Synthesis and In Vitro Evaluation of E- and Z-Geometrical Isomers of PSS232 as Potential Metabotropic Glutamate Receptors Subtype 5 (mGlu₅) Binders

Dedicated to Prof. Scott E. Denmark on the occasion of his 60^th birthday

Abstract

Based on the core structure of [¹¹C]-ABP688, our group developed a novel fluorine-18 labeled PET radiotracer, (E)-[¹⁸F]-PSS232, with significantly improved in vivo properties compared to the earlier fluorine-18 derivative [¹⁸F]-FDEGPECO. The synthetic approach used to obtain PSS232 and the radiolabeling precursor mesylate is disclosed as well as the evaluation of the two geometrical isomers of PSS232. In vitro displacement assays showed higher binding affinity of the E-geometrical isomer (1 nM vs 15 nM, for Z-isomer), which was, for this reason, selected for radiolabeling. One-step radiolabeling conditions (K₂₂₂/¹⁸F^–, DMSO, 95 °C, 10 min) to synthesize (E)-[¹⁸F]-PSS232 from the mesylate via nucleophilic substitution are described. At ambient temperature, (E)-[¹⁸F]-PSS232, with log D _7.4 value of 2.0, was chemically stable over six hours in the presence of sodium ascorbate as a radical scavenger.

To date, the most widely clinically applied positron emission tomography (PET)[1] radiotracer for imaging metabotropic glutamate receptor subtype 5 (mGlu₅) is (E)-3-[(6-methylpyridin-2-yl)ethynyl]cyclohex-2-enone O-[¹¹C]methyl oxime ([¹¹C]-ABP688, [¹¹C]-1, Figure [1]).[2] [3] [4] [5] [6] [7] Despite its excellent in vitro and in vivo properties,[8–10] the clinical application of [¹¹C]-1 is limited to centers with on-site cyclotron facilities due to the short physical half-life of carbon-11 (20 min). With respect to physical half-life, fluorine-18 (110 min) is a preferred radionuclide and efforts have been made to develop the fluorine-containing analogue of [¹¹C]-1. Among others,[11] [12] [13] [14] [15] our group reported on one such analogue, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-[2-(2-[¹⁸F]fluoroethoxy)ethyl] oxime ([¹⁸F]-FDEGPECO, (E)-[¹⁸F]-2),[16] [17] which demonstrated desirable in vitro properties (e.g., binding affinity, specificity, and selectivity for mGlu₅), but a poor signal-to-noise ratio in in vivo PET studies. Due to its in vitro profile, (E)-[¹⁸F]-2 presented a strong basis for further optimization and we rationalized that increased lipophilicity as reflected through log D _7.4 values (1.7 ± 0.1 vs 1.9 ± 0.1 for (E)-[¹⁸F]-2 and (E)-[¹⁸F]-3, respectively)[16] [18] would benefit permeation through the blood-brain barrier (BBB) and potentially lead to an improved signal-to-noise ratio. The (E)-[¹⁸F]-2 analogue, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-[2-(3-[¹⁸F]fluoropropoxy)ethyl] oxime [(E)-[¹⁸F]-PSS223, (E)-[¹⁸F]-3],[18] [19] was evaluated and although (E)-[¹⁸F]-3 was selective and specific for mGlu₅, it underwent rapid defluorination in vivo. In comparison to (E)-[¹⁸F]-2, (E)-[¹⁸F]-3 has a PEGylated side-chain extended by one methylene unit, which presumably was prone to enzymatic oxygenation by cytochrome P450 and subsequent defluorination.[20] In order to prevent the observed­ defluorination, an alternative derivative 3-(pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-[¹⁸F]fluo­roethoxy)propyl] oxime ([¹⁸F]-PSS232, [¹⁸F]-4, Figure [1]) was hypothesized, such that a methylene unit was added next to oxime functionality; in essence reducing the number of methylene units at the end of PEGylated side-chain by one, to maintain the length of the side-chain, which resulted in (E)-[¹⁸F]-4 exhibiting excellent in vivo properties and subsequently being selected as a PET radiotracer for further evaluation in human subjects.[21] In another recent report, we explored a series of α-fluoro derivatives of ABP688 and found a significant difference in the binding affinity of E- and Z-isomers,[22] which prompted us to evaluate this effect between E- and Z-isomers of 4. Herein, we report on the syntheses of these two geometrical isomers (E)-4 and (Z)-4, their in vitro binding affinity determination, and the optimization of radiolabeling method to obtain (E)-[¹⁸F]-4 as well as the in vitro radiochemical stability of our new mGlu₅ PET radiotracer.

The synthesis of geometrical isomers of 4 was envisioned via the hydroxy oximes (E)-10 and (Z)-10 (Scheme [1]), which were prepared using the Sonogashira cross-coupling reaction conditions as previously reported.[16] [19] Oxime (E)-10 was reacted with the bromoethers 8a/8b to afford substituted oxime (E)-11 after cleavage of protecting groups (TBS and TES) with tetrabutylammonium fluoride­. Similar to the synthesis of 3, which we have described previously,[18] alcohol (E)-11 was treated with mesyl chloride to give the mesylate (E)-12 (Scheme [1]; for structure see Table [2]), a common precursor for the synthesis of reference compounds (E)-4 and (E)-[¹⁸F]-4. The synthesis of fluoride (E)-4 was accomplished in 62% yield in the substitution reaction with KF/K₂₂₂. The synthesis of (Z)-4 was accomplished in an analogous manner starting from (Z)-10 and bromoether 8. After the removal of silyl protecting groups, alcohol (Z)-11, was obtained as a 1:9 (NMR ratio) E/Z mixture and mesylated to give (Z)-12 as a 1:7 E/Z mixture. Finally, compound (Z)-4 was obtained as a mixture with an E/Z ratio of 1:5.

