with the highest gene expression in proximal colon, lung, uterus, eye, and bladder [31], the uptake of [ 3 H]PSB-603 observed in the bladder, appendix, and lung was low to moderate ( Figure 2). The high uptake in the liver is likely related to the metabolic degradation of

Synthesis of 5 and 6
The synthesis of the new derivatives 5 and 6 was performed as depicted in Schem 1. The required precursor 4 was synthesized following procedures published in the lite ature (see Scheme S1 in Supplementary Materials) [32][33][34][35][36].
Yan et al. [37] described the synthesis of 7 (for the structure, see Table 2), where the used aniline as amine in a dimethylformamide (DMF)/pyridine mixture. In adaption t the published conditions, DMF was chosen with an excess amount of 2-fluoropyridin-4 amine under thermal heating (125 °C). Because of the low reactivity of the employed am nopyridine, sodium hydride was used as a strong base. After an acidic workup and chro matographic purification, compound 5 was obtained with a yield of 51% (Scheme 1, NM and MS spectra in Figures S1-S4 in Supplementary Materials). The synthesis of 6 was performed according to Yan et al. [37] and Borrmann et al. [13 in DMF with an excess of 4-fluoropiperidine as amine, which was synthesized via a two step synthetic route [38]. This amine could be directly applied in the form of its trifluoro acetic acid (TFA) salt because the addition of an excess of triethylamine (TEA) to the reac tion mixture resulted in the corresponding free base being formed in situ. At a tempera ture of 125 °C, 6 could be obtained with a yield of 44% after purification (Scheme 1, NM

Synthesis of 5 and 6
The synthesis of the new derivatives 5 and 6 was performed as depicted in Scheme 1. The required precursor 4 was synthesized following procedures published in the literature (see Scheme S1 in Supplementary Materials) [32][33][34][35][36].
Yan et al. [37] described the synthesis of 7 (for the structure, see Table 2), where they used aniline as amine in a dimethylformamide (DMF)/pyridine mixture. In adaption to the published conditions, DMF was chosen with an excess amount of 2-fluoropyridin-4-amine under thermal heating (125 • C). Because of the low reactivity of the employed aminopyridine, sodium hydride was used as a strong base. After an acidic workup and chromatographic purification, compound 5 was obtained with a yield of 51% (Scheme 1, NMR and MS spectra in Figures S1-S4 in Supplementary Materials).

Synthesis of 5 and 6
The synthesis of the new derivatives 5 and 6 was performed as depicted in Scheme 1. The required precursor 4 was synthesized following procedures published in the literature (see Scheme S1 in Supplementary Materials) [32][33][34][35][36].
Yan et al. [37] described the synthesis of 7 (for the structure, see Table 2), where they used aniline as amine in a dimethylformamide (DMF)/pyridine mixture. In adaption to the published conditions, DMF was chosen with an excess amount of 2-fluoropyridin-4amine under thermal heating (125 °C). Because of the low reactivity of the employed aminopyridine, sodium hydride was used as a strong base. After an acidic workup and chromatographic purification, compound 5 was obtained with a yield of 51% (Scheme 1, NMR and MS spectra in Figures S1-S4 in Supplementary Materials). The synthesis of 6 was performed according to Yan et al. [37] and Borrmann et al. [13] in DMF with an excess of 4-fluoropiperidine as amine, which was synthesized via a twostep synthetic route [38]. This amine could be directly applied in the form of its trifluoroacetic acid (TFA) salt because the addition of an excess of triethylamine (TEA) to the reaction mixture resulted in the corresponding free base being formed in situ. At a temperature of 125 °C, 6 could be obtained with a yield of 44% after purification (Scheme 1, NMR and MS spectra in Figures S5-S8 in Supplementary Materials). The synthesis of 6 was performed according to Yan et al. [37] and Borrmann et al. [13] in DMF with an excess of 4-fluoropiperidine as amine, which was synthesized via a two-step synthetic route [38]. This amine could be directly applied in the form of its trifluoroacetic acid (TFA) salt because the addition of an excess of triethylamine (TEA) to the reaction mixture resulted in the corresponding free base being formed in situ. At a temperature of

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A 2B , A 2A , A 1 , and A 3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype (Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A 1 and A 3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A 2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7.

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A2B, A2A, A1, and A3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype (Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A1 and A3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7.

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A2B, A2A, A1, and A3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype (Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A1 and A3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7.

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A2B, A2A, A1, and A3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype ( Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A1 and A3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7. Table 2. Determined Ki values of the novel fluorinated compounds 5 and 6 and the published Ki values of PSB-603, 7, and 8 towards the four adenosine receptor subtypes, along with the corresponding calculated selectivity ratios.

