Synthesis of [11C]dehydropravastatin, a PET probe potentially useful for studying OATP1B1 and MRP2 transporters in the liver

Article abstract of DOI:10.1016/j.bmc.2012.04.051

Drug transporters mediate the uptake and elimination of drugs in various organs; therefore, having knowledge of how a transporter functions in the body would play a key role in ensuring drug efficacy in in vivo systems. In this context, we designed and synthesized [¹¹C]dehydropravastatin, a novel PET probe that would be potentially useful for evaluation of the functions of the OATP1B1 and MRP2 transporters, based on the use of palladium(0)-mediated rapid C-[¹¹C]methylation (viz., the rapid cross-coupling between [¹¹C]methyl iodide and a boron intermediate).

Full text of DOI:10.1016/j.bmc.2012.04.051

Bioorganic & Medicinal Chemistry 20 (2012) 3703–3709
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of [11C]dehydropravastatin, a PET probe potentially useful for studying
OATP1B1 and MRP2 transporters in the liver
Ryosuke Ijuin a, Tadayuki Takashima b, Yasuyoshi Watanabe b, Yuichi Sugiyama c, Masaaki Suzuki a,
⇑
a Molecular Imaging Medicinal Chemistry Laboratory, Riken Center for Molecular Imaging Science (CMIS), 6-7-3 Mintojima-minamimachi, Chuo-ku, Kobe, Hyogo, Japan
b Molecular Probe Dynamics Laboratory, Riken Center for Molecular Imaging Science (CMIS), 6-7-3 Mintojima-minamimachi, Chuo-ku, Kobe, Hyogo, Japan
c Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Drug transporters mediate the uptake and elimination of drugs in various organs; therefore, having
knowledge of how a transporter functions in the body would play a key role in ensuring drug efficacy
in in vivo systems. In this context, we designed and synthesized [11C]dehydropravastatin, a novel PET
probe that would be potentially useful for evaluation of the functions of the OATP1B1 and MRP2 trans-
porters, based on the use of palladium(0)-mediated rapid C-[11C]methylation (viz., the rapid cross-cou-
pling between [11C]methyl iodide and a boron intermediate).
Received 26 March 2012
Revised 25 April 2012
Accepted 25 April 2012
Available online 30 April 2012
Keywords:
Positron emission tomography
C–C Coupling
Ó 2012 Elsevier Ltd. All rights reserved.
Rapid reaction
Transporter
1. Introduction
transporter functions in the liver is important for drug develop-
ment4 as well as for diagnoses of hepatic diseases associated with
Positron emission tomography (PET)1–3 is an important molecu-
lar imaging diagnostic technique with excellent disease sensitivity,
and it is routinely used to detect and assess oncologic, neurologic,
and cardiologic abnormalities. This noninvasive molecular imaging
technique is used to study physiology by visualizing the distribu-
tion of radiopharmaceuticals in in vivo vital systems. It is also
expected that PET probes could be applicable to revolutionizing
drug development by introducing a human microdose study at an
early stage (phase 0) to efficiently suppress the large drop-out
(>90%) of drug candidates during clinical trials (phases I–III).
One of the major functions of the liver is to remove various
endogenous and exogenous compounds from the blood circulation.
This clearance process involves the uptake of compounds across
the sinusoidal membrane of the hepatocyte and the efflux across
the bile canalicular membrane; therefore, research of how a
a particular transporter dysfunction.
Among various drug transporters, we focused on organic anion-
transporting polypeptides (OATPs)5,6 and multidrug resistance-
associated protein 2 (MRP2),7 which play roles in the uptake of
drugs into the liver and the canalicular efflux, respectively.8
Pravastatin (1),9 a member of the statin class of drugs, has been
used widely to lower the cholesterol concentration in blood by
inhibiting the action of 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase, which plays a central role in the produc-
tion of cholesterol in the liver. In humans, the uptake of pravasta-
tin into the liver is mediated by the OATP1B1 transporter,10–12 and
the drug is then excreted into the bile by MRP2 without metabo-
lism.13 Therefore, pravastatin would be a good molecular probe
for PET to track and evaluate the functions of these two transport-
ers in vivo.
A comparison of the structures of simvastatin (2)14 and lova-
statin (3),15 both belonging to the statin class of drugs, suggests that
the chiral center at C(20) and the number of methyl groups in the
⇑
Corresponding author. Tel.: +81 78 304 7130; fax: +81 78 304 7131.
E-mail address: suzuki.masaaki@riken.jp (M. Suzuki).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.051

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R. Ijuin et al. / Bioorg. Med. Chem. 20 (2012) 3703–3709
Scheme 2. Synthesis of the boronic acid unit 12. (i) Bromine, CCl4, 0 °C to room
temperature, 16 h, quant.; (ii) DBU, THF, 0 °C to room temperature, 16 h, 85%; (iii)
(BPin)2, PdCl2(dppf)ÅCH2Cl2, KOAc, DMSO, 80 °C, 18 h, 69%; iv) TFA, CH2Cl2, room
temperature, 4 h, 59%, recovery 14%.
methylation reaction between sp2(vinyl)-sp3 carbons using
[
11C]methyl iodide and an organoboron precursor in the 11C-label-
ing step.