Structural assignment of the oxime ether double bond was done by analogy with other oxime ether derivatives (Figure [2]).[22] In particular, strong anisotropic deshielding (described in the comprehensive study by Denmark and co-workers for the series of α-chloroketoximes)[23] by the oxime oxygen on the equatorial protons results in a downfield shift of H_a (these protons are closer through space to the oxime oxygen) and upfield shift of H_cy (these protons are closer through space to the lone pairs on the oxime nitrogen) in the E-isomer and vice versa in the Z-isomer. Further conformation was compounded by invoking the γ-effect[24] [25] and a 7.8 ppm upfield chemical shift was determined in the ¹³C NMR for the sterically compressed carbon atom (C_allylic) in (Z)-4. This analysis was in complete agreement with our observation made previously for α-fluorinated ABP688 analogues.[22] One should note, however, the inversed nomenclature of geometrical isomers in the two series of analogues (Z/E α-fluorinated analogues vs E/Z-4).

For the synthesis of bromoether 8, which was employed in the syntheses of both isomers, an approach similar to that employed for the synthesis of 3 was used.[18] [19] Ethylene glycol was monoprotected with TBS and reacted with an acid chloride to yield ester 7, which was further reduced to bromoether 8 using Et₃SiH and InBr₃ [26] in moderate yield (54%). In agreement with our earlier observation, 8 was isolated as a mixture of TBS and TES silyl enol ethers. The ratio of TBS/TES silyl enol ethers varied, but this did not affect the subsequent coupling.

Reaction of the acid chloride with alcohol 6 required optimization. Under the phase-transfer reaction conditions (Table [1], entries 1 and 2) only traces of the product were formed. Similarly, the reaction of ethylene glycol and bromopropionic acid using a Dean–Stark apparatus gave only trace amounts of the product. In a further attempt (entry 4) nucleophilic substitution was done using Et₃N as a base and a catalytic amount of DMAP. This reaction yielded only 6% of ester 7. Employing Hünig’s base (entry 5) instead of Et₃N afforded 11% of 7. Ester 7 was produced in 22% when bromopropionic acid was used under Mitsunobu conditions (entry 6); however, the reaction was not reproducible. The use of NaH as a base afforded 7 in 32% and 50% yield (entries 7 and 8).

Table 1 Optimization of Reaction Conditions for the Preparation of Ester 7

Entry	R	6 (g)	Base	Additive/Activator	Solvent	Time (h)	Yield (%)a
1	Cl	0.1	NaOH	TBAF·THF	CH2Cl2	1	nd
2	Cl	0.1	NaOH	TBAC	CH2Cl2	2	nd
3	OH	1.5	–	TsOH	toluene	21	nd
4	Cl	0.4	Et3N	DMAP	CH2Cl2	15	6
5	Cl	3.4	i-Pr2NEt	DMAP	CH2Cl2	17	11
6	OH	0.3	–	DIAD/PPh3	THF	70	22
7	Cl	0.1	NaH	–	THF	2	32
8	Cl	3.3	NaH	–	THF	16	50

^a nd: Not determined.

Interestingly, the reaction of alcohol (E)-13, obtained from the reaction of oxime (E)-10 with bromopropanol in 73% yield, with bromofluoroethane failed to afford the desired (E)-4 directly (Scheme [1]) or in reaction with bromoacetyl bromide.

While (E)-4 was completely stable upon storage at –20 °C a neat (Z)-4 mixture with E-isomer (E/Z ratio of 1:5) underwent isomerization. The E/Z ratio changed to 2:1 under the same conditions, over a period of two months and an additional unidentified by-product was observed. Preparative HPLC was performed to isolate (Z)-4 in E/Z NMR ratio of 1:11 and this material was used further for IC₅₀ determination. The observed isomerization suggests that the E-isomer is likely the thermodynamically more stable isomer. Next an experiment was performed in which CDCl₃ solutions at 4 mg/mL of (E)-4 and (Z)-4 were monitored by ¹H NMR over 25 days and observed no change in the former case; however, in the latter the E/Z NMR ratio changed from 1:5 to 1:3. The two solutions were next heated to 60 °C over five hours; once again isomerization was observed for the Z-isomer and the E/Z ratio decreased to 1:2. In this case, no other by-products were detected.

The binding affinity toward mGlu₅ of the two analogues was determined in a displacement assay with [³H]-1 using rat brain homogenates in triplicate.[16] [18] For the determination of nonspecific binding, 2-[(3-methoxyphenyl)ethynyl]-6-methylpyridine (MMPEP) was applied.

The IC₅₀ values estimated from the plotted curves were 1.3 ± 0.1 nM and 15.3 ± 1.4 nM for (E)-4 and (Z)-4, respectively (Figure [3]). While the difference in binding affinity of E- and Z-isomers, in the case of α-fluorinated analogues was 5.5-fold,[22] a 11.8-fold higher binding affinity was determined for (E)-4 when compared to the Z-isomer. This suggests that the configuration of the oxime ether double bond bearing the long lipophilic side-chain plays a significant role in binding to the target mGlu₅. The Ametamey group reported a similar effect for the E- and Z-isomers when the substituent on the oxime ether was 2-fluoropyridine[16] with a 4.8-fold higher binding affinity for the E-isomer. In the same report, only a slight preference for Z-isomer was observed (1.1-fold) when 3-fluoro-4-cyanobenzene constituted the oxime ether.

Due to its favorable IC₅₀ value (comparable to that of ABP688[8] [16]), our attention was focused on the further development of (E)-4. Conditions to establish a successful radiolabeling protocol were next sought involving the nucleophilic substitution with ¹⁸F^–. Mesylate (E)-12, which was stable for months both in solution and neat, was employed as a precursor for this purpose. Radiosynthesis was initially accomplished at 90 °C over 10 minutes in DMF to give (E)-[¹⁸F]-4. The decay corrected radiochemical yields of 13–37% (n = 40) were not significantly improved when the labeling was performed at 95 °C. In a typical experiment 3–12 GBq of the formed product was obtained starting from 30–70 GBq of ¹⁸F-fluoride. Specific activity was 80–400 GBq/μmol at the end of the synthesis. Total synthesis time was 70 minutes from the end of bombardment and the E/Z HPLC ratio was 99:1. Subsequently, it was shown that the quality of images obtained with (E)-[¹⁸F]-4 was not reduced due to 1% of Z-isomer in the product mixture.[21]

For the purposes of translating the manually performed radiosynthesis to a module-based radiolabeling procedure, several different reaction conditions were evaluated (Table [2]), considering solvent, reaction time, and temperature. Generally, it was established that higher reaction temperature resulted in higher conversions to radiolabeled product (Table [2], entries 3 and 5 vs 4 and 6) and reaction times had very little effect (entries 1–6; 20 vs 15 vs 10 min). The highest conversion was accomplished with DMSO as the solvent (entries 3 and 4), which was selected for further application in translating manual synthesis to the module based approach.

