[11C]Enzastaurin, the first design and radiosynthesis of a new potential PET agent for imaging of protein kinase C

Article abstract of DOI:10.1016/j.bmcl.2011.01.100

Enzastaurin (LY317615) is a potent and selective protein kinase C (PKC) inhibitor with an IC₅₀ value of ～6 nM. [¹¹C] Enzastaurin (3-(1-[¹¹C]methyl-1H-indol-3-yl)-4-[1-[1-(2- pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione), a new potential PET agent for imaging of PKC, was first designed and synthesized in 20-25% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled maleic anhydride intermediate followed by the conversion to maleimide.

Full text of DOI:10.1016/j.bmcl.2011.01.100

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1649–1653
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
[11C]Enzastaurin, the first design and radiosynthesis of a new potential PET
agent for imaging of protein kinase C
Min Wang a, Lu Xu a, Mingzhang Gao a, Kathy D. Miller b, George W. Sledge b, Qi-Huang Zheng a,
⇑
a Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
b Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Enzastaurin (LY317615) is a potent and selective protein kinase C (PKC) inhibitor with an IC50 value of
ꢀ6 nM.
11C]Enzastaurin (3-(1-[11C]methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-
1H-indol-3-yl]-1H-pyrrole-2,5-dione), a new potential PET agent for imaging of PKC, was first designed
and synthesized in 20–25% decay corrected radiochemical yield and 370–555 GBq/ mol specific activity
Received 11 January 2011
Accepted 24 January 2011
Available online 31 January 2011
[
l
at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled maleic anhy-
dride intermediate followed by the conversion to maleimide.
Keywords:
[
Protein kinase C (PKC)
Radiosynthesis
11C]Enzastaurin
Ó 2011 Elsevier Ltd. All rights reserved.
Positron emission tomography (PET)
Cancer
Alzheimer’s disease
Protein kinase C (PKC) is a family of enzymes that are involved in
controlling the function of other proteins through the phosphoryla-
tion of hydroxyl groups of serine and threonine amino acid residues
on these proteins. PKC enzymes are activated by signals such as in-
creases in the concentration of diacylglycerol or Ca2+, and thus PKC
enzymes play important roles in several signal transduction cas-
cades.1 PKC expression and activity are associated with various dis-
ease processes, such as Alzheimer’s disease and cancer.2 In cancer,
PKC activation is directly responsible for the vascular endothelial
growth factor (VEGF) signaling that leads to neovascularization.3
VEGF is the most common and direct acting angiogenic factor in can-
cer, and VEGF receptors are overexpressed in tumor-associated
endothelial cells. The PKC family consists of about 10 isozymes.
Enzastaurin (LY317615, 3-(1-methyl-1H-indol-3-yl)-4-[1-[1-(2-
pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-
dione) is a potent and selective inhibitor of PKCb with antiprolifera-
tive activity, IC50 value ꢀ6 nM, originally developed by Eli Lilly.4–7
The studies4–7 indicated Enzastaurin suppresses VEGF-induced
angiogenesis in the rat corneal micropocket assay, decreases micro-
vessel density, and prevents VEGF secretion from human tumor cell
xenografts in nude mice; in addition, prolonged courses of Enzastau-
rin increase chemotherapy or radiation tumor growth delay of
breast, glioma, and small cell lung cancer xenografts. Enzastaurin
is a structurally distinct maleimide, and bisarylmaleimide protein
kinase inhibitors have attracted great interest from synthetic and
medicinal chemists.8,9 Carbon-11-labeled Enzastaurin may serve
as a new probe for the biomedical imaging technique positron emis-
sion tomography (PET), and enable non-invasive monitoring of en-
zyme PKC in diseases.8,9 To radiolabel therapeutic agents as
diagnostic agents for imaging of PKC and monitoring of therapeutic
efficacy of PKC inhibitors, we have first designed and synthesized
[
11C]Enzastaurin
(3-(1-[11C]methyl-1H-indol-3-yl)-4-[1-[1-(2-
pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-
dione) as a new potential PET agent.
There is limited synthetic information regarding the target
compound Enzastaurin appearing in the literature. Wishing to
study this compound in this laboratory, we decided to make our
own material by adopting the available literature methods from
previous works.8–11 As illustrated in Scheme 1, Enzastaurin (5b)
as well as its N-des-methylated compound 5a were synthesized
according to the synthetic protocol developed by Faul et al.10,11
The procedure involved a one-step condensation of substituted
(aryl or indolyl) acetamides with substituted (aryl or indolyl) gly-
oxyl esters in the presence of t-BuOK in THF. Treatment indole-3-
acetamide with concentrated HCl formed hydrochloride salt,
which was reduced with BH3Ápyridine in MeOH to give indolin-3-
acetamide (1) in 49% yield. Condensation of 1 and 1-pyridin-2-
ylmethylpiperidin-4-one, followed by reductive amination with
NaBH(OAc)3 in HOAC provided 2 in 86% yield. The indolin ring of
2
was oxidized by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) in THF to afford its corresponding indole compound 3 in
60% yield. Indole-3-glyoxylate esters 4a–b were prepared by the
reaction of indoles with oxalyl chloride in Et2O, followed by
quench of the intermediate glyoxyl chloride with NaOMe in MeOH
in 73% and 34% yield, respectively. The maleimides 5a and 5b were
⇑
Corresponding author. Tel.: +1 317 278 4671.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.100

1650
M. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1649–1653
O
O
NH2
N
N
N
O
O
NH2
NH2
pyridine
BH3
N
MeOH, conc HCl
N
H
N
H
NaBH(OAc)3, HOAc
1, 49%
N
O
2, 86%
NH2
N
DDQ, THF
N
H
O
N
O
N
3, 60%
O O
N
R
N
OMe
(COCl)2
3, t-BuOK
N
N
R
NaOMe, MeOH
THF
N
R
4a, R=H, 73%
4b, R=Me, 34%
N
5a, R=H, 68%
5b (Enzastaurin, LY317615), R=Me, 50%
Scheme 1. Synthesis of Enzastaurin and N-des-methylated Enzastaurin.
