Synthesis of the tritiated isotopomers of enzastaurin and its N-des-pyridylmethyl metabolite for use in ADME studies

Article abstract of DOI:10.1002/jlcr.1497

Enzastaurin (3-(1-methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4- piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 1), an agent with potential utility in the treatment of solid tumors, is currently in phase II clinical trials. Enzastaurin undergoes metabolism in vitro and in vivo to several products of oxidative metabolism, the major one of which is 3-(1-methyl-1H- indol-3-yl)-4-(1-piperidin-4-yl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (2). In a model study, the attempted synthesis 1-[²H] by reaction of 1 with deuterium gas in the presence of Ir[(COD)(Cy₃P)pyr]PF₆ (Crabtree's catalyst) was unsuccessful. Alternatively, it was decided to prepare tritiated 2 as both a final product and the starting material for the tritiation of 1. We have reported herein a route that was developed for use in the preparation of tritium-labeled 2-[³H] and its successful conversion to 1-[³H]. Copyright

Full text of DOI:10.1002/jlcr.1497

Research Article
Received 12 November 2007,
Revised 19 December 2007,
Accepted 8 January 2008
Published online 21 February 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1497
Synthesis of the tritiated isotopomers of
enzastaurin and its N-des-pyridylmethyl
metabolite for use in ADME studies
Ã
William J. Wheeler, and Dean K. Clodfelter
Enzastaurin (3-(1-methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 1), an
agent with potential utility in the treatment of solid tumors, is currently in phase II clinical trials. Enzastaurin undergoes
metabolism in vitro and in vivo to several products of oxidative metabolism, the major one of which is 3-(1-methyl-1H-indol-
3-yl)-4-(1-piperidin-4-yl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (2). In a model study, the attempted synthesis 1-[2H] by reaction
of 1 with deuterium gas in the presence of Ir[(COD)(Cy3P)pyr]PF6 (Crabtree’s catalyst) was unsuccessful. Alternatively,
it was decided to prepare tritiated 2 as both a final product and the starting material for the tritiation of 1. We have
reported herein a route that was developed for use in the preparation of tritium-labeled 2-[3H] and its successful conversion
to 1-[3H].
Keywords: PKC; AKT; VEGF; tritiated; enzastaurin
H
H
Introduction
N
N
O
O
O
O
Protein kinase C (PKC) is a family of serine-/threonine-specific
kinases, which are involved in signal transduction pathways that
govern a wide variety of physiological processes.1 Vascular
endothelial growth factor (VEGF) is the most common and direct
acting angiogenic factor in cancer patients, and the up-
regulation of VEGF receptors has been observed in tumor-
associated endothelial cells; there are data to support the
premise that PKC activation is directly responsible for the VEGF
signaling that leads to neovascularization.2 Enzastaurin, (3-(1-
methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinyl-methyl)-4-piperidinyl]-
1H-indol-3-yl]-1H-pyrrole-2,5-dione, 1), a selective inhibitor of
PKCb, when dosed to mice-bearing human small cell lung (SW2)
and renal cell (Caki01) tumors, resulted in significant reductions
in plasma VEGF levels, which was observed 5–7 days after the
onset of treatment and persisted for 2–3 weeks after the
termination of therapy.3 This reduction of VEGF was accom-
panied by a decrease in intratumoral vessel density. A dual
center, phase I clinical study was initiated to study the safety
and pharmacokinetics of enzastaurin in patients with solid
tumors; these studies revealed that the half life of enzastaurin
(1) ranged from 9 to 25 h with no significant accumulation.4 The
N-des-pyridylmethyl metabolite 2 had a longer half life and
accumulated upon multiple dosing. Subsequent in vitro studies
have revealed that 1 robustly induced apoptosis of a variety of
human tumor cells by suppressed signaling through the PI3K/
AKT pathway.5
N
N
N
N
CH3
CH3
N
N
H
N
1
2
labeled enzastaurin (1-[14C]) for use in ADME studies in
laboratory animals and humans has been reported.8 In order
to gather more data to support further evaluation of 1, the
tritiated isotopomers (1-[3H] and 2-[3H]) were required. In this
paper, we will discuss the successful efforts to prepare these
tritiated compounds.
Discussion
In spite of the pyridine-directed tritiation reported by Shu et al.