Table 2 Optimization of the Reaction Conditions for the Radiolabeling of Mesylate (E)-12 To Obtain (E)-[¹⁸F]-4 ^a,b

Entry	(E)-12 (mg)	Solvent	Temp (°C)	RCC at 5 min (%)	RCC at 10 min (%)	RCC at 15 min (%)	RCC at 20 min (%)
1	2.0	MeCN	95	17	34	46	48
2	2.0	MeCN	80	42	–	51	66
3	1.8	DMSO	95	44	54	58	55
4	1.8	DMSO	120	76	76	71	73
5	1.8	DMF	95	31	45	51	49
6	1.8	DMF	110	51	60	60	62

^a Manual approach.

^b RCC: Radiochemical conversion.

Compound (E)-[¹⁸F]-4 was found to be prone to radiolysis when produced in amounts larger than 5 GBq for which reason, sodium ascorbate (25 mg/mL) was employed as a radical scavenger in the final formulation solution. After six hours, in the radiotracer solution, more than 95% of parent compound as an E/Z mixture was intact. Also, the activity concentration of investigated samples was of importance, and radiolysis was typically observed when concentration exceeded 500 MBq/mL.

As a measure of lipophilicity log D _7.4 was determined to give a value of 2.0 ± 0.1 for (E)-[¹⁸F]-4 using the shake-flask method, which was comparable to (E)-[¹⁸F]-3 and slightly higher than that measured for (E)-[¹⁸F]-2. The measured log D _7.4 value was within the range established for successful passage through the BBB.[16]

In conclusion, we have prepared two geometrical isomers (E and Z) of novel mGlu₅ PET radiotracer 4, and measured 11.8-fold difference in the binding affinity toward mGlu₅ in favor of the E-isomer. Not only did (Z)-4 have lower binding affinity for mGlu₅, but also underwent isomerization upon storage. The syntheses of the two isomers were accomplished via substitution reactions of bromoether 8 with oximes (E)-10 and (Z)-10, respectively, in six synthetic steps, and 7% and 5% overall yields. Mesylate (E)-12 was used as a precursor for radiolabeling to obtain (E)-[¹⁸F]-4. Compound (E)-[¹⁸F]-4 exhibited desirable log D _7.4 value. We have established practical synthetic approach to prepare radiolabeling precursor mesylate (E)-12 and optimized the radiolabeling protocol to accommodate translation to the module-based synthesis for the purposes of preparing (E)-[¹⁸F]-4 under GMP conditions with the ultimate aim of applying (E)-[¹⁸F]-4 in human clinical practice. The results of the in vivo studies (in rodents and humans) will be reported in due course.

All reactions requiring anhydrous conditions were conducted in flame-dried glass apparatus under an atmosphere of inert gas. All chemicals and anhydrous solvents were purchased from Aldrich or ABCR and used as received unless otherwise noted. Reported density values are for ambient temperature. [³H]-ABP688 (2.405 GBq/μmol, 37 MBq/mL solution in EtOH) was obtained from AstraZeneca­. Oximes (E)-10 and (Z)-10 were prepared as described previously.[16] [19] MMPEP was prepared based on the published procedure.[27]

Preparative chromatographic separations were performed on Aldrich Science silica gel 60 (35–75 μm) and reactions were followed by TLC analysis using Sigma-Aldrich silica gel 60 plates (2–25 μm) with fluorescent indicator (254 nm) and visualized with UV or KMnO₄.

IR spectra were recorded on a JASCO FT/IR 6200 (OmniLab) spectrometer using a CHCl₃ solution of the compound. ¹H and ¹³C NMR spectra were recorded in Fourier transform mode at the field strength specified on Bruker Avance FT-NMR spectrometers.

NMR spectra were obtained from the specified deuterated solvents in 5 mm diameter tubes. Chemical shift in ppm is quoted relative to residual solvent signals calibrated as follows: CDCl₃ δ_H (CHCl₃) = 7.26, δ_C = 77.2; DMSO-d ₆ δ_H (CD₃SOCHD₂) = 2.50, δ_C = 39.5. Standard abbreviations were used for denoting the multiplicities in the ¹H NMR spectra ; coupling constants are reported in Hz. Numbers in parentheses following carbon atom chemical shifts refer to the number of attached hydrogen atoms as revealed by the DEPT spectral editing technique. Electrospray (ES) mass spectra (LRMS) were obtained with a Micromass Quattro micro API LC electrospray ionization and ES mass spectra (HRMS) were obtained with a Bruker FTMS 4.7 T BioAPEXII spectrometer. Electron impact (EI) and chemical ionization (CI) mass spectra (LRMS and HRMS) were obtained with a Waters Micromass AutoSpec Ultima MassLynx 4.0 spectrometer. Ion mass/charge (m/z) ratios are reported as values in atomic mass units. Semi-preparative purification of radiolabeled material was performed on a Merck-Hitachi L6200A system equipped with Knauer variable wavelength detector and an Eberline radiation detector or Smartline system with Pump 1000, Manager 5000, Knauer UV detector, and GabiStar (Raytest) radiodetector using a reverse phase column (C18 Phenomenex Gemini, 5 μm, 250 × 10 mm) and eluting with gradient: 0–5 min 5% aq MeCN, 5–15 min 5–50% aq MeCN, 15–30 min 50% aq MeCN, 30–50 min, 50–90% aq MeCN, 50–65 min, 65% MeCN at flow rate 5 mL/min. Analytical HPLC samples were analyzed by Agilent HPLC 1100 system equipped with UV multi-wavelength detector and Raytest Gabi star radiation detector using reverse phase column (ACE 111-0546, C18, 3 μm, 50 × 4.6 mm) and eluting with 45% aq MeCN at flow rate 1 mL/min.