O
N
O
O
Cl
N
N
N
N
N
N
N
HCl, CH3CN
(COCl)2
DDQ, THF
NaBH(OAc)3, HOAc
N
H
H
Cl
N
N
N
7, 68%
6, 85%
8, 47%
O
O
OH
OH
n-BuLi, PhSO2Cl
THF
N
SO2Ph
9, 84%
N
H
O
O
O
O
O
O
N
N
N
N
N
H
SO2Ph
Et3N, CH2Cl2
aq. NaOH
MeOH
+
8
9
N
N
10, 27%
N
11, 46%
Scheme 2. Synthesis of maleic anhydride precursor.

M. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1649–1653
1651
achieved by condensation of indolyl acetamide 3 with indole-3-
glyoxylate esters 4a–b in the presence of t-BuOK in THF in 68%
and 50% yield, respectively.
As shown in Scheme 2, condensation of indolin and 1-pyridin-
2-ylmethylpiperidin-4-one, followed by reductive amination with
NaBH(OAc)3 in HOAC afforded 6 in 85% yield. The indolin ring of
6 was oxidized by DDQ in THF to yield its corresponding indole
compound 7 in 68% yield. Treatment of 7 in CH3CN with 1.0 M
HCl in Et2O formed monohydrochloride salt, followed by the reac-
tion with oxalyl chloride in CH3CN at 0 °C to generate indole-3-gly-
oxylyl chloride 8 in 47% yield, which was used for next step
reaction without further purification. The indole N-phenylsulfonyl
group was introduced from reaction of indole-3-acetic acid with
benzenesulfonyl chloride in THF at À70 °C to achieve 9 in 84%
yield. Coupling of N-protected indole-3-acetic acid 9 with indo-
lyl-3-glyoxylyl chloride 8 in the presence of Et3N in CH2Cl2 resulted
in maleic anhydride 10 in a very low yield (<2%). Surprisingly, the
yield was increased to 27% when 4 Å molecular sieves were added
to the reaction system, and the reaction time was shortened from
overnight to 5 h. The N-phenylsulfonyl group of 10 was removed
by hydrolysis with 20% NaOH in MeOH to afford the precursor
11 in 46% yield.
Enzastaurin is an agent with potential utility in the treatment of
solid tumors, which is currently in phase II clinical trials. The syn-
thesis of carbon-14-labeled and tritium-labeled Enzastaurin for
use in ADME (absorption, distribution, metabolism, elimination)
studies in laboratory animals and humans has been reported.1 Dif-
ferent from the stable isotopes carbon-14 and tritium, carbon-11
only has a short half-life (T1/2 = 20 min). Hence, the synthetic strat-
egies employed for the carbon-14 and tritium labeled enzastaurins
are unsuitable for the synthesis of carbon-11-labled enzastaurin.
The most straightforward procedure to prepare carbon-11-labled
enzastaurin would entail the 11C-methylation of N-des-methylated
Enzastaurin (5a) with the most commonly used 11C-methylating
reagent,
[ [
11C]methy iodide ([11C]CH3I) or 11C]methyl triflate
([11C]CH3OTf).12,13 However, the pKa value of maleimide is much
lower than that of indole, and methylation would perform mainly
on the maleimide nitrogen, not on the indole nitrogen.1 Consider-
ing that maleic anhydride was mild enough to tolerate a wide
range of substituent functionalities and can be converted to malei-
mide in the presence of ammonia or an ammonia source, we se-
lected its maleic anhydride 11 as the precursor to escape the
competitive methylation of indole nitrogen with maleimide nitro-
gen. We have reported a method for preparation of maleic anhy-
dride by hydrolysis of its corresponding maleimide to synthesize
the precursor of SB-216763.9 Initially, this method prompted us
to hydrolyze maleimide 5a with 2 N KOH in EtOH to prepare
maleic anhydride 11. Unfortunately, it failed to give the desired
compound 11. Therefore, we had to resort to other synthetic strat-
egy to obtain the precursor.
As shown in Scheme 3, an intermediate standard compound 13
was prepared in 11% yield by coupling indolyl-3-glyoxylyl chloride
8 with N-methyl indole-3-acetic acid 12,14 which was obtained by
methylation of 3-indolylacetic acid on nitrogen with CH3I in the
presence of NaH in THF in 65% yield.
Synthesis of the target radiotracer [11C]Enzastaurin ([11C]5b) is
indicated in Scheme 4. Precursor 11 was labeled by [11C]CH3I pre-
pared from [11C]CO2,12,13 in the presence of NaH in acetonitrile
through the N-[11C]methylation8,9 to provide a radiolabeling inter-
mediate [11C]13. Without further purification, [11C]13 was quickly
converted to the target tracer [11C]5b using hexamethyldisilazane
(HMDS) in MeOH/DMF. [11C]5b was isolated from the radiolabeling
mixture by the semi-preparative reversed-phase high performance
liquid chromatography (HPLC) in 20–25% radiochemical yields, de-
cay corrected to end of bombardment (EOB), based on [11C]CO2.
