[11C] Labeled acyclic retinoids, central nervous system activators and methods for their preparation.

Abstract

To provide a 11C-labelled acyclic retinoid that can be synthesized at high yield in a short time and is suitable for elucidating intracerebral kinetics for PET, and a PET probe that uses the retinoid, and production methods for the retinoid and the PET probe.SOLUTION: The present invention discloses a 11C-labelled acyclic retinoid represented by a chemical formula (a) shown below. There is also provided a method for producing the retinoid (a) by a coupling step in which 11C-labelled methyl iodide is cross-coupled with an organic tin compound (b) shown below (in the formula, R1 and R2 each represent an optionally branched alkyl group) in an aprotic solvent in the presence of a palladium complex, a phosphine ligand, and a copper(I) halide.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to 11 The present invention relates to a C-labeled acyclic retinoid, a central nervous system activator, and a method for producing the same. 11 Since the [C]-labeled acyclic retinoid has the ability to penetrate into the brain, it can be suitably used as a PET probe for studying the dynamics of acyclic retinoids in the brain. 11 It contains an acyclic retinoid that is an unlabeled form of [C]-labeled acyclic retinoid, and has the effect of activating the central nervous system by migrating into the brain. Therefore, it can be used as a therapeutic agent for central neurodegenerative diseases or as a lead compound for the development of such a therapeutic agent. Here, "central neurodegenerative diseases" refers to all diseases caused by degeneration of the central nervous system (e.g., Alzheimer's dementia, frontotemporal dementia, dementia with Lewy bodies, Parkinson's disease, progressive supranuclear palsy, Huntington's disease, argyrophilic grain dementia, neurofibrillary tangle senile dementia, brain tumor, etc.), and is a concept that also includes cerebrovascular diseases such as central nervous system degeneration caused by intracerebral hemorrhage. [Background technology]

[0002] All-trans-retinoic acid (ATRA) is a type of cyclic retinoid with a six-membered carbon skeleton, and is known to act as a ligand for the nuclear retinoic acid receptor and exhibit anticancer activity. Furthermore, ATRA and its synthetic analogues have been reported to have ameliorative effects on Alzheimer's disease (AD), attracting much attention.

[0003] [ka]

[0004] In order to develop brain-targeted drugs for AD and other conditions, it is important to analyze the dynamics of candidate compounds in the brain. Positron emission tomography (PET) is an extremely effective means for this purpose. 11reported a study on synthesizing C-labeled ATRA and using it as a PET probe (Non-Patent Document 1).

[0005] but, 11 It became clear that there were many problems to be solved in using C-labeled ATRA as a PET probe, including the following: 1) The number of steps required to synthesize the labeled precursor is large. 2) 11 C-labeled ATRA is unstable under light and labeling reaction conditions. 3) 11 During the C labeling process, multiple geometric isomers are generated, and their separation and purification is cumbersome. 4) Numerous metabolic products are generated in vivo, making analysis of intracerebral dynamics complicated.

[0006] To solve these problems, we developed a compound with a structure similar to ATRA and relatively stable chemical structure. 11 Some C-labeled artificial retinoids have also been synthesized. For example, in Non-Patent Document 2, 11 As C-labeled artificial retinoid 11 C-labeled Am80 was synthesized. However, 11 When C-labeled Am80 was used as a PET probe, no intracerebral transfer was observed, and intracerebral dynamics analysis could not be performed. 11 As a C-labeled artificial retinoid, 11 We synthesized C-labeled CBt-PMN and used it as a PET probe. 11 It has been observed to have a higher brain penetration rate than C-labeled bexarotene. 11 Since the final yield of C-labeled CBt-PMN when totally synthesized is extremely low at 0.33%, PET imaging has only been used for small animals, and no analysis of its kinetics or distribution in the brain has been performed.

[0007] [ka]

[0008] Incidentally, Patent Document 1 describes that peretinoin, a type of acyclic retinoid, can be used as an anticancer drug. However, it has not been known that peretinoin has the ability to enter the brain, nor has it been known that it can activate the central nervous system (CNS) or be used as a drug for central neurodegenerative diseases. In addition, the conventional synthesis of peretinoin involves many steps and is time-consuming. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Unexamined Patent Publication No. 56-140949 [Non-patent literature]

[0010] [Non-Patent Document 1] Suzuki M, Takashima-Hirano M, Ishii H, Watanabe C, Sumi K, Koyama H, Doi H. Bioorg Med Chem Lett. 2014;243:3622-3625. [Non-Patent Document 2] Takashima-Hirano M, Ishii H, Suzuki M. ACS Med Chem Lett. 2012;3:804-807. [Non-Patent Document 3] O. Shibahara, M. Watanabe, S. Yamada, M. Akehi, T. Sasaki, A. Akahoshi, T. Hanada, H. Hirano, S. Nakatani, H. Nishioka, Y. Takeuchi, H. Kakuta, J. Med. Chem. 2017, 60, 7139-7145. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention has been made in consideration of the above-mentioned problems in the conventional art, and has an object to achieve either of the following 1) or 2). 1) It can be synthesized in a short time with good yield, and can be suitably used as a PET probe to clarify dynamics in the brain. 11 To provide a C-labeled acyclic retinoid, a PET probe using the same, and a method for producing the same. 2) To provide a new method for producing acyclic retinoids and a central nervous system activator containing an acyclic retinoid. [Means for solving the problem]

[0012] In order to solve the above-mentioned problems, the present inventors have focused on acyclic retinoids having a chain-like carbon skeleton. Peretinoin, a type of acyclic retinoid, is known as a therapeutic agent for peripheral liver cancer, but there have been no reports of acyclic retinoids being transported into the brain. 11 However, acyclic retinoids have a ring-open structure, and are related in chemical structure. For this reason, it is recommended to use acyclic retinoids instead of cyclic retinoids. 11 We attempted to use it as a PET probe by labeling it with 1C. [ka]

[0013] As a result, it was surprisingly found that acyclic retinoids are transported into the brain, and are therefore suitable for elucidating the dynamics of their activity in the brain. 11 C-labeled acyclic retinoids have the advantage that they are easier to synthesize and more chemically stable than cyclic retinoids.

[0014] That is, the present invention 11 The C-labeled acyclic retinoid is a compound represented by the following chemical formula (a) (i.e., 11 C-labeled peretinoin), or an ester or salt thereof. [ka]

[0015] The PET probe of the present invention is 11 The present invention is characterized by containing a C-labeled acyclic retinoid. According to the results of the inventors' tests, this PET probe has brain migration properties and can be used as a PET probe for elucidating brain dynamics. 11 C-labeled acyclic retinoids were 11 It has a faster brain penetration rate than C-labeled acyclic retinoids and is particularly excellent as a PET probe.

[0016] The present invention 11 C-labeled acyclic retinoids are 11 Methyl iodide labeled with C and the following organotin compound (b) (where -COOR 1 is an ester group, R 2 represents an alkyl group which may have a branch.) in the presence of a palladium complex, a phosphine ligand, and a cuprous halide. Furthermore, the ester group of the coupling product can be hydrolyzed in the same reaction vessel to easily produce a carboxylic acid. A strong base such as potassium hydroxide or sodium hydroxide can be used for the hydrolysis, and after the hydrolysis, an acid such as formic acid is added to produce a free carboxylic acid. [ka]

[0017] In this manufacturing method, a phosphine ligand is coordinated in an unsaturated manner to a palladium complex to create an active reaction field, and then, the palladium complex to which the phosphine ligand is coordinated is 11 C-labeled methyl iodide undergoes oxidative addition to [ 11 C] A palladium complex is formed in which a phosphine ligand is coordinated to CH3PdI. On the other hand, the organotin compound (b) undergoes a metal exchange reaction with cuprous halide to give a highly nucleophilic organocuprophere. and,[ 11 C]CH3PdI is coordinated with a phosphine ligand, and the palladium complex undergoes a substitution reaction with an organocopper compound, followed by reductive elimination to give [ 11 The coupling reaction with C]CH3 is completed.

[0018] The palladium complex is not particularly limited, and for example, a palladium zero-valent complex obtained by reducing Pd(OAc)2, Pd2(dba)3, etc. can be used. In the present specification, the phosphine ligand refers to a compound having three organic substituents bonded to a trivalent phosphorus atom and further having an unshared electron pair. Specific examples of the phosphine ligand include trialkylphosphines such as trimethylphosphine, trioctylphosphine, tributylphosphine, and dimethyloctylphosphine, tri(o-tolyl)phosphine, tricyclohexylphosphine, trixylylphosphine, trimesitylphosphine, tris(tetramethylphenyl)phosphine, diphenyl-p-chlorophenylphosphine, tris(p-methoxyphenyl)phosphine, diphenylethylphosphine, dimethylphenylphosphine, bis(diphenylphosphino)methane, 1,2-bis(diphenylphosphino)ethane, dioctyloctoxyphosphine, dibutylbutoxyphosphine, diphenylphenoxyphosphine, ditolyltolyloxyphosphine, dixylylxylyloxyphosphine, diphenyl ... Examples of the phosphine derivatives include phenylethoxyphosphine, diethylphenoxyphosphine, octyldioctoxyphosphine, butylbutoxyphosphine, phenyldiphenoxyphosphine, tolylditolyloxyphosphine, xylyldixylyloxyphosphine, phenyldiethoxyphosphine, ethyldiphenoxyphosphine, trioctyl phosphite, tributyl phosphite, dimethyloctyl phosphite, tricyclohexyl phosphite, triphenyl phosphite, tritolyl phosphite, trixylyl phosphite, diphenylethyl phosphite, dimethylphenyl phosphite, 1,3-bis(diphenylphosphino)propane (dppp), (2-biphenylyl)dicyclohexylphosphine, and tri-(1-naphthyl)phosphine.