Specific radioactivity of (E)-[¹⁸F]-4 was determined from a calibration curve prepared with decreasing concentrations of (E)-4.

2-[(tert-Butyldimethylsilyl)oxy]ethanol (6)

To a flame-dried flask charged with anhyd DMF (87 mL) was added 1,2-ethyleneglycol (5; 2.7 mL, 3.0 g, 48.3 mmol, d = 1.113) at r.t. under N₂ atmosphere. The resulting colorless solution was treated with i-Pr₂NEt (78.0 mL, 59.0 g, 459 mmol, d = 0.755) in one portion and the pale yellow biphasic mixture was vigorously stirred. This mixture was further treated with a solution of tert-butyldimethylchlorosilane (7.3 g, 48.3 mmol) in DMF (52 mL) dropwise over 68 min during which time the mixture turned cloudy and it was allowed to stir for 19 h. After this time, the mixture was partitioned between H₂O (100 mL) and Et₂O (180 mL) and the two layers were well shaken and separated. The aqueous phase was extracted with Et₂O (2 × 180 mL). The combined organic extracts were washed with 2 M aq HCl (2 × 180 mL; Caution! vigorous bubbling), sat. aq NaHCO₃ (1 × 180 mL), brine (1 × 180 mL), dried (Na₂SO₄), and concentrated in vacuo to give a pale yellow oil. The crude product was purified by chromatography on a silica gel column (eluting with 20% EtOAc–pentane) to afford 6 (2.93 g, 16.6 mmol, 34%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃): δ = 3.73–3.70 (m, 2 H), 3.66–3.62 (m, 2 H), 2.05 (t, J = 6.2 Hz, 1 H), 0.91 (s, 9 H), 0.08 (s, 6 H).

¹³C NMR (100 MHz, CDCl₃): δ = 64.3 (2), 63.9 (2), 26.1 (3, 3 C), 18.5 (0), –5.1 (3, 2 C).

MS (ES+): m/z = 177 (M + H)⁺.

HRMS (EI+): m/z calcd for C₈H₂₁O₂Si: 177.1305; found: 177.1308.

2-[(tert-Butyldimethylsilyl)oxy]ethyl 3-Bromopropanoate (7)

A stirred solution of 6 (3.27 g, 18.6 mmol) in anhyd THF (160 mL) was treated with NaH (60% suspension in oil, 1.1 g, 27.9 mmol) and the resulting heterogeneous pale yellow mixture was allowed to stir for 60 min at r.t. under N₂ atmosphere. After this time, the mixture was further treated dropwise with 3-bromopropionyl chloride (1.9 mL, 3.2 g, 18.6 mmol, d = 1.7) over 3 min and the resulting almost colorless mixture was allowed to stir for 16 h. After this time, the mixture was poured over sat. aq NH₄Cl (120 mL) and ice (150 g) in a separatory funnel and the mixture was diluted with EtOAc (280 mL) and the two layers were well shaken and separated. The aqueous phase was washed with EtOAc (2 × 280 mL). The combined organic extracts were washed with brine (200 mL), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by chromatography on a silica gel column (eluting with a gradient of 5% Et₂O–pentane to 10% Et₂O–pentane) to afford 7 (2.87 g, 9.22 mmol, 50%) as a colorless oil.

IR (neat): 2954, 2929, 2857, 1740, 1472, 1254, 1130, 1108, 834, 777 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 4.20 (ddm, J = 6.2, 4.0 Hz, 2 H), 3.82 (ddm, J = 6.1, 3.8 Hz, 2 H), 3.58 (t, J = 6.9 Hz, 2 H), 2.94 (t, J = 6.9 Hz, 2 H), 0.89 (s, 9 H), 0.07 (s, 6 H).

¹³C NMR (100 MHz, CDCl₃): δ = 66.4 (2), 61.3 (2), 37.9 (2), 26.0 (3, 3 C), 25.9 (2), 18.5 (0), –5.1 (3, 2 C).

MS (ES+): m/z = 312 (M + H)⁺.

[2-(3-Bromopropoxy)ethoxy](tert-butyl)dimethylsilane (8a)

A stirred solution of 7 (418 mg, 1.34 mmol) in CHCl₃ (1.3 mL) was treated with InBr₃ (24.0 mg, 0.07 mmol) in one portion under N₂ atmosphere. Et₃SiH (0.86 mL, 623.4 mg, 5.36 mmol, d = 0.728) was then added dropwise over 1 min and the resulting heterogeneous mixture was allowed to heat at 60 °C (oil bath temperature) for 6 h in a flask equipped with a condenser. Immediately upon heating, the mixture turned creamy and yellow. After this time, the mixture was allowed to cool to r.t. and then diluted with H₂O (5 mL) and CH₂Cl₂ (10 mL), and the two layers were well shaken and separated. The aqueous phase was further extracted with CH₂Cl₂ (2 × 10 mL). The combined organic extracts were washed with brine (1 × 15 mL), dried (Na₂SO₄), and concentrated in vacuo to give a brown oily residue (1.5 g). The crude product was purified by chromatography on a silica gel column (eluting with 5% EtOAc–pentane) to afford 8a (219 mg, 0.74 mmol, 54%) as a mixture with [2-(3-bromopropoxy)ethoxy]triethylsilane (8b) in a 2:1 ratio.

¹H NMR (400 MHz, CDCl₃): δ = 3.76 (tm, J = 5.0 Hz, 4 H, 8a and 8b), 3.60 (tm, J = 5.9 Hz, 4 H, 8a and 8b), 3.54–3.50 (m, 8 H, 8a and 8b), 2.13–2.07 (m, 4 H, 8a and 8b), 0.96 (t, J = 8.0 Hz, 9 H, 8b), 0.90 (s, 9 H, 8a) 0.61 (q, J = 7.6 Hz, 6 H, 8b), 0.07 (s, 6 H, 8a).

