Masaaki Suzuki a,b,⇑, Misato Takashima-Hirano b, Hideki Ishii c, Chika Watanabe d, Kengo Sumi d, Hiroko Koyama d, Hisashi Doi b,e
a Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology Center, 35, Gengo Morioka-cho, Obu-shi, Aichi 474-8511, Japan
b RIKEN Center for Molecular Imaging Science (CMIS), 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
c Molecular Imaging Center, National Institute of Radiological Science (NIRS), 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
d Division of Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1184, Japan
e RIKEN Center for Life Science Technologies (CLST), 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
a b s t r a c t
Retinoids are a class of chemical compounds which include both natural dietary vitamin A (retinol) metabolites and active synthetic analogs. Both experimental and clinical studies have revealed that retinoids regulate a wide variety of essential biological processes. In this study, we synthesized 11C-labeled all-trans-retinoic acid (ATRA), the most potent biologically active metabolite of retinol and used in the treatment of acute promyelocytic leukemia. The synthesis of 11C-labeled ATRA was accomplished by a combination of rapid Pd(0)-mediated C-[11C]methylation of the corresponding pinacol borate precursor prepared by 8 steps and hydrolysis. [11C]ATRA will prove useful as a PET imaging agent, particularly for elucidating the improved therapeutic activity of ATRA (natural retinoid) for acute promyelocytic leukemia by comparing with the corresponding PET probe [11C]Tamibarotene (artificial retinoid).Retinoids are a class of chemical compounds which include both natural dietary vitamin A (retinol) metabolites and active synthetic analogs.1 Both experimental and clinical studies have revealed that retinoids regulate a wide variety of essential biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, apoptosis, and homeostasis, as well as several associated disorders. Retinoids mediate their biological effects through binding to nuclear receptors known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear hormone receptor superfamily. Retinoids (including all-trans-retinoic acid (ATRA) (1), 13-cis-retinoic acid (2), 9-cis-retinoic acid (3)) and their synthetic analogs (Etretinate (4), acyclic retinoid (5), and Tamibarotene (6)) are therapeutic agents used for applications in dermatology and oncology (Fig. 1).2 ATRA (1), the most potent biologically active metabolite of vitamin A which only binds to RARs, is used clinically in the treatment of acute promyelocytic leukemia(APL). The relationship of ATRA and its receptors to cancer, metabolicdisease, and neurodegenerative diseases such as Alzheimer’sand Parkinson’s have been reported by many groups, but themechanisms involved are still poorly understood.3 Morin et al. previouslyreported the synthesis of 123I-iodinated analogues of ATRAfor SPECT imaging.4 Thus, in vivo imaging of ATRA should prove very useful in helping to understand the relationship between retinoids, their receptors and the progression of a variety of diseases. Herein, we describe the radiolabeling of ATRA for positron emission tomography (PET) imaging. PET is a powerful noninvasive molecular imaging technique that provides high sensitivity, good spatial resolution, and accurate quantification, and therefore, PET is widely used in diagnostic medicine and has the potential to significantly benefit drug discovery.5 Labeled small molecules can provide clinical distribution data to facilitate compound selectionat an early stage of the drug development or can offer valuableinformation for disease diagnosis by probing drug-receptorenzyme occupancies and disease mechanisms across multipletherapeutic areas (for example CNS, oncology, metabolic andimmunological disorders). Radionuclides used in PET are comprisedof short-lived radioisotopes including 11C, 13N, 15O, and18F. Of these, carbon-11 (half-life = 20.4 min) is one of the mostideal isotopes for PET research, because carbon is found in allorganic molecules. In this context, our group has been workingon the development of Pd(0)-mediated rapid C-[11C]methylationreactions using stannyl or boron compounds as precursors.6 Basedon our method which allows the rapid introduction of a 11C-methylgroup onto a vinyl carbon, we describe herein the first 11C-labeling