Scheme 1. Synthesis of the alcoholic intermediate 7. (i) NaOMe, MeOH, reflux,
60 h; (ii) H2O, HCl, 0 °C; (iii) (EtO)2P(O)CN, Et3N, THF, 1.5 h, 0 °C to room
temperature; (iv) TBDMSCl, imidazole, DMF, 2 h, 0 °C to room temperature, overall
yield 32%.
2. Results and discussion
2.1. PET probe synthesis
The syntheses of dehydropravastatin (4) and radioactive [11C]
dehydropravastatin ([11C]4) were performed via the deconstruc-
tion–reconstruction of an ester moiety, as shown in Schemes 1–
4. In Scheme 1, pravastatin sodium salt undergoes methanolysis
with NaOMe to remove an ester moiety, and this is followed by
lactonization using diethyl cyanophosphonate treatment in the
presence of triethylamine and the protection of hydroxy groups
by a tert-butyldimethylsilyl (TBS) group to render the alcoholic
intermediate 7.16
As shown in Scheme 2, the acid unit was synthesized as follows.
Commercially available tert-butyl methacrylate (8) was dibromi-
nated with bromine in CCl4, at 0 °C to room temperature, to give
intermediate 9 quantitatively, which was then debrominated with
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) at 0 °C in tetrahydrofu-
ran (THF), giving vinylic bromide (10) in 85% yield.17,18 The vinyl
bromide (10) was subsequently converted to 3-pinacol boronated
methacrylate (11) in 69% yield, which underwent de-esterification
with trifluoroacetic acid to give the desired boronated acid (12) in
59% yield.19
ester moiety of the statin structure do not have much influence on
the efficacies of these drugs. Therefore, we instead chose dehydro-
pravastatin (4) because it lacks a chiral center at C(20), thus making
it a suitable candidate for the radiolabeling in terms of both its eas-
ier synthetic accessibility compared with that of pravastatin and it
being an adequate target for our new synthetic methodology to
synthesize a 11C-labeled PET molecular probe.
Short-lived radionuclides such as 11C and 18F, with physical half-
lives of 20.4 and 109.8 min, respectively, are usually used for radio-
labeling probes for PET. The carbon atom is an essential element in
organic molecules such as drugs, and therefore, 11C labeling is the
most important method for the synthesis of a PET probe. In this con-
text, we have developed various types of palladium(0)-mediated
rapid C-[11C]methylations onto organic frameworks, based on the
cross-coupling reaction of methyl iodide and the corresponding
organostannanes or organoboron compounds.
The labeling of a highly functionalized complex molecule is a
fascinating and challenging subject of our rapid C-[11C]methylation
reaction, and its realization proves the high potentiality of the new
methodology. We describe herein the synthesis of [11C]20,30-dehy-
dropravastatin, which was well executed by our rapid C-[11C]
The reconstruction of the whole dehydropravastatin structure
was conducted as follows. The alcohol 7 was esterified with com-
mercially available tiglic acid using diethyl chlorophosphate and
Scheme 3. Synthesis of 20,30-dehydropravastatin (4) and boronic ester precursor (16) for [11C]dehydropravastatin formation. (i) Tiglic acid, (EtO)2P(O)Cl, 4-pyrrolidino-
pyridine, Et3N, benzene, 0 °C for 30 min, then canulation to substrate, reflux, 2 h, 86%; (ii) TBAF, AcOH, THF, 0 °C to room temperature (rt), 90%; (iii) 0.1 M NaOH, H2O, 1,4-
dioxane, room temperature, 15 min, quant.; (iv) 12, (EtO)2P(O)Cl, 4-pyrrolidinopyridine, Et3N, benzene, 0 °C to reflux, 2 h, 86%; (v) TBAF, THF, room temperature.

R. Ijuin et al. / Bioorg. Med. Chem. 20 (2012) 3703–3709
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Scheme 4. Synthesis of [11C]dehydropravastatin ([11C]-4). (i) 11CO2, LAH, HI; (ii) Pd2(dba)3, P(o-toly)3, K2CO3, THF, 65 °C, 5 min; (iii) TBAF, 60 °C, 2 min; iv) 0.1 M NaOH, room
temperature, 1 min.
triethylamine to give the ester 13 in 86% yield. The reaction gave a
7:3 mixture of (E), (Z) isomers, which were treated by normal-phase
high-performance liquid chromatography (HPLC) to give the pure
(E) isomer 13.20 The resulting isomer 13 was deprotected by treat-
ment with tetrabutylammonium fluoride (TBAF) in the presence of
a large amount of acetic acid in order to neutralize the basicity of
the tetrabutylammonium ion to give compound 14 in 90% yield.21
Without acetic acid, the desired compound 14 was not obtained; in-
stead, an undesired aromatic compound 15 was produced via dehy-
drogenation. The deprotected lactone structure in compound 14 was
opened by sodium hydroxide to give dehydropravastatin (4) in
quantitative yield.
