Synthesis and evaluation of [123I]-indomethacin derivatives as COX-2 targeted imaging agents

Article abstract of DOI:10.1002/jlcr.1615

A novel series of iodinated indomethacin derivatives was synthesized, and evaluated as selective inhibitors of COX-2. Two candidate compounds N-(p-iodobenzyl)-2-(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl) acetamide (3) and 1-(piodobenzyl)-5-met

Full text of DOI:10.1002/jlcr.1615

Research Article
Received 30 November 2008,
Revised 29 April 2009,
Accepted 2 May 2009
Published online 24 July 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1615
Synthesis and evaluation of [123I]-
indomethacin derivatives as COX-2
targeted imaging agents
Md. Jashim Uddin,a Brenda C. Crews,a Anna L. Blobaum,a
Philip J. Kingsley,a Kebreab Ghebreselasie,a Sam S. Saleh,a
Jeffrey A. Clanton,b Ronald M. Baldwin,b,c and Lawrence J. Marnetta
Ã
A novel series of iodinated indomethacin derivatives was synthesized, and evaluated as selective inhibitors of COX-2. Two
candidate compounds N-(p-iodobenzyl)-2-(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamide (3) and 1-(p-
iodobenzyl)-5-methoxy-2-methyl-3-indoleacetic acid (9) possessed optimum properties suitable for potential in vivo
imaging. Arylstannane precursors for radioiododestannylation were synthesized in 70–85% yield from the iodo compounds
by reaction with hexabutylditin and tetrakis(triphenylphosphine)palladium(0) in refluxing dioxane. Radioiododestannyla-
tion was conducted by reaction with carrier-added Na[123I] in the presence of Chloramine-T in an EtOAc/H2O binary system
under acidic conditions (pH 3.5), allowing direct isolation of the labeled products by separation of the organic phase.
Radioiodinated products [123I]3 and [123I]9 were recovered in a decay-corrected radiochemical yield of 86–87% and
radiochemical purity of 98–99%.
Keywords: Iodine-123; indomethacin; iododestannylation; two-phase reaction; COX-2
p-bromobenzyl group yields inhibitors that display a high degree
of COX-2-selectivity.17,18 As a prelude to the evaluation of these
Introduction
classes of inhibitors for single photon emission computerized
tomography imaging, we synthesized a series of iodo derivatives
and evaluated their COX-2 inhibitory properties against purified
proteins and in intact cell assays. The incorporation of iodine into
both types of indomethacin derivatives yielded compounds that
were active against purified COX-2 but many of them did not
inhibit the enzyme in intact cells. The most promising compounds
identified in cell-based assays were used as targets for stannyla-
tion-based introduction of 123I. The chemistry reported herein
includes an efficient two-phase method for 123I incorporation that
There are two cyclooxygenase (COX) genes in humans, which
are responsible for the conversion of polyunsaturated fatty acids
to prostaglandins.1 COX-1 is constitutively expressed and, for
the most part, appears to play a role in tissue homeostasis,
whereas COX-2 is induced in response to a broad range of
physiological and pathophysiological stimuli.2–5 COX-2 mRNA
and protein are detectable in a significant percentage of
inflammatory and premalignant lesions,6–8 and an even higher
percentage of malignant tumors (e.g. colon adenocarcinoma,
esophageal adenocarcinoma).9,10 Studies have shown that the
expression of COX-2 is an early event in tumorigenesis and that
it plays a role in tumor progression.11 Thus, COX-2 is a potential
target for imaging a variety of cancers using radiolabeled COX-2
inhibitors, and some approaches toward this goal have been
reported.12
18F- and 123I-labeled COX-2 inhibitors based on the diarylhetero-
cycle class of inhibitors have been synthesized and selective
uptake has been reported in COX-2-expressing cells.13–16 Such
selective uptake has not been rigorously demonstrated in vivo
although promising preliminary data have been reported with an
123I-labeled celecoxib derivative.16 Other classes of COX-2
inhibitors may be more effective scaffolds for the development
of targeted radiological imaging agents based on their potency,
selectivity or pharmacokinetic properties. Our laboratory and
others have reported that conversion of the nonselective COX
inhibitor, indomethacin, into amide derivatives or replacement of
the p-chlorobenzoyl moiety on the indole nitrogen with a
A complete description of the experimental methods and compound
charcterization is available online at http://www.interscience.wiley.com
aA. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of
Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical
Biology, Center for Molecular Toxicology and Vanderbilt-Ingram Cancer Center,
Vanderbilt University School of Medicine, Nashville, Tennessee 37232–0146, USA
bDepartment of Radiology and Radiological Sciences, Vanderbilt University
Medical Center, Nashville, Tennessee 37232–0146, USA
cVanderbilt Institute of Imaging Sciences, Vanderbilt University Medical Center,
Nashville, Tennessee 37232–0146, USA
*Correspondence to: Lawrence J. Marnett, A. B. Hancock, Jr., Memorial
Laboratory for Cancer Research, Department of Biochemistry, Chemistry and
Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Molecular
Toxicology and Vanderbilt-Ingram Cancer Center, Vanderbilt University School
of Medicine, Nashville, Tennessee 37232–0146, USA.