[0019] Examples of aprotic solvents that can be used include formamide-based solvents such as N,N-dimethylformamide and N,N-diethylformamide, acetamide-based solvents such as N,N-dimethylacetamide and N,N-diethylacetamide, pyrrolidone-based solvents such as N-methyl-2-pyrrolidone and N-vinyl-2-pyrrolidone, sulfoxide-based solvents such as dimethyl sulfoxide and diethyl sulfoxide, hexamethylphosphoramide, γ-butyrolactone, etc. Among these, formamide-based solvents, acetamide-based solvents, and pyrrolidone-based polar solvents are preferred.

[0020] It is also preferable to add at least one of carbonate, alkali metal fluoride, and alkali metal salt of ascorbic acid in the coupling step. In this case, the trialkyltin halide by-produced in the coupling step reacts with at least one of carbonate, alkali metal fluoride, and alkali metal salt of ascorbic acid, and is neutralized or precipitated (precipitated as trialkylstannyl carbonate in the case of carbonate, as trialkylstannyl fluoride in the case of alkali metal fluoride, and as ascorbate in the case of alkali metal salt of ascorbic acid) and is removed from the reaction system. This promotes the metal exchange reaction from Sn to Cu, and can also increase the yield.

[0021] The central nervous system activator of the present invention contains, as an active ingredient, an acyclic retinoid (common name: peretinoin) represented by the following chemical formula (c), or an ester or salt thereof. [ka]

[0022] The present invention 11 C-labeled acyclic retinoids have the ability to penetrate into the brain. 11Peretinoin, or its ester or salt, which is not C-labeled, also has the ability to enter the brain. Furthermore, according to the results of the inventors' experiments, peretinoin, which is an acyclic retinoid represented by the above chemical formula (c), and its ester improve the motor function of mice after cerebral hemorrhage and suppress the activity of microglia around the hematoma. From these facts, peretinoin, which is an acyclic retinoid represented by the above chemical formula (c), or its ester or salt, can be used as a central nervous system (CNS) activator. Furthermore, it can be used as a drug for treating central neurodegenerative diseases, or as a lead compound for developing a drug for treating central neurodegenerative diseases, promoting the development of new diagnostic and therapeutic drugs.

[0023] The method for producing the acyclic retinoid of the present invention comprises the steps of: 1 is an alkyl group) and the carboxylic acid ester represented by the following chemical formula (e) are reacted with each other to obtain the acyclic retinoid represented by the chemical formula (f) (where -COOR is an alkyl group). 2 is an ester group). [ka]

[0024] The phosphonic acid ester of chemical formula (d) used in this manufacturing method can be obtained easily and in high yield by using trans,trans-farnesol as a starting material, converting its hydroxyl group to a chloride, and further performing the Michaelis-Arbuzov reaction using trimethyl acetate. The carboxylic acid ester of chemical formula (e) can be obtained by converting hydroxyacetone into an E isomer hydroxyester by Wittig olefin synthesis, and then oxidizing this to obtain the carboxylate while maintaining the E configuration. According to the manufacturing method of the present invention, the phosphonic acid ester of the following chemical formula (d) and the aldehyde of chemical formula (e) are bonded via a carbon-carbon double bond by the HWE reaction, whereby the acyclic retinoid of chemical formula (f) can be produced in fewer steps than the manufacturing method described in Patent Document 1.

[0025] Furthermore, peretinoin can be obtained in high stereoselective yield by hydrolysis of the acyclic retinoid of formula (f) thus obtained. A strong base such as potassium hydroxide or sodium hydroxide can be used for the hydrolysis. After hydrolysis, an acid such as formic acid is added to convert the compound into a free carboxylic acid. Effect of the Invention

[0026] The production method of the present invention 11 The C-labeled acyclic retinoid has sufficient radioactivity to obtain PET images and can be used as a PET probe. 11 C-labeled acyclic retinoids are 11 Since the C labeling site is metabolically stable, it provides a high-quality PET probe with high reproducibility. Furthermore, since hydrolysis of the ester group after the coupling step proceeds quickly and easily, it can be synthesized using a general automated radiopharmaceutical synthesis device. In addition, since it has brain-translocating properties, it will be a useful molecular tool in the development of drugs for treating central neurodegenerative diseases, such as promoting the development of new intracerebral cancer diagnostic and therapeutic drugs of acyclic retinoids targeting glioma, a cancer of the central nervous system. In addition, the central nervous system activator of the present invention has the effect of activating the central nervous system by being transferred into the brain, and therefore can be used as a therapeutic agent for central nervous degenerative diseases or as a lead compound for the development of such therapeutic agents. [Brief description of the drawings]

[0027] [Figure 1] PET image (a) and graph of brain radioactivity (b) when 11C-labeled peretinoin [11C]-3 was administered to a rat. [Diagram 2] PET image (a) and graph of brain radioactivity (b) when 11C-labeled peretinoin [11C]-3 was administered to a cynomolgus monkey. [Diagram 3] PET image (a) and graph of brain radioactivity (b) when 11C-labeled acyclic retinoid [11C]-4c was administered to rats. [Figure 4] FIG. 1 shows a PET image (a) and a graph of brain radioactivity (b) when 11C-labeled acyclic retinoid [11C]-4c was administered to a cynomolgus monkey. [Diagram 5] PET image (a) and graph of brain radioactivity (b) when 11C-labeled acyclic retinoid [11C]-4b was administered to rats. [Figure 6] PI fluorescence images showing the inhibitory effect of peretinoin 3 on thrombin-induced brain tissue damage. [Figure 7] 1 is a graph showing the rate of thrombin-induced cerebral cortical cell death. [Figure 8] 1 is a graph showing the percentage of striatal tissue atrophy induced by thrombin. [Figure 9] PI fluorescence images showing the effect of K252a on the protective effect of peretinoin 3 on brain tissue. [Figure 10] 1 is a graph showing the rate of cerebral cortical cell death induced by K252a. [Figure 11] 1 is a graph showing the percentage of striatal tissue atrophy induced by K252a. [Figure 12] 1 is a graph showing changes in mouse body weight during motor function tests. [Figure 13] 1 is a graph showing the results of a beam walking test. [Figure 14] 1 is a graph showing the results of a modified limb reflex test. [Figure 15] 13 is a graph showing images of microglia in brain tissue after completion of a motor function test and their evaluation. [Figure 16] 13 is a graph showing images of neurons and an assessment of neuronal loss after completion of a motor function test. [Figure 17] This is an image showing the induction of differentiation from stem cells to nerve cells. [Figure 18] 1 is a graph evaluating the responsiveness to NMDA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Acyclic retinoids 4a, 4b, 4c and peretinoin 3 (see chemical formula below) and their 11 C-labeled compound [ 11 C]-4a, [ 11 C]- 4b, [ 11 C]-4c and [ 11 C]-3 was synthesized by the following procedure. Acyclic retinoids 4a, 4b, 4c and peretinoin 3 are the acyclic retinoids contained in the central nervous system activator of the present invention. 11 C]-4a, [ 11 C]-4b, [ 11 C]-4c and [ 11 C]-3 is the compound of the present invention. 11 It is a C-labeled acyclic retinoid. Details are given below. [ka]

[0029] (Chemicals, analytical equipment, etc. used in the examples) All chemicals and solvents, except THF, were purchased from Sigma-Aldrich Japan, Wako Pure Chemical Industries, Tokyo Chemical Industry, Kanto, and Nacalai Tesque and used as received without further purification. 1 H, 13 C, and 19 F nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM 400-400 spectrometer. Chemical shifts were referenced to TMS at 0.00 ppm and to CHCl3 at 0.00 ppm. 1 H NMR: 7.26; 13 The C NMR was 77.0. The abbreviations s, d, t, q, and m indicate singlet, doublet, triplet, quartet, and multiplet, respectively. High-resolution mass spectra (HRMS) were measured on a PE Biosystems Mariner system and a JEOL JMS-700 / GI. 11 C]CO2 was measured using a YPRIS HM-18 cyclotron (Sumitomo Heavy Industries, Ltd.). 14 N(p, α) 11It was produced by the C nuclear reaction. 11 C]Methyl iodide and 11 A proprietary automated radiolabeling system was used to prepare C-labeled compounds, which consisted of heating the reaction mixture, dilution, HPLC injection, fractional collection, and evaporation. Radioactivity was quantified with an ATOMLABTM500 dose calibrator (Biodex Medical Systems, Inc.). 11 The analytical HPLC system used for [C]methylated products was equipped with an amplifier and bias power supply 925-SCINT (AMETEK Co., Ltd.), a linear count rate meter 7131-1 (Ohyo Koken Kogyo Co. Ltd.), a Shimadzu system controller (SPD-10Avp), an online degasser (DGU-12A), a solvent delivery unit (LC-10ATvp), a column oven (CTO-10A), a photodiode array detector (SCL-10Avp), and software (LC Solution). The columns used for analytical and preparative HPLC were CAPCELL PAK C18 4.6 × 250 mm and CAPCELL PAK C18 MG 5 μm 10 × 250 mm (Shiseido Co., Ltd.).