Another more reactive radiolabeled precursor [11C]CH3OTf12,13
was also used in the radiolabeling reaction of the precursor 11,
but the radiochemical yield was much lower than that of
CH2COOH
CH2COOH
NaH, CH3I
N
N
H
THF
CH3
12, 65%
[
[
11C]CH3I. This suggested it is better to use [11C]CH3I for the N-
11C]methylation of indole nitrogen, which is consistent with the
O
O
O
results from our previous works.8,9 The synthesis was performed
in an automated multi-purpose 11C-radiosynthesis module, allow-
ing measurement of specific activity during synthesis.15,16 The spe-
N
N
cific activity of [11C]5b was in a range of 370–555 GBq/
l
mol at EOB
CH3
Et3N, CH2Cl2
measured by the on-the-fly technique using semi-preparative
12
8
+
HPLC during synthesis16 and 185–278 GBq/
lmol at the end of syn-
N
thesis (EOS) determined by analytical HPLC,17 respectively. Chem-
ical purity and radiochemical purity were determined by analytical
HPLC.17 The chemical purity of the precursor 11, intermediate 13
and reference standard 5b was >95%. The radiochemical purity of
the target tracer [11C]5b was >99% determined by radio-HPLC
N
13, 11%
Scheme 3. Synthesis of maleic anhydride intermediate.
through
c-ray (PIN diode) flow detector, and the chemical purity
O
O
O
H
O
N
O
N
11CH3
N
HMDS
MeOH/DMF
NaH, CH3CN
11C]CH3I
N
11CH3
N
[
11
N
N
N
N
[
11C]13
[
11C]5b ([11C]Enzastaurin, [11C]LY317615), 20-25%
Scheme 4. Synthesis of [11C]Enzastaurin.

1652
M. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1649–1653
of the target tracers [11C]5b was >93% determined by reversed-
phase HPLC through UV flow detector.
(LC–MS) analysis was performed on an Agilent system, consisting of an 1100
series HPLC connected to
spectrometer configured
a
diode array detector and
for positive-ion/negative-ion
a
1946 D mass
electrospray
The synthetic information of Enzastaurin was limited in the lit-
erature. Thus, the experimental details and characterization data
for compounds 1–13 and for the tracer [11C]5b are given.18
In summary, [11C]Enzastaurin was first designed and synthe-
sized as a new potential PET agent for imaging of PKC in various
diseases. An automated multi-purpose 11C-radiosynthesis module
of our own design for fully automated synthesis of [11C]Enzastau-
rin has been built, featuring the measurement of specific activity
by the on-the-fly technique. The radiosynthesis employed a one-
pot two-step reaction via N-[11C]methylation radiolabeling on in-
dole nitrogen of the precursor incorporated efficiently with the
most commonly used [11C]methylating agent, [11C]CH3I, produced
by gas-phase production of [11C]methyl bromide ([11C]CH3Br) from
our laboratory. The target tracer was isolated and purified by a
semi-preparative HPLC procedure in moderate radiochemical
yields, short overall synthesis time, and high specific activity.
These results facilitate the potential preclinical and clinical PET
studies of [11C]Enzastaurin in animals and humans.
ionization. The high resolution mass spectra (HRMS) were obtained using a
Waters/Micromass LCT Classic spectrometer. Chromatographic solvent
proportions
are
indicated
as
volume:volume
ratio.
Thin-layer
chromatography (TLC) was run using Analtech silica gel GF uniplates
(5 Â 10 cm2). Plates were visualized under UV light. Preparative TLC was run
using Analtech silica gel UV 254 plates (20 Â 20 cm2). Normal phase flash
column chromatography was carried out on EM Science silica gel 60 (230–400
mesh) with a forced flow of the indicated solvent system in the proportions
described below. All moisture- and/or air-sensitive reactions were performed
under a positive pressure of nitrogen maintained by a direct line from a
nitrogen source. Analytical HPLC was performed using
(Phenomenex)
C-18 column, 4.6 Â 250 mm; 3:1:1 CH3CN/MeOH/
20 mM, pH 6.7 phosphate (buffer solution) mobile phase; flow rate 1.5 mL/
min; and UV (254 nm) and -ray (PIN diode) flow detectors. Semi-preparative
HPLC was performed using a YMC-Pack ODS-A, S-5
m, 12 nm, 10 Â 250 mm
C-18 column; 3:1:1 CH3CN/MeOH/20 mM, pH 6.7 phosphate (buffer solution)
mobile phase; 5.0 mL/min flow rate; UV (254 nm) and -ray (PIN diode) flow
detectors. Sterile Millex-GS 0.22 m vented filter unit was obtained from
Millipore Corporation, Bedford, MA.
a
Prodigy
5 lm
c
l
c
l
(b) 2-(Indolin-3-yl)acetamide (1). A suspension of indole-3-acetamide (7.75 g,
44.5 mmol) in MeOH (60 mL) was cooled to 0 °C. 12 N HCl (21 mL, 252 mmol)
was added dropwise, keeping the reaction temperature below 25 °C. After
cooling to 0 °C, borane-pyridine complex (15 mL, 150 mmol) was added
dropwise. The reaction mixture was stirred at room temperature for 1 h.