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(tert-Butyldimethylsilyl)oxy]ethoxy}propyl) Oxime [(E)-9a] and (E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(Triethylsilyl)oxy]ethoxy}propyl) Oxime [(E)-9b]

To a flame-dried flask charged with (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone oxime [(E)-10; 141 mg, 0.66 mmol] was added anhyd DMF (5 mL). The clear pale yellow mixture was treated with NaH (63 mg of 60% suspension in oil, 1.58 mmol) and the resulting bright yellow heterogeneous mixture was stirred for 47 min at r.t. under N₂ atmosphere. The resulting orange mixture of the deprotonated oxime was then treated with a solution of 8a/8b (2:1 ratio, respectively, 217 mg, 0.73 mmol) in DMF (5 mL) dropwise over 20 min during which time the mixture turned brown and was allowed to stir further for 2.5 h. After this time, the crude mixture was quenched with sat. aq NaHCO₃ (15 mL) and diluted with H₂O (10 mL) and Et₂O (45 mL). The two layers were well shaken and separated. The aqueous phase was extracted with Et₂O (2 × 45 mL). The combined organic extracts were washed with H₂O (3 × 35 mL), brine (1 × 40 mL), dried (Na₂SO₄), and concentrated in vacuo to give the crude mixture as a brown oily residue (699 mg). Low-resolution­ mass spectrometry confirmed the presence of the desired product. The crude mixture was used in the next step without purification.

MS (ES+): m/z = 430 (M + H)⁺.

(Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(tert-Butyldimethylsilyl)oxy]ethoxy}propyl) Oxime [(Z)-9a] and (Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-{2-[(Triethylsilyl)oxy]ethoxy}propyl) Oxime [(Z)-9b)

An analogous approach to that described for (E)-9a and (E)-9b was employed to prepare the crude mixture as a brown oily residue (327 mg). Without purification the crude mixture was used in the next step.

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Hydroxy­ethoxy)propyl] Oxime [(E)-11]

To the crude mixture of (E)-9a and (E)-9b (285 mg, 0.66 mmol) in a round-bottomed flask was added anhyd THF (13 mL) at r.t. under N₂ atmosphere. The resulting clear orange mixture was further treated with TBAF solution in THF (1.33 mL, 1.33 mmol, c = 1 M) dropwise over 4 min and the resulting brown mixture was allowed to stir at r.t. under N₂ for 61 min. After this time, the mixture was partitioned between H₂O (30 mL) and EtOAc (40 mL) and the two layers were well shaken and separated. The aqueous phase was extracted with EtOAc (2 × 40 mL, slow separation of phases). The combined organic extracts were washed with brine (1 × 40 mL), dried (Na₂SO₄), and concentrated in vacuo to give a brown oil (779 mg). The crude product was purified by chromatography on a silica gel column (eluting with gradient 90% EtOAc–pentane to 100% EtOAc) to give (E)-11 (134.7 mg, 0.43 mmol, 64%) as a pale yellow oil.

IR (neat): 3401, 2934, 2869, 2205, 1581, 1463, 1429, 1121, 1059, 866, 779 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 8.58 (ddd, J = 4.8, 1.7, 0.9 Hz, 1 H), 7.65 (td, J = 7.7, 1.8 Hz, 1 H), 7.44 (dt, J = 7.8, 1.0 Hz, 1 H), 7.22 (ddd, J = 7.6, 4.9, 1.2 Hz, 1 H), 6.57 (t, J = 1.6 Hz, 1 H), 4.21 (t, J = 6.2 Hz, 2 H), 3.72 (br t, J = 4.0 Hz, 2 H), 3.57 (t, J = 6.4 Hz, 2 H), 3.56–3.54 (m, 2 H), 2.54 (br m, J = 6.4 Hz, 2 H), 2.39 (td, J = 5.9, 1.6 Hz, 2 H), 2.26 (br s, 1 H), 1.96 (quint, J = 6.3 Hz, 2 H), 1.79 (quint, J = 6.4 Hz, 2 H).

¹³C NMR (100 MHz, CDCl₃): δ = 155.5 (0), 150.2 (1), 143.4 (0), 136.3 (1), 131.2 (1), 127.4 (1), 127.3 (0), 123.0 (1), 92.0 (0), 90.0 (0), 72.0 (2), 71.2 (0), 67.9 (2), 61.9 (2), 29.8 (2), 29.5 (2), 22.4 (2), 20.9 (2).

MS (ES+): m/z = 315 (M + H)⁺.

HRMS (ESI): m/z calcd for C₁₈H₂₃N₂O₃: 315.1703; found: 315.1707.

(Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Hydroxy­ethoxy)propyl] Oxime [(Z)-11]

An analogous approach to that described for (E)-11 was employed to prepare the title compound (180.8 mg, 0.57 mmol, 75%) as a mixture with the E-isomer in NMR E/Z ratio of 1:9. The mixture was identified by ¹H NMR spectrum (only peaks corresponding to Z-isomer are listed; several peaks are overlapped) and used in the next step.

¹H NMR (400 MHz, CDCl₃): δ = 8.61 (ddd, J = 4.9, 1.7, 0.9 Hz, 1 H), 7.67 (td, J = 7.8, 1.8 Hz, 1 H), 7.47 (dt, J = 7.8, 1.0 Hz, 1 H), 7.25 (ddd, J = 7.6, 4.9, 1.1 Hz, 1 H), 7.20 (br t, J = 1.8 Hz, 1 H), 4.19 (t, J = 6.1 Hz, 2 H), 3.74 (br t, J = 4.3 Hz, 2 H), 3.62–3.57 (m, 4 H), 2.45 (td, J = 6.0, 1.8 Hz, 2 H), 2.42–2.39 (m, 2 H), 1.96 (quint, J = 6.2 Hz, 2 H), 1.89 (quint, J = 6.1 Hz, 2 H).