It is expected that dehydropravastatin sodium (4) would be a
good substrate for the OATP transporter, similar to natural pravasta-
tin sodium (the detailed in vitro and in vivo data will be shown in a
separate biological paper). With the consideration of such potential
biological information in mind, we continued to label the dehydro-
pravastatin (4) with a 11C radionuclide.
The alcoholic intermediate 7 was reacted with the boronated
acrylic acid 12 under the same conditions as the synthesis of com-
pound 13, giving boronic ester 16, a precursor for 11C labeling.
Since compound 16 was very unstable under TBAF conditions,
we selected it as a substrate for the 11C-labeling reaction without
TBS group deprotection.
Thus, boronated ester 16 was reacted with [11C]methyl iodide in
the presence of Pd2(dba)3, P(o-toly)3, and K2CO3 (in 1:3:4 molar
ratio) at 65 °C for 5 min in THF to give a mixture of the desired
11C-incorporated 13 ([11C]13) and the further dehydroxylated
(undesired) [11C]15, which were detected by HPLC using a UV
and radio detector.22,23 Successive treatments of the resulting mix-
ture with a solution of TBAF in THF, and then with an aqueous
NaOH solution, gave a mixture that included the desired [11C]4.
It should be noted that it was difficult to deprotect silyl groups
using TBAF in dimethylformamide (DMF). Such deprotection in
THF gave the desired [11C]4, in addition to another radioactive
Figure 1. Color-coded PET images of the abdominal regions of rats after administration of [11C]4. Coronal maximum intensity projection PET images of radioactivity in the
abdominal region were captured at 1, 5, 10, 20, 30, and 60 min in control rats (A), in rats treated with rifampicin at an infusion rate of 1.5 l
mol minꢀ1 kgꢀ1 (B), and in Mrp2
hereditary-deficient rats (C) after intravenous administration of [11C]4.

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R. Ijuin et al. / Bioorg. Med. Chem. 20 (2012) 3703–3709
byproduct that was identified by liquid chromatography—mass
P2O5 (Merck, SICAPENT). NMR spectra were recorded on a JEOL
AL-400 spectrometer (400 MHz for 1H, 100 MHz for 13C). Chemical
shifts are reported in d parts per million referenced to an internal
tetramethylsilane standard for 1H NMR. Chloroform-d1 (d 77.0 for
13C) was used as an internal reference for 13C NMR. 1H and 13C
NMR spectra were recorded in CDCl3, CD3OD. Mass spectra were
recorded on a Thermofisher Scientific LCQ Fleet mass spectrome-
ter. High resolution mass spectra were recorded on a JEOL JMS-
T100LC mass spectrometer. Optical rotation was recorded on a
JASCO P-2100 polarimeter. Melting point was measured by an
AS-ONE ATM-01 melting point apparatus.
spectrometry to be the dehydrated structure [11C]17.
2.2. PET study of [11C] 20,30-dehydropravastatin ([11C]4)
PET images of the radioactivity in the abdominal region over
time, following administration of [11C]4 (22 1 MBq/body) to rats,
are shown in Figure 1. Radioactivity was identifiable in the liver
and kidneys within 2 min after the radiotracer administration, at
which point the radioactivity began to decrease rapidly. On the
other hand, the radioactivity was localized mainly in the intestine
(via the bile excreted into the intestine), and some radioactivity
was also observed in the urinary bladder (via the urine excreted
into the urinary bladder) by 60 min (Fig. 1a). The radioactivity
excreted into the bile or the urine increased until the end of the scan
and reached 67.9 8.9% and 1.51 0.33% of the original dose,
respectively. When rifampicin, a typical OATP inhibitor, was co-
administered with [11C]4, the distribution of the radioactivity in
the liver and that excreted into the bile was decreased (Fig. 1b).
In addition, the total radioactivity in the bile of Mrp2 hereditary-
deficient rats was reduced as compared with that in control animals
(Fig. 1c). The radiometabolite of [11C]4 was detected in the blood,
liver, and bile sampled within 20 min after radiotracer administra-
tion, and its composition was much lower than that of [11C]4 in
blood sampled at 2 min. [11C]4 formed the major portion of the
radioactivity in the blood, liver, and bile sampled at 20 min, but
the existence of its radiometabolite was not negligible in rats. In
bile, at least four metabolites were detected, three of which were
polar than unchanged compound 4. LC/MS/MS analysis revealed
that ions with a m/z of 437.3 (+16 Da) and a m/z of 455.3 (+34 Da)
were found in the extracts of bile sampled after IV injection of com-
pound 4. It is speculated that a m/z 437.3 may be an oxidative form
of compound 4, and a m/z 455.3 may be a dihydrodiol form in the
alkene position of compound 4, However, the identification of these
metabolites was not pursued further as it accounted for a very small
proportion of the dose. In addition, in vitro metabolism using the
hepatocyte suspension system has shown that the metabolic stabil-
ity in human hepatocytes is much higher than that in rat hepato-
cytes (data not shown). Thus, [11C]4 has great potential to be a
suitable PET probe to evaluate the functions of OATPs (ideally
OATP1B1) and MRP2 in hepatobiliary excretion. Further investiga-
tion using [11C]4 for evaluating the functions of these drug trans-
porters in rats and in vitro transport study is ongoing.