After cooling to 0 °C, water (75 mL) was added slowly. The mixture was
basified to pH 8.0 with 5 N NaOH and then extracted with EtOAc. The
combined organic layer was washed with saturated NaHCO3, brine, dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was
purified by column chromatography (20:1 EtOAc/MeOH) to afford 1 (3.8 g,
49%) as a white solid: mp 88–90 °C; 1H NMR (DMSO-d6) d 7.36 (br s, 1H), 7.00
(d, J = 7.0 Hz, 1H), 6.90 (td, J = 7.5, 0.5 Hz, 1H), 6.85 (br s, 1H), 6.51 (td, J = 7.5,
0.5 Hz, 1H), 6.47 (d, J = 7.5 Hz, 1H), 5.43 ((br s, 1H), 3.55–3.47 (m, 2H), 3.03–
3.05 (m, 1H), 2.50–2.45 (m, 1H), 2.25–2.20 (m, 1H).
(c) 2-(1-(1-(Pyridin-2-ylmethyl)piperidin-4-yl)indolin-3-yl)acetamide (2). To a
stirred solution of 1 (3.15 g, 17.9 mmol) in HOAc (15 mL) was added 1-pyridin-
2-ylmethylpiperidin-4-one (3.74 g, 19.7 mmol) in one portion at room
temperature. After cooling to 0 °C, NaBH(OAc)3 (5.72 g, 27.0 mmol) was
added in portions. The reaction mixture was stirred at room temperature for
3 h. The mixture was cooled, diluted with water and basified to pH 11.0 with
5 N NaOH, then extracted with EtOAc. The combined organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was first purified by column chromatography (100:10:1 EtOAc/
Acknowledgments
This work was partially supported by the Breast Cancer
Research Foundation and Indiana Genomics Initiative (INGEN) of
Indiana University, which is supported in part by Lilly Endowment
Inc. The authors would like to thank Dr. Bruce H. Mock and Barbara
E. Glick-Wilson for their assistance in production of [11C]CH3I. 1H
NMR spectra were recorded on a Bruker Avance II 500 MHz NMR
spectrometer in the Department of Chemistry and Chemical Biol-
ogy at Indiana University Purdue University Indianapolis (IUPUI),
which is supported by a NSF-MRI grant CHE-0619254.
References and notes
MeOH/NH4OH) to obtain
a yellow sticky oil, which was triturated with
1. Wheeler, W. J.; Clodfelter, D. K. J. Labelled. Compd. Radiopharm. 2008, 51, 175.
2. Bradshaw, D.; Hill, C. H.; Nixon, J. S.; Wilkinson, S. E. Agents Actions 1993, 38,
135.
minimal cold 9:1 EtOAc/MeOH to afford 2 (5.41 g, 86%) as a white solid: mp
52–53 °C; 1H NMR (DMSO-d6) d 8.48 (d, J = 4.5 Hz, 1H), 7.76 (td, J = 1.5, 7.5 Hz,
1H), 7.45 (d, J = 7.5 Hz, 1H), 7.37 (br s, 1H), 7.25 (t, J = 6.0 Hz, 1H), 6.70–6.96 (m,
2H), 6.88 (br s, 1H), 6.51 (t, J = 7.5 Hz, 1H), 6.45 (d, J = 7.5 Hz, 1H), 3.61 (s, 2H),
3.50–3.47 (m, 2H), 2.99 (t, J = 7.0 Hz, 1H), 2.91 (d, J = 5.5 Hz, 2H), 2.48–2.50 (m,
2H), 2.25–2.15 (m, 3H), 1.66–1.58 (m, 4H).
(d) 2-(1-(1-(Pyridin-2-ylmethyl)piperidin-4-yl)-1H-indol-3-yl)acetamide (3).
A suspension of 2 (3.90 g, 11.1 mmol) in THF (60 mL) was cooled to 0 °C
under N2 atmosphere. A solution of DDQ (2.78 g, 12.2 mmol) in THF (25 mL)
was added dropwise. The reaction mixture was stirred at room temperature
overnight. The reaction mixture was diluted with water (100 mL) and 5%
NaHCO3 (100 mL). THF was removed in vacuo and the solid precipitated from
the solution. After collected by filtration, and rinsed with water and EtOAc, the
solid was reslurried in 5:1 EtOAc/MeOH at 50 °C for 15 min, cooled to room
temperature, filtered, and rinsed with minimal 5:1 EtOAc/MeOH to afford 3
(2.33 g, 60%) as a pale brown solid: mp 202–203 °C; 1H NMR (DMSO-d6) d 8.51
(s, 1H), 7.79 (t, J = 7.0 Hz, 1H), 7.55–7.48 (m, 3H), 7.34–7.28 (m, 3H), 7.11 (t,
J = 7.5 Hz, 1H), 6.70 (t, J = 7.0 Hz, 1H), 6.84 (br s, 1H), 4.34 (m, 1H), 3.68 (s, 2H),
3.46 (s, 2H), 2.98 (d, J = 9.5 Hz, 2H), 2.32 (m, 2H), 2.04–1.94 (m, 4H); MS (ESI, m/
z): 349 ([M+H]+, 100%).
3. McMahon, G. Oncologist 2000, 5, 3.
4. Raab, M. S.; Breitkreutz, I.; Tonon, G.; Zhang, J.; Hayden, P. J.; Nguyen, T.;
Fruehauf, J. H.; Lin, B. K.; Chauhan, D.; Hideshima, T.; Munshi, N. C.; Anderson,
K. C.; Podar, K. Blood 2009, 113, 1513.
5. Spalding, A. C.; Zeitlin, B. D.; Wilder-Romans, K.; Davis, M. E.; Nor, J. E.;
Lawrence, T. S.; Ben-Josef, E. Transl. Oncol. 2008, 1, 195.
