The First Convergent Synthesis of 23,23-Difluoro-25-hydroxyvitamin D3 and Its 24-Hydroxy Derivatives: Preliminary Assessment of Biological Activities

In this paper, we report an efficient synthetic route for the 23,23-difluoro-25-hydroxyvitamin D3 (5) and its 24-hydroxylated analogues (7,8), which are candidates for the CYP24A1 main metabolites of 5. The key fragments, 23,23-difluoro-CD-ring precursors (9–11), were synthesized starting from Inhoffen-Lythgoe diol (12), and introduction of the C23 difluoro unit to α-ketoester (19) was achieved using N,N-diethylaminosulfur trifluoride (DAST). Preliminary biological evaluation revealed that 23,23-F2-25(OH)D3 (5) showed approximately eight times higher resistance to CYP24A1 metabolism and 12 times lower VDR-binding affinity than its nonfluorinated counterpart 25(OH)D3 (1).


Introduction
The introduction of fluorine atom(s) into biologically active compounds has been widely used in the development of pharmaceuticals, with the expected effects of increasing metabolic stability and improving binding affinity to target proteins [1][2][3][4]. Vitamin D 3 is no exception. Fluorinated vitamin D 3 analogues have been synthesized to extend the biological half-life and modulate the binding affinity to the vitamin D receptor (VDR), and their biological activities have been evaluated [5]. Among them, the side-chain fluorination of vitamin D 3 has been vigorously pursued because hydroxylation of the side-chain C23 or C24 position and several subsequent oxidation steps by the metabolic enzyme CYP24A1 are the main deactivation processes of 25-hydroxyvitamin D 3 [25(OH)D 3 ] (1) (Scheme 1) [6][7][8]. Falecalcitriol (2), which has been clinically approved for the treatment of secondary hyperparathyroidism [9][10][11], is one such vitamin D 3 analogue, and it contains a hexafluoroisopropanol unit in the side chain ( Figure 1).
In recent years, new aspects of vitamin D 3 function have been discovered [12][13][14][15], and syntheses of various vitamin D 3 analogues have been carried out [16,17]. As a result, the importance of developing comprehensive and straightforward synthetic methods for fluorinated vitamin D 3 analogues has increased. However, the synthetic methods reported so far are limited and mainly use sterol skeletons as starting materials, followed by photochemical transformation and thermal isomerization (Scheme 2) [5]. This strategy leads to a limited number of vitamin D derivatives even after multi-step synthesis with low chemical yields.

Results and Discussion
The retrosynthetic analysis is shown in Scheme 3. The CD-rings (9)(10)(11) were synthesized from Inhoffen-Lythgoe diol (12), and the introduction of the difluoro unit to the C23 position was performed using a difluorination reaction of 19 with DAST [21][22][23]. Stereoselective introduction of the hydroxy group to the C24 position was achieved using Sharpless asymmetric dihydroxylation of 28 [24,25]. The A-ring phosphine oxide (14) was prepared from vitamin D 3 [26].
In Schemes 4 and 5, the synthesis of the 23,23-difluoro-CD-ring moiety (9) is described. Selective protection of the C8 secondary hydroxy group of 12 with a TBS group, followed by oxidation at the C22 primary alcohol by TPAP/NMO, afforded aldehyde (16) [27]. The aldehyde was subjected to the Horner-Emmons reagent (17) under basic conditions to create triethylsilyl enol ether (18), which was converted to α-ketoester (19) by selective desilylation of the silyl enol ether unit using TBAF in the presence of acetic acid [23]. The C23-difluoro unit was constructed using DAST toward α-ketoester (19), and the obtained difluoro methyl ester (20) was converted to a Weinreb amide (13) (Scheme 4).
The structure of 7, including stereochemistry at the C24 position, was clarified with X-ray crystallography ( Figure 3).