(E)-2-[3-({[3-(Pyridin-2-ylethynyl)cyclohex-2-en-1-ylidene]amino}oxy)propoxy]ethyl Methanesulfonate [(E)-12]

A stirred solution of (E)-11 (134 mg, 0.43 mmol) in anhyd THF (4.3 mL) was treated with Et₃N (120 μL, 87.0 mg, 0.86 mmol, d = 0.726) in one portion and MsCl (40 μL, 58.0 mg, 0.51 mmol, d = 1.477) was then added dropwise (addition time <1 min) and the resulting pale yellow mixture was allowed to stir at r.t. under N₂ atmosphere. After 21 min, another portion of MsCl (20 μL, 29.0 mg, 0.26 mmol) was added and the mixture was stirred for an additional 15 min. After this time, the mixture was partitioned between H₂O (20 mL) and EtOAc (30 mL) and the two layers were well shaken and separated. The aqueous phase was extracted with EtOAc (2 × 35 mL). The combined organic extracts were washed with H₂O (3 × 30 mL), brine (1 × 35 mL), dried (Na₂SO₄), and concentrated in vacuo to give a pale yellow oil. The crude product was purified by chromatography on a silica gel column (eluting with 80% EtOAc–pentane) to afford (E)-12 (161.4 mg, 0.41 mmol, 97%) as a pale yellow oil.

IR (neat): 2935, 2872, 1580, 1561, 1352, 1175, 1127, 779, 742 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 8.58 (ddd, J = 4.8, 1.5, 0.9 Hz, 1 H), 7.65 (td, J = 7.8, 1.8 Hz, 1 H), 7.44 (dt, J = 7.8, 1.0 Hz, 1 H), 7.22 (ddd, J = 7.6, 4.8, 1.2 Hz, 1 H), 6.55 (br t, J = 1.6 Hz, 1 H), 4.37–4.35 (m, 2 H), 4.18 (t, J = 6.3 Hz, 2 H), 3.71–3.69 (m, 2 H), 3.58 (t, J = 6.4 Hz, 2 H), 3.05 (s, 3 H), 2.53 (br m, J = 6.4 Hz, 2 H), 2.39 (td, J = 6.1, 1.6 Hz, 2 H), 1.95 (quint, J = 6.3 Hz, 2 H), 1.79 (quint, J = 6.4 Hz, 2 H).

¹³C NMR (100 MHz, CDCl₃): δ = 155.5 (0), 150.3 (1), 143.4 (0), 136.3 (1), 131.2 (1), 127.4 (1), 127.3 (0), 123.0 (1), 92.0 (0), 90.0 (0), 71.0 (2), 69.3 (2), 68.7 (2), 68.3 (2), 37.8 (3), 29.6 (2), 29.5 (2), 22.3 (2), 20.9 (2).

MS (ES+): m/z = 393 (M + H)⁺.

HRMS (ESI): m/z calcd for C₁₉H₂₅N₂O₅S: 393.1479; found: 393.1477.

(Z)-2-[3-({[3-(Pyridin-2-ylethynyl)cyclohex-2-en-1-ylidene]amino}oxy)propoxy]ethyl Methanesulfonate [(Z)-12]

An analogous approach to that described for (E)-12 was employed to prepare the title compound (129.6 mg, 0.33 mmol, 58%) as a mixture with the E-isomer in NMR E/Z ratio of 1:7. The mixture was identified by ¹H NMR spectrum (only peaks corresponding to Z-isomer are listed; several peaks are overlapped) and used in the next step.

¹H NMR (400 MHz, CDCl₃): δ = 8.61 (ddd, J = 4.8, 1.7, 0.9 Hz, 1 H), 7.68 (td, J = 7.7, 1.8 Hz, 1 H), 7.47 (br t, J = 7.8, 1.0 Hz, 1 H), 7.25 (ddd, J = 7.7, 4.9, 1.2 Hz, 1 H), 7.17 (br t, J = 1.8 Hz, 1 H), 4.39–4.37 (m, 2 H), 4.15 (t, J = 6.2 Hz, 2 H), 3.73–3.71 (m, 2 H), 3.61 (t, J = 6.4 Hz, 2 H), 3.07 (s, 3 H), 2.45 (td, J = 6.1, 1.8 Hz, 2 H), 2.41–2.38 (m, 2 H), 1.96 (quint, J = 6.3 Hz, 2 H), 1.89 (quint, J = 6.2 Hz, 2 H).

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Fluoro­ethoxy)propyl) Oxime [(E)-4]

A stirred solution of Kryptofix-222^® (K₂₂₂, 251 mg, 0.67 mmol) and KF (38.7 mg, 0.67 mmol) in anhyd MeCN (5.5 mL) was treated with a solution of (E)-12 (130.7 mg, 0.33 mmol) in anhyd MeCN (5.5 mL) dropwise over 7 min at r.t. under N₂ atmosphere during which time the heterogeneous mixture turned brown. The flask was equipped with a condenser and the mixture was placed in an oil bath and heated to 80 °C over 65 min. After this time, the mixture was allowed to cool to r.t. and quenched with sat. aq NH₄Cl (20 mL). The mixture was diluted further with H₂O (10 mL) and EtOAc (50 mL) and the two layers were well shaken and separated. The aqueous phase was extracted with EtOAc (2 × 50 mL). The combined organic extracts were washed with H₂O (3 × 30 mL), brine (40 mL), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by chromatography on a silica gel column (eluting with 50% EtOAc–pentane) to afford (E)-4 (65.4 mg, 0.21 mmol, 62%) as a pale yellow oil.

IR (neat): 2948, 2872, 2201, 1580, 1462, 1428, 1130, 1048, 778, 739 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 8.59 (br m, J = 4.4 Hz, 1 H), 7.66 (td, J = 7.7, 1.8 Hz, 1 H), 7.44 (br t, J = 7.8, 1.0 Hz, 1 H), 7.22 (ddd, J = 7.6, 4.9, 1.1 Hz, 1 H), 6.57 (t, J = 1.6 Hz, 1 H), 4.56 (dm, J = 47.7 Hz, 2 H), 4.21 (t, J = 6.3 Hz, 2 H), 3.69 (dm, J = 29.7 Hz, 2 H), 3.60 (t, J = 6.4 Hz, 2 H), 2.54 (br d, J = 6.4 Hz, 2 H), 2.40 (td, J = 6.2, 1.5 Hz, 2 H), 1.98 (quint, J = 6.4 Hz, 2 H), 1.80 (quint, J = 6.4 Hz, 2 H).

