Synthesis of 11C-labeled retinoic acid, [11C]ATRA, via an alkenylboron precursor by Pd(0)-mediated rapid C-[11C] methylation

Article abstract of DOI:10.1016/j.bmcl.2014.05.041

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).

Full text of DOI:10.1016/j.bmcl.2014.05.041

Accepted Manuscript
Synthesis of 11C-Labeled Retinoic Acid, [11C]ATRA, via an Alkenylboron Pre-
cursor by Pd(0)-mediated Rapid C-[11C]Methylation
Masaaki Suzuki, Misato Takashima-Hirano, Hideki Ishii, Chika Watanabe,
Kengo Sumi, Hiroko Koyama, Hisashi Doi
PII:
S0960-894X(14)00541-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.041
BMCL 21655
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2014
6 May 2014
8 May 2014
Please cite this article as: Suzuki, M., Takashima-Hirano, M., Ishii, H., Watanabe, C., Sumi, K., Koyama, H., Doi,
H., Synthesis of 11C-Labeled Retinoic Acid, [11C]ATRA, via an Alkenylboron Precursor by Pd(0)-mediated Rapid
C-[11C]Methylation, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.05.041
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Synthesis of 11C-Labeled Retinoic Acid,
[11C]ATRA, via an Alkenylboron Precursor by
Pd(0)-mediated Rapid C-[11C]Methylation
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Masaaki Suzuki*a, b, Misato Takashima-Hiranob, Hideki Ishiic, Chika Watanabed, Kengo
Sumid, Hiroko Koyamad, Hisashi Doib, e
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a
Center for Development of Advanced Medicine for Dementia, National Center for
Geriatrics and Gerontology Center
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35, Gengo Morioka-cho, Obu-shi, Aichi 474-8511, JAPAN
RIKEN Center for Molecular Imaging Science (CMIS)
b
6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, JAPAN
Molecular Imaging Center, National Institute of Radiological Science (NIRS)
4-9-1 Anagawa, Inage-ku, Chiba 263-8555, JAPAN
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Division of Regeneration and Advanced Medical Science
Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1184, JAPAN
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RIKEN Center for Life Science Technologies (CLST)
6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, JAPAN
*Correspondence Author: E-mail: suzukims@ncgg.go.jp
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Abstract
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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).
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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
(Figure 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, metabolic disease, and
neurodegenerative diseases such as Alzheimer’s and Parkinson’s have been reported by many
groups, but the mechanisms involved are still poorly understood.[3] Morin et al. previously
reported the synthesis of 123I-iodinated analogues of ATRA for 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.
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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
selection at an early stage of the drug development or can offer valuable information for
disease diagnosis by probing drug-receptor enzyme occupancies and disease mechanisms
across multiple therapeutic areas (for example CNS, oncology, metabolic and immunological
disorders). Radionuclides used in PET are comprised of short-lived radioisotopes including
11C, 13N, 15O, and 18F. Of these, carbon-11 (half-life = 20.4 min) is one of the most ideal
isotopes for PET research, because carbon is found in all organic molecules. In this context,
our group has been working on the development of Pd(0)-mediated rapid C-[11C]methylation
reactions using stannyl or boron compounds as precursors.[6] Based on our method which
allows the rapid introduction of a 11C-methyl group onto a vinyl carbon, we describe herein
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the first C-labeling of ATRA using Pd(0)-mediated rapid C-[11C]methylation of an alkenyl
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boron precursor.
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ATRA has three vinyl methyl groups at C-5, C-9, and C-13. Of these, we decided to label a
C-5 methyl of ATRA because the labeling at the C-9 and C-13 positions might yield
geometric isomers when using a transition metal.
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Synthesis of an alkenyl boron precursor for [11C]ATRA involving two kinds of Horner-
Wadsworth-Emmons (HWE) olefinations is shown in Scheme 1. Retinoids consisting of
polyene structures are quite sensitive to light and heat and they tend to isomerize or
decompose.[7] Therefore, the reactions were carried out under dim light beginning with the
first HWE olefination. The derived geometric isomers were separated by column
chromatography. The starting material 2-carboethoxy-3,3-dimethylcyclohexanone (7) was
prepared according to Steiner’s procedure.[8] Compound 7 was converted to an enol triflate 8
in 70% yield by treatment with NaH in the presence of triflic anhydride. Then, the ester of 8
was converted to the alcohol 9 in 72% yield with diisobutylaluminum hydride (DIBAL-H),
and then quantitatively oxidized to aldehyde 10 by treatment with tetrapropylammonium
perruthenate (TPAP) and N-methylmorpholine-N-oxide (NMO). The first HWE olefination
was accomplished for 10 in 40% yield by using triethyl 3-methyl4-phosphonocrotonate (16)
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pretreated with BuLi in the presence of DMPU. Then, the derived ester 11 was reduced to
alcohol 12 in 85% yield by DIBAL-H in diethyl ether, which was very unstable at room
temperature even in the dark. Therefore, 12 was quickly converted to the pinacol borate
derivative 13 in 41% yield by Miyaura’s method.[9] The resulting alcohol 13 was oxidized by
TPAP-NMO in dichloromethane to quantitatively afford the aldehyde 14. The second HWE
olefination of 14 using the anion of 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
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limit decomposition. The 13-cis-pinacol borate precursor for [11C]13-cis-RA was isolated by
reverse-phase HPLC separation.
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Radiosynthesis of [11C]ATRA using rapid C-[11C]methylation followed by 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 conducted using
[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 hydrolysis of the ethyl ester at 100 °C for 2 min to afford the desired
[11C]ATRA ([11C]1) in 14% yield (HPLC analytical yield). To complete the hydrolysis within