Experimental Section
1 H and 13 C-NMR spectra were recorded on JEOL AL-400 NMR (400 MHz) and ECP-600 NMR (600 MHz) spectrometers (Tokyo, Japan). 1 H-NMR spectra were referenced with (CH 3 ) 4 Si (δ 0.00 ppm) or CHCl 3 (δ 7.26 ppm) as an internal standard. 13 C-NMR spectra were referenced with deuterated solvent (δ 77.0 ppm for CDCl 3 ). IR spectra were recorded on a JASCO FT-IR-800 Fourier transform infrared spectrophotometer (Tokyo, Japan). Highresolution mass spectra were obtained on a SHIMADZU LCMS-IT-TOF mass spectrometer (Kyoto, Japan) with an electrospray ionization (ESI) method or atmospheric pressure chemical ionization (APCI). Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Tokyo, Japan). Column chromatography was performed on silica gel 60N (Kanto Chemical Co., Inc., 40-50 µm, Tokyo, Japan) or silica gel 60 (Merck, 0.040-0.063 mm, Tokyo, Japan). All experiments were performed under anhydrous conditions under an atmosphere of argon unless otherwise stated. The supporting information of 1 H and 13 C NMR spectra of all new compounds: 5, 7-11, 13, 20, 23, 24, 26, 28-30, and 32-36, as well as 19 F NMR spectra of compounds: 5, 7, and 8 is available at the link in Supplementary Materials. was added at −40 • C; the mixture was stirred at the same temperature for 20 min, and a solution of 16 [27] (977.0 mg, 3.01 mmol) in THF (3.5 mL) was added. The reaction mixture was stirred at 0 • C for 20 min. After the reaction was quenched with H 2 O at 0 • C, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The crude residue was roughly purified by flash column chromatography on silica gel (hexanen:EtOAc = 3:1, 1% Et 3 N) to obtain the crude coupling product 18, and it was used for the next reaction without further purification.
To the above coupling product 18 in CH 2 Cl 2 (7 mL), AcOH (1.03 mL) and tetrabutylammonium fluoride (4.8 mL, 1 M THF solution, 4.8 mmol) were added at 0 • C under air, and the mixture was stirred at room temperature for 15 min. After the reaction was quenched with H 2 O at room temperature, the mixture was extracted with CH 2 Cl 2 three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was partially purified by flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain a crude residue of 19 (1.10 g).
To the solution of the above crude residue of 19 (1.10 g), CH 2 Cl 2 (6.5 mL) was slowly added to N,N-diethylaminosulfur trifluoride (DAST) (2.8 mL, 3.4 g, 21.1 mmol) at 0 • C, and the mixture was stirred at room temperature for 16 h. The mixture was cooled to 0 • C, and MeOH and H 2 O were slowly added. The mixture was extracted with CH 2 Cl 2 three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was partially purified by flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain 20 (747.0 mg, 59%, three steps) as a colorless oil. To the solution of compound 20 (1.01 g, 2.41 mmol) and Me(MeO)NH·HCl (934.3 mg, 9.58 mmol) in THF (20 mL), isopropyl magnesium chloride (19.0 mL, 1 M in THF, 19.0 mmol) was added at 0 • C, and the mixture was stirred at the same temperature for 23 h. After the reaction was quenched with water and aqueous saturated NH 4 Cl, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 6:1) to obtain 13 (933.9 mg, 87%) as a colorless oil. To the solution of compound 13 (251.5 mg, 0.56 mmol) in THF (20 mL), isopropenyl magnesium bromide (2.25 mL, 0.5 M in THF, 1.12 mmol) was added at 0 • C, and the mixture was stirred at room temperature for 1 h. After the reaction was quenched with water, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 20:1) to obtain the crude product 21.
NaBH 4 (45.1 mg, 1.19 mmol) was added to the solution of the above crude 21 and CeCl 3 ·6H 2 O in EtOH (3 mL) and MeOH (3 mL) at 0 • C, and the mixture was stirred at the same temperature for 5 min. After the reaction was quenched with water, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The crude 22 was used for the next reaction without further purification.
N,N-Dimethyl-4-aminopyridine (464.5 mg, 3.68 mmol) and Ac 2 O (1.85 g, 2 mL, 18.1 mmol) were added to the solution of the crude 22 in pyridine (4 mL) at 0 • C, and the mixture was stirred at room temperature for 10 min. After the reaction was quenched with water at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 20:1) to obtain 23 (mixture of diastereomers) (201.8 mg, 76%, three steps) as a colorless oil.