The [11C]methylation was conducted in a lead-shielded hot cell
with remote control of all operations. [11C]Carbon dioxide was
produced by a 14N(p, 11C reaction using a CYPRIS HM-12S cyclo-
a)
tron (Sumitomo Heavy Industries) and then converted into
[
11C]methyl iodide by treatment with lithium aluminum hydride
followed by hydriodic acid using an original automated synthesis
system for 11C-labeling in RIKEN Center for Molecular Imaging
Science. The obtained [11C]methyl iodide was used for the palla-
dium(0)-mediated rapid [11C]methylation. The analytical HPLC
system used for the [11C]methylated product consisted of an Aloka
radioanalyzer (RLC-700) and a Shimadzu HPLC system with a
system controller (CBM-20A), an online degasser (DGU-20A3), a
solvent delivery unit (LC-20AB), a column oven (CTO-20AC), a pho-
todiode array detector (SPD-M20A), and software (LC-Solution).
The columns used for analytical and semipreparative HPLC were
COSMOSIL C18 MS-II 4.6 ꢁ 100 mm and 10 ꢁ 250 mm (Nacalai
Tesque). The radioactivity was quantified with an ATOMLAB™
300 dose calibrator (Aloka).
4.1.2. Synthesis of tert-butyl 2,3-dibromo-2-methylpropanoate
(9)
A solution of tert-butyl methacrylate (8.15 mL, 50 mmol) in CCl4
was cooled to 0 °C and added 2.65 mL of bromine (51 mmol) and
stirred for 16 h at room temperature. The reaction mixture was
then quenched with saturated sodium bisulfate and diluted with
ether. The organic phase was extracted with ether three times
and dried over Na2SO4. Combined organic layer was concentrated
under reduced pressure to afford 15 g (quant. as light yellow oil)
of desired compound 9.
3. Conclusion
1H NMR (CDCl3)
d 4.18(dd, J = 0.49, 9.75 Hz, 1H), 3.70
(d, J = 9.75 Hz, 1H), 1.98(d, J = 0.49 Hz, 3H), 1.51(s, 9H).
13C NMR (CDCl3) d 167.0, 82.8, 56.6, 38.2, 27.2, 25.9.
Mass spectra (EI) 289 (M+4-CH3), 287 (M+2-CH3), 285 (M-CH3),
231 (M+4-OtBu), 229 (M+2-OtBu), 227 (M-OtBu), 203 (M+4-OtBu-
CO), 201 (M+2-OtBu-CO), 119 (M-OtBu-CO).
In conclusion, 20,30-dehydropravastatin (4) and radiolabeled
[
11C]4 were designed and synthesized using palladium(0)-medi-
ated C-methylation and rapid C-[11C]methylation, respectively. In
radiosynthesis, the cross-coupling reaction and deprotection of
the TBS group as well as hydrolysis were carried out in a single
container so that the loss of intermediates in the transfer process
could be minimized. The physicochemical properties of [11C]4
were as follows: (1) total radioactivity, 1.2 GBq; (2) radio purity,
>99%; chemical purity, 98%; (3) radio chemical yield, 64%; decay
HRMS (EI) m/z Calcd for C7H11O281Br2: 288.9085. Found
288.9088 [M-CH3], m/z Calcd for C7H11O279Br81Br: 286.9105.
Found 286.9102 [M-CH3], m/z Calcd for C7H11O279Br2: 284.9125.
Found 284.9135 [M-CH3].
correct yield, 19.7%; specific radioactivity, 79 GBq/lmol. The total
synthesis time was 34 2 min. The application of [11C]4 to PET
4.1.3. Synthesis of (E)-tert-butyl 3-bromo-2-methylacrylate (10)
studies on animals and humans will be reported in due course.
4. Experimental section
4.1. General
A solution of tert-butyl 2,3-dibromo-2-methylpropanoate (3.03 g,
10 mmol) in THF was cooled at to 0 °C and added DBU (1.65 mL,
11 mmol) and stirred for 16 h at room temperature. The reaction
mixture was diluted with ether and then washed with water. The
All manipulations were carried out under an argon atmosphere
unless otherwise noted. Argon gas was dried by passage through

R. Ijuin et al. / Bioorg. Med. Chem. 20 (2012) 3703–3709
3707
organic phase was washed with 5% of citric acid three times, satu-
4.1.6. Synthesis of (E)-(1S,3S,7S,8S)-3-((tert-
butyldimethylsilyl)oxy)-8-(2-((2R,4R)-4-((tert-
butyldimethylsilyl)oxy)-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-
7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2-methylbut-
2-enoate (13)
rated sodium hydrogen carbonate solution twice, and brine. The
organic layer was dried over Na2SO4 and concentrated under
reduced pressure to afford 1.86 g (8.46 mmol, 85% as colorless
oil) of desired compound 10.