[0030] < 11 Synthesis of C-labeled acyclic retinoids Acyclic retinoid 14a and acyclic retinoid 14b were synthesized by the following synthetic method. [ka]

[0031] Synthesis of (2E,6E)-3,7,11-trimethyl-2,6,10-(dodecatrienyl)phosphonic acid diethyl ester 14b A 50 mL round-bottom flask was charged with trans,trans-farnesyl bromide 13 (2.7 mL, 10 mmol), triethyl phosphite (1.9 mL, 11 mmol), and dry toluene (20 mL). The mixture was refluxed for 20 h and then cooled to room temperature. The solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / acetone = 5:1 to 1 / 2) to give compound 14b (2.82 g, 8.23 ​​mmol, 82%) as a yellow oil. 1 H NMR (400 MHz, CDCl3), 1.30 (t, J = 7.0 Hz, 6H, 2CH3), 1.59 (s, 6H, 2CH3), 1.65 (d, J = 3.6 Hz, 3H, CH3), 1.67 (s, 3H, CH3), 1.89-2.14 (m, 8H, 4CH2), 2.56 (dd, J = 7.6, 2 J( 1 H- 31 P) = 21.6 Hz, 2H, CH2), 4.00-4.16 (m, 4H, 2CH2), 5.03-5.13 (m, 2H, 2C=CH), 5.14-5.24 (m, 1H, C=CH); 13 C NMR (100 MHz, CDCl3), 16.06, 16.40, 16.56 (d, 3 J( 13 C- 31 P) = 5.7 Hz, 2C), 17.76, 25.76, 26.49, 26.53, 26.46 (d, 1 J( 13 C- 31 P) = 139.2 Hz), 26.81, 39.78, 61.81 (d, 2 J( 13 C- 31 P) = 5.8 Hz, 2C), 112.51 (d, 2 J( 13 C- 31 P) = 11.5 Hz), 123.88, 124.39, 131.39, 135.37, 140.34 (d, 3 J( 13 C-31 P) = 13.4 Hz); HR-MS (EI+,100% acetone): m / z: calcd for C 19 H 35 O3P ([M-C4H9] + ) 342.2324; found, 342.2319.

[0032] Compound 14a was obtained in the same manner as in the synthesis of compound 14b, except that trimethyl phosphite was used instead of triethyl phosphite.

[0033] Compounds 4a, 4b, and 4c were synthesized by the following synthesis method. [ka]

[0034] Synthesis of acyclic retinoids 4a and 4b Synthesis of (2E, 4E, 6E, 10E)-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid ethyl ester 4b (E)-3-Methyl-4-oxo-2-butenoic acid ethyl ester was synthesized from (E)-4-hydroxy-3-methyl-2-butenoic acid ethyl ester (72.2 mg, 0.500 mmol) based on the literature (GE Magoulas, et.al. J. Med. Chem. 2011, 46, 721-737.). The (E)-3-methyl-4-oxo-2-butenoic acid ethyl ester thus obtained was used without purification. Compound 14b (171 mg, 0.500 mmol) and dry THF (2 mL) were placed in a 30 mL two-necked round-bottom flask and cooled to -78 ° C in a dry ice / acetone bath, and potassium bis(trimethylsilyl)amide (1.0 M THF solution, 500 μL, 0.5 mmol) was added at this temperature. After the reaction mixture was stirred for 1 h, (E)-3-methyl-4-oxo-2-butenoic acid ethyl ester dissolved in THF (2 mL) at -78 °C was added dropwise. After stirring at room temperature for 1 h, the resulting mixture was cooled to 0 °C, saturated aqueous NH4Cl solution (2 mL) was added, and extracted with diethyl ether (3 × 5 mL). The organic layer was washed with water (10 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The crude product was purified by silica gel column chromatography {hexane / ethyl acetate = 50:1} to give acyclic retinoid 4b (43 mg, 0.13 mmol, 26%) as a yellow oil. 1H NMR (400 MHz, CDCl3), 1.27 (t, J = 7.2 Hz, 3H, CH3), 1.58 (s, 3H, CH3), 1.59 (d, J = 0.8 Hz, 3H, CH3), 1.66 (d, J = 0.8 Hz, 3H, CH3), 1.84 (d, J = 0.8 Hz, 3H, CH3), 1.90-2.20 (m, 8H, 4CH2), 2.31 (d, J = 1.2 Hz, 3H, CH3), 4.15 (q, J = 7.2 Hz, 2H, CH2), 5.00-5.20 (m, 2H, 2C=CH), 5.73 (s, 1H, C=CH), 5.95 (d, J = 11.2 Hz, 1H, C=CH), 6.16 (d, J = 15.6 Hz, 1H, C=CH), 6.83 (dd, J = 15.6 11.2, Hz, 1H, C=CH); 13 C NMR (100 MHz, CDCl3), 13.88, 14.44, 16.10, 17.24, 17.77, 25.78, 26.49, 26.77, 39.76, 40.33, 59.67, 118.12, 123.62, 124.35, 125.02, 131.10, 131.43, 133.52, 135.70, 143.97, 153.09, 167.33; HR-MS (EI+, 100% acetone): m / z: calcd for C 22 H 34 O2([M-C4H9] + ) 330.2559; found, 330.2561.

[0035] (2E, 4E, 6E, 10E)-3,7,11,15-Turnus-2,4,6,10,14-Turnus acid メチルエステル4a synthesis Compound (4a) was synthesized from compound 14b (35.5 mg, 0.104 mmol) and (E)-3-methyl-4-oxo-2-butenoic acid methyl ester ((E)-4-hydroxy-3-methyl-2-butenoic acid) using a procedure similar to that for the synthesis of acyclic retinoid (4b), to give compound 4a (18.0 mg, 56.9 μmol, 55%) as a yellow oil. 1 H NMR (400 MHz, CDCl3), 1.58 (s, 3H, CH3), 1.59 (d, J = 0.8 Hz, 3H, CH3), 1.66 (s, 3H, CH3), 1.84 (s, 3H, CH3), 1.90-2.20 (m, 8H, 4CH2), 2.32 (d, J = 0.8 Hz, 3H, CH3), 3.69 (s, 3H, CH3), 5.00-5.20 (m, 2H, 2C=CH), 5.73 (s, 1H, C=CH), 5.95 (d, J = 11.6 Hz, 1H, C=CH), 6.17 (d, J = 14.8 Hz, 1H, C=CH), 6.83 (dd, J = 14.8, 11.6 Hz, 1H, C=CH); 13 C NMR (100 MHz, CDCl3), 13.90, 16.10, 17.26, 17.77, 25.78, 26.49, 26.77, 39.76, 40.33, 51.01, 117.58, 123.61, 124.35, 125.00, 131.29, 131.45, 133.43, 135.71, 144.15, 153.43, 167.75; HR-MS (EI+, 100% acetone): m / z: calcd for C 21 H 32 O2(M + ) 316.2402; found, 316.2415.

[0036] Synthesis of (2E,4E,6E,10E)-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid benzyl ester 4c 4,5-didehydrogeranylgeranoic acid (18.2 mg, 60.2 μmol) and dry DMF (600 μL) were placed in a 5 mL test tube, and K2CO3 (12 mg, 90 μmol) and benzyl bromide (7.8 μL, 11 mg, 66 μmol) were added. After stirring at 50 °C for 2 h, the resulting mixture was cooled to room temperature. The mixture was extracted with diethyl ether (3 × 3 mL), and the extract was washed with saturated saline and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography {hexane / acetone = 100:1} to give compound 4c (16.8 mg, 42.8 μmol, 71%) as a yellow oil. 1 H NMR (400 MHz, CDCl3), 1.58 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.84 (s, 3H, CH3), 1.90-2.20 (m, 8H, 4CH2), 2.34 (s, 3H, CH3), 5.02-5.14 (m, 2H, 2C=CH), 5.15 (s, 2H, CH2), 5.79 (s, 1H, C=CH), 5.95 (d, J = 11.2 Hz, 1H, C=CH), 6.16 (d, J = 15.2 Hz, 1H, C=CH), 6.89 (dd, J = 15.2, 11.2 Hz, 1H, C=CH), 7.26-7.42 (m, 5H, C6H5); 13 C NMR (100 MHz, CDCl3), 14.01, 16.11, 17.27, 17.77, 25.78, 26.49, 26.77, 39.76, 40.33, 65.61, 117.63, 123.60, 124.34, 125.01, 128.12, 128.23 (2C), 128.59 (2C), 131.42 (2C), 133.40, 135.73, 136.57, 144.30, 153.89, 167.05; HR-MS (EI+, 100% acetone): m / z: calcd for C 27 H 36 O2(M +) 392.2715; found, 392.2734.

