C-Labeling of the C ( 1 )-C ( 10 ) Dihydroxy Acid Moiety for the Study on the Synthesis of Kulokekahilide-2 PET Tracer

11C-labeled C1-C10 partial structure of kulokekahilide-2 (1) was successfully synthesized based on Pd0-mediated rapid C-[11C]methylation using [11C]methyl iodide and pinacol alkenylboronate. The preparation of organoboron intermediate via olefin cross-metathesis is also a crucial procedure for the synthesis of 11C-labeling C1-C10 dihydroxy acid moiety of 1.

Positron emission tomography (PET), which uses specific probes radiolabeled with short-lived positronemitting radionuclides ( 11 C, 13 N, 15 O, 18 F etc.), is a powerful non-invasive molecular imaging technique usable for highly accurate diagnoses and investigation of the in vivo biochemistry of bioactive compounds.In addition, it is strongly hoped that PET would be applied as a human microdosing study to an early-stage of drug development [10].Carbon-11 (half-life = 20.4min) is one of the most meritorious isotopes for PET research because carbon is included in all organic molecules.In recent years, efficient labeling methods of the 11 C radioisotope into organic frameworks have continuously been developed by Suzuki et al. using palladium(0)-mediated rapid C-[ 11 C]methylation (5 min reactions) consisting of the cross-coupling reactions of [ 11 C]methyl iodide and the stannyl or boron substrates [11] [12].Actually, the rapid cross-coupling reactions have successfully been applied for the syntheses of various disease-oriented PET tracers [13]- [16].Our interest has been intrigued by extending the 11 C labeling reactions to complex natural product.Described herein in the synthesis of 11

Results and Discussion
Kulokekahilide-2 has several possible positions for 11 C radiolabeling.Prior to actual synthesis of 11 C-incorporated 1, we investigated a model study using a partial structure.Here, we are particularly interested in introducing 11 C onto the methylene group of 2 as a 11 CH 3 by our rapid cross-coupling reaction [12], [17] between sp 2  vinyland sp 3 -carbon atoms using an organostannyl or boron precursor 3 (Scheme 1) where key step of synthetic strategy involves the preparation of precursor 3 derived from methyl ester 2.
Before synthesizing 11 C-labeled 4a, we prepared the nonradioactive molecule 4b.Thus, the intermediary compound 5 was synthesized from (S)-3-propionyl-4-isopropyl-2-oxazolidinone according to the reported procedure [1].Protection of the secondary alcohol 5 gave PMB ether 6 in 49% yield.Subsequent deprotection of the TBS group at C7 in 6 afforded the desired compound 4b in 78% yield after column chromatography on silica gel (Scheme 2).
The synthesis of the organostannyl or boron compound 3 started with an acylation of commercially available ox-azolidinone 7 with the treatment of LDA and EtCOCl to give aldol coupling precursor [18].Subsequent an-  ti-selective aldol reaction with methacrylaldehyde to provide the aldol product 8, which was converted into aldehyde 9 in three steps.Then the BF 3 •OEt 2 mediated Mukaiyamaaldol reaction of 9 with silyl ketene acetal 10 afforded methyl ester (5R)-11 as a single diastereomer.Successful Moffatt oxidation of 11 to give ketone 12, and subsequent reduction of the resulting keto group in 12 with NaBH 4 stereoselectively (S/R = 22/1) proceeded to give the desired alcohol (5S)-13 in 69% yield.PMB protection of the secondary hydroxy substituent at C5 in 14, followed by cleavage of the TBS protecting group at C7 with HF•Py, accomplishing the synthesis of 1,1-disubstituted alkene 2 (Scheme 3).
As the key step for synthesis of organostannane or organoboron precursor of 2, cross metathesis was chosen because it has become a powerful and convenient synthetic technique for the preparation of functionalized alkenes in organic chemistry [19].With this concern, hydroxy 1,1-disubstituted alkene 15 as a model compound for screening the most effective Ru complexes and cross partners in metathesis.Cross metathesis using 15 prepared by Grignard addition reaction [20] was investigated under various reaction conditions: Grubbs secondgeneration (G-II) [21] [22] or the Hoveyda-Grubbs second-generation complex (HG-II) [23] in CH 2 Cl 2 , benzene, and toluene at reflux or microware irradiation, and the use of an excess amount of cross partners such as vinylstannane 16a, or vinyl boronates (16b and 16c).These results are summarized in Table 1.Cross metathesis of 15 with vinylstannane 16a using HG-II (1.0 equiv) in CH 2 Cl 2 or toluene at reflux did not give the desired organostannane precursor 17a presumably due to highly sterically hindered Sn(n-C 4 H 9 ) 3 group in 16a (Table 1, entries 1-2).The lower sterically hinderedvinyl dioxaborolane 16b compared with tetramethyl vinyl boronate 16c also did not afford the corresponding organoboron compound 17b with the use of HG-II or G-II catalysts in thermal or microware heating conditions [24]- [31] (entries 3-5).Grubbs II-catalyzed cross metathesis of 15 with vinyl pinacol boronate 16c (4.0 equiv) in benzene at refluxdid not afford desired 17c (entry 6).By contrast, when 15 was treated with more robust and powerful HG-II (25 mol%) in CH 2 Cl 2 at reflux for one day, we observed a small amount of organoboron precursor 17c (entry 7).The increase of the catalyst to a stoichiometric amount under the same reaction conditions to give 17c in 35% yield (entry 8) as a single E-isomer as judged by the NOE observation.
We envisioned here that the E-selective olefin cross metathesis using HG-II catalyst in the reaction of 15 and vinyl pinacol boronate 16c could be applied for synthesis of the organoboron derivative of 1,1-disubstituted alkene 2, which is crucial precursor for the synthesis of the 11 C-labeling dihydroxy acid moiety of 1.However, contrary to our expectation, cross metathesis using 2 did not proceed under above reaction conditions (Table 1, entry 8) with the notice of complete recovery of 2.The reaction was further conducted under more forcing the reaction conditions, giving the desired (E)-organoboron derivative 3 in 14% yield along with recovered 2 in 32% yield (Scheme 4).The geometry of the newly formed double bond was decided by NOE observation as shown below.
By using the precursor 3, we examined the Pd 0 -mediate rapid C-[ 11 C]methylations protocol [12] for preparing the 11 C-labeled partial structure of 1 under cold conditions (Scheme 4).The methylation of 3 was conducted  following the standard procedure: CH 3 I dissolved in DMF was added to a solution of 3, Pd 2 (dba) 3 , P(o-tolyl) 3 , and K 2 CO 3 in DMF, and the resulting mixture was heated at 70˚C for 5 min, then purified on ODS to give 4b in 78% yield.Based on the above-mentioned protocol, we preformed the synthetic of 11 C-labeled Dtda methyl ester 4a, Thus, 3/Pd 2 (dba) 3 /P(o-tolyl) 3 /K 2 CO 3 (2:1:4:10) dissolved in DMF under argon was mixed with [ 11 C]CH 3 I, prepared as previously described [32], the solution was heated at 70˚C for 5 min (Scheme 5).After the mixture was poured into a separate vial containing a solution of ascorbic acid in acetonitrile, the resulting reaction mixture was submitted to HPLC, and then purified by reverse phase semi-preparative HPLC to give desired [ 11 C]Dtdamethyl ester 4a in 72% as reverse phase HPLC analytical yield (Figure 2).Total synthesis time including HPLC purification was 33 min.The radioactivity of isolated 4a was 315 MBq and the radiochemical purity was >99%.The chemical identity of 4a was confirmed by co-injection with the authentic sample of 4b by analytical HPLC.