E-mail: larry.marnett@vanderbilt.edu
J. Label Compd. Radiopharm 2009, 52 387–393
Copyright r 2009 John Wiley & Sons, Ltd.

Md. J. Uddin et al.
generates labeled compounds in high yield and purity without the series of iodinated indomethacin derivatives was generated
need for further chromatographic purification.
based on p-halobenzyl substitution on the indole nitrogen; the
p-bromobenzyl analog has been reported to be a selective
COX-2 inhibitor.18 We synthesized 1-(p-iodobenzyl)-5-methoxy-
2-methyl-3-indoleacetic acid (9) by direct alkylation of
5-methoxy-2-methyl-3-indoleacetic acid with p-iodobenzylbro-
mide in the presence of NaH in 67% yield (Scheme 2).
Results
Chemistry and pharmacology
A series of indole amide derivatives was synthesized by coupling
indomethacin with different amines (Scheme 1). For example, selectivity for inhibition of recombinant human COX-2 and
Each of the compounds was tested for its potency and
N-(p-iodobenzyl)-2-(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-in- purified sheep COX-1 as previously described.17 (Table 1).
dol-3-yl)acetamide (3) was synthesized by
a reaction of Compounds also were evaluated for their ability to inhibit
indomethacin with 4-iodobenzylamine using ethyl-1-[3-(di- COX-2 in intact cells using the mouse macrophage cell line
methylamino)propyl]-3-ethylcarbodiamide (EDCI), 1-hydroxy- RAW264.7. Although all of the indole amide compounds (1–8)
benzotriazole hydrate (HOBt), N,N-diisopropylethylamine were potent and selective inhibitors of purified recombinant
(DIEA), N,N-dimethylformamide (DMF) in 77% yield. A second COX-2, only 3 inhibited COX-2 in intact cells. In contrast to
Cl
H
N
I
1: R =
I
I
H
N
R-H, EDCI, DIEA,
Me
2: R =
3: R =
O
Cl
N
O
HOBt, DMF, 25 oC, 16 h
R
N
H
I
1-5
MeO
Me
O
N
O
N
N
N
4: R =
5: R =
OH
N
O O
S
Cl
MeO
N
I
Indomethacin
H
H2N R1
N
BOC
a)
EDCI, HOBt
6: R1 = (CH2)2, R2 = CO
7: R1 = (CH2)4, R2 = SO2
8: R1 = C6H4, R2 = CO
DIPEA, DMF, 25 oC, 16 h
Me
O
I
N
O
HCl gas, CH2Cl2, 25 oC, 1 h
Ar R2 Cl
b)
c)
NH
R1
R2
TEA, CH2Cl2
25 oC, 12-16 h
MeO
N
H
6-8
Scheme 1. Synthesis of iodinated compounds 1–8.