To a solution of sodium formate (155.8 mg, 2.29 mmol) and Pd(PPh 3 ) 4 (423.2 mg, 0.37 mmol) in dioxane (1.5 mL), nBu 3 P (345.5 mg, 427.0 µL, 1.77 mmol) was added at room temperature, and the mixture was stirred at 90 • C for 10 min. Acetate 23 (201.8 mg, 0.43 mmol) was dissolved in dioxane (1.5 mL), and the solution was added to the mixture. After being stirred at the same temperature for 17 h, the reaction mixture was quenched with H 2 O at room temperature. The mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was partially purified by flash column chromatography on silica gel (hexane:EtOAc = 100:1), and repurification by flash column chromatography on silica gel (hexane only) provided 24 (111.9 mg, 63%) as a colorless oil. mCPBA (286.1 mg, 1.66 mmol) was added to the mixture of 24 (111.9 mg, 0.27 mmol) and NaHCO 3 (131.9 mg, 1.57 mmol) in CH 2 Cl 2 (2 mL) at 0 • C, and the mixture was stirred at the same temperature for 100 min under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 20:1) to obtain the crude epoxide. LiAlH 4 (14.0 mg, 0.37 mmol) was added to the solution of the above crude epoxide in Et 2 O (3 mL) at 0 • C, and the mixture was stirred at the same temperature for 15 min, and then at room temperature for 20 min. After the reaction was quenched with MeOH, water, and saturated aqueous potassium sodium tartrate, the mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The crude alcohol was used for the next reaction without further purification.
p-Toluenesulfonic acid monohydrate (981.0 mg, 5.16 mmol) was added to the solution of the above crude alcohol in MeOH (20 mL), and the mixture was stirred at room temperature for 25 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 2:1) to obtain 9 (63.8 mg, 74%, three steps) as a colorless oil. To the solution of compound 13 (933.9 mg, 2.09 mmol) in THF (10 mL), isopropyl magnesium chloride (6.3 mL, 1 M in THF, 6.26 mmol) was added at 0 • C, and the mixture was stirred at room temperature for 1 h. After the reaction was quenched with water and aqueous saturated NH 4 Cl at 0 • C, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain the crude product 25.
NaBH 4 (45.1 mg, 1.19 mmol) was added to the solution of the above crude 25 in EtOH (3 mL) at 0 • C, and the mixture was stirred at the same temperature for 5 min. After the reaction was quenched with water, the mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 10:1) to obtain 26 (mixture of diastereomers) (563.4 mg, 62%, two steps) as a colorless oil. Trifluoromethanesulfonic anhydride (366.8 mg, 213 µL, 1.30 mmol) was added to the solution of 26 (467.3 mg, 1.08 mmol) and 2,6-di-tert-butylpyridine (464.5 mg, 3.68 mmol) in CH 2 Cl 2 (4 mL) at 0 • C, and the mixture was stirred at the same temperature for 75 min. After the reaction was quenched with water and aqueous saturated NH 4 Cl at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The crude 27 was used for the next reaction without further purification.
1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) (600 µL) was added to the solution of the above crude 27 in CH 2 Cl 2 (4 mL) at room temperature and the mixture was stirred at the same temperature for 15 h. After the reaction was quenched with water and aqueous saturated NH 4 Cl at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane only) to obtain 28 (269.8 mg, 60%, two steps) as a colorless oil. The mixture of AD-mix α (605.3 mg) in tBuOH (4 mL) and H 2 O (4 mL) was stirred at 0 • C for 20 min, and 28 (55.9 mg, 0.13 mmol) was added to the mixture at 0 • C with stirring at the same temperature for 4 h, and then at 4 • C for 20 h under air. After the reaction was quenched with water, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 1:1) to obtain 29 (53.4 mg, 88%) as a colorless oil. The mixture of AD-mix β (704.8 mg) in tBuOH (4 mL) and H 2 O (4 mL) was stirred at 0 • C for 30 min, and 28 (51.8 mg, 0.13 mmol) was added to the mixture at 0 • C with stirring at the same temperature for 5 h, and then at 4 • C for 19 h under air. After the reaction was quenched with water, the mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 2:1) to obtain 30 (53.8 mg, 96%) as a white powder. To the solution of 29 (53.4 mg, 0.12 mmol) in MeOH (10 mL), p-toluenesulfonic acid monohydrate (222.8 mg, 1.17 mmol) was added, and the mixture was stirred at room temperature for 39 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times.