[0037] Organotin compounds 15a, 15b and 15c were synthesized according to the following synthesis route. [ka]

[0038] Synthesis of (Z)-4-(tert-butyl-dimethylsilyloxy)-3-bromo-2-butenoic acid ethyl ester 9b (2Z)-3-Bromo-4-hydroxy-2-butenoic acid ethyl ester (8b) was synthesized according to the published literature procedure (M. Shengming, L. Xiyan, Org. Synth. 1995, 72, 112-115.). 2 A 50 mL round-bottom flask was charged with 5a (1.56 g, 7.46 mmol) and dry DMF (10 mL), cooled to 0° C., and imidazole (663 mg, 9.75 mmol) and tert-butyldimethylsilyl chloride (1.24 g, 8.25 mmol) were added. After stirring at room temperature for 4 h, the reaction mixture was quenched with water (10 mL) and extracted with diethyl ether (3×10 mL). The organic layer was washed with water (30 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was subjected to silica gel column chromatography {hexane / ethyl acetate=10:1} to give compound 9b (2.40 g, 7.41 mmol, 99%) as a yellow oil. 1 H NMR (400 MHz, CDCl3), 0.10 (s, 6H, 2CH3), 0.92 (s, 9H, 3CH3), 1.30 (t, J = 7.2 Hz, 3H, CH3), 4.22 (q, J = 7.2 Hz, 2H, CH2), 4.29 (d, J = 2.4 Hz, 2H, CH2), 6.69 (t, J = 2.4 Hz, 1H, C=CH); 13C NMR (100 MHz, CDCl3), -5.38 (2C), 14.30, 18.38, 25.84 (3C), 60.66, 68.50, 117.16, 139.42, 164.59; HR-MS (EI+, 100% acetone): m / z: calcd for C8H 14 79 BrO3Si ([M-C4H9] + ) 264.9896; found, 264.9905.

[0039] Synthesis of (Z)-4-(tert-butyl-dimethylsilyloxy)-3-(tributylstannyl)-2-butenoic acid ethyl ester 10b A 50 mL two-neck round-bottom flask was charged with copper cyanide (537 mg, 6 mmol) and dry THF (10 mL), and n-butyllithium solution (1.89 M in hexane, 3.17 mL, 6.00 mmol) was added dropwise at -78 °C, and the mixture was stirred for 15 min. Bis(tributyltin) (3.03 mL, 6.00 mmol) was added dropwise to the mixture at -78 °C. After stirring at 0 °C for 1 h, a solution of bromide 9b (646 mg, 2.00 mmol) in dry THF (10 mL) was added to the resulting mixture of copper ate complexes at -78 °C, and the mixture was stirred for 30 min. The mixture was quenched with brine, NH4Cl aq (10 mL), and 28% NH4OH at -78 °C. The resulting mixture was extracted with diethyl ether (3 × 10 mL), and the extract was washed with water (30 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure. The crude product was subjected to silica gel column chromatography {hexane / ethyl acetate=150:1} to give compound 10b (995 mg, 1.87 mmol, 94%) as a yellow oil. 1H NMR (400 MHz, CDCl3), 0.07 (s, 6H, 2CH3), 0.86 (t, J = 7.2 Hz, 9H, 3CH3), 0.90-0.98 (m, 6H, 3CH2), 0.92 (s, 9H, 3CH3), 1.20-1.34 (m, 9H, 3CH2and CH3), 1.35-1.51 (m, 6H, 3CH2), 4.18 (q, J = 7.2 Hz, 2H, CH2), 4.26-2.26 (m, 2H, CH2), 6.56-6.82 (m, 1H, C=CH); 13 C NMR(100 MHz, CDCl3),-5.32 (2C), 10.89 (3C), 13.81 (3C), 14.43, 18.61, 26.05 (3C), 27.48 (3C), 29.29 (3C), 60.32, 68.70, 124.70, 168.40, 171.93; HR-MS (EI+, 100% acetone): m / z: calcd for C 20 H 41 O3Si 120 Sn ([M-C4H9] + ) 477.1847; found, 477.1824.

[0040] Synthesis of (Z)-4-hydroxy-3-(tributylstannyl)-2-butenoic acid ethyl ester (11b) In a 50 mL round-bottom flask, 10b (687 mg, 1.29 mmol) and dry DMSO (4 mL) were added, and 18-crown-6-ether (1.03 g, 3.90 mmol) and potassium fluoride (226 mg, 3.89 mmol) were added at 0 °C. After stirring at room temperature for 3.5 h, phosphate buffer (pH 7.4) was added at 0 °C, and the mixture was extracted with diethyl ether (3 × 10 mL). The extract was washed with water (30 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was separated by silica gel column chromatography {hexane / ethyl acetate = 15:1} to give compound 11b (425 mg, 1.01 mmol, 78%) as a yellow oil. 1H NMR (400 MHz, CDCl3), 0.86 (t, J = 7.2 Hz, 9H, 3CH3), 0.90-1.07 (m, 6H, 3CH2), 1.20-1.34 (m, 9H, 3CH2and CH3), 1.34-1.54 (m, 6H, 3CH2), 1.61 (t, J = 6.0 Hz, 1H, OH), 4.18 (q, J = 7.2 Hz, 2H, CH2), 4.44-4.49 (m, 2H, CH2), 6.53-6.80 (m, 1H, C=CH); 13 C NMR (100 MHz, CDCl3), 10.97 (3C), 13.79 (3C), 14.40, 27.48 (3C), 29.25 (3C), 60.47, 68.56, 124.77, 168.06, 172.25; HR-MS (EI+, 100% acetone): m / z: calcd for C 14 H 27 O3 120 Sn ([M-C4H9] + ) 363.0982; found, 363.1002.

[0041] Synthesis of (2E,4E,6E,10E)-7,11,15-trimethyl-3-(tributylstannyl)-2,4,6,10,14-hexadecapentaenoic acid ethyl ester (15b) A 20 mL round-bottom flask was charged with 11b (168 mg, 0.401 mmol) and hexane (3 mL). To the solution was added MnO2 (1.04 g, 12.0 mmol). The resulting suspension was stirred at 40 °C for 1.5 h and then passed through a Celite pad. The filtrate was evaporated to give (Z)-4-oxo-3-(tributylstannyl)-2-butenoic acid ethyl ester (12b) as a yellow oil. 12b was used without further purification. A 30 mL two-neck round-bottom flask was charged with 1 (137 mg, 0.328 mmol) and dry THF (2 mL). The solution was cooled to -78 °C in a dry ice / acetone bath and potassium bis(trimethylsilyl)amide (1.0 M in THF, 400 μL, 0.4 mmol) was added at this temperature. The reaction mixture was stirred for 1 h and 12b dissolved in THF (2 mL) at -78 °C was added dropwise. After stirring at RT for 1 h, the resulting mixture was cooled to 0 °C, phosphate buffer (pH 7.4) was added, and extracted with diethyl ether (3 × 5 mL). The organic layer was washed with water (10 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure. The crude product was subjected to silica gel column chromatography (hexane only) to give compound 15b (18.6 mg, 30.7 μmol, 7.7%) as a yellow oil. 1H NMR (400 MHz, CDCl3), 0.86 (t, J = 7.4 Hz, 9H, 3CH3), 0.91-1.12 (m, 6H, 3CH2), 1.20-1.35 (m, 9H, 3CH2, CH3), 1.35-1.57 (m, 6H, 3CH2), 1.59 (s, 3H, CH3), 1.60 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.82 (s, 3H, CH3), 1.90-2.20 (m, 8H, 4CH2), 4.17 (q, J = 7.4 Hz, 2H, CH2), 5.00-5.20 (m, 2H, 2C=CH), 5.92 (d, J = 10.8 Hz, 1H, C=CH), 6.44 (d, J = 15.6 Hz, 1H, C=CH), 6.46 (s, 1H, C=CH), 6.71 (dd, J = 15.6, 11.4 Hz, 1H, C=CH); 13 C NMR (100 MHz, CDCl3), 12.43 (3C), 13.79 (3C), 14.46, 16.10, 17.24, 17.77, 25.78, 26.57, 26.79, 27.48 (3C), 29.21 (3C), 39.78, HR-MS (EI+, 100% acetone): m / z: calcd for C 29 H 49 O2 120 Sn ([M-C4H9] + ) 549.2755; found, 549.2747.