I
I
H
Me
N
O
BrH2C
I
H2N (CH2)2
EDCI, HOBt
R
Me
N
O
OH
Me
NaH, DMF, 25 oC, 1 h
N
O
N
(CH2)2
R
DIEA, DMF, 25 oC, 16 h
H
MeO
OH
10: R = OH
11: R = F
(5-Methoxy-2-methyl-
3-indoleacetic acid)
MeO
9
MeO
I
I
I
MeOH, H2SO4
reflux 16 h
10% NaOH/MeOH,
reflux 6 h
MeI, LiHMDS
THF, -78 oC 1h
Me
Me
N
O
Me
N
O
9
N
O
OH
OMe
OMe
Me
Me
13
MeO
MeO
12
14
MeO
Scheme 2. Synthesis of iodinated compounds 9–14.
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 387–393

Md. J. Uddin et al.
Table 1. COX-1 and COX-2 enzyme inhibition assay data against purified COX enzymes or LPS-activated RAW264.7
macrophages.
I
Cl
I
Me
Me
Me
O
N
O
N
N
O
O
R
R
R
Me
MeO
MeO
MeO
1-8
13-14
9-12
Compound No.
R
IC50 (mM)a
IC50 (mM) intact cellsb
COX-1
COX-2
0.12
H
1
2
466
441
441
N
I
I
H
466
466
0.06
0.12
N
N
I
3
4
1
H
I
466
466
0.45
0.28
41
N
N
N
O
O
S
5
41
N
N
I
H
N
N
6
7
466
466
0.21
0.10
41
41
H
O
I
H
N
S
N
H
O
O
I
I
H
N
H
N
C
8
9
10
466
466
466
0.17
0.40
466
41
0.08
n.d.
O
OH
OH
F
N
H
N
11
12
13
14
466
466
466
466
466
466
466
1.71
n.d.
n.d.
n.d.
1.49
H
OMe
OMe
OH
aOvine COX-1 (44 nM) or human COX-2 (66 nM) was preincubated with inhibitors at 251C for 17 min show at 371C for 3 min
followed by the addition of [1–14C]arachidonic acid (50 mM) at 371C for 30 s. All assays were conducted in duplicate. IC50 values
were determined by incubating several concentrations of inhibitors dissolved in dimethylsulfoxide (DMSO).
bCells (6.2 ꢀ 106 cells/T25 flask) were activated with lipopolysaccharide (200 ng/mL) and b-interferon (10 U/mL) in serum-free
DMEM for 7 h and then treated with inhibitor (0–2 mM) for 30 min at 371C. Exogenous arachidonate metabolism was determined
by adding [1–14C]arachidonic acid (20 mM) for 15 min at 371C. IC50 values are the average of two independent determinations.
Cell line assay results were not determined (n.d.) with compounds that did not inhibit purified COX-2.
amides and esters of indomethacin, amides and esters of the methyl ester of compound 14, also did not inhibit purified
p-iodobenzyl analog did not inhibit COX-2. We attempted protein.
to increase the potency by introducing a methyl group a
to the carboxyl group. This substitution is present in many
COX inhibitors, such as naproxen and flurbiprofen but had not
Radiochemistry
Compounds 3 and 9 showed the most promising properties as
potential imaging agents and were chosen to label with 123I.
Aryltributylstannanes 15 and 16 were generated from the iodo
been tested in the N-benzyl series of indomethacin analogs.
Unfortunately, compound 14 was somewhat less potent than
the unsubstituted compound 9. Compound 13, which is a
J. Label Compd. Radiopharm 2009, 52 387–393
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Md. J. Uddin et al.
Pd(Ph3P)4
Sn2(n-Bu3)2
Dioxane, reflux 16 h
R
(CH2)
SnBu3
R
(CH2)
3,9
I
15, 16
Cl
Na123I, Chloramine-T,
EtOAc/H2O,
1N-HCl, 25 oC, 4 min
Me
Me
O
N
O
[
123I]-3: R =
N
H
R
(CH2)
123I]-3, [123I]-9
123I
[
123I]-9: R =
OMe
O
[
N
OH
MeO
Scheme 3. Radiochemical synthesis of [123I]3 and [123-I]9.
