Preparation of a First 18F-Labeled Agonist for M1 Muscarinic Acetylcholine Receptors

M1 muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve terminals of all forebrain regions and have been implicated in the cognitive decline associated with Alzheimer’s disease and other CNS pathologies. Consequently, major efforts have been spent in the development of subtype-selective positron emission tomography (PET) tracers for mAChRs resulting in the development of several 11C-labeled probes. However, protocols for the preparation of 18F-labeled mAChR-ligands have not been published so far. Here, we describe a straightforward procedure for the preparation of an 18F-labeled M1 mAChR agonist and its corresponding pinacol boronate radiolabeling precursor and the non-radioactive reference compound. The target compounds were prepared from commercially available aryl fluorides and Boc protected 4-aminopiperidine using a convergent reaction protocol. The radiolabeling precursor was prepared by a modification of the Miyaura reaction and labeled via the alcohol-enhanced Cu-mediated radiofluorination. The developed procedure afforded the radiotracer in a non-decay-corrected radiochemical yield of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) on a preparative scale. Taken together, we developed a straightforward protocol for the preparation of an 18F-labeled M1 mAChR agonist that is amenable for automation and thus provides an important step towards the routine production of a 18F-labeled M1 selective PET tracer for experimental and diagnostic applications.


Preparation of the Reference Compound 1 and Pinacol Boronate Precursor 8 for Radiolabeling
Initially the modified procedure of Budzik et al. [9] was applied in order to prepare the non-radioactive ligand 1 and the intermediate 8 (Scheme 1). This synthetic route consists of nitration of the respective 2-fluoro-5-halotoluene followed by the selective nucleophilic substitution of the fluorine substituent in ortho-position to the nitro group with 4-N-Boc-aminopiperidine, reduction of the nitro group, cyclization to the benzimidazolone, N-Boc deprotection and, finally, reductive alkylation of the resulting intermediate with 4-tetrahydropyranone. The nitration step was performed as described by Kher et al. [10], except that the reaction time was increased from 30 min to 16 h, affording known 4a and 4b in yields of 64% and 72%, respectively. The subsequent steps furnished the corresponding o-phenylendiamines, 6a and 6b, in 70% and 29% yield, respectively. Cyclization of 6b using triphosgene instead of the originally proposed 1,1 -carbonyldiimidazole, afforded benzimidazolone 7 in almost quantitative yield. Unexpectedly, deprotection of this intermediate by HCl in EtOAc or trifluoroacetic acid (neat or in CH 2 Cl 2 ) gave rise to a number of by-products. Reductive alkylation of the respective crude amine afforded 8 in only 6% yield. In order to circumvent the problems mentioned above, 1-(tetrahydro-2H-pyran-4-yl)piperidine-4-amine (10) was prepared by reductive alkylation of 4-N-Boc-aminopiperidine (9) with tetrahydro-4H-pyran-4-one using NaBH 3 CN as reductant and Bu 4 NBr as phase transfer catalyst followed by N-Boc deprotection and was allowed to react with 4a or 4b (Scheme 2) affording the corresponding o-nitroanilines 11a and 11b in good yields. Reduction of the nitro group in 11a using Raney nickel and hydrazine hydrate proceeded smoothly, furnishing the corresponding o-phenylenediamine intermediate in 63% yield. In contrast, application of the same procedure to the bromo-substituted nitroaniline 11b afforded only traces of the product. Among several reductants tested, powdered Zn/NH 4 Cl in a mixture of EtOH and EtOAc according to the modified procedure of Tsukinoki and Tsuzuki [11] enabled the preparation of the desired intermediate in an excellent (>90%) yield. Subsequent cyclization of the prepared o-phenylenediamines with triphosgene afforded ligand 1 and substituted imidazolone 8 in 14% and 33% yield over four steps, respectively. intermediate by HCl in EtOAc or trifluoroacetic acid (neat or in CH2Cl2) gave rise to a number of byproducts. Reductive alkylation of the respective crude amine afforded 8 in only 6% yield.
In order to circumvent the problems mentioned above, 1-(tetrahydro-2H-pyran-4-yl)piperidine-4-amine (10) was prepared by reductive alkylation of 4-N-Boc-aminopiperidine (9) with tetrahydro-4H-pyran-4-one using NaBH3CN as reductant and Bu4NBr as phase transfer catalyst followed by N-Boc deprotection and was allowed to react with 4a or 4b (Scheme 2) affording the corresponding onitroanilines 11a and 11b in good yields. Reduction of the nitro group in 11a using Raney nickel and hydrazine hydrate proceeded smoothly, furnishing the corresponding o-phenylenediamine intermediate in 63% yield. In contrast, application of the same procedure to the bromo-substituted nitroaniline 11b afforded only traces of the product. Among several reductants tested, powdered Zn/NH4Cl in a mixture of EtOH and EtOAc according to the modified procedure of Tsukinoki and Tsuzuki [11] enabled the preparation of the desired intermediate in an excellent (>90%) yield. Subsequent cyclization of the prepared o-phenylenediamines with triphosgene afforded ligand 1 and substituted imidazolone 8 in 14% and 33% yield over four steps, respectively. The boronic acid pinacol ester 2 was prepared in 48% yield by the Miyaura reaction, using a modification of the protocol described by Ishiyama et al. [12] (Scheme 3). The Pd content determined by ICP/MS amounted to 1.2 ± 0.1 ppm.