[0042] Synthesis of (2E,4E,6E,10E)-7,11,15-trimethyl-3-(tributylstannyl)-2,4,6,10,16-hexadecapentaenoic acid methyl ester 15a and ethyl ester 15b 15a was synthesized from (2E,6E)-3,7,11-trimethyl-2,6,10-(dodecatrienyl)phosphonic acid dimethyl ester (14a) (11.7 mg, 37.2 μmol) and (Z)-4-oxo- (13.8 mg, 34.2 μmol) (7.9 mg, 13.4 μmol, 39%). 15b was also synthesized from 3-(tributylstannyl)-2-butenoic acid methyl ester ((Z)-4-hydroxy-3-(tributylstannyl)-2-butenoic acid methyl ester (127 mg, 0.313 mmol) by a similar procedure to give 15b as a yellow oil (10.0 mg, 17.0 μmol, 15%). 1 H NMR (400 MHz, CDCl3), 0.86 (t, J = 7.4 Hz, 9H, 3CH3), 0.93-1.10 (m, 6H, 3CH2), 1.24-1.33 (m, 6H, 3CH2), 1.42-1.51 (m, 6H, 3CH2), 1.585 (s, 3H, CH3), 1.594 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.82 (s, 3H, CH3), 1.93-2.26 (m, 8H, 4CH2), 3.72 (s, 3H, CH3), 5.06-5.10 (m, 2H, 2C=CH), 5.92 (d, J = 10.8 Hz, 1H, C=CH), 6.44 (d, J = 15.6 Hz, 1H, C=CH), 6.46 (s, 1H, C=CH), 6.71 (dd, J = 15.6, 11.4 Hz, 1H, C=CH); 13 C NMR (100 MHz, CDCl3), 12.40 (3C), 13.81 (3C), 16.10, 17.26, 17.77, 25.80, 26.55, 26.79, 27.48 (3C), 29.21 (3C), 39.80, 40.29, HR-MS (EI+, 100% acetone): m / z: calcd for C 28 H47 O3 120 Sn ([M-C4H9] + ) 535.2598; found, 535.2603.

[0043] Synthesis of (2E,4E,6E,10E)-7,11,15-trimethyl-3-(tributylstannyl)-2,4,6,10,14-hexadecapentaenoic acid 16 A 5 mL test tube was charged with a mixture of 15a and 15b (1:1 molar ratio, 22.7 mg, 38.4 μmol), methanol (200 μL), and 2-propanol (200 μL). After adding 10 M KOH in CH3OH / H2O (2:1, v / v), 100 μL, 1.00 mmol, the reaction mixture was stirred at 100 °C for 30 min. After neutralization with formic acid (1 mL), the resulting mixture was extracted with diethyl ether (3 × 3 mL), and the extract was washed with ethanol (10 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure. The crude product was subjected to silica gel column chromatography {hexane / ethyl acetate = 10:1} to give a yellow oil (15.6 mg, 27.0 μmol, 71%). 1 H NMR (400 MHz, CDCl3), 0.86 (t, J = 7.4 Hz, 9H, 3CH3), 0.91-1.12 (m, 6H, 3CH2), 1.20-1.35 (m, 6H, 3CH2), 1.35-1.54 (m, 6H, 3CH2), 1.59 (s, 3H, CH3), 1.60 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.83 (s, 3H, CH3), 1.90-2.20 (m, 8H, 4CH2), 5.02-5.20 (m, 2H, 2C=CH), 5.93 (d, J = 11.6 Hz, 1H, C=CH), 6.47 (d, J = 16.0 Hz, 1H, C=CH), 6.50 (s, 1H, C=CH), 6.71 (dd, J = 16.0, 11.6 Hz, 1H, C=CH), 8.82-9,78 (br s, 1H, COOH).

[0044] Synthesis of (2E,4E,6E,10E)-7,11,15-trimethyl-3-(tributylstannyl)-2,4,6,10,14-hexadecapentaenoic acid benzyl ester 15c In a 5 mL test tube, (2E,4E,6E,10E)-7,11,15-trimethyl-3-(tributylstannyl)-2,4,6,10,14-hexadecapentaenoic acid (15.6 mg, 27.0 μmol) and dry DMF (200 μL). To the solution was added K2CO3 (5.60 mg, 40.5 μmol) and benzyl bromide (3.50 μL, 5.08 mg, 29.7 μmol). After stirring at 50 °C for 1 h, the resulting mixture was cooled to room temperature. The mixture was extracted with diethyl ether (3 × 3 mL), and the extract was washed with brine (10 mL) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography {hexane / acetone = 100:1} to give compound 15c (13.5 mg, 20.2 μmol, 75%) as a yellow oil. 1 H NMR (400 MHz, CDCl3), 0.86 (t, J = 7.6 Hz, 9H, 3CH3), 0.91-1.12 (m, 6H, 3CH2), 1.20-1.35 (m, 6H, 3CH2), 1.35-1.54 (m, 6H, 3CH2), 1.58 (s, 3H, CH3), 1.59 (d, J = 0.8 Hz, 3H, CH3), 1.66 (s, 3H, CH3), 1.81 (s, 3H, CH3), 1.90-2.19 (m, 8H, 4CH2), 5.02-5.14 (m, 2H, 2C=CH), 5.17 (s, 2H, CH2), 5.91 (d, J = 11.6 Hz, 1H, C=CH), 6.44 (d, J = 15.2 Hz, 1H, C=CH), 6.52 (s, 1H, C=CH), 6.72 (dd, J = 15.2, 11.6 Hz, 1H, C=CH), 7.27-7.42 (m, 5H, C6H5); 13C NMR (100 MHz, CDCl3), 12.45 (3C), 13.83 (3C), 16.10, 17.28, 17.77, 22.80, 26.55, 26.79, 27.50 (3C), 29.22 (3C), 39.80, 40.31, 66.10, 123.72, 124.37, 125.44, 127.29, 128.18, 128.32 (2C), 128.59 (2C), 131.43, 132.63, 135.64, 136.16, 136.38, 143.25, 168.23, 169.37; HR-MS (EI+, 100% acetone): m / z: calcd for C 34 H 51 O2 120 Sn ([M-C4H9] + ) 611.2911; found, 611.2915.

[0045] The following synthesis method is used: 11 C]-4a, [ 11 C]-4b,[ 11 C]-4c and [ 11 C]-3 was synthesized. [ka]

[0046] Synthesis of (2E,4E,6E,10E)-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid ethyl ester 4b by C-methylation with non-radioactive methyl iodide A 1 mL Schlenk flask was charged with argon and charged with [Pd2(dba)3] (11.5 mg, 12.6 μmol), P(o-tolyl)3 (27.7 mg, 91.1 μmol), CuBr (6.50 mg, 45.3 μmol), and CsF (17.3 mg, 114 μmol). A solution of stannyl precursor 15b (13.3 mg, 22.0 μmol) in DMF (2 mL) was transferred to the Schlenk flask through a stainless steel cannula at -10 °C, and methyl iodide (220 μL, 0.10 M in DMF, 22 μmol) was added. The mixture was heated at 60 °C for 1 h, and the resulting mixture was quenched with water and extracted with diethyl ether (3 × 3 mL). The extracted organic layers were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by silica gel column chromatography {hexane / ethyl acetate=100:1} to give acyclic retinoid 4b as a pale yellow oil (5.80 mg, 17.5 μmol, 80%). 1 H NMR (400 MHz, CDCl3), 1.27 (t, J = 7.2 Hz, 3H, CH3), 1.58 (s, 3H, CH3), 1.59 (d, J = 0.8 Hz, 3H, CH3), 1.66 (d, J = 0.8 Hz, 3H, CH3), 1.84 (d, J = 0.8 Hz, 3H, CH3), 1.90-2.20 (m, 8H, 4CH2), 2.32 (d, J = 0.8 Hz, 3H, CH3), 4.15 (q, J = 7.2 Hz, 2H, CH2), 5.00-5.17 (m, 2H, 2C=CH), 5.73 (s, 1H, C=CH), 5.95 (d, J = 11.2 Hz, 1H, C=CH), 6.16 (d, J = 15.6 Hz, 1H, C=CH), 6.83 (dd, J = 15.6, 11.2 Hz, 1H, C=CH).