compounds by reaction with hexabutylditin and tetrakis(triphenyl- Student’s t-test at a significance level of 0.01 and 0.001. Intact
phosphine)palladium(0) in refluxing 1,4-dioxane. Radioiodination of compound 9 was identified in the COX-2 expressing nude mouse
the tributylstannyl derivatives was conducted in an EtOAc/H2O tumor (3 nmol/g tissue). This result suggests that compound 9
binary-phase system by reaction with carrier-added [Na123I] in the remains intact in the biological environment for a long enough
presence of Chloramine-T and aqueous 1 M HCl. Separation of the period of time (3 h) to be taken up by the tumor.
organic layer and evaporation afforded radiolabeled compounds
[
123I]3 or [123I]9 in a decay-corrected radiochemical yield of 86–87%
and a radiochemical purity of 98–99%. A radio-TLC analysis of [123I]-9
is shown in Figure 2(a) and a radio-TLC analysis of Na123I in Figure
2(b). HPLC analysis of the product showed the only radioactive peak
to co-elute with standard iodo compound. The final specific activity
of the radiolabeled products was 491 Ci/mmol (Scheme 3).
Enzyme–inhibitor binding kinetics
Compounds like celecoxib and indomethacin are potent COX-2
inhibitors because they are slow, tight-binding inhibitors. They
establish a rapid equilibrium with a loosely bound enzyme–
inhibitor complex then slowly convert to a much more tightly
bound complex. Their rate of dissociation from the tightly bound
complex is very low (Equation (1)). Compounds 3 and 9 also are
slow, tight-binding inhibitors. Figure 1(a) shows the time and
concentration dependent inhibition of hCOX-2 by compound 9.
The inhibition of hCOX-2 proceeded rapidly and the inhibition
plateau for compound 9 at high concentrations approached 0%
activity remaining; this indicates a very slow rate of reversal of
inhibition. Figure 1(b) displays a plot of the single exponential rate
constants for inhibition as a function of concentration, which
allows the determination of the equilibrium constant for initial
association (KI =kꢁ1/k1). Compound 9 had a KI value similar in
nature to indomethacin (KI =11.571.5 versus 13.272.3 mM),
indicating a similar affinity for the enzyme with the two
compounds. The association rate (k2) of compound 9 with
hCOX-2 was identical to indomethacin (k2 = 0.061 sꢁ1) and the
In vivo uptake and retention of compound 9 by COX-2
expressing tumors
The utility of compounds such as 3 and 9 for COX-2 targeted
imaging depends on their stability in vivo. As a preliminary attempt
to evaluate their in vivo stability, we assayed the uptake of
compound 9 into a COX-2 expressing tumor. Female nude mice
with 1483 human head and neck squamous cell carcinoma
xenograft tumors (0.670.1 cm3) on the left flank were injected
retroorbitally at a dose of 2 mg/kg compound 9 formulated with
DMSO (16%)/EtOH (33%)/propylene glycol (17%)/warm sterile saline
34% (371C). At 3 h post injection, the mice (n= 3) were anesthetized.
Blood samples were quickly taken by cardiac puncture into a 1.5 mL
heparinized tube on ice, followed by removal of the liver and tumor.
The collected tissues were rinsed briefly in ice-cold PBS, blotted dry,
weighed and snap-frozen in liquid nitrogen. The blood samples
were centrifuged at 41C at 6000 rpm for 5 min, and the plasma was
transferred to clean tubes and frozen at ꢁ801C. The tissue was
homogenized in 100mM Tris buffer, pH 7.0, and then an aliquot of
the homogenate was mixed with 1.2 ꢀ volume of acetonitrile and
kept at ꢁ201C until phases formed. The acetonitrile was removed
and the samples were dried, reconstituted and analyzed by reversed
phase HPLC-UV using a Phenomenex 10 ꢀ 0.2 cm C18 or a Discussion
Phenomenex 7.5 ꢀ 0.2 cm Synergi Hydro-RP column held at 401C.
dissociation rate (kꢁ2
) of compound 9 was very slow
(kꢁ2 = 0.0022 sꢁ1). Thus, once associated with COX-2, compound
9 (and compound 3, not shown) remains very tightly associated.
k1
k2
E þ I
Ð
½EIꢂ
Ð
EIꢃ
ð1Þ
kꢁ1
kꢁ2
The samples were quantified against a standard curve prepared
from tissue homogenates of undosed control animals. The statistical
comparisons of the experimental results were performed by
Two of the iodine-containing amide derivatives of indomethacin
demonstrated selective COX-2 inhibitory activity (3 and 9),
which is consistent with the model that was proposed for
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Md. J. Uddin et al.