¹³C NMR (100 MHz, CDCl₃): δ = 155.5 (0), 150.3 (1), 143.4 (0), 136.3 (1), 131.4 (1), 127.4 (1), 127.1 (0), 123.0 (1), 91.9 (0), 90.1 (0), 84.2 (2), 83.3 (d, J = 168 Hz, 2), 71.2 (2), 70.2 (d, J = 19.8 Hz, 2), 68.5 (2), 29.7 (2), 29.5 (2), 22.4 (2), 21.0 (2).

¹⁹F NMR (376 MHz, CDCl₃): δ = –222.7 to –223.1 (m).

MS (ES+): m/z = 318 (M + H)⁺.

HRMS (ESI): m/z calcd for C₁₈H₂₂FN₂O₂: 317.1660; found: 317.1660.

(Z)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-Fluoro­ethoxy)propyl] Oxime [(Z)-4]

An analogous approach to that described for the synthesis of (E)-4 was employed to prepare the title compound (64.3 mg, 0.20 mmol, 62%); pale yellow oil.

IR (neat): 2950, 2927, 2873, 1582, 1462, 1428, 1131, 1048, 885, 778 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 8.61 (br s, 1 H), 7.67 (td, J = 7.8, 1.5 Hz, 1 H), 7.47 (br d, J = 7.7 Hz, 1 H), 7.25–7.23 (m, 1 H), 7.19 (br t, J = 1.6 Hz, 1 H), 4.56 (dm, J = 47 Hz, 2 H), 4.17 (t, J = 6.2 Hz, 2 H), 3.70 (dm, J = 29 Hz, 2 H), 3.62 (t, J = 6.5 Hz, 2 H), 2.45 (td, J = 6.1, 1.7 Hz, 2 H), 2.41–2.38 (m, 2 H), 1.98 (quint, J = 6.3 Hz, 2 H), 1.89 (quint, J = 6.3 Hz, 2 H).

¹³C NMR (100 MHz, CDCl₃): δ = 151.9 (0), 150.4 (1), 143.2 (0), 136.4 (1), 130.6 (0), 127.6 (1), 123.6 (1), 123.3 (1), 89.9 (0, 2 C),[28] 83.3 (d, J = 168 Hz, 2), 70.9 (2), 70.2 (d, J = 19 Hz, 2), 68.5 (2), 30.6 (2), 29.6 (2), 27.7 (2), 22.4 (2).

¹⁹F NMR (376 MHz, CDCl₃): δ = –223.2 to –223.7 (m).

MS (ES+): m/z = 318 (M + H)⁺.

HRMS (ESI): m/z calcd for C₁₈H₂₂FN₂O₂: 317.1660; found: 317.1660.

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(3-Hydroxypropyl) Oxime [(E)-13]

To a flame-dried flask charged with (E)-10 (200 mg, 0.94 mmol) was added DMF (12 mL) at r.t. under N₂ atmosphere. The resulting pale yellow clear solution was treated with NaH (60% suspension in oil, 45 mg, 1.13 mmol) and the heterogeneous mixture was allowed to stir for 50 min during which time it turned orange. The mixture was then further treated with a solution of 3-bromopropanol (90 μL, 144 mg, 1.03 mmol, d = 1.6) in DMF (7 mL) dropwise over 12 min and the resulting brown mixture was allowed to stir for 2 h. After this time, the crude mixture was partitioned between H₂O (30 mL) and EtOAc (60 mL) and the two layers were well shaken and separated. The aqueous phase was extracted with EtOAc (2 × 60 mL). The combined organic extracts were washed with H₂O (3 × 30 mL), brine (40 mL), dried (Na₂SO₄), and concentrated to give the crude product, which was purified by chromatography on a silica gel column (eluting with gradient of 50% to 80% EtOAc–pentane) to give (E)-13 (187.2 mg, 0.69 mmol, 73%) as a pale yellow oil.

IR (neat): 3372, 2940, 2874, 2197, 1581, 1464, 1355, 1059, 865, 779 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 5.73 (dm, J = 4.8 Hz, 1 H), 7.66 (td, J = 7.7, 1.8 Hz, 1 H), 7.45 (dt, J = 7.8, 1.0 Hz, 1 H), 7.22 (ddd, J = 7.6, 4.9, 1.1 Hz, 1 H), 6.57 (tm, J = 1.6 Hz, 1 H), 4.27 (t, J = 5.9 Hz, 2 H), 3.76 (dd, J = 10.9, 5.5 Hz, 2 H), 2.54 (dd, J = 6.4 Hz, 2 H), 2.40 (td, J = 6.2, 1.6 Hz, 2 H), 1.93 (quint, J = 6.0 Hz, 2 H), 1.80 (quint, J = 6.4 Hz, 2 H).
¹³C NMR (100 MHz, CDCl₃): δ = 155.7 (0), 150.3 (1), 143.3 (0), 136.3 (1), 131.0 (1), 127.7 (0), 127.4 (1), 123.0 (1), 92.2 (0), 89.9 (0), 72.0 (2), 60.5 (2), 32.5 (2), 29.5 (2), 22.4 (2), 20.9 (2).

MS (ES+): m/z = 271 (M + H)⁺.

HRMS (ESI): m/z calcd for C₁₆H₁₉N₂O₂: 271.1441; found: 271.1430.