(Figure 1)9, treatment of 1 (as a model for tritiation) with
deuterium gas in the presence of 0.5 or 1.0 eq. (two
experiments) of Ir[(COD)(Cy3P)pyr]PF6 in methylene chloride
Oral dosing of 1 to xenograft-bearing mice (HCT116 colon Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN
46285, USA
carcinoma and U87MG glioblastoma) suppressed tumor growth.
In Phase II clinical studies, enzastaurin showed some promise in
*Correspondence to: William J. Wheeler, Eli Lilly and Company, Lilly Corporate
Center, Indianapolis, IN 46285, USA.
the treatment of recurrent glioblastoma (25% of 92 patients).6
The synthesis of unlabeled enzastaurin7 as well as carbon-14- E-mail: isotopicsolutions@comcast.net
J. Label Compd. Radiopharm 2008, 51 175–181
Copyright r 2008 John Wiley & Sons, Ltd.

W. J. Wheeler and D. K. Clodfelter
(Crabtree’s catalyst) yielded only unreacted starting material (no (Scheme 2). Using careful silica gel chromatography, the mixture
deuterium incorporation).
of 4 and 5 was separated. Had the alkylation of 3 yielded even a
Several alternative methods were evaluated for the use in the small amount of material methylated only on the indole
tritiation of 1. Most certainly, 2-[3H] could be synthesized using nitrogen, it would have provided a means for the synthesis of
chemistry described by Faul et al.; however, this would require 2-[3H], albeit not the most ideal solution. Based on the results
the alkylation of a suitable indole precursor with CT3I, followed shown in Scheme 2, methylation of 5 with CT3I might be useful
by several subsequent radioactive steps.7 Since both 1-[3H] and if 4-[3H] could be easily converted to 2-[3H]. In the synthesis of
2-[3H] were required, the most efficient path forward would be ruboxistaurin, another PKC inhibitor, previously under clinical
to synthesize 1-[3H] from 2-[3H]. Pursuant to this option, trial for the treatment of diabetic retinopathy, the N-methyl
reaction of 2 with the HBr salt of 2-bromo-methylpyridine in the maleimide (6) was converted to the unsubstituted maleimide (8)
presence of Hunig’s base yielded 1 in 42% yield (Scheme 1). via the corresponding anhydride (7) as shown in Scheme 3.10
With the means of synthesizing 1 from 2 in hand, the remaining
task became the synthesis of 2-[3H].
Upon further consideration, it seemed probable that starting
from the anhydride obtained from 3 (or 5) would avoid an
The most straightforward scenario for the synthesis of 2-[3H] unnecessary step, thus affording 2-[3H] in only three steps from
would entail the alkylation of 3 (or an analog of 3) with CT3I. Not readily available starting material. Hydrolysis of the maleimides
surprisingly (maleimide pKa (DMSO) = 10.0 vs indole = 21.0), to ring-opened maleic acid was sluggish (in spite of reports that
alkylation of 3 yielded a mixture of regioisomers 4 and 5 competitive hydrolysis to the anhydride was observed in the
synthesis of 1 when a basic pH was not minimized in the work-
up7) and never went to completion as was previously shown in
the hydrolysis of 6. Overnight reaction of 3 and 5 with excess
(22%)
3H
H
potassium hydroxide (10% aqueous) in refluxing dioxane failed
to go to completion; the reaction mixture was extracted with
EtOAc (to remove unreacted imide) and the aqueous layer was
acidified to yield 10 in 36 and 47%, respectively (Scheme 4).
Treatment of 9 (t- BOC analog of 2) in the same manner gave 11
in 36% yield.
(42%)
N
N
3H
CH3
(18%)
3H
N
O
O
N
3H
(18%)
CF3
Figure 1. Tritium incorporation resulting from overnight treatment of 1 mg of the
substrate (and 1 mg of two other similar compounds) with 3.13 eq. of Crabtree’s
catalyst/4.6 Ci of tritium in methylene chloride.