[0047] Rapid C-[ 11 C]methylation 11 C] Synthesis of 4c [Pd2(dba)3] (1.0 mg, 1.1 μmol), P(o-tolyl)3 (2.4 mg, 8 μmol), CuBr (0.6 mg, 4 μmol), and CsF (1.5 mg, 10 μmol) in DMF (400 μL) were mixed to give the stannyl precursor 15c (1.1 mg, 1.6 μmol) in [ 11 C] The mixture was cooled to below -10 °C while waiting for the preparation of CH3I. 11 C] formed from CO2 using the conventional LiAlH4 method [ 11 [C] CH3I was trapped in the reaction mixture, cooled, then rapidly heated to 65 °C, left for 2 min, and then bubbled with N2 gas for 2 min. The reaction mixture was cooled for 10 s to avoid bumping. After dilution with CH3CN (600 μL), the mixture was passed through a fine filter F (F162, Forte Grow Medical co., ltd.) with quartz glass wool (Tosho co., ltd.) and analyzed by preparative HPLC (mobile phase, CH3CN only; column, CAPCELL PAK C18 UG 120 5 μm, 10 mm (inner diameter) × 250 mm, flow rate, 5 mL / min, UV detection, 280 nm, retention time, 7.37-8.31 min). The HPLC analytical yield was calculated to be 97% from the peak area of ​​radio-HPLC of the reaction mixture. The desired fraction was collected in a flask, and the organic solvent was removed under reduced pressure. The desired radiotracer was dissolved in a 0.25% solution of polysorbate 80 in saline (3.0 mL). The total synthesis time, including purification by HPLC and preparation of the radiopharmaceutical formulation for intravenous administration, was 31 min. The isolated radioactivity was 2.87 GBq at the end of the synthesis, with a specific activity of 144 GBqμmol -1 The decay-corrected radiochemical yield was 57%, which was determined by the amount of [ 11 C] Calculated based on the radioactivity of CH3I. 11The identity of [C] 4c was confirmed by co-injection with an authentic sample of non-radiolabeled 4c by analytical HPLC (mobile phase, CH3CN only; column, CAPCELL PAK C18, 4.6 mm (id) × 250 mm; flow rate, 1 mL / min; UV detection, 280 nm; retention time, 8.0 min). Chemical and radiochemical purity analyzed at 280 nm were > 99% and > 97%, respectively.

[0048] Rapid [ 11 C]methylation 11 Synthesis of C-3 [Pd2(dba)3] (1.0 mg, 1.1 μmol), P(o-tolyl)3 (2.4 mg, 8 μmol), CuBr (0.6 mg, 4 μmol), CsF (1.5 mg, 10 μmol), and the stannyl precursor 15a (0.9 mg, 1.5 μmol) in DMF (300 μL) were reacted to give [ 11 C] The mixture was cooled to below -10°C while waiting for the preparation of CH3I. 11 C] formed from CO2 using the conventional LiAlH4 method [ 11C] CH3I was trapped in the reaction mixture, cooling was stopped, then it was rapidly heated to 65 °C and left for 2 min before bubbling N2 gas for 2 min. The reaction mixture was cooled for 10 s to avoid bumping. 8 M KOHaq. (500 μL) solution was added to the mixture, after which N2 gas was bubbled at 100 °C for 6 min. After cooling for 10 s, the reaction mixture was diluted with CH3CN / CH3CN / H2O (60:36:4 v / v / v, 1 mL) containing sodium ascorbate (2.2 mg, 11 μmol), and the mixture was purified using a fine filter F (F162, Forte Grow Medical co., ltd.) and quartz glass wool (Tosho co., ltd.) and preparative HPLC (mobile phase, CH3CN / 0.2%HCOOH in water = 90:10; column, CAPCELL PAK C18 UG 120 5 μm, 10(id)×250 mm, flow rate, 5 mL / min, UV detection, 280 nm, retention time, 8:45-9:32 min). The HPLC analytical yield (%) was calculated from the radio-HPLC peak area of ​​the reaction mixture. The desired fraction was collected in a flask, and the organic solvent was removed under reduced pressure. The desired radiotracer was dissolved in a 0.25% solution of polysorbate 80 in saline (3.0 mL). The total synthesis time, including HPLC purification and preparation of the radiopharmaceutical formulation for intravenous administration, was 40 min. The isolated radioactivity was 0.94 GBq at the end of the synthesis, with a specific activity of 186 GBq μmol -1 The decay-corrected radiochemical yield was 25%, which was trapped in solution. 11 C] was calculated based on the radioactivity of CH3I. 11 The chemical identity of [C]3 was confirmed by co-injection with a sample of non-radiolabeled 3 by analytical HPLC (mobile phase, CH3CN / 0.2% HCOOH in water = 90:10; column, CAPCELL PAK C18, 4.6(id) × 250 mm; flow rate, 1 mL / min; UV detection, 280 nm; retention time, 7.7 min). The chemical and radiochemical purity analyzed at 280 nm were 83% and 99%, respectively.

[0049] The above synthesis was carried out.11 C]-4a, 11 C]-4b, 11 C]-4c and 11 The radioactivity, molar activity of the radioactivity, and decay-corrected radiochemical yield of C]-3 are shown in Table 1. From this table 11 C]-4a, 11 C]-4b, 11 C]-4c and 11 C]-3 are found to have sufficient radioactivity for use as a PET probe.

Table 1

[0050] <Synthesis of peretinoin 3> 11 Peretinoin 3 not labeled with C was synthesized by applying the Horner-Wadsworth-Emmons (HWE) reaction, which enables the formation of an E-isomer-selective olefin, from phosphate 14a, which was the precursor when synthesizing C]-3. 11 First, trans,trans-farnesol 16 was converted to chloride 17 (yield 51%), and then phosphate 14a was synthesized in a yield of 64% by the Michaelis-Arbuzov reaction using trimethyl phosphite. On the other hand, aldehyde 20, which is the other compound for the synthesis of the target acyclic retinoid, was synthesized as follows. That is, hydroxyester 19 of the E-isomer was synthesized in a yield of 57% by the Wittig olefin synthesis using hydroxyacetone 18 as a starting material, and aldehyde 20 that retained the E-configuration was synthesized in a yield of 43% by oxidation using manganese dioxide.

Chemical formula

[0051] On the other hand, aldehyde 20, which is the other compound for the synthesis of the target acyclic retinoid, was synthesized as follows. That is, hydroxyester 19 of the E-isomer was synthesized in a yield of 57% by the Wittig olefin synthesis using hydroxyacetone 18 as a starting material, and aldehyde 20 that retained the E-configuration was synthesized in a yield of 43% by oxidation using manganese dioxide.

Chemical formula

[0052] The phosphate 14a synthesized by the above synthesis method was combined with the aldehyde 20 via a carbon-carbon double bond by the HWE reaction to obtain peretinoin 3. That is, potassium hexamethyldisilazide (KHMDS) was selected as a base to prepare the ylide from 14a, and the methyl ester 4a of 4,5-didehydro GGA (peretinoin) was synthesized under the conditions of 14a / KHMDS / 14 (1:1:1 molar ratio), and this was hydrolyzed with a strong base to synthesize the desired peretinoin 3 in 26% yield in three steps. [ka]

[0053] The synthesis procedure and analytical data of peretinoin 3 are described in detail below. (2E,6E)-1-Chloro-3,7,11-trimethyldodeca-2,6,10-triene (17) 1 H NMR (400 MHz, CDCl3): δ= 1.60 (s, 6H, 2CH3), 1.68 (s, 3H CH3), 1.73 (s, 3H, CH3), 1.98-2.11 (m, 8H, 4CH2), 4.10 (d, J = 7.6 Hz, CH2), 5.06-5.14 (m, 2H, olefine), 5.45 (t, J = 7.6 Hz, olefine).

[0054] Dimethyl ((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)phosphonate (14a) 1H NMR (400 MHz, CDCl3): δ= 1.60 (s, 6H, 2CH3), 1.66 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.95-2.10 (m, 8H, 4CH2), 2.58 (dd, J = 22.0, 7.6 Hz, CH2), 3.74 (d, J = 10.4 Hz, 6H, 2CH3O), 5.08-5.20 (m, 3H, olefines); 13 C NMR (100 MHz, CDCl3): δ = 16.00, 16.26 (d, J P-C = 1.9 Hz) 17.67, 25.45 (d, J P-C = 139.2 Hz), 25.69 (2C), 26.35 (d, J P-C = 3.8 Hz), 26.70, 39.69, 52.57 (d, J P-C = 6.7 Hz, 2C), 111.98 (d, J P-C = 10.5 Hz), 123.72, 124.28, 131.32, 135.32, 140.57 (d, J P-C = 14.3 Hz).

[0055] Methyl (E)-4-hydroxy-3-methylbut-2-enoate (19) Hydroxyacetone (6.16 mL, 6.65 g, 89.8 mmol), benzene (90 mL), and (triphenylphosphoranylidene)methyl acetate (30.0 g, 89.8 mmol) were added to a 300-mL recovery flask in that order at room temperature, and the mixture was heated under reflux for 24 h. After concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3 / 1) to give the desired product 19 (6.63 g, 51.0 mmol, 57%) as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ= 2.10 (s, 3H, CH3), 3.72 (s, 3H, CH3), 4.15-4.16 (m, 2H, CH2), 5.99-6.01 (m, 1H, olefine);13 C NMR (100 MHz, CDCl3): δ = 15.60, 51.01, 67.04, 113.24, 157.50 167.19.