Figure 1. Kinetics of the time-dependent inhibition of hCOX-2 by compound 9.
Assays were performed as described in Experimental Procedures for time-
dependent COX inhibition assays. Representative data are expressed as percent
activity of the uninhibited control with nonlinear regression curves. The purified
enzymes were pre-incubated with inhibitor at 371C for various times (0, 0.5, 1, 3, 5,
10, 15, 30, 45, 60 and 120 min) prior to the addition of substrate (50 mM). (a) Data in
this panel are shown to 15 min on the x-axis to illustrate the kinetics observed at
shorter pre-incubation times. (b) Data in this panel represents the secondary plot
of kobs versus inhibitor concentration used to generate values for KI, k2 and kꢁ2
Figure 2. (a) Radio-TLC of [123I]9 reaction mixture organic phase on silica gel 60
using n-hexane: ethyl acetate (3:1 v/v). (b) Radio-TLC of Na123I.
Experimental section
Silica gel column chromatography was performed using Sorbent
˚
using the following equation: kobs = ((k2Ã[I])/(KI1[I]))1kꢁ2
.
silica gel standard grade, porosity 60 A, particle size 32–63 mm
(230 ꢀ 450 mesh), surface area 500–600 m2/g, bulk density
0.4 g/mL, pH range 6.5–7.5, purchased from Sorbent Technolo-
gies (Atlanta, GA). All other reagents, purchased from the Aldrich
Chemical Company (Milwaukee, WI), were used without further
purification. 1H NMR was taken on a Bruker AV-I console
operating at 400.13 MHz. Experimental conditions included
2048 ꢀ 512 data matrix, 13 ppm sweep width, recycle delay
of 1.5 s and 4 scans per increment. Mass spectrometric analyses
binding of similar amides of indomethacin to the COX-2
enzyme.17 A striking observation from this study is that
substitution of the p-chlorobenzoyl group of indomethacin
with a p-iodobenzyl group generates compound 9, which is a
potent and selective COX-2 inhibitor. The molecular basis of
this efficacy is yet to be determined. A four-fold decrease
in potency was documented with the a–methylated product
14. Although all of the indomethacin amides were potent
COX-2 inhibitors against purified proteins, only compounds
were performed on
a ThermoElectron TSQ 7000 triple
quadrupole instrument in positive or negative ion mode using
electrospray ionization. Radio TLC was carried out on
polyester-backed silica gel 60 plates, eluted with n-hexane:ethyl
acetate (3:1 v/v) and analyzed on a Bioscans AR-2000 (Bioscan,
Inc., Washington, DC). The purity and radiochemical purity of the
final products were determined by HPLC on a Nucleosil C18
column (4.6 ꢀ 250 mm) using a gradient of 50–90% CH3CN/H2O)
with UV detection at 290 nm.
3
and 9 inhibited COX-2 in activated macrophages so
attention focused on these two compounds. Both compounds
were slow, tight-binding inhibitors that associate tightly with
COX-2. In addition, compound 9 exhibited sufficient metabolic
stability to enable it to accumulate in a COX-2 expressing tumor
in vivo.
Radiolabeling of the compounds via iododestannylation of
the corresponding tributylstannyl precursor proceeded
smoothly in the two-phase reaction mixture, which allowed
isolation of radiochemically pure products by simple separation
of the organic phase. Tonnesen et al. reported a number of two-
phase reaction systems, which proved unsatisfactory due to
slow and incomplete incorporation of the radionuclide.19
In conclusion, we report the identification of iodinated
indomethacin derivatives that are selective inhibitors of purified
and cellular COX-2 in a slow, tight-binding manner. We also
report a two-phase radioiodination (123I) protocol that is rapid,
efficient and allows direct isolation of highly purified material
without chromatography.