Preparation of [ 18 F]1
Radiolabeling of the Bpin ester 2 was performed according to the modified protocol for Cumediated alcohol-enhanced radiofluorination [13][14][15][16][17] (Scheme 4). [ 18 F]Fluoride was loaded onto an anion exchange resin and eluted with a solution of Et4NHCO3 in MeOH. After evaporation of MeOH, [ 18 F]Et4NF/Et4NHCO3 was taken up in a solution of 2 and Cu(py)4(OTf)2 in 2:1 DMA/nBuOH and the reaction mixture was heated at 110 °C for 10 min under Ar or air to afford the 18 F-labeled ligand [ 18 F]1 in excellent (>90%) radiochemical conversions (RCCs). The crude tracer obtained after concentration of the reaction mixture under reduced pressure was purified by HPLC and formulated as a ready-touse solution. On a preparative scale [ 18 F]1 was produced in a non-decay corrected radiochemical yield (n.d.c RCY) of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) within 90-100 min ( Figure  1). Besides the molar activity, which is dependent on the activity amount, the carrier amount per The boronic acid pinacol ester 2 was prepared in 48% yield by the Miyaura reaction, using a modification of the protocol described by Ishiyama et al. [12] (Scheme 3). The Pd content determined by ICP/MS amounted to 1.2 ± 0.1 ppm.
Molecules 2020, 25, x 3 of 12 intermediate by HCl in EtOAc or trifluoroacetic acid (neat or in CH2Cl2) gave rise to a number of byproducts. Reductive alkylation of the respective crude amine afforded 8 in only 6% yield. In order to circumvent the problems mentioned above, 1-(tetrahydro-2H-pyran-4-yl)piperidine-4-amine (10) was prepared by reductive alkylation of 4-N-Boc-aminopiperidine (9) with tetrahydro-4H-pyran-4-one using NaBH3CN as reductant and Bu4NBr as phase transfer catalyst followed by N-Boc deprotection and was allowed to react with 4a or 4b (Scheme 2) affording the corresponding onitroanilines 11a and 11b in good yields. Reduction of the nitro group in 11a using Raney nickel and hydrazine hydrate proceeded smoothly, furnishing the corresponding o-phenylenediamine intermediate in 63% yield. In contrast, application of the same procedure to the bromo-substituted nitroaniline 11b afforded only traces of the product. Among several reductants tested, powdered Zn/NH4Cl in a mixture of EtOH and EtOAc according to the modified procedure of Tsukinoki and Tsuzuki [11] enabled the preparation of the desired intermediate in an excellent (>90%) yield. Subsequent cyclization of the prepared o-phenylenediamines with triphosgene afforded ligand 1 and substituted imidazolone 8 in 14% and 33% yield over four steps, respectively. The boronic acid pinacol ester 2 was prepared in 48% yield by the Miyaura reaction, using a modification of the protocol described by Ishiyama et al. [12] (Scheme 3). The Pd content determined by ICP/MS amounted to 1.2 ± 0.1 ppm.

Preparation of [ 18 F]1
Radiolabeling of the Bpin ester 2 was performed according to the modified protocol for Cumediated alcohol-enhanced radiofluorination [13][14][15][16][17] (Scheme 4). [ 18 F]Fluoride was loaded onto an anion exchange resin and eluted with a solution of Et4NHCO3 in MeOH. After evaporation of MeOH, [ 18 F]Et4NF/Et4NHCO3 was taken up in a solution of 2 and Cu(py)4(OTf)2 in 2:1 DMA/nBuOH and the reaction mixture was heated at 110 °C for 10 min under Ar or air to afford the 18 F-labeled ligand [ 18 F]1 in excellent (>90%) radiochemical conversions (RCCs). The crude tracer obtained after concentration of the reaction mixture under reduced pressure was purified by HPLC and formulated as a ready-touse solution. On a preparative scale [ 18 F]1 was produced in a non-decay corrected radiochemical yield (n.d.c RCY) of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) within 90-100 min ( Figure  1). Besides the molar activity, which is dependent on the activity amount, the carrier amount per