1H NMR (CDCl3) d 7.41(q, J = 1.6 Hz, 1H), 1.96(d, J = 1.6 Hz, 3H),
1.49(s, 9H).
13C NMR (CDCl3) d 164.0, 135.4, 121.6, 81.4, 27.9, 15.5.
Mass spectra (EI) 222 (M+2), 220 (M) 149, (M+2-OtBu), 147 (M-
OtBu), 121 (M+2-OtBu-CO), 119 (M-OtBu).
HRMS (EI) m/z Calcd for C4H4O181Br: 148.9425. Found 148.9423
[M-OtBu], m/z Calcd for C4H4O179Br: 146.9445. Found 148.9444
[M-OtBu].
To a solution of 89 mg (0.6 mmol) of 4-pyrrolidinopyridine in
0.6 mL of benzene was added 112
and 40 mg (0.4 mmol) of tiglic acid at 0 °C. Then, 86
l
L (0.8 mmol) of triethylamine
4.1.4. Synthesis of (E)-tert-butyl 2-methyl-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (11)
lL (0.5 mmol)
of diethyl chlorophosphate was added to the resulting solution. The
mixture was stirred at room temperature for 30 min. A solution of
(4R, 6R)-6-{2-[(1S, 2S, 6S, 8S, 8aR)-1,2,6,7,8,8a-hexahydro-6-tert-
butyldimethylsilyloxy-8-hydroxy-2-methyl-1-naphthyl]ethyl}tet-
rahydro-4-tert-butyldimethylsilyloxy-2H-pyran-2-one (110 mg;
0.2 mmol) dissolved in 0.2 mL of benzene was added to the reaction
mixture, and the mixture was refluxed for a further 2.5 h. At the end
of this time, the reaction mixture was cooled to room temperature,
and diluted with ethyl acetate. The diluted mixture was washed
with 5% citric acid three times, saturated sodium hydrogen carbon-
ate two times, and brine. The organic phase was dried over Na2SO4
and concentrated under reduced pressure. The resulting oil was
purified by silica gel column chromatography, to give 108 mg
A solution of (E)-tert-butyl 3-bromo-2-methylacrylate (221 mg,
1 mmol) and KOAc (196 mg, 2 mmol) and bis (pinacolato) diboron
(508 mg, 2 mmol) in 3.5 mL of 1,4-dioxane was added 10 mg of
PdCl2(dppf)ÅCH2Cl2, and then reaction mixture was heated to
80 °C for 16 h. The reaction mixture was diluted with water and ex-
tracted with ether 4 times. Combined organic layer was washed
with brine, dried over Na2SO4, and concentrated. Resulting crude
was purified by SiO2 column chromatography to afford 184 mg
(68.6% as white solid) of desired compound 11.
1H NMR (CDCl3) d 6.43(q, J = 1.2 Hz, 1H), 2.11(d, J = 1.2 Hz, 3H),
1.48(s, 9H), 1.29(s, 12H).
(172 lmol, 86% as colorless oil) of desired compound 13.
1H NMR (CDCl3) d 6.77(m, 1H), 5.99(d, 1H, J = 9.77 Hz), 5.85(dd,
1H, J = 5.85, 9.77 Hz), 5.49(br s, 1H), 5.41(br s, 1H), 4.57(m, 1H),
4.42(m, 1H), 4.26(m, 1H), 2.34–2.40(m, 5H), 1.26–1.85(m, 15H),
0.88(s, 18H), 0.88(s, 3H), 0.07(s, 12H).
13C NMR (CDCl3) d 167.9, 150.1, 84.3, 84.3, 81.3, 28.8, 28.8, 28.8,
25.7, 25.7, 25.7, 25.7, 17.8.
MS (ESI) 291 (M+Na).
HRMS (TOF) m/z Calcd for C14H25B1O4Na1: 291.1746. Found
291.1743 [M+Na].
13C NMR (CDCl3) d 170.7, 167.9, 137.6, 135.6, 135.0, 129.1,
128.1, 127.6, 76.5, 70.1, 66.1, 64.0, 39.7, 38.0, 37.5, 37.2, 33.3,
31.5, 18.7, 18.4, 14.9, 14.2, 12.5, ꢀ4.47.
½
a 2d5
+69.6 (c = 18.9, CHCl3).
ꢂ
MS (ESI) 655 (M+Na), 633(M+H).
HRMS (TOF) m/z Calcd for C35H60O6Si2Na1: 655.3826. Found
655.3826 [M+Na].
Melting point 62 °C.