[0056] Methyl (E)-3-methyl-4-oxobut-2-enoate (20) 13 (6.45 g, 49.6 mmol) was added to a 1-L recovery flask and dissolved in hexane (500 mL), then manganese dioxide (129 g, 1.48 mol) was added and stirred at room temperature for 1 hour. The oxidizing agent was then removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3 / 1) to obtain the target compound 20 (2.75 g, 21.5 mmol, 43%) as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ= 2.18 (d, 1.2 Hz, 3H, CH3), 3.84 (s, 3H, CH3), 6.52 (t, 1.2 Hz, 1H, olefine), 9.57 (s, 1H, CHO); 13 C NMR (100 MHz, CDCl3): δ = 10.85, 52.00, 135.00, 150.70, 165.92, 194.43.

[0057] (2E,4E,6E,10E)-3,7,11,15-tetramethylhexadeca-2,4,6,10,14-pentaenoic acid (3) A solution of 14a (6.12 g, 19.5 mmol) in THF (170 mL) was added to two 250-mL Schlenk flasks under an argon atmosphere, and potassium hexamethyldisilazane (1.0 M in THF, 19.5 mL, 19.5 mmol) was added at -78 °C. After stirring for 30 min, 20 (2.49 g, 19.5 mmol) dissolved in THF (30 mL) was added at -78 °C, and the mixture was stirred for 15 h while gradually warming to room temperature. Then, saturated aqueous ammonium chloride solution (100 mL) was added under ice cooling, and the mixture was extracted with ethyl acetate (3 × 40 mL). The organic layer was then washed with saturated saline (100 mL) and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 50 / 1) to obtain the crude product (3.91 g). The crude product (3.91 g) was added to a 100-mL eggplant flask and dissolved in 2-propanol (24 mL), then 10 M potassium hydroxide solution (40 mL) was added and the mixture was heated to reflux for 1.5 hours. Formic acid (32 mL) was then added under ice cooling, and the mixture was concentrated under reduced pressure, followed by extraction with ethyl acetate (3× 20 mL). The organic layer was then washed with saturated saline (40 mL) and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was dissolved in hexane (8.7 ml) and crystallized at −20 °C. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10 / 1) and combined with the product obtained by recrystallization to obtain peretinoin 3 (1.50 g, 4.97 mmol, 26%, 3 steps) as pale yellow needle crystals. 1H NMR (400 MHz, CDCl3): δ= 1.60 (s, 3H, CH3), 1.61 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.86 (s, 3H, CH3), 1.98-2.16 (m, 8H, 2CH2), 2.34 (s, 3H, CH3), 5.09-5.10 (m, 2H, olefines), 5.77 (s, 1H, olefine), 5.98 (d, J = 10.8 Hz, 1H, olefine), 6.21 (d, J = 14.8 Hz, 1H, olefine), 6.90 (dd, J = 15.2, 10.8Hz, 1H, olefin), 9.35-11.97 (br s, 1H, COOH).

[0058] < 11 Pharmacokinetic study of C-labeled acyclic retinoids Synthesized by the method described above 11 C-labeled acyclic retinoid 4c and 11 C-labeled peretinoin 3 was administered intravenously (iv) as a PET probe to Sprague-Dawley rats and cynomolgus monkeys, and PET images were taken.

[0059] · 11 When C-labeled peretinoin 3 was administered 11 The PET images of C-labeled peretinoin 3 administered to rats are shown in Figure 1(a), and those of C-labeled peretinoin 3 administered to cynomolgus monkeys are shown in Figure 2(a). 11 C-labeled peretinoin 3 was found to penetrate into the brain of both rats and cynomolgus monkeys. In addition, the measurement of the intensity of radioactivity in the brain over time shown in Figure 1(b) and Figure 2(b) showed that 11 C-labeled peretinoin 3 begins to migrate into the brain within a few minutes, and its concentration in the brain continues to increase over time, accumulating to a concentration 1.3 times higher in rats and 3.0 times higher in cynomolgus monkeys than when it is distributed uniformly throughout the body. 11It was revealed that C-labeled peretinoin 3 is a substance that rapidly penetrates the blood-brain barrier and accumulates in the brain. 11 C-labeled peretinoin 3 was found to be particularly suitable as a PET probe for brain imaging.

[0060] · 11 When C-labeled acyclic retinoid 4c was administered 11 PET images of C-labeled acyclic retinoid 4c administered to rats are shown in Figure 3(a), and PET images of C-labeled acyclic retinoid 4c administered to cynomolgus monkeys are shown in Figure 4(a). 11 C-labeled acyclic retinoid 4c was found to be transported into the brain in both animal species. 11 It was found that C-labeled acyclic retinoid 4c can be transported into the brain and can be used as a PET probe for brain imaging. In addition, the measurement of radioactivity in the brain over time, shown in Figure 3(b) and Figure 4(b), revealed that the benzyl ester 11 C-labeled acyclic retinoid 4c is the free carboxylic acid 11 Compared to C-labeled peretinoin 3, it was found that it was difficult for it to penetrate into the brain immediately after administration, and that it began to penetrate into the brain after 10 minutes or more had passed. The reason for this is that it is a benzyl ester. 11 C-labeled acyclic retinoid 4c is the free carboxylic acid 11 It is presumed that it is metabolized to C-labeled peretinoin 3 and then transported to the brain.

[0061] · 11 When C-labeled acyclic retinoid 4b was administered 11 The PET image of C-labeled acyclic retinoid 4b administered to a rat is shown in Figure 5a. 11 It was found that C-labeled acyclic retinoid 4b can penetrate into the rat brain, is useful as a PET probe, and can be used for brain imaging. In addition, the measurement of intracerebral radioactivity shown in Figure 5(b) indicates that the ethyl ester form 11C-labeled acyclic retinoid 4b is a free carboxylic acid 11 Compared with C-labeled peretinoin 3, it was found that it was difficult to migrate into the brain immediately after administration, and the migration into the brain began after more than 10 minutes. The reason is that it is an ethyl ester form 11 C-labeled acyclic retinoid 4b is a free carboxylic acid 11 It was presumed that it might migrate into the brain after being metabolized to C-labeled peretinoin 3

[0062] <The effects of peretinoin 3> 11 The effects of peretinoin 3, an unlabeled acyclic retinoid, were evaluated

[0063] (Inhibitory effect on thrombin toxicity) Thrombin is known to induce cerebral cortical cell death and striatal tissue atrophy. Therefore, we performed an experiment to evaluate thrombin toxicity. Specifically, 300 μm-thick coronal sections were prepared from the brains of 2-3-day-old Wistar / ST rats, and fan-shaped tissue pieces containing the cerebral cortex and striatum were excised and placed on a porous membrane (Millicell-CM, Millipore) and cultured at the air-liquid interface under 34°C / 5% CO2 conditions (medium composition: 50% minimal essential medium, 25% Hanks' balanced salt solution, 25% horse serum). On the 12th day of culture, the medium was replaced with serum-free medium (medium composition: 75% minimal essential medium, 25% Hanks' balanced salt solution) containing 5 μg / ml propidium iodide (PI), and drug treatments such as peretinoin were started. 24 hours later, thrombin (100 U / ml) was added to the medium. 72 hours after the start of thrombin treatment, PI fluorescence images were obtained using a fluorescence microscope (Keyence), and the degree of cell death was evaluated using the fluorescence intensity of the cortical region as an index (100 μM NMDA-treated sections were prepared for each experiment for 72 hours, and the PI fluorescence intensity was standardized as 100%). In addition, the area of ​​the striatal region in each tissue slice was measured from bright-field images of the cultured tissue slices obtained before and after thrombin treatment, and the tissue atrophy rate due to thrombin treatment was quantified.

[0064] ·Results PI fluorescence images taken 72 hours after the start of thrombin treatment are shown in FIG. 6. From this figure, it was found that when thrombin was administered, the fluorescence weakened as the concentration of peretinoin 3 was increased to 10 μM, 20 μM, and 30 μM, and that brain tissue damage caused by thrombin was suppressed by peretinoin 3. The percentage of thrombin-induced cerebral cortical cell death and the percentage of striatal tissue atrophy calculated from these images are shown in FIG. 7 and FIG. 8. From the graphs in FIG. 7 and FIG. 8, it was found that thrombin-induced cerebral cortical cell death and striatal tissue atrophy were significantly suppressed by peretinoin 3 in a concentration-dependent manner in the cerebral cortex-striatum tissue sections.

[0065] (Evaluation using K252a) K252a (3 μM) is known to inhibit TrkB, a receptor for the neurotrophic factor BDNF. Therefore, we investigated how K252a affects the cytoprotective effect of peretinoin 3 using the same method as that used to examine its effect on thrombin toxicity.