6. Keyes, K. A.; Mann, L.; Sherman, M.; Galbreath, E.; Schirtzinger, L.; Ballard, D.;
Chen, Y. F.; Iversen, P.; Teicher, B. A. Cancer Chemother. Pharmacol. 2004, 53, 133.
7. Tabatabai, G.; Frank, B.; Wick, A.; Lemke, D.; von Kürthy, G.; Obermüller, U.;
Heckl, S.; Christ, G.; Weller, M.; Wick, W. Ann. Neurol. 2007, 61, 153.
8. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Hutchins, G. D.; Zheng, Q.-H.
Nucl. Med. Biol. 2010, 37, 763.
9. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Hutchins, G. D.; Zheng, Q.-H.
Bioorg. Med. Chem. Lett. 2011, 21, 245.
10. Faul, M. M.; Grutsch, J. L.; Kobierski, M. E.; Kopach, M. E.; Krumrich, C. A.;
Staszak, M. A.; Udodong, U.; Vicenzi, J. T.; Sullivan, K. A. Tetrahedron 2003, 59,
7215.
11. Faul, M. M.; Kobierski, M. E.; Kopach, M. E. J. Org. Chem. 2003, 68, 5739.
12. Jewett, D. M. Int. J. Radiat. Appl. Instrum. A 1992, 43, 1383.
13. Mock, B. H.; Mulholland, G. K.; Vavrek, M. J. Nucl. Med. Biol. 1999, 26, 467.
14. Roy, S.; Eastman, A.; Gribble, G. W. Org. Biomol. Chem. 2006, 4, 3228.
15. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd. Radiopharm. 2005, 48,
S225.
(e) Methyl 2-(1H-indol-3-yl)-2-oxoacetate (4a). To a stirred solution of indole
(2.0 g, 17.1 mmol) in Et2O (20 mL) was added oxalyl chloride (1.5 mL,
17.2 mmol) dropwise at 0 °C. After the resultant yellow slurry was stirred at
0 °C for 0.5 h, it was cooled to À78 °C. A solution of NaOMe in MeOH (25 wt %,
7.8 mL, 34.1 mmol) was added to this slurry at À78 °C. The reaction mixture
was allowed to warm to room temperature and quenched by addition of water
(10 mL). The solid was collected by filtration, rinsed with water and dried to
give 4a (2.53 g, 73%) as a yellow solid: mp 160 °C (dec.); 1H NMR (DMSO-d6) d
12.42 (s, 1H), 8.45 (d, J = 3.5 Hz, 1H), 8.16 (dd, J = 1.5, 6.0 Hz, 1H), 7.55 (dd,
J = 1.5, 6.0 Hz, 1H), 7.32–7.27 (m, 2H), 3.89 (s, 3H).
(f) Methyl 2-(1-methyl-1H-indol-3-yl)-2-oxoacetate (4b). To a stirred solution
of 1-methyl-1H-indole (5.0 g, 38.2 mmol) in Et2O (50 mL) was added oxalyl
chloride (3.5 mL, 40.1 mmol) dropwise at 0 °C. After the resultant yellow slurry
was stirred at 0 °C for 0.5 h, it was cooled to À78 °C. A solution of NaOMe in
MeOH (25 wt %, 22.5 mL, 98.5 mmol) was added to this slurry at À78 °C. The
reaction mixture was allowed to warm to room temperature and quenched by
addition of water (25 mL). The solid was collected by filtration, rinsed with
water, Et2O and dried to give 4b (2.83 g, 34%) as a tan solid: mp 80–81 °C; 1H
NMR (DMSO-d6) d 8.50 (s, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H),
7.38–7.31 (m, 2H), 3.92 (s, 3H), 3.90 (s, 3H).
16. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd.
Radiopharm. 2005, 48, S224.
17. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671.
18. (a) General. All commercial reagents and solvents from Sigma–Aldrich and
Fisher Scientific were used without further purification.
prepared according to a literature procedure,13 and [11C]CH3I was prepared
from NaI column (NaI mixed with
11C]CH3Br by passing through
Carbograph™-2 60/80, Grace, 1:1 w/w) at 300 °C. Melting points were
[
11C]CH3Br was
[
a
determined on
a MEL-TEMP II capillary tube apparatus and were
uncorrected. 1H NMR spectra were recorded on Bruker Avance II 500 MHz
NMR spectrometers using tetramethylsilane (TMS) as an internal standard.