of ATRA using Pd(0)-mediated rapid C-[11C]methylation of an alkenylboron precursor.ATRA has three vinyl methyl groups at C-5, C-9, and C-13. Ofthese, we decided to label a C-5 methyl of ATRA because the labelingat the C-9 and C-13 positions might yield geometric isomerswhen using a transition metal.Synthesis of an alkenyl boron precursor for [11C]ATRA involvingtwo kinds of Horner–Wadsworth–Emmons (HWE) olefinations isshown in Scheme 1. Retinoids consisting of polyene structuresare quite sensitive to light and heat and they tend to isomerizeor decompose.7 Therefore, the reactions were carried out underdim light beginning with the first HWE olefination. The derivedgeometric isomers were separated by column chromatography.The starting material 2-carboethoxy-3,3-dimethylcyclohexanone(7) was prepared according to Steiner’s procedure.8 Compound 7was converted to an enol triflate 8 in 70% yield by treatment withNaH in the presence of triflic anhydride. Then, the ester of 8 wasconverted to the alcohol 9 in 72% yield with diisobutylaluminumhydride (DIBAL-H), and then quantitatively oxidized to aldehyde10 by treatment with tetrapropylammonium perruthenate (TPAP)and N-methylmorpholine-N-oxide (NMO). The first HWE olefinationwas accomplished for 10 in 40% yield by using triethyl 3-methyl4-phosphonocrotonate (16) pretreated with nBuLi in thepresence of DMPU. Then, the derived ester 11 was reduced to alcohol12 in 85% yield by DIBAL-H in diethyl ether, which was very unstable at room temperature even in the dark. Therefore, 12was quickly converted to the pinacol borate derivative 13 in 41%yield by Miyaura’s method.9 The resulting alcohol 13 was oxidizedby TPAP-NMO in dichloromethane to quantitatively afford thealdehyde 14. The second HWE olefination of 14 using the anionof 16 generated by NaH in THF afforded 15 in 36% yield from 13.The pinacol borate precursor 15 was quickly stored at _78 to_30 _C in the dark to limit decomposition. The 13-cis-pinacolborate precursor for [11C]13-cis-RA was isolated by reverse-phaseHPLC separation.Radiosynthesis of [11C]ATRA using rapid C-[11C]methylation followedby rapid hydrolysis was also carried out under dim light.10[11C]H3I was prepared as previously described.11 Palladium(0)-mediated rapid [11C]methylation of the precursor 15 was conductedusing [11C]H3I in the presence of Pd2(dba)3, P(o-tolyl)3,K2CO3 (1:4:9) in DMF at 65 _C for 4 min followed by basic hydrolysisof the ethyl ester at 100 _C for 2 min to afford the desired[11C]ATRA ([11C]1) in 14% yield (HPLC analytical yield). To completethe hydrolysis within 2 min, heating of the reaction mixturewas required. The radioactivity of the product was often significantlydecreased and unknown compounds arising from decompositionwere observed in the HPLC radiochromatogram of thereaction mixture. We considered that these phenomena were asa result of radiolysis. Therefore, sodium ascorbate, a radical scavengerused to prevent radiolysis, was added as a base instead of
K2CO3 to prevent product decomposition by radiolysis during thereaction and work-up.12 Consequently, rapid C-[11C]methylationin the presence of sodium ascorbate as a base proceeded smoothlyand rapid hydrolysis consistently afforded more than 1 GBq of thedesired [11C]1 with >99% radiochemical purity in 36% yield (HPLCanalytical yield) (Scheme 2). Total synthetic time including HPLCpurification and formulation was 32 min. The decay-correctedradiochemical yield based on [11C]H3I was 25%. Additionally, thedecomposition of the product was not observed in the presenceof sodium ascorbate and the radiochemical purity was maintained>99% at 90 min after the completion of synthesis. In this reaction,13-cis-[11C]retinoic acid ([11C]2) and 9-cis-[11C]retinoic acid([11C]3) were also formed (Fig. 2). Other RI peaks were not identifiedhowever several peaks with retention times less than 10 minwere also observed in the absence of hydrolysis and thus, are consideredto be decomposition products.In summary, the synthesis of 11C-labeled ATRA was accomplishedby a combination of rapid Pd(0)-mediated C-[11C]methylationand hydrolysis. We hope that [11C]ATRA will prove useful as aPET imaging agent, particularly for elucidating the improved therapeuticactivity of Tamibarotene (6) for acute promyelocytic leukemia13by comparing with the corresponding PET probe [11C]6previously reported by our group.14,15
AcknowledgmentsThis work was supported in part by a consignment expensefor the Molecular Imaging Program on ‘Research Base forExploring New Drugs’ from the Ministry of Education, Culture,Sports, Science and Technology (MEXT) of Japan and in partby JSPS KAKENHI Grant Number (A) 13371294. We thank ProfessorYuichi Hashimoto from the University of Tokyo, Japan,for his valuable advice on in vivo molecular imaging studiesof retinoids and the use of related artificial compounds asdrugs. We would also like to thank Mr. Masahiro Kurahashi(Sumitomo Heavy Industry Accelerator Service Ltd.) for operatingthe cyclotron.References and notes
1. (a) Barua, A. B.; Furr, H. C. Mol. Biotechnol. 1998, 10, 167; (b) Germain, P.;Chambon, P.; Eichele, G.; Evans, R. M.; Lazar, M. A.; Leid, M.; de Lera, A. R.;Lotan, R.; Mangelsdorf, D. J.; Gronemeyer, H. Pharmacol. Rev. 2006, 58, 712; (c)Werner, E. A.; Deluca, H. F. Am. J. Physiol. Endocrinol. Metab. 2002, 282, 672.