4.1.5. Synthesis of (E)-2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)acrylic acid (12)
4.1.7. Synthesis of (E)-(1S,3S,7S,8S)-3-hydroxy-8-(2-((2R,4R)-4-
hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-7-methyl-
1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2-methylbut-2-enoate
(14)
A solution of (E)-tert-butyl 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)acrylate (268 mg, 1 mmol) in 5 mL of CH2Cl2
was added 0.5 mL of trifluoroacetic acid at 0 °C and then stirred
at room temperature for 2 h. The reaction mixture was purified
by PLC (hexane/EtOAc 1/1) to afford 126 mg (59% as light yellow
powder) of desired compound 12 and 37 mg (14%) of the starting
boron substrate.
To a solution of 40 mg (63 lmol) (E)-(1S,3S,7S,8S)-3-((tert-butyldi-
1H NMR (CD3OD) d 11.1(br, 1H), 6.40(q, J = 1.2 Hz, 1H), 2.09(d,
J = 1.2 Hz, 3H), 1.29(s, 12H).
methylsilyl)oxy)-8-(2-((2R,4R)-4-((tert-butyldimethylsilyl)oxy)-6-
oxotetrahydro-2H-pyran-2-yl)ethyl)-7-methyl-1,2,3,7,8,8a-hexa-
hydronaphthalen-1-yl 2-methylbut-2-enoate in 0.5 mL of THF was
13C NMR (CD3OD) d 171.8, 145.6, 83.3, 83.3, 24.4, 24.4, 24.4,
24.4, 16.3.
added 86 lL (1.5 mmol) of acetic acid and 1 mL (1 mmol) of 1.0 M
MS (ESI) 213 (M+H) [positive mode], 211(Mꢀ1) [negative
tetrabuthylammonium fluoride THF solution at 0 °C. The mixture
was then stirred for 14 h at room temperature. At the end of this
time, the mixture was diluted with ethyl acetate, and washed with
saturated sodium hydrogen carbonate two times and brine. The
mode].
HRMS (TOF) m/z Calcd for
211.1150 [MꢀH].
C10H16B1O4: 211.1143. Found
Melting point 152 °C.

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organic phase was dried with Na2SO4 and concentrated under re-
duced pressure. The resulting oil was purified by silica gel column
chromatography to give 23 mg (56.9 lmol, 90% as white powder)
of benzene was added diethyl chlorophosphate (130 lL;
0.75 mmol) at 0 °C for 30 min. The reaction mixture was added
to the solution of (4R, 6R)-6-{2-[(1S, 2S, 6S, 8S, 8aR)-1,2,6,7,8,8a-
hexahydro-6-tert-butyldimethylsilyloxy-8-hydroxy-2-methyl-1-
naphthyl]ethyl}tetrahydro-4-tert-butyldimethylsilyloxy-2H-pyr-
an-2-one (165 mg; 0.3 mmol) was added to the reaction mixture,
and then the mixture was refluxed for a further 2.5 h. At the end
of this time, the reaction mixture was cooled to room temperature,
and then diluted with ethyl acetate. The diluted mixture was
washed with 5% citric acid three times, saturated sodium hydrogen
carbonate two times, and brine. The organic phase was dried over
Na2SO4 and concentrated under reduced pressure. The resulting oil
was purified by silica gel column chromatography to give 192 mg
(86.0% as light yellow solid) of desired compound 16.
of desired compound 14.
1H NMR (CDCl3) d 6.78(m, 1H), 6.01(d, 1H, J = 9.51 Hz), 5.89(dd,
1H, J = 5.83, 9.51 Hz), 5.59(br s, 1H), 5.44(br s, 1H), 4.60(m, 1H),
4.42(m, 1H), 4.35(m, 1H), 2.57–2.75(m, 3H), 2.34–2.42(m, 2H),
1.90(m, 1H), 1.29–1.79(m, 15H), 0.91(d, 3H, J = 7.07 Hz).
13C NMR (CDCl3) d 170.2, 167.5, 137.7, 135.6, 135.2, 128.4,
127.3, 126.0, 75.8, 69.3, 64.9, 62.5, 30.2, 38.4, 37.6, 36.7, 36.5,
35.8, 32.5, 30.9, 23.4, 14.3, 13.5, 11.9.
½
a 2d5
+266.4 (c = 0.50, CHCl3).
ꢂ
MS (ESI) 427 (M+Na), 831(2 M+Na).
HRMS (TOF) m/z Calcd for C23H32O6Na1: 427.2097. Found
427.2105 [M+Na].
1H NMR (CDCl3) d 6.41(s, 1H), 5.97(d, 1H, J = 9.76 Hz), 5.84(dd,
1H, J = 5.85, 9.76 Hz), 5.47(br s, 1H), 5.41(br s, 1H), 4.56(m, 1H),
4.41(m, 1H), 4.26(m, 1H), 2.35–2.57(m, 5H), 2.17(s, 2H), 2.12(s,
3H), 1.85–1.64(m, 4H), 1.30–1.26(m, 15H), 0.88(s, 18H), 0.88(s,
3H), 0.06(s, 12H).