(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-¹⁸F-Fluoroethoxy)propyl] Oxime [(E)-[¹⁸F]-4]

[¹⁸F]-Fluoride was produced via nuclear ¹⁸O(p, n)¹⁸F reaction in cyclotron, which involved proton bombardment of ¹⁸O-enriched water (940 μL in a medium volume liquid target) for 70 min at a target current of 15–30 μAh. [¹⁸F]-Fluoride was obtained as an aqueous solution, which was immediately passed through QMA cartridge [preconditioned with 0.5 M aq K₂CO₃ (1 × 5 mL), then H₂O (1 × 5 mL) and dried in air] and the trapped [¹⁸F]-fluoride was eluted from the cartridge with 0.25% wt Kryptofix-222^® solution (1 mL) in basic (0.05% wt K₂CO₃) aq MeCN (75% vv) into a tightly closed reaction vial to give 30–70 GBq. The solvents were evaporated in vacuo (130 mbar) with a gentle stream of N₂ gas at 110 °C over 5 min. To the resulting solid residue was added anhyd MeCN (1 mL) and the mixture was azeotropically dried in vacuo (130 mbar) with a gentle stream of N₂ at 110 °C over 3 min and this was repeated twice more. After this time, the solid residue was additionally dried in vacuo (100 mbar) at r.t. over 1 min to afford 20–60 GBq of activity. The resulting solid Kryptofix-222^®/[¹⁸F^–] complex was treated with a solution of (E)-12 (2.80 mg, 7.13 μmol) in anhyd DMF (0.3 mL) and the resulting dark brown mixture was allowed to heat at 90 or 95 °C (in a metal heating block) over 10 min during which time the reaction mixture turned light brown. The crude mixture was diluted with 50% aq (v/v) MeCN (2 mL) and purified by semi-preparative HPLC (Phenomenex C18 Gemini 5 μm, 10 × 250 mm column, eluting with 5% aq MeCN for 10 min, then a gradient of 5% to 50% aq MeCN for 10 min, and finally 50% aq MeCN for 30 min at 5 mL/min) and the desired product was collected over 1 min (retention time: 31.0 min) and immediately diluted with H₂O (10 mL) to give 4–15 GBq of activity. The aqueous solution was passed through a C18 cartridge [preconditioned with EtOH (1 × 5 mL), then H₂O (1 × 5 mL) and dried in air] and the cartridge was washed with H₂O (2 × 1 mL). The product was eluted from the C18 cartridge with EtOH (1 × 0.3 mL) in a sterile vial containing 50% aq PEG 200 (5 mL) to afford 3–12 GBq of the radiolabeled title compound (13–37% decay corrected yield: 95–99% HPLC purity, 80–400 GBq/μmol specific activity).

Pharmacological Evaluation

Determination of log D_7.4

The determination of log D value was performed using the shake-flask method as previously reported.[16] [29] Formulated solution of (E)-[¹⁸F]-4 (ca. 1 MBq) was partitioned between phosphate buffer (pH 7.4) saturated with octan-1-ol (700 μL) and octan-1-ol saturated with phosphate (pH 7.4) buffer (700 μL). Washed octan-1-ol phase was divided in five individual aliquots (100 μL) and each was diluted with phosphate (pH 7.4) buffer saturated with octan-1-ol (100 μL) and the two phases were shaken and radioactivity in each phase was measured in a γ-counter.

Competition Binding Assay

Brain membranes were prepared from Sprague-Dawley rat brains as described previously.[16] [22] Frozen membranes were thawed on ice and pelleted at 45000 g at 4 °C for 5 min. The membranes were washed twice with HEPES buffer (30 mM HEPES, 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, pH 8 at 4 °C) and resuspended in HEPES buffer at a protein concentration of 1.3 mg/mL. The binding assay was performed as previously described.[16] In brief, brain membranes (0.1 mg protein) were incubated in triplicates at ambient tempearture with 2 nM [³H]-1 and (E)-4 or (Z)-4 at concentrations between 10 pM and 100 μM in a total volume of 0.2 mL HEPES. Compound (E)-4 or (Z)-4 was diluted from a 1 mM ethanolic (50%) solution. The corresponding EtOH concentrations did not affect [³H]-1 binding (data not shown). Unspecific binding of [³H]-1 was estimated with 100 μM MMPEP. After 45 min, the samples were filtered and the filters containing the membranes with bound [³H]-1 were measured in a β-counter (Beckman LS6500). To estimate IC₅₀, bound [³H]-1 (B, pmol per mg protein) was fitted with Excel solver to

B = B_min + {(B_max – B_min)/[1 + (C/IC₅₀)]}

where C is the total (E)-4 or (Z)-4 concentration; B_max is the maximal B, that is, the plateau in the B/C plot at low log C; and B_min is the minimal B, that is, the plateau at high log C. The inhibition constant K _i of (E)-4 or (Z)-4 was estimated from IC₅₀ and K _d of 1 (1.7 ± 0.2 nM) with the Cheng–Prusoff equation. For each compound three independent experiments were performed in triplicate.

Abbreviations

mGlu₅: Metabotropic glutamate receptor subtype 5; PET: positron emission tomography; [¹¹C]-ABP688: (E)-3-[(6-methylpyridin-2-yl)ethynyl]cyclohex-2-enone O-[¹¹C]methyl oxime; [³T]-ABP688: (E)-3-[(6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone O-[³T]methyl oxime; [¹⁸F]-FDEGPECO: (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-[2-(2-[¹⁸F]fluoroethoxy)ethyl] oxime; [¹⁸F]-PSS223: (E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-[2-(3-[¹⁸F]fluoropropoxy)ethyl] oxime; [¹⁸F]-PSS232: (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-[3-(2-[¹⁸F]fluoroethoxy)propyl] oxime; MMPEP: 2-[(3-methoxyphenyl)ethynyl]-6-methylpyridine; PBS: phosphate buffer in saline; IC₅₀: half maximal inhibitory concentration; K _i : inhibition constant; TBS: tert-butyldimethylsilyl; TES: triethylsilyl, TBAF: tetrabutylammonium fluoride; TBAC: triethylbenzylammonium chloride; DIAD: diisopropylazodicarboxylate.

Acknowledgment

The authors would like to acknowledge Dr. Thomas Nauser and Mr. Mathias Nobst for technical assistance in the radiochemistry lab. Mr. Patrick Dennler (PSI) is acknowledged for suggesting application of InBr₃/Et₃SiH method for the ester reduction. Dr. Mark A. Sephton­ (ZHAW) is acknowledged for proofreading the manuscript and discussions on NMR assignments. Prof. P. A. Schubiger, Drs. Cindy A. Wanger-Baumann, Thomas Betzel, Adrienne Müller, and Miss Cindy R. Fischer are acknowledged for many useful discussions.

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  CrossrefPubMedSearch in Google Scholar
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