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-9168.11/18/8; approval date: 12 June 2013). Animal experiments were performed with female CD-1 mice, 10-12 weeks old, obtained from the Experimental Centre of the Faculty of Medicine (MEZ) at the Leipzig University, Leipzig, Germany.
[ 3 H]PSB-603 (specific activity 2.92 TBq/mmol, Pharmaron formerly Quotient Bioresearch (Radiochemicals) Ltd, Cardiff, UK; the precursor was synthesized at the University of Bonn, Germany [13]) was dissolved in 100 µL of sterile isotonic saline and administered as a bolus injection via the tail vein of the restrained animal. At

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A2B, A2A, A1, and A3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype ( Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A1 and A3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7. Table 2. Determined Ki values of the novel fluorinated compounds 5 and 6 and the published Ki values of PSB-603, 7, and 8 towards the four adenosine receptor subtypes, along with the corresponding calculated selectivity ratios.

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A2B, A2A, A1, and A3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype ( Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A1 and A3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7.

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-

Affinity towards Adenosine Receptor Subtypes
For the two novel fluorinated xanthine derivatives, the binding affinity towards the adenosine receptor subtypes A2B, A2A, A1, and A3 was determined in competition binding assays by incubation of the appropriate standard radioligand with membrane preparations of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, respectively, expressing the corresponding human receptor subtype ( Table 2).
The selectivity of 5 and 6 towards the two adenosine receptor subtypes A1 and A3 were increased compared to those of the nonfluorinated derivatives 7 and 8. Regarding the adenosine A2A receptor, the introduction of an electron-withdrawing functionality at the piperidine ring, such as the fluorine atom in 6, showed a negative effect on the selectivity, while the introduction of electron-donating nitrogen in 5 showed an improvement of the selectivity compared to aryl compound 7.

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-

Organ Distribution Studies of [ 3 H]PSB-603 in Mice
All animal studies followed the international guidelines on animal care, and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (Reg.-Nr.: TVV 08/13; Reference number: 24-9168.11/18/8; approval date: 12 June 2013). Animal experiments were performed with female CD-1 mice, 10-12 weeks old, obtained from the Experimental Centre of the Faculty of Medicine (MEZ) at the Leipzig University, Leipzig, Germany.  [13]) was dissolved in 100 µL of sterile isotonic saline and administered as a bolus injection via the tail vein of the restrained animal. At 3 min (n = 3; dose: 0.18 ± 0.02 µM), retro-orbital blood samples were obtained from the anesthetized animals. Immediately afterwards, organs of interest were isolated and blood plasma was obtained by centrifugation of the whole blood sample (15,000 rpm, room temperature, 1 min). All samples were weighed and dissolved in SOLVABLE (PerkinElmer Life and Analytical Sciences, Rodgau, Germany) for 5 h at 50 • C, overnight at 30 • C, and a further 5 h at 50 • C. By adding H 2 O 2 , decolorization of the samples was achieved. The samples were left to cool down to room temperature before the addition of 15 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Rodgau, Germany). The samples were shaken overnight, and the radioactivity was measured in a Beckman Liquid Scintillation Counter LS 6500 (Beckman Coulter, Fullerton, CA, USA). For each sample, the percentage of the injected dose was calculated and normalized according to the tissue weight (% ID/g tissue), followed by normalization according to the injected dose and the weight of the animal [(% ID/g tissue)/(100%/g animal)] to calculate the standardized uptake value (SUV) for each tissue.

General Methods and Materials
Analysis of all compounds was performed by MS, HPLC, TLC, and NMR spectroscopy. The spectra ( 1 H, 13 C, 19 F-NMR, and MS) for the final compounds are given in the Supplementary Materials.
Analytical thin-layer chromatography was performed on silica gel-coated plates (Macherey-Nagel, ALUGRAM SIL G/UV254). The spots were identified using a UV lamp (254 nm) or by spraying a solution of 0.1% ninhydrin in ethanol/water 1/10 or dipping into a KMnO 4 solution (3 g KMnO 4 , 20 g K 2 CO 3 , 0.25 mL glacial acid, 300 mL water). The retention factor (R f ) is the ratio of the distance of the compound to the starting point and the total distance of the solvent front from the starting point. For purification of the final products, flash column chromatography was used with silica gel ZEOsorb 60/40-63 µm from Apollo Scientific Ltd. and silica gel 40-63 µm from VWR Chemicals. The chemical purity of the final compounds was ≥96% and was controlled by HPLC using a 150 × 3 mm Reprosil-Pur Basic HD 3 µm column (Dr. Maisch GmbH, Germany). These analytical chromatographic separations were performed on a Dionex Ultimate 3000 system incorporating an LPG-3400SD pump, an autosampler WPS-3000 TSL, a column compartment TCC-3000SD, a diode array detector DAD3000 (monitoring from 254 to 720 nm), and a low-resolution mass spectrometer MSQ 3000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). A mixture of acetonitrile (ACN) and aqueous 20 mM NH 4 OAc was used as eluent in a linear gradient system (0-5 min at 25% ACN, 5-45 min up to 95% ACN, 45-55 min at 95% ACN, 55-57 min up to 25% ACN, and 57-65 min at 25%) with a flow of 0.6 mL/min.
All clogP values and chemical names of compounds were generated by ChemDraw Professional 19.0.1.28.