Chemistry
N-(p-Iodobenzyl)-2-(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
3-yl)acetamide (3)
General procedure: To a stirred solution of indomethacin (3.57 g,
10 mmol) in DMF was added p-iodobenzylamine (2.33 g,
10 mmol), HOBt (2.02 g, 15 mmol), DIPEA (3.88 g, 30 mmol), EDCI
(2.10 g, 11 mmol) at 251C. The resultant mixture was stirred for
16 h at 251C. Removal of solvent in vacuo afforded a residue, to
which 100 mL water was added and extracted with EtOAc
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Md. J. Uddin et al.
(3 ꢀ 75 mL). The organic layer was collected, washed with water fm, 6H, H-2 (3XCH2)g, 1.43–1.49 fm, 6H, H-3 (3XCH2)g, 2.23 [s,
and evaporated in vacuo. The crude product was purified using 3H, CH3], 3.57 (s, 2H, CH2CO), 3.72 (s, 3H, OCH3), 5.26 [s, 2H, CH2
silica gel column chromatography (35:7:1, CHCl3:MeOH:NH4OH) (benzyl)], 6.67 (dd, J = 9.2, 2.4 Hz, 1H, indolyl H-6), 6.75 (d,
to give 3 as yellow solid (4.41 g, 77%) m.p. 200.51C. 1H NMR J = 8.6 Hz, 2H, p-tributylstannylbenzyl H-2, H-6), 6.98 (d,
(500 MHz, DMSO-d6) d 2.20 (s, 3H, CH3), 3.55 (s, 2H, CH2CO), 3.71 J = 2.4 Hz, 1H, indolyl H-4), 7.20 (d, J = 9.2 Hz, 1H, indolyl H-7),
(s, 3H, OCH3), 4.20 (d, J = 6 Hz, 2H, CH2Ar), 6.70 (dd, J = 9.0, 2.2 Hz, 7.42 (d, J = 8.6 Hz, 2H, p-tributylstannylbenzyl H-3, H-5), Mass
1H, indolyl H-6), 6.95 (d, J = 9.0 Hz, 1H, indolyl H-7), 7.06 (d, (ESI) m/z [M1H]1 calcd for 600.24; found 600.21.
J = 8.4 Hz, 2H, p-iodobenzyl H-2, H-6), 7.08 (d, J = 2.4 Hz, 1H,
indolyl H-4), 7.58–7.66 (m, 4H, p-chlorobenzoyl H-3, H-5 and Radiochemistry
p-iodobenzyl H-3, H-5), 7.70 (d, J = 8.7 Hz, 2H, p-chlorobenzoyl
H-2, H-6), 8.47–8.52 (m, 1H, NHCOCH2). Mass (ESI) m/z [M1H]1
calcd 573.04; found 573.13.
N-(p-[123I]iodobenzyl)-2-(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamide ([123I]3)
1-(p-Iodobenzyl)-5-methoxy-2-methyl-3-indoleacetic acid (9)
To a stirred solution of compound 15 (2.63 mg in 1 mL EtOAc)
was added Na[123I] (10 mCi in 500 mL 0.01N-NaOH), NaI (0.3 mg
in 500 mL H2O), chloramine-T trihydrate (0.8 mg in 500 mL in H2O)
and aqueous 1N-HCl (300 mL). After stirring the reaction mixture
for 4 min at room temperature, water (1 mL) followed by EtOAc
(1 mL) was added. The organic layer was collected, washed with
water, and evaporated using argon or air-flow to give [123I]3
(8.6 mCi, radiochemical yield 86% and radiochemical purity
99%).
To a cold (01C) stirred slurry of NaH (300 mg, 12.5 mmol) in DMF
(50 mL) was added 5-methoxy-2-methyl-3-indoleacetic acid
(1.1 g, 5 mmol). After stirring for 1 h at 01C, p-iodobenzyl
bromide (1.8 g, 6 mmol) was added and stirred 1 h at room
temperature. The reaction mixture was then poured into a
crushed ice/water mixture (100 g) and acidified with 10%
aqueous HCl (pH 4). The resultant solid was filtered, washed
(with cold water) and dried under vacuum. The crude product
was purified using silica gel column chromatography (35:7:1,
CHCl3:MeOH:NH4OH) to give 9 as a yellow solid (1.4 g, 67%) m.p.