2 minutes, heating of the reaction mixture was required. The radioactivity of the product was
often significantly decreased and unknown compounds arising from decomposition were
observed in the HPLC radiochromatogram of the reaction mixture. We considered that these
phenomena were as a result of radiolysis. Therefore, sodium ascorbate, a radical scavenger
used to prevent radiolysis, was added as a base instead of K2CO3 to prevent product
decomposition by radiolysis during the reaction and work-up.[12] Consequently, rapid C-
[11C]methylation in the presence of sodium ascorbate as a base proceeded smoothly and rapid
hydrolysis consistently afforded more than 1 GBq of the desired [11C]1 with >99%
radiochemical purity in 36% yield (HPLC analytical yield) (Scheme 2). Total synthetic time
including HPLC purification and formulation was 32 min. The decay-corrected radiochemical
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yield based on [11C]H3I was 25%. Additionally, the decomposition of the product was not
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observed in the presence of 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 (Figure 2). Other RI peaks
were not identified however several peaks with retention times less than 10 min were also
observed in the absence of hydrolysis and thus, are considered to be decomposition products.
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In summary, the synthesis of 11C-labeled ATRA was accomplished by a combination of
rapid Pd(0)-mediated C-[11C]methylation and hydrolysis. We hope that [11C]ATRA will
prove useful as a PET imaging agent, particularly for elucidating the improved therapeutic
activity of Tamibarotene (6) for acute promyelocytic leukemia[13] by comparing with the
corresponding PET probe [11C]6 previously reported by our group.[14, 15]
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Acknowledgements
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This work was supported in part by a consignment expense for the Molecular Imaging
Program on “Research Base for Exploring New Drugs” from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan and in part by JSPS KAKENHI
Grant Number (A) 13371294. We thank Professor Yuichi Hashimoto from the University of
Tokyo, Japan, for his valuable advice on in vivo molecular imaging studies of retinoids and
the use of related artificial compounds as drugs. We would also like to thank Mr. Masahiro
Kurahashi (Sumitomo Heavy IndustryAccelerator Service Ltd.) for operating the cyclotron.
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H.; Hosoya, T.; Långström, B.; Watanabe, Y. TrAC, Trends Anal. Chem. 2004, 23, 595;
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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.;
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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.
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2012, 85, 1233; (m) Suzuki, M.; Takashima-Hirano, M.; Koyama, H.; Yamaoka, T.;
Sumi, K.; Nagata, H.; Hidaka, H.; Doi, H. Tetrahedron 2012, 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 Advances 2013, 3, 9391. For review see (o) Suzuki, M.; Doi, H. J. Synth.
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C.-H. Hsieh), InTech, open access publisher, 2012, pp. 115; (q) Suzuki, M.; Doi, H.;
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10. A mixture of 15 (6.0 mg, 13.6 µmol), Pd2(dba)3 (2.9 mg, 3.17 µmol), P(o-tolyl)3 (3.8 mg,
12.7 µmol), and 0.2 M Na L-ascorbate aq. (50 µL, 10 µmol) in DMF (400 µL) was placed
in the reaction vessel under dim light. [11C]H3I was transported by a stream of helium (30
mL/min) and trapped in the mixture at 30 °C for 2 min. It was heated at 65 °C for 4 min
and then a solution of 6 M KOH in methanol-water (2:1, 600 µL) was added. It was
heated at 100 °C for 2 min and then it was acidified (pH2) by HCOOH (500 µL) and
diluted with acetonitrile (500 µL). The resulting mixture was injected onto a preparative
HPLC (mobile phase, acetonitrile:0.2% HCOOH in water = 90:10; column, Cholester
(COSMOSIL), 10 (i.d.) × 250 mm, 5 µm; flow rate, 5 mL/min; UV detection, 360 nm;
retention time, 13 min). The desired fraction was collected into a flask containing 25%
ascorbic acid (200 µL), and the organic solvent was removed under reduced pressure. The
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desired radiotracer was dissolved in a mixture of polysorbate 80, propylene glycol, and
saline (1:9:90 v/v/v, 4 mL). The total synthesis time including HPLC purification and
radiopharmaceutical formulation for intravenous administration was 32 min. The isolated
radioactivity was 1.5 GBq and the specific radioactivity was 44 GBq/µmol. The decay-
corrected radiochemical yield based on [11C]H3I was 25%. The chemical identity of
[11C]1 was confirmed by co-injection with an authentic sample 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 µm; flow rate, 1 mL/min; UV
detection, 360 nm; retention time, 6.1 min). The chemical purity analyzed at 360 nm was
92% and the radiochemical purity was greater than 98%. The identification of minor
products, [11C]2 and [11C]3, were also conducted by co-injection with the corresponding
authentic samples, 2 and 3 obtained from the isomerization reaction of 1 under the similar
conditions as used for rapid C-methylation.
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11. Långström, B.; Antoni, G.; Gullberg, P.; Halldin, C.; Malmborg, P.; Nagren, K.; Rimland,
A.; Svard, H. J. Nucl. Med. 1987, 28, 1037.
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Kilbourn, M. R. Appl. Radiat. Isot. 2009, 67, 88.
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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. Blood 1997, 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.
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Figure 1. Structures of retinoic acids and related compounds
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Scheme 1. Synthesis of precursor
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Scheme 2. Radiosynthesis of [11C]ATRA (11C-incorporated all-trans-retinoic acid)
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Figure 2. Representative chromatogram of [11C]1 purification. UV absorption: 360 nm
UV
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[11C]2
[11C]1
[11C]3
RI
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Time, min
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Graphical abstract
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