Chemical shift data for the proton resonances were reported in parts per
million (ppm, d scale) relative to internal standard TMS (d 0.0), and coupling
constants (J) were reported in hertz (Hz). Liquid chromatography–mass spectra

M. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1649–1653
1653
(g) 3-(1H-Indol-3-yl)-4-(1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-1H-indol-3-
yl)-1H-pyrrole-2,5-dione (5a). To stirred suspension of (200 mg,
(875 mg, 2.78 mmol) and Et3N (1.16 mL, 8.34 mmol) in CH2Cl2 (30 mL)
containing 4 Å molecular sieves was added 8 (1.10 g, 2.63 mmol) in portions
at 0 °C. After the reaction mixture was stirred at room temperature for 5 h, the
solvent was evaporated in vacuo. The residue was purified by column
chromatography (1:1.5 hexanes/acetone) to afford 10 (417 mg, 27%) as a red
solid: mp 108–109 °C; 1H NMR (DMSO-d6) d 8.52 (d, J = 4.5 Hz, 1H), 8.14 (s,
1H), 8.04 (dd, J = 1.0, 8.5 Hz, 2H), 7.95 (d, J = 8.5 Hz, 1H), 7.92 (s, 1H), 7.81 (td,
J = 1.5, 7.5 Hz, 1H), 7.78–7.75 (m, 1H), 7.64 (t, J = 8.0 Hz, 2H), 7.60 (d, J = 8.5 Hz,
1H), 7.49 (d, J = 7.5 Hz, 1H), 7.30–7.24 (m, 2H), 7.06–7.03 (m, 1H), 6.92–6.88
(m, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.40 (t, J = 7.5 Hz, 1H),
4.49–4.44 (m, 1H), 3.68 (s, 2H), 2.94 (d, J = 1.0 Hz, 2H), 2.37–2.32 (m, 2H), 1.99–
1.79 (m, 4H); MS (ESI, m/z): 643 ([M+H]+, 100%); HRMS (ESI, m/z): Calcd for
a
3
0.57 mmol) and 4a (168 mg, 0.83 mmol) in THF (5 mL) was added 1.0 M t-
BuOK in THF(1.15 mL, 1.15 mmol) at 0 °C. The reaction mixture was stirred at
room temperature overnight. The mixture was partitioned between EtOAc and
5% NaHCO3. The organic layer was washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was purified by
preparative TLC plate (100:5 CH2Cl2/MeOH) to afford 5a (194.5 mg, 68%) as an
orange solid: mp 259 °C (dec.); 1H NMR (DMSO-d6) d 11.68 (s, 1H), 10.91 (s,
1H), 8.51 (d, J = 4.0 Hz, 1H), 7.78–7.77 (m, 2H), 7.69 (s, 1H), 7.56 (d, J = 8.5 Hz,
1H), 7.48 (d, J = 7.5 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.28–7.26 (m,1H), 7.07–6.96
(m, 3H), 6.75 (t, J = 7.5 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 6.61 (t, J = 7.5 Hz, 1H),
4.43 (m, 1H), 3.67 (s, 2H), 2.94 (d, J = 7.5 Hz, 2H), 2.32 (m, 2H), 1.91 (m, 4H); MS
(ESI, m/z): 502 ([M+H]+, 100%).
C
37H31N4O5S ([M+H]+) 643.2015. Found: 643.2009.
(n) 3-(1H-Indol-3-yl)-4-(1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-1H-indol-3-
yl)furan-2,5-dione (11). To a stirred suspension of 10 (280 mg, 0.44 mmol) in
MeOH (5 mL) was added 20% NaOH (4 mL). The reaction mixture was
heated at reflux for 4 h. MeOH was evaporated in vacuo, and the residue was
poured into ice-water, and acidified to pH 2.0 with 2 N HCl. The solid was
collected by filtration, and rinsed with EtOAc and Et2O to afford 11 (101 mg,
46%) as a red solid: mp 268 °C (dec.); 1H NMR (DMSO-d6) d 11.95 (d, J = 2.5 Hz,
1H), 8.50 (d, J = 4.0 Hz, 1H), 7.92 (d, J = 3.0 Hz, 1H), 7.80–7.76 (m, 2H), 7.64 (d,
J = 8.5 Hz, 1H), 7.45 (t, J = 8.5 Hz, 2H), 7.28–7.26 (m, 1H), 7.14–7.10 (m, 2H),
7.08–7.04 (m, 1H), 6.85 (t, J = 7.5 Hz, 1H), 6.72–6.68 (m, 2H), 4.48–4.44
(m, 1H), 3.65 (s, 2H), 2.94 (d, J = 7.5 Hz, 2H), 2.31 (t, J = 6.5 Hz, 2H), 1.99–1.84
(m, 4H); HRMS (ESI, m/z): Calcd for C31H27N4O3 ([M+H]+) 503.2083. Found:
503.2079.
(o) 2-(1-Methyl-1H-indol-3-yl)acetic acid (12). To a stirred suspension of NaH
(60% dispersion in mineral oil, 3.0 g, 75 mmol) in THF (60 mL) was added a
solution of indole-3-acetic acid (2.63 g, 15 mmol) in THF (25 mL) at 0 °C. After
stirring at 0 °C for 0.5 h, a solution of methyl iodide (3.1 mL, 50 mmol) in THF
(20 mL) was added. The reaction mixture was stirred at room temperature
overnight, and then cooled to 0 °C. MeOH (4 mL) was added slowly to destroy
excess hydride with vigorous stirring, followed by cold water until a clear
yellow solution resulted. Et2O (50 mL) was added. The aqueous layer was
separated, acidified with 6 N HCl and extracted with CH2Cl2. The combined
organic layer was dried over anhydrous Na2SO4, filtered and concentrated to
(h) 3-(1-Methyl-1H-indol-3-yl)-4-(1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-
1H-indol-3-yl)-1H-pyrrole-2,5-dione (Enzastaurin, 5b). To
a
stirred
suspension of 3 (200 mg, 0.57 mmol) and 4b (150 mg, 0.69 mmol) in THF
(5 mL) was added 1.0 M t-BuOK in THF (1.15 mL, 1.15 mmol) at 0 °C. After the
reaction mixture was stirred at room temperature overnight, it was partitioned
between EtOAc and 5% NaHCO3. The organic layer was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue
was purified by preparative TLC plate (100:5 CH2Cl2/MeOH) to afford 5b
(147 mg, 50%) as a red solid: mp 154–156 °C; 1H NMR (DMSO-d6) d 10.93 (s,
1H), 8.50 (d, J = 4.0 Hz, 1H), 7.88 (s, 1H), 7.78 (t, J = 7.5 Hz, 1H), 7.76 (s, 1H), 7.57
(d, J = 8.5 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.27 (t,
J = 6.0 Hz, 1H), 7.08–7.02 (m, 3H), 6.77 (t, J = 7.5 Hz, 1H), 6.62 (t, J = 7.5 Hz, 1H),
6.56 (d, J = 8.0 Hz, 1H), 4.43–4.45 (m, 1H), 3.86 (s, 3H), 3.65 (s, 2H), 2.92 (d,
J = 10 Hz, 2H), 2.30 (m, 2H), 1.91–1.84 (m, 4H); MS (ESI, m/z): 516 ([M+H]+,
100%); HRMS (ESI, m/z): Calcd for C32H30N5O2 ([M+H]+) 516.2400. Found:
516.2389.