237.41C. 1H NMR (500 MHz, DMSO-d6) d 2.23 (s, 3H, CH3), 3.57 (s,
2H, CH2CO), 3.72 (s, 3H, OCH3), 5.29 [s, 2H, CH2 (benzyl)], 6.66
(dd, J = 9.2, 2.4 Hz, 1H, indolyl H-6), 6.74 (d, J = 8.6 Hz, 2H,
4-iodobenzyl H-2, H-6), 6.97 (d, J = 2.4 Hz, 1H, indolyl H-4), 7.20
(d, J = 9.2 Hz, 1H, indolyl H-7), 7.62 (d, J = 8.6 Hz, 2H, 4-iodobenzyl
H-3, H-5), Mass (ESI) m/z [M1H]1 calcd 434.03; found 434.18.
1-(p-[123I]iodobenzyl)-5-methoxy-2-methyl-3-indoleacetic acid (123I-9)
To a stirred solution of 16 (20 mg) in EtOAc (500 mL) was added
Na[123I] (17.2mCi in 100 mL 0.01N-NaOH), NaI (4.5 mg in 100 mL
H2O), chloramine-T tri hydrate (7.7mg in 100mL in H2O) and
aqueous 1N-HCl (300mL). After vortexing the reaction mixture for
4min at room temperature, water (1mL) followed by EtOAc (1mL)
was added. The organic layer was collected, washed with water,
and evaporated using argon or air-flow to afford [123I]9 (14.9 mCi,
radiochemical yield 87% and radiochemical purity 98%).
Tin-chemistry
Acknowledgement
N-(p-Tributylstannylbenzyl)-2-f1-(p-chlorobenzoyl)-5-methoxy-2-methyl-
1H-indol-3-ylg acetamide (15)
We are grateful to Dr Carol Rouzer for critical reading of this
manuscript and to the National Institutes of Health for financial
support of this work (CA128323).
Supporting Information Available: A complete description of
the experimental methods and compound characterization. This
material is available online at http://www3.interscience.wiley.com.
General procedure: To a stirred solution of N-f4-iodobenzylg-2-
1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-ylacetamide
(3) (1.5 g, 2.6 mmol) in 1,4 dioxane (30 mL) was added
tetrakis(triphenylphosphine)palladium(0) (150 mg, 0.13 mmol)
followed by hexabutylditin (1 mL). The reaction mixture was
refluxed for 7 h. After cooling to room temperature, the solvent
was removed in vacuo and product was purified using silica gel
column chromatography (5:3, n-Hexane: EtOAc) to give the title
1
References
compound (15) as a yellow solid (1.6 g, 70%) m.p. 109.71C. H
NMR (500 MHz, DMSO-d6) d 0.81–0.85 f(m, 9H, H-1 (3XCH3)g,
0.99–1.1 f(m, 6H, H-4 (3XCH2)g, 1.24–1.32 f m, 6H, H-2 (3XCH2)g,
1.44–1.50 fm, 6H, H-3 (3XCH2)g, 2.24 (s, 3H, CH3), 3.55 (s, 2H,
CH2CO), 3.71 (s, 3H, OCH3), 4.20 (d, J = 6 Hz, 2H, CH2Ar), 6.70 (dd,
J = 9.0, 2.2 Hz, 1H, indolyl H-6), 6.74 (d, J = 8.6 Hz, 2H,
p-tributylstannylbenzyl H-2, H-6), 6.95 (d, J = 9.0 Hz, 1H, indolyl
H-7), 7.08 (d, J = 2.4 Hz, 1H, indolyl H-4), 7.40 (d, J = 8.6 Hz, 2H,
p-tributylstannylbenzyl H-3, H-5), 7.58–7.66 (m, 4H, p-chloro-
benzoyl H-3, H-5), 7.70 (d, J = 8.7 Hz, 2H, p-chlorobenzoyl H-2,
H-6), 8.47–8.52 (m, 1H, NHCOCH2). Mass (ESI) m/z [M1H]1 calcd
737.25; found 737.29.