Alkylation of 10 with methyl iodide followed by chromato-
graphy on silica gel yielded 11 in 54% yield (Scheme 5). Reaction
H
N
H
N
O
O
O
O
2-bromopyridine HBr
i-Pr2NEt, DMF
42%
N
N
N
N
CH3
CH3
N
N
H
N
2
1
Scheme 1
CH3
N
H
N
O
O
O
O
1. NaH/DMF
2. CH3I
N
H
N
R
N
N
N
N
O
O
O
O
3
4;
R = CH3
5; R = H
Scheme 2
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 175–181

W. J. Wheeler and D. K. Clodfelter
CH3
N
X
O
O
N
O
O
1. NaOH/reflux
2. HCl
N
N
N
O
O
OTr
OTr
6
7, X = O
8, X = NH
HMDS/CH2Cl2
CH3OH
Scheme 3
12
R''
N
O
O
O
O
O
6
5
17
18
19
1. KOH, dioxane
2. HCl
4
9
23
3
N
N
20
29
N
N
1
24
R'
R'
25
26
28
N
R
N
R
Scheme 4
Peak assignments were made using gradient selected COSY
(gCOSY), HSQC and HMQC experiments. Chemical shifts are
reported in parts per million (ppm) downfield from tetramethyl-
silane. ES-MS spectra were run on a Waters Micromass ZQ single
quadrapole mass spectrometer. Exact masses were determined
on a Premier Micromass High Resolution Q-TOF mass spectro-
meter coupled with Acquity UPLC. Flash chromatography was
performed on a Biotage system using silica gel cartridges. Thin
R
R0
R00
R
R0
3
5
9
t-BOC
t-BOC
t-BOC
H
H
H
CH3
H
10
11
t-BOC
t-BOC
H
CH3
CH3
of 11 with hexamethyldisilazide in DMF/CH3OH yielded 9 in 95% layer chromatography was conducted on EMD Chemicals, Inc.,
yield (Scheme 5). Reaction of 9 with 12 N HCl in refluxing CH3OH silica gel 60 F254 plates (5 ꢀ 10 cm, 250 mm). The RCP was
yielded 2 in 57% yield. Reaction of the sodium salt (prepared by assessed by radio-HPLC.
reaction with NaH/THF) of 10 with [3H]-methyl nosylate yielded
11-[3H] which was converted, after purification by HPLC, to 9-
Synthesis of 3-(1-methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinyl-
[3H] by reaction with HMDS/DMF/CH3OH.y De-protection of 9-
methyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 1
[3H] by reaction with 9:2 TFA/water followed by HPLC
purification yielded 2-[3H] with a specific activity of 80 Ci/mmol
A DMF (2 mL) solution of 2 (10.0 mg, 24 mmol) was treated with
2-bromomethylpyridine hydrobromide (12.0 mg, 48 mmol) and
Hunig’s base (9.0 mg, 13.0 mL, 72 mmol), and the reaction mixture
was stirred at room temperature overnight. Thin layer chroma-
tography (silica gel, CH2Cl2/CH3OH/NH4OH, 90:10:1) showed loss
of 2 and the formation of 1. The reaction mixture was poured
into a mixture of CH2Cl2 and saturated sodium bicarbonate. The
aqueous layer was extracted with CH2Cl2 (2 ꢀ 15 mL) and the
combined organic layers were washed with saturated brine,
dried (anhydrous MgSO4) and concentrated in vacuo. The red
residue was purified by Biotage chromatography on silica gel,
eluting with 5 mL fractions of CH2Cl2/CH3OH/NH4OH (90:10:1).
Fractions 5–8 were combined and concentrated to yield 1
(5.4 mg, 45%)
(the radiochemical purity (RCP) was 97.4%).
Reaction of 2-[3H] with 2-bromomethylpyridine in DMF/
Hunig’s base followed by HPLC purification (Scheme 6) yielded
1-[3H] with a specific activity of 82 Ci/mmol (the radiochemical
purity was 98.2%).
Experimental
NMR spectra were obtained on a Varian Mercury-400 or Violin-
Inova-500 spectrometer at 400 or 500 (1H) and 100 (13C) MHz
(numbering system for the NMR’s are shown in structure 10).
yThe synthesis and purification of 1 and 2-[3H] was conducted by
Amersham International Plc, Whitchurch, Cardiff, Wales, UK, using
methods supplied by the authors.