Preparation of [ 18 F]1
Radiolabeling of the Bpin ester 2 was performed according to the modified protocol for Cu-mediated alcohol-enhanced radiofluorination [13][14][15][16][17] 4 (OTf) 2 in 2:1 DMA/nBuOH and the reaction mixture was heated at 110 • C for 10 min under Ar or air to afford the 18 F-labeled ligand [ 18 F]1 in excellent (>90%) radiochemical conversions (RCCs). The crude tracer obtained after concentration of the reaction mixture under reduced pressure was purified by HPLC and formulated as a ready-to-use solution. On a preparative scale [ 18 F]1 was produced in a non-decay corrected radiochemical yield (n.d.c RCY) of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) within 90-100 min (Figure 1). Besides the molar activity, which is dependent on the activity amount, the carrier amount per batch was measured (please refer to [18] for further discussion). The latter amounted . The Cu content, measured by ICP/MS, amounted to 3.4 ± 0.1 µg/batch and was below any level of concern according to the ICH Guideline of Elemental Impurities (Q3D) [19].
Molecules 2020, 25, x 4 of 12 batch was measured (please refer to [18] for further discussion). The latter amounted to 25.2 nmol/batch and the molar activity to 30.8 GBq/µmol (measured for 770 MBq [ 18 F]1; refer to the Supplementary Materials for more details). The Cu content, measured by ICP/MS, amounted to 3.4 ± 0.1 µg/batch and was below any level of concern according to the ICH Guideline of Elemental Impurities (Q3D) [19].    1 µg/batch and was below any level of concern according to the ICH Guideline of Elemental Impurities (Q3D) [19].

Nuclear Magnetic Resonance Spectroscopy (NMR)
Unless otherwise stated, all NMR-Spectra were measured in CDCl 3 . 1 H-NMR spectra were obtained with a Bruker DPX Avance 300 (Bruker, Rheinstetten, Germany). 1 H chemical shifts are reported in ppm relative to residual peaks of deuterated solvents. The observed signal multiplicities are characterized as follows: s = singlet, d = doublet, t = triplet, m = multiplet and q = quartet. Coupling constants are reported in Hertz (Hz). 13 C-NMR spectra [additional APT (Attached Proton Test)]: Bruker DPX Avance 300 (75 MHz). 13 C chemical shifts are reported in ppm relative to residual peaks of deuterated solvents. 1 H-, 13 C-and 19 F-NMR spectra are provided in the Supplementary Materials.

Chemistry
All reactions were carried out with magnetic stirring. Air or moisture sensitive reagents were handled under argon using either a glovebox or a Schlenk line. Organic extracts were dried over anhydrous MgSO 4 . Solutions were concentrated under reduced pressure at 40-50 • C using a rotary evaporator (Bruker, Rheinstetten, Germany).

Deprotection of 7
Procedure A An excess of 4 m HCl in EtOAc was added to a solution of 7 (0.27 g, 0.66 mmol) in CH 2 Cl 2 (2 mL) and the resulting mixture was stirred at ambient temperature for 1 h. After removal of volatiles under reduced pressure and addition of 0.1 m NaOH (50 mL), the resulting mixture was extracted with CH 2 Cl 2 (3 × 30 mL). The combined organic fraction was dried and concentrated under reduced pressure to afford the crude amine hydrochloride, which was used for the next step without any purification and characterization. Yield: 0.18 g, 0.6 mmol (90% crude). Appearance: white solid.
Procedure B TFA (30 mL) was slowly added to a solution of 7 (1.55 g, 3.78 mmol) in CH 2 Cl 2 (30 mL) and the reaction mixture was stirred at ambient temperature for 1 h. After concentration under reduced pressure and addition of saturated NaHCO 3 (30 mL) the resulting mixture was extracted with CH 2 Cl 2 (3 × 30 mL). The combined organic fraction was dried and concentrated under reduced pressure to afford the crude amine trifluoroacetate, which was used for the next step without any purification and characterization. Yield: 1.44 g, ≤3.78 mmol (100% crude). Appearance: white solid.

Conclusions
We developed a straightforward and efficient protocol for the manual preparation of [ 18 F]1, a potentially M 1 selective PET-probe for in vivo studies. The protocol is amenable to automation owing to its simplicity, and thus provides an important step towards the routine production of 18 F-labeled M 1 selective PET tracers for experimental and diagnostic applications.

Conflicts of Interest:
The authors declare no conflict of interest.