[1] W. L. Smith, D. L. DeWitt, R. M. Garavito, Annu. Rev. Biochem. 2000,
69, 145–182.
[2] D. L. DeWitt, W. L. Smith, Proc. Natl. Acad. Sci. USA. 1998, 85, 1412–1416.
[3] D. A. Kujubu, B. S. Fletcher, B. C. Varnum, R. W. Lim,
H. R. J. Herschman, Biol. Chem. 1991, 266, 12866–12872.
[4] J.-Y. Fu, J. L. Masferrer, K. Seibert, A. Raz, P. Needleman, J. Biol.
Chem. 1990, 265, 16737–16740.
[5] M. C. Allison, A. G. Howatson, C. J. Torrance, F. D. Lee,
R. I. N. Russell, Engl. J. Med. 1992, 327, 749–754.
[6] C. E. Eberhart, R. J. Coffey, A. Radhika, F. M. Giardiello,
S. Ferrenbach, R. N. BuBois, Gastroenterology, 1994, 107, 1183–1188.
[7] M. M. Taketo, Inflamm. Res. 1998, 47, 112–116.
[8] S. I. Abdalla, P. Lao-Sirieix, M. R. Novelli, L. B. Lovat, I. R. Sanderson,
R. C. Fitzgerald, Clin. Cancer Res. 2004, 10, 4784–4792.
[9] H. Sano, Y. Kawahito, R. L. Wilder, A. Hashiramoto, S. Mukai, K. Asai,
S. Kimura, H. Kato, M. Kondo, T. Hla, Cancer Res. 1995, 55, 3785–3789.
[10] V. N. Shirvani, R. Ouatu-Lascar, B. S. Kaur, M. B. Omary,
G. Triadafilopoulos, Gastroenterology 2000, 118, 487–496.
1-(p-Tributylstannylbenzyl)-5-methoxy-2-methyl-3-indoleacetic acid (16)
Using the same procedure as for 15 provided 16 as a yellow
gummy mass (85%). 1H NMR (500 MHz, DMSO-d6) d 0.80–0.84
f(m, 9H, H-1 (3XCH3)g, 0.98–1.0 f(m, 6H, H-4 (3XCH2)g, 1.23–1.31 [11] M. M. J. Taketo, Natl. Cancer Inst. 1998, 90, 1529–1536.
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 387–393

Md. J. Uddin et al.
[12] E. F. J. D. Vries, A. V. Waarde, A. R. Buursma, W. Vaalburg, J. Nucl. [17] A. S. Kalgutkar, B. C. Crews, S. W. Rowlinson, A. B. Marnett,
Med. 2003, 44, 1700–1706.
K. R. Kozak, R. P. Remmel, L. J. Marnett, Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 925–930.
[13] T. Ishikawa, N. K. Jain, M. M. Taketo, H. R. Herschman, Mol. Imaging
Biol. 2006, 8, 171–187.
[14] T. J. McCarthy, A. U. Sheriff, M. J. Graneto, J. J. Talley, M. J. Welch,
J. Nucl. Med. 2002, 43, 117–124.
[15] G. W. Kabalka, A. R. Mereddy, H. M. Schuller, J. Label. Compd.
Radiopharm. 2005, 48, 295–300.
[18] W. C. Black, C. Bayly, M. Belley, C.-C. Chan, S. Charleson, D. Denis,
J. Y. Gauthier, R. Gordon, D. Guay, S. Kargman, C. K. Lau, Y. Leblanc,
J. Mancini, M. Quellet, D. Percival, P. Roy, K. Skorey, P. Tagari,
P. Vickers, E. Wong, L. Xu, P. Prasit, Bioorg. Med. Chem. Lett. 1996, 6,
725–730.
[16] H. M. Schuller, G. Kabalka, G. Smith, A. Meredy, M. Akula,
M. Cekanova, Chem. Med. Chem. 2006, 1, 603–610.
[19] G. L. Tonnesen, R. N. Hanson, D. E. Seitz, Int. J. Appl. Radiat. Isot.
1981, 32, 171–173.
J. Label Compd. Radiopharm 2009, 52 387–393
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org