ES-MS: [M1H]1 m/z = 516; [M1Na]1 m/z = 538. This material
was identical in all respects with authentic material.7 HPLC:
J. Label Compd. Radiopharm 2008, 51 175–181
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

W. J. Wheeler and D. K. Clodfelter
H
N
O
O
O
O
O
HMDS/DMF
CH3OH, heat
N
N
N
N
N
CH3
CH3
N
O
O
O
O
9
(95%)
11
(54%)
12N HCl
CH3OH/reflux
1. NaH/DMF
2. CH3I
H
N
O
O
10
N
N
CH3
N
H
1. NaH/THF
2
(57%)
2. [3H]-methyl nosylate
3. HMDS/DMF/CH3OH
4. TFA/CH3OH/H2O
H
N
O
O
N
N
CT3
N
H
2-[3H]
Scheme 5
H
N
H
N
O
O
O
O
2-bromomethyl-
N
N
N
N
N
pyridine/DMF
i-Pr2NEt
CT3
CT3
N
H
N
2-[3H]
1-[3H]
Scheme 6
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 175–181

W. J. Wheeler and D. K. Clodfelter
Zorbax SB-C8 (4.6 ꢀ 250 mm, 5 mm) at 1 mL/min at ambient imide-Me), 3.86 (s, 3H, indole-Me), 3.89–4.16 (m, 2H, 26 and 28
temperature with UV detection at 220 nm and gradient elution eq-H), 4.61 (tt, J = 3.5 and 12.2, 1H, 24 ax-H), 6.52 (d, J = 8.1 Hz,
(Solvent A: 10% CH3CN/90% H2O/0.5% H3PO4, v/v; Solvent B: 1H, 6-H), 6.62 (t, J = 8.1 Hz, 1H, 5-H), 6.80 (t, J = 8.1 Hz, 1H, 18-H),
90% CH3CN/10% H2O/0.5% H3PO4, v/v): 0–5 min: 10% B; 7.04 (t, J = 8.1 Hz, 1H, 4-H), 7.06 (t, J = 8.3, 1H, 19-H), 7.11 (d,
5–35 min: 70% B; 35–45 min: 70% B; 45–46 min: 10% B; J = 7.3 Hz, 1H, 17-H), 7.42 (d, J = 8.1 Hz, 1H, 3-H), 7.59 (d,
46–60 min: 10% B. The sample was dissolved in 40% CH3CN/ J = 8.1 Hz, 1H, 20-H), 7.63 (s, 1H, 23-H) and 7.89(s, 1H, 9-H).
60% H2O/0.5% H3PO4 (v/v) and the injection volume was 20 mL.
1, RT = 22.25 min and 2, RT = 20.15 min.
HRMS: [M1H]1; m/z calculated for C32H35N4O4 539.2658,
found 539.2637.
Synthesis of 4-[3-4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydrofur-
an-3-yl]indol-1-yl]-piperidine-1-carboxylic acid, tert.-butyl
ester, 10
Synthesis of 4-[3-[4-(1H-indol-3-yl)-1-methyl-2,5-dioxo-1H-
pyrrol-3-yl]-indol-1-yl]]-piperidine-1-carboxylic acid, tert.-
butyl ester, 5
A dioxane solution (5 mL) of 5 (0.250 g, 0.486 mmol) was
treated with 10% potassium hydroxide (10 mL) and stirred
at reflux overnight. The initial deep red color of the solution
gradually lightened to a pale red. The reaction mixture was
poured into water and extracted with EtOAc (5 ꢀ 25 mL);
the aqueous layer was acidified to pH 2.4 with HCl (5 N)
and then extracted with EtOAc (5 ꢀ 40 mL). The combined
organic extracts were washed with saturated brine, dried
(anhydrous MgSO4) and concentrated in vacuo to yield 10
(0.114 g, 46%). This material was a single spot on TLC (silica gel,
7:3 Et2O/heptane), Rf = 0.35. This material was mixed with some
less pure material (0.052 g) from a previous run and purified by
silica gel chromatography, eluting with 20 mL fractions of 7:3
Et2O/heptane; fractions 16–25 were concentrated in vacuo
(0.101 g) and recrystallized from Et2O to yield 10 as a red
crystalline solid (0.078 g).
A solution of 3 (1.0 g, 1.95 mmol) was dissolved in anhydrous
DMF (30 mL) and stirred under argon. The mixture was treated
with a 60% mineral oil dispersion of sodium hydride (0.066 g,
1.65 mmol); the color deepened and stirring was continued for
0.5 h. Methyl iodide (0.426 g, 0.187 mL, 3.0 mmol) was added, the
solution cleared to ruby red and stirring was continued for 1 h.
TLC (silica gel, CH2Cl2/CH3OH, 95:5) showed loss of 3. The
reaction mixture was poured into saturated NH4Cl and extracted
with ethyl acetate. The aqueous layer was extracted with ethyl
acetate; the combined organic layers were washed with
saturated brine, dried (anhydrous MgSO4) and concentrated
in vacuo. The oily residue was re-dissolved in acetonitrile and
washed with hexanes (to remove the mineral oil). The
acetonitrile was removed in vacuo. ES-MS of the residue showed
peaks at 525 ([M1H]1 of 5) as well as 542 ([M1NH4]1 of 5) and
556 ([M1NH4]1 of 4). The residue was purified by silica gel
chromatography, eluting with CH2Cl2; fractions 78–90 were a
mixture of 4 and 5 and were re-chromatographed eluting with
pentane/Et2O. Fractions 81–160 were combined, concentrated
and crystallized from Et2O to yield 5 (0.292 g, 29%) as red
needles.