[0066] ·Results Figure 9 shows PI fluorescence images after the experiment under the conditions of control, administration of thrombin only, administration of thrombin + peretinoin 3, administration of thrombin + peretinoin 3, administration of thrombin + peretinoin 3 + K252a, administration of thrombin + K252a, and administration of K252a only. Furthermore, the percentage of cerebral cortical cell death and the percentage of striatal tissue atrophy obtained from these images are shown in Figure 10 and Figure 11, respectively. The following was found from Figures 9, 10, and 11. That is, 1) the protective effect of peretinoin 3 on cerebral cortical cells was blocked by K252a, suggesting the involvement of the BDNF-TrkB signaling system. 2) The inhibitory effect of peretinoin 3 on striatal atrophy was not blocked by K252a, suggesting the involvement of a mechanism other than the BDNF-TrkB signaling system. These results suggest that peretinoin 3 exerts its cytoprotective effects in the cerebral cortex and striatum through different mechanisms of action.

[0067] (Effects on cerebral hemorrhage pathology) Male ICR mice aged 8-10 weeks were placed in a stereotaxic apparatus under triple anesthesia, and 0.035 U of collagenase VII (Sigma) was injected into the striatum at a fixed coordinate (2.3 mm lateral to the midline, 0.2 mm posterior to the bregma suture, and 3.5 mm inferior to the skull) over a period of 2.5 minutes using a 30-gauge needle to induce bleeding. The skull surface was then repositioned with dental cement, and the animals were kept in the care of the animals. Peretinoin 3 (40 mg / kg) was suspended in water with 0.5% carboxymethylcellulose and administered intragastrically using an oral gavage three times at 3, 27, and 51 hours after the induction of bleeding. For comparison, 0.5% carboxymethylcellulose without peretinoin 3 was also administered intragastrically in the same manner. The degree of motor dysfunction in mice was evaluated by performing two types of motor function tests (beam walking test and modified limb-placing test) 6, 24, 48, and 72 hours after the induction of hemorrhage. After the motor function test 72 hours later, mice were perfused under anesthesia and their brains were removed and frozen coronal sections with a thickness of 16 μm were prepared. Microglia, astrocytes, and neurons in brain tissue were identified by immunohistochemistry using antibodies against Iba1, GFAP, and NeuN, respectively, and the effect of peretinoin 3 was evaluated by quantifying the number of cells or the immunoreactive area in a certain area.

[0068] (Result) Figure 12 shows the changes in body weight during the test. Here, vehicle refers to the administration of 0.5% carboxymethylcellulose that does not contain peretinoin 3. This figure shows that peretinoin 3 suppresses weight loss in mice after cerebral hemorrhage. In addition, in the beam-walking test, as shown in FIG. 13, administration of peretinoin 3 reduced the fall rate, increased the walking width, and increased the score. Furthermore, the modified limb-placing test (DEHA) showed a decrease in deficit score with administration of peretinoin 3, as shown in Figure 14. These results indicate that peretinoin 3 significantly reduces motor dysfunction 24 to 72 hours after hemorrhage.

[0069] In addition, the results of immunohistochemical observation of brain tissue after the motor function test are shown in Figures 15 and 16. Figure 15 shows that peretinoin 3 significantly suppresses the accumulation of activated microglia at the periphery of the hematoma. Figure 16 shows that peretinoin 3 suppresses the loss of nerve cells within the hematoma. These results suggest the possibility of peretinoin 3 as a treatment for cerebral hemorrhage.

[0070] <Experiment demonstrating differentiation induction from stem cells to nerve cells> To examine whether peretinoin 3 induces differentiation of stem cells into neurons like ATRA, the following experiment was performed. Human pluripotent embryonal carcinoma-derived NTERA-2cl.D1 [NT2 / D1] cells (ATCC CRL-1973) (hereafter referred to as "NT2 cells") were cultured in a maintenance culture medium (high glucose D-MEM (Sigma-Aldrich, D6429), 10% FBS, 100 U / ml penicillin / streptomycin) at 37°C and 5% CO2 for 3-4 days until confluent on a 10 cm plastic dish. NT2 cells were detached with Accutase (Innovative Cell Technologies, #AT104), subcultured, and maintained in culture for 50 days. The medium was changed three times a week. NT2 cells were cultured at 1.5 × 10 6 ~2.5×10 6The cells were dispersed in Petri dishes for low-adherence microorganisms (STAR ​​SDish9015, Rikaken) at a concentration of 100 μM / dish and cultured in suspension on a shaker (100 rpm) at 37°C and 5% CO2. After one day, 1 μM or 10 μM ATRA was added to induce differentiation and form spheroids. After 14 days, the spheroids were collected and plated on a 10 cm dish coated with 5 μg / ml poly-D-lysine (PDL) (Sigma-Aldrich) / laminin (LAM) (iMatrix-511, Nippi), and cultured at 37°C and 5% CO2. From the next day, three types of cell division inhibitors (10 μM uridine, 10 μM floxuridine, and 1 μM AraC) were added and cultured. After three days, the cells were detached and cultured at 0.1 × 10 6 The cells were then replated onto PDL / LAM-coated 35-mm dishes at a concentration of 100 / dish and cultured for 4 days in the presence of three types of cell division inhibitors to induce differentiation into neurons.

[0071] After differentiation induction, the cells were maintained and cultured in BrainPhys™ Neuronal Medium (STEMCELL Technologies) containing 0.2% NeuroCult™ SM1 Neuronal Supplement at 37°C and 5% CO2 for 2 to 3 months. Half of the medium was replaced three times a week. As a result, cells positive for the neuronal marker MAP2 were obtained, and these were named NT2-N cells.

[0072] To confirm the expression of synaptic receptors in mature cells, calcium influx via NMDA-type glutamate receptors was measured as follows. The fluorescent calcium indicator Fluo-8 AM (AAT Bioquest) was dissolved in 0.1 μM glycine-containing Mg-free HHBS (Hepes-buffered Hanks' balanced salt solution (1.26 mM CaCl2, 5.33 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.34 mM Na2HPO4, 5.56 mM D-glucose, 20 mM HEPES, pH 7.4)) (4 μM, 2 μM, respectively). This solution (1 ml) was loaded into NT2-N cells under 37 °C, 5% CO2 conditions (2 hours, 30 minutes, respectively), and fluo8 was loaded into the cells. After that, the extracellular fluid was replaced with 0.1 μM glycine-containing Mg-free HHBS, and the cells were left in the incubator (37°C, 5% CO2) of a live cell time-lapse imaging device (BioStation IM-Q, Nikon) for more than 1 hour and used for the following measurements. For the NT2-N cells placed in the device, 5 to 10 fields of view to be analyzed were manually set, and the fluorescence intensity was recorded for each field at an excitation wavelength of 480 nm / fluorescence wavelength of 520 nm every 2 seconds for 5 minutes. In addition, after a certain time had passed from the start of recording, 100 μL of the 100 μM NMDA solution (0.1 μM glycine-containing Mg-free HHBS) prepared above was administered.

[0073] After 2-3 months of incubation, a portion of the specimen was fixed in 4% paraformaldehyde in PBS (30 minutes at room temperature). After fixation, the specimen was carefully washed with PBS and treated with 1% Triton-X100 in PBS for 10 minutes. After treatment, the specimen was thoroughly washed with PBS, blocked with 1% BSA / 3% normal goat serum in PBS, immersed in anti-MAP2 antibody (1:1000, Millipore #AB5622) and anti-Nestine antibody (1:1000, ab22035) solutions, washed again, and stained with fluorescently labeled secondary antibodies (Jackson Immun Laboratory, 115-545-146 and 111-585-144, 1:5000 each).

[0074] ·Results As shown in Figure 17, administration of 10 μM peretinoin 3, like ATRA, increased the number of cells expressing the neuronal marker MAP2 without expressing the neural progenitor marker nestin, and differentiation into neurons was confirmed. In addition, calcium imaging shown in Figure 18 suggested that the neurons induced to differentiate with ATRA and peretinoin 3 expressed NMDA-type glutamate receptors, which led to the formation of synapses.

[0075] The present invention is not limited to the above-mentioned embodiments and examples. Various modifications that do not depart from the scope of the claims and that are easily conceivable by those skilled in the art are also included in the present invention. [Industrial Applicability]

[0076] The present invention 11 C-labeled cyclic retinoids can be synthesized in a short time with good yield and can be suitably used as PET probes to elucidate their dynamics in the brain. The central nervous system activator of the present invention can also be used as a therapeutic agent for central nervous degenerative diseases or as a lead compound therefor.

Claims

【Request 1】 A compound represented by the following chemical formula (a), or an ester or salt thereof: 11 C-labeled acyclic retinoids. 【Chemistry 1】 【Request 2】 The method of claim 1 11 PET probe containing a C-labeled acyclic retinoid. 【Request 3】 In an aprotic solvent, 11 Methyl iodide labeled with C and the following organotin compound (b) (wherein -COOR 1 is an ester group, R 2 represents an alkyl group which may have a branch.) in the presence of a palladium complex, a phosphine ligand, and cuprous halide. 11 Method for producing C-labeled acyclic retinoids. 【Chemistry 2】

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