Melting point 156 °C.
4.1.8. Synthesis of sodium (3R,5R)-3,5-dihydroxy-7-
((1S,2S,6S,8S)-6-hydroxy-2-methyl-8-(((E)-2-methylbut-2-
enoyl)oxy)-1,2,6,7,8,8a-hexahydronaphthalen-1-yl)heptanoate
(4)
13C NMR (CDCl3) d 170.3, 167.2, 147.3, 135.1, 134.4, 127.7,
127.7, 127.2, 83.7, 83.7, 76.3, 70.5, 65.5, 63.5, 39.3, 37.6, 36.9,
36.7, 36.6, 32.9, 31.0, 25.9, 25.9, 25.9, 25.9, 25.7, 25.7, 25.7, 24.8,
24.8, 23.6, 18.2, 18.0, 17.1, 13.7, ꢀ4.57, ꢀ4.66, ꢀ4.87, ꢀ4.91.
½
a 2d5
+93.0 (c = 0.60, CHCl3).
ꢂ
MS (ESI) 744(M+H), 767 (M+Na)
HRMS (TOF) m/z Calcd for C40H69BO8Si2Na: 767.4529. Found
767.4526 [M+Na].
Melting point 63 °C.
4.1.10. Radiosynthesis of sodium (3R,5R)-3,5-dihydroxy-7-
((1S,2S,6S,8S)-6-hydroxy-2-methyl-8-((1-[11C]-(E)-2-methylbut-
2-enoyl)oxy)-1,2,6,7,8,8a-hexahydronaphthalen-1-
yl)heptanoate [11C]-4
To a solution of 23 mg (56.9
(2-((2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-7-
methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2-methylbut-2-
enoate in 1 mL of 1,4-dioxane and 480 L of water was added
569 L (56.9 mol) of 0.1 N NaOH solution. The mixture was stir-
lmol) (E)-(1S,3S,7S,8S)-3-hydroxy-8-
l
l
l
red for 30 min at room temperature. At the end of this time, reac-
tion mixture was freeze dried to give 27 mg of white powder. The
powder was purified by HPLC giving 23 mg of pure compound 4.
1H NMR (CD3OD) d 6.77(m, 1H), 5.99(d, 1H, J = 9.51 Hz), 5.88(m,
1H), 5.51(br s, 1H), 5.38(br s, 1H), 4.26(m, 1H), 4.05(m, 1H), 3.65(br
s, 1H), 2.20–2.53(m, 6H), 1.78(s, 3H), 1.77(s, 3H), 1.22–1.70(m, 7H),
0.91(d, 3H, J = 7.1 Hz).
13C NMR (CD3OD) d 180.4, 169.1, 138.7, 136.8, 136.7, 130.0,
128.6, 72.3, 71.3, 69.4, 68.1, 65.5, 45.5, 44.9, 39.0, 38.5, 37.1,
35.4, 32.4, 25.0, 14.4, 14.0, 12.1.
[
11C]Methyl iodide formed from [11C]CO2 according to the estab-
lished method24 was trapped in a solution of (E)-(1S,3S,7S,8S)-3-
((tert-butyldimethylsilyl)oxy)-8-(2-((2R,4R)-4-((tert-butyldimeth-
ylsilyl)oxy)-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-7-methyl-1,2,
½
a 2d5
+131.3 (c = 0.44, EtOH).
MS (ESI) 421 (M-Na) [negative mode].
ꢂ
3,7,8,8a-hexahydronaphthalen-1-yl
2-methyl-3-(4,4,5,5-tetra-
(2.0 mg; 2.7 mol),
mol), and
L) at room temperature.
The resulting mixture was heated to 65 °C for 5 min. The reaction
mixture was cooled to 0 °C. Then 200 L of 1 M TBAF solution was
added to the reaction mixture and heated to 60 °C for 2 min. The
reaction vessel was cooled by air to room temperature and 400
of 0.1 M NaOH solution was added, and then placed 1 min. After
dilution with 400 L of 10 mM NH4OAc, the mixture was injected
HRMS (TOF) m/z Calcd for C23H33O7: 421.2226. Found 421.2221
methyl-1,3,2-dioxaborolan-2-yl)acrylate
l
[M-Na].
Pd2(dba)3 (2.0 mg; 2.2
lmol), P(o-toly)3 (2.0 mg; 6.6 l
Melting point 134 °C.
K2CO3 (1.2 mg; 8.7 mol) in THF (300 l
l
l
4.1.9. Synthesis of (E)-(1S,3S,7S,8S)-3-((tert-
butyldimethylsilyl)oxy)-8-(2-((2R,4R)-4-((tert-
butyldimethylsilyl)oxy)-6-oxotetrahydro-2H-pyran-2-yl)ethyl)-
7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2-methyl-3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (16)
lL
l
onto a semipreparative HPLC column with a mobile phase consisting
of 10 mM NH4OAc and CH3CN (70:30) under the flow rate of 5 mL/
min with equipped UV detection at 230 nm. The [11C]4 retention
time was 12.5 min. The desired fraction was collected in a flask
and the organic solvent was removed under reduced pressure.