1H NMR (CDCl3/DMSO/d6): d 1.34 (s, 9H, OC(CH3)3), 1.56
(dd, J = 3.8 and 11.5, 1H, 25 or 29 ax-H), 1.61 (dd, J = 3.8 and 11.5,
1H, 25 or 29 ax-H), 1.89 (d, J = 11.5 Hz, 2H, 25 and 29eq-H),
2.77 (t, J = 12.3 Hz, 2H, 26 and 28 ax-H), 4.12 (m, 2H, 26 and 28
ax), 4.26 (tt, J = 3.2 and 8.3 Hz, 1H, 24 ax-H), 6.59 (d, J = 6.8 Hz, 1H,
6-H), 6.61 (t, J = 7.7 Hz, 1H, 5-H), 6.78 (t, J = 7.9 Hz, 1H, 18-H),
6.94 (ddd, J = 1.8, 6.1 and 7.9 Hz, 1H, 4-H), 7.04 (t, J = 7.5 Hz, 1H,
19-H), 7.13 (d, J = 8.1 Hz, 1H, 17-H), 7.27 (d, J = 8.1 Hz, 1H, 20-H),
7.28 (d, J = 7.1 Hz, 1H, 3-H), 7.44 (s, 1H, 23-H), 7.75 (d, J = 3.3 Hz,
1H, 9-H) and 10.78 ppm (bs, 1H, NH); 13C NMR (CDCl3/DMSO/d6):
d 28.33 (C(CH3)3), 31.94 (25 and 29-C), 43.16 (26 and 28-C),
53.90 (24-C), 79.96 (Me3C), 105.33 (8-C), 105.41 (15-C), 109.66
(20-C), 112.05 (3-C), 120.21 (6-C), 120.78 (18-C), 121.83 (5-C),
122.40 (4-C), 122.38 (17-C), 122.50 (19-C), 124.15 (7-C),
126.77 (16-C), 127.66 (14-C), 128.84 (10-C), 128.97 (23-C),
134.20 (9-C), 135.62 (21-C), 136.52 (2-C), 154.27 (CO-t-Bu)
166.54 (11 or 13-C) and 166.66 ppm (11 or 13-C); ES-MS:
[M1NH4]1, m/z = 529.
ES-MS: ([M1H]1, m/z = 525; [M1NH4]1 m/z = 542; 1H NMR
(CDCl3/DMSO/d6): d 1.51 (s, 9H, OC(CH3)3), 1.77 (dd, J = 3.9 and
12.7 Hz, 1H, 25 or 29 ax-H), 1.82 (dd, J = 3.9 and 12.7 Hz, 1H, 25 or
29 ax-H), 2.05 (d, J = 12.7 Hz, 2H, 25 and 29 eq-H), 2.91 (t,
J = 12.6 Hz, 2H, 26 and 28 ax-H), 3.22 (s, 3H, imide-Me), 4.30 (m,
2H, 26 and 28 eq-H), 4.37 (tt, J = 3.9 and 11.8 Hz, 1H 24 ax-H),
6.76 (d, J = 7.4 Hz, 1H, 5-H), 6.87 (d, J = 7.4 Hz, 1H, 6-H), 6.88 (t,
J = 7.4, 1H, 18-H), 7.10 (t, J = 7.4 Hz, 1H, 4-H), 7.16 (t, J = 7.4, 1H,
19-H), 7.22 (d, J = 8.3 Hz, 1H, 17-H), 7.36 (d, J = 7.9 Hz, 1H, 3-H),
7.37 (d, J = 7.9 Hz, 1H, 20-H), 7.61 (s, 1H, 23-H), 7.80(d, J = 3.1 Hz,
1H, 9-H) and 8.60 ppm (bs, 1H, NH); 13C NMR (CDCl3/DMSO/d6): d
24.11 (N–C), 28.41 (C(CH3)3), 32.12 (25 and 29-C), 43.21 (26 and
28-C), 53.78 (24-C), 80.03 (Me3C), 106.39 (15-C), 107.27 (8-C),
109.28 (3-C), 111.17 (20-C), 120.31 (5-C), 120.45 (18-C), 122.06 (6-
C), 122.17 (19-C), 122.57 (17-C), 126.35 (16-C), 127.06 (14-C),
127.83 (10-C), 128.14 (23-C), 128.24 (9-C), 135.70 (21-C), 135.88
(2-C), 154.51 (CO-t-Bu) and 172.38 (11 and 13-C).