The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 1:2) to obtain 10 (36.1 mg, 91%, in two steps) as a white powder. To the solution of 30 (48.9 mg, 0.11 mmol) in MeOH (5 mL) and CH 2 Cl 2 (5 mL), p-toluenesulfonic acid monohydrate (204.1 mg, 1.07 mmol) was added, and the mixture was stirred at room temperature for 66 h under air. After the reaction was quenched with H 2 O and saturated aqueous NaHCO 3 at room temperature, the mixture was extracted with CH 2 Cl 2 three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 1:2) to obtain 11 (30.4 mg, 83%, in two steps) as a white powder. 4-Methylmorpholine N-oxide (36.6 mg, 0.31 mmol) was added to the solution of 9 (63.8 mg, 0.20 mmol) in CH 2 Cl 2 (2 mL), and the mixture was cooled to 0 • C. TPAP (37.8 mg, 0.11 mmol) was added to the mixture, and the mixture was stirred at 0 • C for 1 h. The reaction was diluted with an excess amount of Et 2 O. The mixture was directly purified by flash column chromatography on silica gel (Et 2 O only) to obtain the crude ketone (31), and this was used for the next reaction without further purification.
TESCl (211.0 mg, 234 µL, 1.4 mmol) was added to the 0 • C cooled solution of the above crude ketone (31) and imidazole (130.6 mg, 1.92 mmol) in CH 2 Cl 2 (5 mL), and the mixture was stirred at room temperature for 28 h. After the reaction was quenched with H 2 O at 0 • C, the mixture was extracted with CH 2 Cl 2 three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 20:1-10:1) to obtain 32 (76.1 mg, 88%, in two steps) as a colorless oil. Pyridinium p-toluenesulfonate (PPTS) (19.3 mg, 0.08 mmol) was added to a solution of 10 (45.7 mg, 0.14 mmol) in anisaldehyde dimethyl acetal (1.5 mL) at room tempera-ture, and the mixture was stirred at the same temperature for 18 h. After the reaction was quenched with water and saturated aqueous NaHCO 3 , the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was partially purified by flash column chromatography on silica gel (hexane:EtOAc = 4:1-2:1), and repurification by flash column chromatography on silica gel (hexane:EtOAc = 3:1) provided 33 (mixture of diastereomers) (31.3 mg, 50%) as a colorless oil. Pyridinium p-toluenesulfonate (PPTS) (158.6 mg, 0.63 mmol) was added to a solution of 11 (36.1 mg, 0.11 mmol) in anisaldehyde dimethyl acetal (3 mL) at room temperature, and the mixture was stirred at the same temperature for 18 h. After the reaction was quenched with water and saturated aqueous NaHCO 3 , the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was partially purified by flash column chromatography on silica gel (hexane only-hexane:EtOAc = 4:1-2:1), and repurification by flash column chromatography on silica gel (hexane:EtOAc = 4:1) provided 34 (mixture of diastereomers) (22.9 mg, 61%) as a colorless oil.    5 mL) was added to the reaction mixture, and the mixture was stirred at −78 • C for 1 h. After the reaction was quenched with H 2 O at the same temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 20:1) to obtain the crude coupling product (52.0 mg), and it was used for the next reaction without further purification.
Tetrabutylammonium fluoride (710 µL, 1 M THF solution, 0.71 mmol) was added to the solution of the above crude coupling product (52.0 mg) in THF (4 mL), and the mixture was stirred at room temperature for 16 h. After the reaction was quenched with H 2 O and aqueous saturated NH 4 Cl at room temperature, the mixture was extracted with EtOAc three times. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 1:1) to afford 5 (30.8 mg, 100%, in two steps) as a white powder.  (1 mL) was added to the reaction mixture, and the mixture was stirred at −78 • C for 2 h. After the reaction was quenched with H 2 O at the same temperature, the mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 5:1) to obtain the crude coupling product (25.5 mg), and it was used for the next reaction without further purification.