2. (a) Takai, K.; Okuno, M.; Yasuda, I.; Matsuhima-Nishiwaki, R.; Uematsu, T.;Tsurumi, H.; Shiratori, Y.; Muto, Y.; Moriwaki, H. Interbirology 2005, 48, 39; (b)Kagechika, H.; Kawachi, E.; Hashimoto, Y.; Himi, T.; Shudo, K. J. Med. Chem.1988, 31, 2182.
3. (a) Drager, U. C. Sci. STKE 2006, 324; (b) Altucci, L.; Leibowitz, M. D.; Ogilvie, K.M.; de Lera, A. R.; Gronemeyer, H. Nat. Rev. Drug Disc. 2007, 6, 793; (c) Shudo,K.; Fukawsawa, H.; Nakagomi, M.; Yamagata, N. Curr. Alzheimer Res. 2009, 6,302.
4. Morin, H.; Li, C. Tetrahedron Lett. 2004, 45, 5673.
5. (a) Phelps, M. E. PET: Molecular Imaging and Its Biological Applications; Springer:New York, 2004; (b) Fowler, J. S.; Wolf, A. P. Acc. Chem. Res. 1997, 30, 181; (c)Ametamey, S. M. Chem. Rev. 2008, 108, 1501; (d) Miller, P. W.; Long, N. J.; Vilar,R.; Gee, A. D. Angew. Chem., Int. Ed. 2008, 47, 8998.
6. Parts 1–14: (a) Suzuki, M.; Doi, H.; Bj鰎kman, M.; Andersson, Y.; L錸gstr鰉, B.;Watanabe, Y.; Noyori, R. Chem. Eur. J. 1997, 3, 2039; (b) Suzuki, M.; Noyori, R.;L錸gstr鰉, B.; Watanabe, Y. Bull. Chem.Soc. Jpn. 2000, 73, 1053; (c) Suzuki, M.;Doi, H.; Kato, K.; Bj鰎kman, M.; L錸gstr鰉, B.; Watanabe, Y.; Noyori, R.Tetrahedron 2000, 56, 8263; (d) Bj鰎kman, M.; Doi, H.; Resul, B.; Suzuki, M.;Noyori, R.; Watanabe, Y.; L錸gstr鰉, B. J. Labelled Compd. Radiopharm. 2000, 43,1327; (e) Hosoya, T.; Wakao, M.; Kondo, Y.; Doi, H.; Suzuki, M. Org. Biomol.Chem. 2004, 2, 24; (f) Suzuki, M.; Doi, H.; Hosoya, T.; L錸gstr鰉, B.; Watanabe,Y. TrAC, Trends Anal. Chem. 2004, 23, 595; (g) Hosoya, T.; Sumi, K.; Doi, H.;Wakao, M.; Suzuki, M. Org. Biomol. Chem. 2006, 4, 410; Patent: Suzuki, M.;Hosoya, T. JP 2005-306670, WO2007/046258.; (h) Patent: Suzuki, M.; Doi, H.;Tsukada, H. JP 2006-229169, WO2008/023780; Doi, H.; Ban, I.; Nonoyama, A.;Sumi, K.; Kuang, C.; Hosoya, T.; Tsukada, H.; Suzuki, M. Chem.-Eur. J. 2009, 15,4165.; (i) Suzuki, M.; Sumi, K.; Koyama, H.; Siqin; Hosoya, T.; Takashima-Hirano, M.; Doi, H. Chem.-Eur. J. 2009, 15, 12489; Patent: Suzuki, M.; Doi, H.;Koyama, H. JP 2008-335250, WO2010/074272.; (j) Kanazawa, M.; Furuta, K.;Doi, H.; Mori, T.; Minami, T.; Ito, S.; Suzuki, M. Bioorg. Med. Chem. Lett. 2011, 21,2017; (k) Koyama, H.; Siqin; Zhang, Z.; Sumi, K.; Hatta, Y.; Nagata, H.; Doi, H.;Suzuki, M. Org. Biomol. Chem. 2011, 9, 4287; (l) Doi, H.; Goto, M.; Suzuki, M.Bull. Chem. Soc. Jpn. 2012, 85, 1233; (m) Suzuki, M.; Takashima-Hirano, M.;Koyama, H.; Yamaoka, T.; Sumi, K.; Nagata, H.; Hidaka, H.; Doi, H. Tetrahedron2012, 68, 2336; (n) Koyama, H.; Zhang, Z.; Ijuin, R.; Siqin; Son, J.; Hatta, Y.;Ohta, M.; Wakao, M.; Hosoya, T.; Doi, H.; Suzuki, M. ACS Adv. 2013, 3, 9391; Forreview see (o) Suzuki, M.; Doi, H. J. Synth. Org. Chem. Jpn. 2010, 68, 1195; (p)Suzuki, M.; Koyama, H.; Takashima-Hirano, M.; Doi, H. In Positron EmissionTomography-Current Clinical and Research Aspects; Hsieh, C.H., Ed.; InTech,open access publisher, 2012; p 115; (q) Suzuki, M.; Doi, H.; Koyama, H.; Zhang,Z.; Hosoya, T.; Onoe, H.; Watanabe, Y. Chem. Rec. 2014, in press.