HRMS: [MꢁH]1; m/z calculated for C30H28N3O5 510.2029,
found 510.2005.
Synthesis of 4-[3-4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydrofur-
an-3-yl]indol-1-yl]-piperidine-1-carboxylic acid, tert.-butyl
ester, 10
HRMS: [MꢁH]1, m/z calculated for C31H31N4O4 523.2345,
found 523.2359.
Fractions 41–60 were concentrated in vacuo and A dioxane solution (10 mL) of 3 (0.250 g, 0.490 mmol) was
crystallized from Et2O/heptane to yield 4 (4-[3-[1-methyl-4- treated with 10% potassium hydroxide (10 mL) and stirred at
(1-methyl-1H-indol-3-yl)-2,5-dihydro-1H-pyrrol-3-yl]indol-1-yl] reflux overnight. The work-up of the reaction mixture as
piperidine-1-carboxylic acid tert.-butyl ester) as a red solid described above yielded 10 (0.091 g, 36%) as a red crystalline
(0.045 g, 4%).
solid. TLC (silica gel, 7:3 Et2O/heptane) showed a single spot at
ES-MS: [M1NH4]1 m/z = 556; 1H NMR (DMSO/d6): d 1.39 (s, 9H, Rf = 0.35, which co-eluted with the material prepared from 5 as
OC(CH3)3), 1.59 (dd, J = 3.6 and 11.7 Hz, 1H, 25 or 29 ax-H), 1.65 described above.
(dd, J = 3.6 and 11.7 Hz, 1H, 25 or 29 ax-H), 1.87 (d, J = 12.6 Hz,
NMR (CDCl3): superimposable with the spectrum obtained
2H, 25 and 29eq-H), 2.84 (m, 2H, 26 and 28 ax-H), 3.03 (s, 3H, from 10 prepared from 5; ES-MS: [M1Na]1, m/z = 534.
J. Label Compd. Radiopharm 2008, 51 175–181
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

W. J. Wheeler and D. K. Clodfelter
purified directly on an Ultrasphere ODS column (10 ꢀ 250 mm)
eluting with a CH3OH/water/triethylamine gradient system to
yield 11-[3H].
Synthesis of 4-[3-[4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
acid,
dihydrofuran-3-yl]indol-1-yl]piperidine-1-carboxylic
tert.-butyl ester, 11
A dioxane solution (10 mL) of 9 (0.250 g, 0.476 mmol) was
treated with was treated with 10% potassium hydroxide (10 mL)
and stirred at reflux overnight. The work-up of the reaction
mixture as described above, yielded 11 (60 mg, 24%) as a red
crystalline solid. TLC (silica gel, 7:3 Et2O/heptane) showed a
single spot at Rf = 0.49.
Synthesis of 4-[3-[4-(1-methyl-1H-indol-3-yl)-2,5-dihydro-
1H-pyrrolo-3-yl]indol-1-yl]piperidine-1-carboxylic acid tert.-
butyl ester (9)
A DMF (2 mL) of 11 (20 mg, 38 mmol) was blanketed with argon
and treated with hexamethyldisilazane (100 mL, 0.480 mmol),
followed by the addition of CH3OH (10 mL). The resulting mixture
was stirred at 50–551C overnight. An aliquot of the reaction
mixture was treated with EtOAc/H2O, TLC (7:3 Et2O/heptane),
and showed a spot to spot conversion to the desired product.
The remainder of the reaction mixture was allowed to cool to
room temperature and was concentrated in vacuo. The residue
was dissolved in EtOAc and was washed with water. The EtOAc
solution was washed with brine, dried (anhydrous MgSO4) and
concentrated to yield 9 (19 mg, 95%); ES-MS: [M1H]1, m/z = 525,
[M1NH4]1, m/z = 542.