(i) 1-(1-(Pyridin-2-ylmethyl)piperidin-4-yl)indoline (6). To a stirred solution of
indoline (4.0 g, 33.6 mmol) in HOAc (20 mL) was added 1-pyridin-2-
ylmethylpiperidin-4-one (7.02 g, 36.9 mmol) in one portion at room
temperature. After cooling to 0 °C, NaBH(OAc)3 (10.7 g, 50.3 mmol) was
added in portions. The reaction mixture was stirred at room temperature for
5 h. The mixture was cooled, diluted with water, basified to pH 11.0 with 5 N
NaOH, and then extracted with EtOAc. The combined organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was first purified by column chromatography (500:10:1 EtOAc/
about 10 mL. Hexane was added slowly until
a brown solid completely
precipitated out. The crude solid was recrystallized from EtOH to give 12
(2.11 g, 65%) as a tan solid: mp 121–123 °C (lit.14 127–128 °C); 1H NMR (CDCl3)
d 7.58 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.23–7.21 (m, 1H), 7.14–7.11
(m, 1H), 7.03 (s, 1H), 3.79 (s, 2H), 3.75 (s, 3H).
MeOH/NH4OH) to afford 6 (8.37 g, 85%) as a pale brown solid: mp 89–90 °C; 1
H
NMR (DMSO-d6) d 8.48 (dd, J = 0.5, 5.0 Hz, 1H), 7.76 (td, J = 1.5, 7.5 Hz, 1H), 7.44
(d, J = 8.0 Hz, 1H), 7.26–7.25 (m, 1H), 6.98 (d, J = 7.5 Hz, 1H), 6.95 (t, J = 7.5 Hz,
1H), 6.50 (t, J = 7.0 Hz, 1H), 6.43 (d, J = 8.0 Hz, 1H), 3.60 (s, 2H), 3.38–3.35 (m,
1H), 3.32 (m, 3H), 2.91 (d, J = 7.0 Hz, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.17–2.12 (m,
2H), 1.65–1.62 (m, 4H); MS (ESI, m/z): 294 ([M+H]+, 100%).
(p) 3-(1-Methyl-1H-indol-3-yl)-4-(1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-
1H-indol-3-yl)furan-2,5-dione (13). To
a stirred solution of 12 (25 mg,
0.13 mmol) and Et3N (0.05 mL, 0.33 mmol) in CH2Cl2 (1 mL) containing 4 Å
molecular sieves was added 8 (50 mg, 0.11 mmol) in portions at 0 °C. After the
reaction mixture was stirred at room temperature for 4 h, the solvent was
evaporated in vacuo. The residue was purified by column chromatography
(1:1.5 hexanes/acetone) to afford 13 (6.0 mg, 11%) as a red solid: mp 121–123
°C (dec.); 1H NMR (DMSO-d6) d 8.51 (d, J = 4.0 Hz, 1H), 8.02 (s, 1H), 7.83–7.78
(m, 1H), 7.76(s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.46 (d,
J = 7.5 Hz, 1H), 7.32–7.28 (m, 1H), 7.17–7.10 (m, 3H), 6.87(t, J = 7.5 Hz, 1H),
6.71 (t, J = 7.5 Hz, 1H), 6.60 (d, J = 8.0 Hz, 1H), 4.50–4.45 (m, 1H), 3.90 (s, 3H),
3.68 (s, 2H), 2.93 (d, J = 11.5 Hz, 2H), 2.39–2.32 (m, 2H), 1.92–1.85 (m, 4H); MS
(ESI, m/z): 517 ([M+H]+, 100%); HRMS (ESI, m/z): Calcd for C32H29N4O3 ([M+H]+)
517.2240. Found: 517.2249.
(j) 1-(1-(Pyridin-2-ylmethyl)piperidin-4-yl)-1H-indole (7).
A solution of 6
(8.36 g, 28.5 mmol) in THF (80 mL) was cooled to 0 °C under N2 atmosphere.
A solution of DDQ (7.18 g, 31.6 mmol) in THF (40 mL) was added dropwise.
After stirring at room temperature for 4 h, the reaction mixture was diluted
with water, basified to pH 11.0 with 5 N NaOH, and then extracted with EtOAc.
Saturated NaHCO3 was added to help the separation of two phases. The organic
layer was washed with brine, dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified was purified by column
chromatography (500:10:1–500:20:2 EtOAc/MeOH/NH4OH) to afford 7 (5.66 g,
68%) as a pale brown solid: mp 106–108 °C; 1H NMR (DMSO-d6) d 8.51 (dt,
J = 1.0, 4.0 Hz, 1H), 7.76 (td, J = 2.0, 7.5 Hz, 1H), 7.54–7.50 (m, 4H), 7.28–7.26
(m, 1H), 7.13–7.10 (m, 1H), 7.02–6.99 (m, 1H), 6.44 (d, J = 3.0 Hz, 1H), 4.40–
4.34 (m, 1H), 3.68 (s, 2H), 2.98 (d, J = 12.0 Hz, 2H), 2.32 (t, J = 11.0 Hz, 2H),
2.06–1.91 (m, 4H); MS (ESI, m/z): 292 ([M+H]+, 100%).