7. (a) Curley, R. W., Jr.; Fowble, J. W. Photochem. Photobiol. 1988, 47, 831; (b)Urbach, J.; Rando, R. R. Biochem. J. 1994, 299, 459.
8. Steiner, U.; Willhalm, B. Helv. Chim. Acta 1952, 35, 1752.
9. Takagi, J.; Takahashi, K.; Ishiyama, T.; Miyaura, N. J. Am. Chem. Soc. 2002, 124,8001.
10. A mixture of 15 (6.0 mg, 13.6 lmol), Pd2(dba)3 (2.9 mg, 3.17 lmol), P(o-tolyl)3(3.8 mg, 12.7 lmol), and 0.2 M Na L-ascorbate aq (50 lL, 10 lmol) in DMF(400 lL) was placed in the reaction vessel underdim light. [11C]H3I wastransported by a stream of helium (30 mL/min) and trapped in the mixture at30 _C for 2 min. It was heated at 65 _C for 4 min and then a solution of 6 M KOHin methanol–water (2:1, 600 lL) was added. It was heated at 100 _C for 2 minand then it was acidified (pH 2) by HCOOH (500 lL) and diluted withacetonitrile (500 lL). The resulting mixture was injected onto a preparativeHPLC (mobile phase, acetonitrile: 0.2% HCOOH in water = 90:10; columnCholester (COSMOSIL), 10 (i.d.) _250 mm, 5 lm; flow rate, 5 mL/min; UVdetection, 360 nm; retention time, 13 min). The desired fraction was collectedinto a flask containing 25% ascorbic acid (200 lL), and the organic solvent wasremoved under reduced pressure. The desired radiotracer was dissolved in amixture of polysorbate 80, propylene glycol, and saline (1:9:90 v/v/v, 4 mL).The total synthesis time including HPLC purification and radiopharmaceuticalformulation for intravenous administration was 32 min. The isolatedradioactivity was 1.5 GBq and the specific radioactivity was 44 GBq/lmol.he decay-corrected radiochemical yield based on [11C]H3I was 25%. Thechemical identity of [11C]1 was confirmed by co-injection with an authenticsample of all-trans-retinoic acid using analytical HPLC (mobile phase,acetonitrile:0.2% HCOOH in water = 90:10; column, GeminiNX(Phenomenex), 4.6 (i.d.) _150 mm, 5 lm; flow rate, 1 mL/min; UV detection,360 nm; retention time, 6.1 min). The chemical purity analyzed at 360 nm was92% and the radiochemical purity was greater than 98%. The identification ofminor products, [11C]2 and [11C]3, were also conducted by co-injection withthe corresponding authentic samples, 2 and 3 obtained from the isomerizationreaction of 1 under the similar conditions as used for rapid C-methylation.
11. L錸gstr鰉, B.; Antoni, G.; Gullberg, P.; Halldin, C.; Malmborg, P.; Nagren, K.;Rimland, A.; Svard, H. J. Nucl. Med. 1987, 28, 1037.
12. (a) Fukumura, T.; Nakao, R. Y.; Yamaguchi, M.; Suzuki, K. Appl. Radiat. Isot.2004, 61, 1279; (b) Scott, P. J. H.; Hockley, B. G.; Kung, H. F.; Machanda, R.;Zhang, W.; Kilbourn, M. R. Appl. Radiat. Isot. 2009, 67, 88.
13. Tobita, T.; Takeshita, A.; Kitamura, K.; Ohnishi, K.; Yanagi, M.; Hiraoka, A.;Karasuno, T.; Takeuchi, M.; Miyawaki, S.; Ueda, R.; Naoe, T.; Ohno, R. Blood1997, 90, 967.
14. Takashima-Hirano, M.; Ishii, H.; Suzuki, M. ACS Med. Chem. Lett. 2012, 3, 804.
15. Molecular imaging studies will be reported in a separate paper.