1
ES-MS showed a [M1NH4]1 at m/z = 543; H NMR (CDCl3): d
1.48 (s, 9H, OC(CH3)3), 1.72 (dd, J = 4.1, 12.4 Hz, 1H, 25 or 29 ax-
H), 1.78 (dd, J = 4.1, 12.4 Hz, 1H, 25 or 29 ax-H), 2.03 (d,
J = 12.4 Hz, 2H, 25 and 29 eq-H), 2.89 (t, J = 13.2 Hz, 2H, 26 and 28
ax-H), 3.88 (s, 3H, indole-Me), 4.29 (d, J = 11.3, 2H, 26 and 28 eq-
H), 4.38 (tt, J = 3.9 and 11.8 Hz, 1H 24 ax-H), 6.73 (dd, J = 1.6 and
7.9 Hz, 1H, 6-H), 6.76 (td, J = 0.9 and 7.9 Hz, 1H, 5-H), 6.93 (ddd,
J = 0.9, 2.1 and 7.9 Hz, 1H, 18-H), 7.15 (ddd, J = 1.7, 6.6 and 8.2,
1H, 4-H), 7.19 (ddd, J = 1.0, 7.2 and 8.2, 1H, 19-H), 7.28 (d,
J = 8.2 Hz, 1H, 17-H), 7.32 (d, J = 8.5 Hz, 1H, 20-H), 7.38 (d,
J = 8.2 Hz, 1H, 3-H), 7.60 (s, 1H, 23-H) and 7.83 (s, 1H, 9-H); 13C
NMR (CDCl3): d 28.41 (C(CH3)3), 32.47 (indole-CH3), 32.49 (25 and
29-C), 43.16 (26 and 28-C), 53.96 (24-C), 80.14 (Me3C), 104.96
(8-C), 105.73 (15-C), 109.67 (C-20), 109.87 (3-C), 120.61 (5-C),
120.96 (18-C), 122.51 (6-C), 122.70 (4-C, 17-C, 19-C), 125.01 (7-C),
126.12 (16-C), 127.44 (14-C), 128.10 (10-C), 128.97 (23-C), 134.20
(9-C), 135.70 (21-C), 137.1 (2-C), 154.6 (CO-t-Bu), 166.62 (11 or 13-
C) and 166.92 (11 or 13-C).
Synthesis of 3-(1-methyl-1H-indol-3-yl)-4-(1-piperidin-4-yl-
1H-indol-3-yl)-pyrrole-2,5-dione (2)
A CH3OH solution (2 mL) of 9 (19 mg, 36 mmol) was treated with
HCl (12 N, 4 mL, 38 mmol) and stirred at reflux for 1 h. Stirring at
room temperature was continued overnight. TLC (silica gel, 7:3
Et2O/heptane) showed some unreacted 9 remained. The mixture
was heated to reflux and stirred an additional 2 h; TLC showed
complete loss of 9. TLC (silica gel, CH2Cl2/CH3OH/NH4OH,
90:10:1) showed material which co-eluted with an authentic
sample of 2.7 The reaction mixture was diluted with EtOAc and
was washed with saturated aqueous NaHCO3 and brine. The
organic layer was dried (anhydrous MgSO4) and concentrated in
vacuo to yield 2 (10 mg, 57%). This material was identical in all
respects to authentic 2, prepared as described by Faul et al.7
HRMS: [M1H]1; m/z calculated for C31H32N3O5 526.2342,
found 526.2347.
Synthesis of 4-[3-[4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-
acid,
dihydrofuran-3-yl]indol-1-yl]piperidine-1-carboxylic
tert.-butyl ester, 11
A mixture of 10 (38 mg, 74 mmol) and sodium hydride (60%
mineral oil dispersion, 1.1 eq., 3.2 mg, 81 mmol) was dissolved in
anhydrous THF (10 mL (under argon) and stirred for 0.5 h. To the
resulting solution was added a THF (2 mL) solution of methyl 4-yl-1H-indol-3-yl)-pyrrole-2,5-dione (2-[3H3])
Synthesis of 3-(1-methyl-[3H3]-1H-indol-3-yl)-4-(1-piperidin-
iodide (1.1 eq., 12 mg, 5 mL, 81 mmol). The resulting solution was
A DMF solution (1 mL) of 11-[3H] was treated with hexamethyl-
stirred overnight at room temperature. The mixture was
acidified with HCl (1 N) and diluted with EtOAc/water. The
organic layer was washed with saturated brine, dried (anhydrous
MgSO4) and concentrated in vacuo. The residue was triturated
with heptane to remove the mineral oil; the heptane was
extracted with CH3CN. The CH3CN extract was added the
original residue and concentrated in vacuo. TLC (silica gel, 7:3
Et2O/heptane) of the residual product showed a major spot
running slightly ahead of the starting material as well as two
very minor impurities. The red semi-solid was purified by
Biotage on silica gel, eluting with 5 mL fractions of Et2O/heptane
(7:3). Fractions 12–28 were concentrated to yield 11 (21 mg,
54%).
disilazane (100 mL, 0.480 mmol) and CH3OH (10 mL) and heated at
501C for 1 h and then stirred at room temperature overnight.