Acknowledgements
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.We thank Dr. Masakatsu Kanazawa (Central Research Lab.Hamamatsu Photonics K. K.), Ms. Mawatari Aya (RIKEN Center for Life Science Technologies) for experimental assistance and Mr. Masahiro Kurahashi (Sumitomo Heavy Industry Accelerator Service Ltd.) for operating the cyclotron.

General
Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-ECX400P spectrometer (400 MHz for 1 H), and the chemical shifts in δ (parts per million) were referenced to the solvent peaks of δ H 7.26 for CHCl 3 .HR ESI-TOF-MS spectra were measured on an Applied Biosystems Mariner Biospectrometry Workstation using ABN as a calibration standard in the positive mode.Microwave irradiation was carried out in a Biotage Initiator™ (Tokyo, Japan) using a sealed vessel.[ 11 C]Carbon dioxide was produced by a 14 N(p, α) 11 C reaction by using a Sumitomo CYPRIS HM-12S cyclotron (Sumitomo Heavy Industries, Tokyo, Japan), and then converted to [ 11 C]methyl iodide by treatment with lithium aluminum hydride followed by hydriodic acid using an automated synthesis system (Cupid, Sumitomo Heavy Industries).The obtained [ 11 C]methyl iodide was used for palladium(0)-mediated rapid [ 11 C]methylation shown in Scheme 5.The synthesis of 11 C-labeled Dtda methyl ester 4a in Scheme 5 was conducted in a lead-shielded hot-cell with remote control of all operations in RIKEN CLST.Purification with HPLC was performed on a GL Science system (Tokyo, Japan).Radioactivity was quantified with an ATOMLAB™ 300 dose calibrator (Aloka, Tokyo, Japan).Analytical HPLC was performed on a Shimadzu system (Kyoto, Japan), and effluent radioactivity was measured with an RLC700 radio analyser  (Aloka).The column used for analytical and semi-preparative HPLC was Develosil ODS-HG-5 (Nomura Chemical, Japan).

Synthesis of Organoboron Precursor 3
Alcohol 2 was prepared from 14 by deprotection of the second TBS ether using HF•Py as a colorless oil. 1
C-incorporated C(1)-C(10) partial structure in [ 11 C]kulokekahilide-2 focused on the 11 C labeling at C10 carbon of 1 using a combination of olefin cross metathesis (CM) and rapid C-[ 11 C]methylation.