The above crude residue was dissolved in MeOH (5 mL), and AG 50W-X4 resin H + form (183.1 mg) was added. The mixture was stirred for 3 h, insoluble materials were filtered off, and the solution was diluted with H 2 O and saturated aqueous NaHCO 3 . The mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography (hexane:EtOAc = 1:1) to afford 7 (10.8 mg, 41%, two steps) as a white solid. Crystal of 7 was obtained by dissolving 7 in EtOH and allowing the solvent to slowly evaporate at room temperature (colorless needles). All measurements were taken on a Rigaku Raxis Rapid imaging plate area detector with graphite monochromated Cu-Kα radiation. The data were collected at a temperature of -100 • C. The structure was solved by direct-method SIR97 and expanded using Fourier techniques. The non-hydrogen atoms were refined anisotropically. All calculations were performed using the Crystal Structure (Crystal Structure 4.2.2) crystallographic software package except for refinement, which was performed using SHELXL97.  After stirring for 20 min, a solution of 36 (28.2 mg, 0.06 mmol) in THF (1 mL) was added to the reaction mixture, and the mixture was stirred at −78 • C for 100 min. After the reaction was quenched with H 2 O at the same temperature, the mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc = 4:1) to obtain the crude coupling product (41.6 mg), and it was used for the next reaction without further purification.

Accession Codes of Compound
The above crude residue was dissolved in MeOH (5 mL), and AG 50W-X4 resin H + form (188.3 mg) was added. The mixture was stirred for 3 h 10 min, insoluble materials were filtered off, and the solution was diluted with H 2 O and saturated aqueous NaHCO 3 . The mixture was extracted with EtOAc three times. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by flash column chromatography (hexane:EtOAc = 1:1) to afford 8 (19.6 mg, 63%, two steps) as a white powder.

Metabolism of 25(OH)D 3 (1) and 5 by Recombinant hCYP24A1
The metabolism of 25(OH)D 3 and its analogue 5 by CYP24A1 was analyzed using the membrane fraction prepared from recombinant Escherichia coli cells expressing human CYP24A1, as described in our previous study [29]. Briefly, the reaction mixture containing 0.02 µM of human CYP24A1, 2.0 µM of adrenodoxin (ADX), 0.2 µM of NADPHadrenodoxin reductase (ADR), 1 mM of EDTA, 1 mM of NADPH, and 5.0 µM of each substrate in 100 mM Tris-HCl (pH 7.4) was incubated at 37 • C for 5 or 15 min. The metabolites were extracted with four volumes of CHCl 3 -CH 3 OH (3:1) and analyzed by HPLC under the following conditions: column, CAPCELL PAK C18 UG120 (5 µm) (4.6 × 250 mm) (SHISEIDO, Tokyo, Japan); UV detection, 265 nm; flow-rate, 1.0 mL min −1 ; column temperature, 40 • C; mobile phase, CH 3 CN: a linear gradient of 20-100% CH 3 CN aqueous solution per 25 min and 100% CH 3 CN for 10 min. The binding affinity of each analogue for hVDR was evaluated using the in vitro system based on the split-luciferase technique described in our previous study [30]. Briefly, 50 µL of cell lysate prepared from recombinant E. coli expressing split-luciferase vitamin D biosensor protein was added to each well of a 96-well plate and left for 10 min at room temperature. Then, 50 µL of the Luciferin solution containing 20 mM of MgSO 4 , 2 mM of D-luciferin, and 4 mM of adenosine triphosphate in 25 mM Tris-HCl (pH 7.4) were injected into each well and incubated for 15 min at room temperature. The luminescence (photon counts) was measured using a luminometer (Infinite 200 Pro 96-microplate luminometer, Tecan). The relative hVDR binding affinity of each analogue was evaluated based on the concentration at which the luminescence showed 50% of the maximum value. Funding: This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (No. 22K14688 to F.K., No. 18K06556 to A.K., and No. 19H02889 to T.S.).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.