The crude reaction mixture was concentrated in vacuo to yield
9-[3H]. The residue was re-dissolved in 9:1 TFA/H2O (2 mL) and
stirred at room temperature for 1 h and then evaporated to
dryness. The crude product was purified on an Ultrasphere ODS
column (10 ꢀ 250 mm) eluting with a water/acetonitrile/TFA
gradient system to yield 2-[3H] (65 mCi, 8.1% overall for the
three steps). The specific activity (as determined by mass
spectrometry) was 80 Ci/mmol; the RCP was 97.4%.
Synthesis of 3-(1-methyl-[3H3]-1H-indol-3-yl)-4-(1-piperidin-
4-yl-1H-indol-3-yl)-pyrrole-2,5-dione (1-[3H])
NMR (CDCl3): superimposable with the spectrum obtained
from 11 prepared from 9; ES-MS: [M1Na]1, m/z = 548.
A DMF solution (0.5 mL) of 2-[3H] (55 mCi, 0.69 mmol) was
treated with 2-bromomethylpyridine (0.47 g, 27 mmol) and di-iso-
propyl-ethylamine (10 mL, 57 mmol) and stirred at room tem-
perature for 3 h. The reaction mixture was concentrated in vacuo
and purified by HPLC chromatography on a YMC Pack C8
column (4.6 ꢀ 250 mm), eluting with a water/acetonitrile/TFA
gradient system to yield 1-[3H] (23 mCi, 42%). The specific
activity (as determined by mass spectrometry) was 82 Ci/mmol;
the RCP was 98.2%.
Synthesis of 4-[3-[4-(1-methyl-[3H3]-1H-indol-3-yl)-2,5-dihy-
drofuran-3-yl]indol-1-yl]piperidine-1-carboxylic acid tert.-
butyl ester (11-[3H])
Sodium hydride (1 mg) was added to a THF solution of 10 (5 mg,
9.77 mmol); the resulting solution was stirred for 5 min at room
temperature and then added to methyl-[3H3] nosylate (800 mCi,
10 mmol) and stirred overnight. The crude reaction mixture was
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 175–181

W. J. Wheeler and D. K. Clodfelter
[4] R. S. Herbst, D. E. Thornton, M. S. Kies, V. Sinha, S. Flanagan, C. A.
Cassidy, M. A. Carducci, Proc. Am. Soc. Clin. Oncol. 2002, 21, 326.
[5] J. R. Graff, A. M. McNulty, K. R. Hanna, B. W. Konicek, R. L. Lynch,
S. N. Bailey, C. Banks, A. Capen, R. Goode, J. E. Lewis, L. Sams, K.
L. Huss, R. M. Campbell, P. W. Iverson, B. L. Neubauer, T. J. Brown,
L. Musib, S. Geeganage, D. Thornton, Cancer Res. 2005, 65,
7462–7469.
[6] H. A. Fine, L. Kim, C. Royce, D. Draper, I. Haggarty, H. Ellinzano, P.
Albert, P. Kinney, L. Musib, D. Thornton, J. Clin. Oncol. 2005, 23, 16S.
[7] M. M. Faul, J. L. Grutsch, M. E. Kobierski, M. E. Kopach,
C. A. Krumrich, M. A. Staszak, U. Udodong, J. T. Vicenzi, K. A.
Sullivan, Tetrahedron 2003, 59, 7215–7229.
Acknowledgement
The authors would like to express their appreciation to Dr Chad
E. Hadden and Mr Ronald J. Lantz of Drug Disposition, Lilly
Research Laboratories, Eli Lilly and Company, Lilly Corporate
Center, Indianapolis, IN 46285, for their help in NMR interpreta-
tion and the determination of the high-resolution mass spectra.
[8] W. J. Wheeler, F. H. White, J. W. Kennington, D. D. O’Bannon,
E. L. Mattiuz, E. A. Stoddard, D. K. Clodfelter, Abstract 16th of the
Central US IIS Meeting, Chesterfield, MO, USA, 2003.
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Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org