For the radio pharmaceutical formulation of [11C]4 for use in a
PET study, [11C]4 was dissolved with a 4 mL of saline. The total syn-
thesis time from end of bombardment (EOB) was 34 min. The de-
cay-corrected radiochemical yield was 19.7% (n = 3) with specific
A solution of (E)-2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)acrylic acid (121 mg; 0.57 mmol) and 4-pyrrolidinopyri-
dine (134 mg; 0.9 mmol) and Et3N (168 lL; 1.2 mmol) in 0.8 mL
radioactivity (SA) of 79 GBq/l
mol (n = 3). We assigned [11C]4 by

R. Ijuin et al. / Bioorg. Med. Chem. 20 (2012) 3703–3709
3709
LC-MS using the remaining [12C]4. Observed peak was 421 (M-Na)
in negative mode.
and its metabolite was calculated as a percentage of the total
amount radioactivity.
4.2. Imaging
Acknowledgements
4.2.1. Experimental animals
This work was supported in part by the Research & Develop-
ment of Life Science Fields responding to the need of society,
Molecular Imaging Research Program of the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan (2005–
2010). Part of this study was also supported by the Research Pro-
ject for the ‘Establishment of Evolutional Drug Development with
the Use of Microdose Clinical Trial’ sponsored by the New Energy
and Industrial Technology Development Organization (NEDO).
We thank Daiichi-Sankyo Co. Ltd. for providing the pravastatin so-
dium. We thank Mr. Masahiro Kurahashi (Sumitomo Heavy Indus-
try Accelerator Service Ltd.) for operating the cyclotron, and Ms.
Yumiko Katayama, Mr. Yasuhiro Wada, Ms. Emi Hayashinaka,
and Mr. Takashi Okauchi of the RIKEN Center for Molecular Imag-
ing Science for their expert technical assistance.
Male Sprague-Dawley (SD) rats weighing 180–242 g and male
Mrp2 hereditary-deficient Eisai hyperbilirubinemic rats (EHBRs)
weighing 287 g were purchased from Japan SLC Inc. The animals
were kept in a temperature- and light-controlled environment
and had ad libitum access to standard food and tap water. All
experimental protocols were approved by the Ethics Committee
on Animal Care and Use of the Center for Molecular Imaging Sci-
ence in RIKEN, and were performed in accordance with the Princi-
ples of Laboratory Animal Care (NIH publication No. 85-23, revised
1985).
4.2.2. PET studies
Rats were anesthetized with a mixture of 1.5% isoflurane and ni-
trous oxide/oxygen (7/3) and then placed on the PET scanner gan-
try (MicroPET Focus 220, Siemens Co., Ltd, Knoxville, TN, USA). The
PET scanner has a spatial resolution of 1.4 mm FWHM at the center
of the field of view, which is 220 mm in diameter with an axial ex-
tent 78 mm in length. After intravenous bolus injection of [11C]4
(approximately 22 1 MBq per animal) via a venous catheter in-
serted into the tail vein, a 60 min emission scan was performed.
The chemical amount of [11C]4 for the bolus injection was calcu-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.051.
References and notes
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fused at a rate of 1.5 lmol/min/kg for at least 90 min before the
administration of [11C]4, and then a constant infusion was kept un-
til the end of the PET scan.
Radiometabolite analysis of rat blood, liver, and bile was per-
formed separately from the PET study to evaluate the composition
of the radioactivity in the tissue. Equal volumes of acetonitrile
were added to an aliquot of the samples (blood, liver homogenate,
and bile) and then the resulting mixture was centrifuged at
12,000 rpm for 2 min at 4 °C. The supernatant was diluted with
HPLC mobile phase, and then analyzed for radioactive components
by using an HPLC system (Shimadzu Corporation) with a coupled
NaI(Tl) positron detector UG-SCA30 (Universal Giken, Kanagawa,
Japan) to measure intact radiotracer and its metabolites. Chro-
matographic separation was carried out using a Waters Atlantis
T3 column (4.6(i.d.) ꢁ 50 mm; Waters, Milford, MA). The flow
was 2.0 mL/min at initial conditions of 80% solvent A (5% acetoni-
trile in 10 mM ammonium acetate) and 20% solvent B (90% aceto-
nitrile in 10 mM ammonium acetate). Analytes were eluted using
the following gradient conditions: 0–0.5 min: 0% solvent B in sol-
vent A; 0.5–2.5 min: 0–100% solvent B in solvent A; 2.5–4.5 min:
100% solvent B. Following the elution, the column was returned
to 0% solvent B in solvent A over 2 min. The elution was monitored
by UV absorbance at 254 nm and coupled NaI positron detection.
The amount of radioactivity associated with intact radiotracer