(q) 3-(1-[11C]Methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-
1H-indol-3-yl]-1H-pyrrole-2,5-dione ([11C]Enzastaurin, [11C]5b). [11C]CO2 was
produced by the 14N(p, 11C nuclear reaction in small volume (9.5 cm3)
a)
aluminum gas target (CTI) from 11 MeV proton cyclotron on research purity
nitrogen (+1% O2) in a Siemens radionuclide delivery system (Eclipse RDS-111).
(k) 4-(3-(2-Chloro-2-oxoacetyl)-1H-indol-1-yl)-1-(pyridin-2-ylmethyl)piper-
idinium chloride (8). To
a
stirred suspension of
7
(2.0 g, 6.87 mmol) in
The proton-beam current was 55
precursor 11 (0.1–0.3 mg) was dissolved in CH3CN (300
l
A, and the irradiation time was 30 min. The
L). To this solution
CH3CN (60 mL) was added 1.0 M HCl in Et2O (7.0 mL, 7.0 mmol) at 0 °C. After
the white slurry was stirred at room temperature for 1 h, it was filtered and
dried to afford a HCl salt (0.95 g) as an off-white solid. The HCl salt was added
to CH3CN (10 mL), and the suspension was cooled to À10 °C. Oxalyl chloride
(0.5 mL, 5.80 mmol) was added dropwise. After the resultant yellow slurry was
stirred at 0 °C for 2 h, it was filtered, rinsed with cold CH3CN and Et2O, and
dried to afford 8 (1.35 g, 47%) as a yellow solid. This compound was used for
next step without purification.
(l) 2-(1-(Phenylsulfonyl)-1H-indol-3-yl)acetic acid (9). To a stirred solution of
indole-3-acetic acid (5.26 g, 30.0 mmol) in THF (150 mL) was added 2.5 M n-
BuLi in hexane (25.6 mL, 64.0 mmol) dropwise under nitrogen atmosphere at
À70 °C. After stirring at same temperature for 1 h,
l
was added NaH (1 mg). The mixture was transferred to a small reaction vial.
No-carrier-added (high specific activity) [11C]CH3I that was produced by the
gas-phase production method14 from
[ [
11C]CO2 through 11C]CH4 and
[
11C]CH3Br with NaI column was passed into the reaction vial at 0 °C until
radioactivity reached a maximum (ꢀ2 min), and then the reaction vial was
isolated and heated at 45 °C for 4 min to produce 3-(1-[11C]methyl-1H-indol-3-
yl)-4-(1-(1-(pyridin-2-ylmethyl)piperidin-4-yl)-1H-indol-3-yl)furan-2,5-dione
([11C]13). Then, a solution of HMDS (2
lL) and MeOH (2 lL) in DMF (300 lL)
was introduced to the reaction vial. The reaction mixture was sealed and
heated at 80 °C for 8 min. The contents of the reaction vial were diluted with
NaHCO3 (1 mL, 0.1 M), and injected onto the semi-preparative HPLC column
with 2 mL injection loop. The product fraction was collected, the solvent was
removed by rotatory evaporation under vacuum, and the final product, [11C]5b,
was formulated in saline, sterile-filtered through a sterile vented Millex-GS
a solution of
benzenesulfonyl chloride (3.8 mL, 30.0 mmol) in THF (50 mL) was added
slowly. The reaction mixture was then allowed to warm to room temperature,
and stirred overnight. The solvent was evaporated in vacuo, and the residue
was dissolved in CH2Cl2, and washed with 1 N HCl. The organic layer was
washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was
evaporated in vacuo, and the crude product was recrystallized from EtOAc/
hexanes to give 9 (7.9 g, 84%) as a brown solid: mp 165–167 °C; 1H NMR
(DMSO-d6) d 12.5 (s, 1H), 7.97–7.92 (m, 3H), 7.74 (s, 1H), 7.70–7.67 (m, 1H),
7.59 (t, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.37–7.34 (m, 1H), 7.28–7.25 (m,
1H), 3.71 (s, 2H).
0.22 lm cellulose acetate membrane, and collected into a sterile vial. Total
radioactivity was assayed and total volume was noted for tracer dose
dispensing. The overall synthesis, purification and formulation time was 35–
40 min from EOB. Retention times in the analytical HPLC system were: tR
11 = 5.21 min, tR 13 = 4.06 min, tR 5b = 7.79 min, tR
11C]5b = 7.79 min. Retention times in the semi-preparative HPLC system
were: tR 11 = 6.18 min, tR 13 = 5.51 min, tR 5b = 11.15 min, tR
11C]13 = 5.51 min, and tR 11C]5b = 11.15 min. The radiochemical yields were
20–25% decay corrected to EOB, based on [11C]CO2.
[
11C]13 = 4.06 min, and tR
[
(m) 3-(1-(Phenylsulfonyl)-1H-indol-3-yl)-4-(1-(1-(pyridin-2-ylmethyl)piper-
idin-4-yl)-1H-indol-3-yl)furan-2,5-dione (10). To
[
[
a stirred solution of 9