Synthesis of carbon-11 labeled triphenylacetamides as novel potential PET melanoma cancer imaging agents

Article abstract of DOI:10.1055/s-2006-942442

New carbon-11 labeled triphenylacetamides (TPAs), N-(4-[ ¹¹C]methoxyphenyl)-2,2,2-triphenyl-acetamide ([¹¹C]MTA) and 3-phenyl-(R)-2-(2,2,2-triphenylacetylamino)propionic acid [ ¹¹C]methyl ester ([¹¹C]PAME), were

Full text of DOI:10.1055/s-2006-942442

PAPER
2301
Synthesis of Carbon-11 Labeled Triphenylacetamides as Novel Potential PET
Melanoma Cancer Imaging Agents
S
ynthesis of Carbon-11
i
Label
n
edTripheny
g
a
Department of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Fax +1(317)2789711; E-mail: qzheng@iupui.edu
b
c
Department of Chemistry, Texas A&M University, College Station, TX 77843, USA
Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 11 February 2006; revised 20 March 2006
MTA 1a and PAME 1b, respectively. Demethylation of
MTA with aluminum trichloride and ethanethiol2 yielded
desmethylated phenol precursor 2a. The hydrolysis of
methyl ester PAME under basic conditions gave carboxy-
Abstract: New carbon-11 labeled triphenylacetamides (TPAs),
N-(4-[11C]methoxyphenyl)-2,2,2-triphenyl-acetamide ([11C]MTA)
and 3-phenyl-(R)-2-(2,2,2-triphenylacetylamino)propionic acid
[11C]methyl ester ([11C]PAME), were designed and synthesized as
potential positron emission tomography (PET) melanoma cancer lic acid precursor 2b. The overall chemical yields for
imaging agents. The single crystal structure of the potent anti-mel-
MTA and PAME were 92% and 90% (two steps), while
anoma agent, N-(4-methoxyphenyl)-2,2,2-triphenylacetamide
2a and 2b formed in 83% and 86% (three steps), respec-
(MTA) is reported.
tively.
Key words: triphenylacetamides, N-(4-[11C]methoxyphenyl)-2,2,2-
The synthesis of target radiotracers N-(4-[11C]methoxy-
triphenylacetamide,
3-phenyl-(R)-2-(2,2,2-triphenylacetyl-
phenyl)-2,2,2-triphenylacetamide ([11C]MTA, [11C]1a)
and 3-phenyl-(R)-2-(2,2,2-triphenylacetylamino)propion-
ic acid [11C]methyl ester ([11C]PAME, [11C]1b) is shown
in Scheme 2. [11C]MTA was synthesized through O-
[11C]methylation of phenol precursor 2a using [11C]meth-
yl triflate ([11C]CH3OTf)3 and purified by HPLC4 in 30–
35% radiochemical yields, based on 11CO2, decay correct-
ed to end of bombardment (EOB), 30–35 minutes overall
synthesis and formulation time from EOB, >99% radio-
chemical purity, and 1.5–1.8 Ci/mmol specific radioactiv-
ity at end of synthesis (EOS). [11C]PAME was
synthesized through O-[11C]methylation of carboxylic
acid precursor 2b using [11C]CH3OTf and purified by
C-18 solid-phase extraction (SPE)5 in 40–45% radio-
chemical yields, based on 11CO2, decay corrected to EOB,
20–25 minutes overall synthesis and formulation time
from EOB, >95% radiochemical purity, and 1.0–1.5 Ci/
mmol specific radioactivity at EOS. The method for deter-
mining the specific radioactivity of radiotracers is an au-
tomated measurement using HPLC, which has been
developed in our previous work.6,7
amino)propionic acid [11C]methyl ester, positron emission tomog-
raphy, melanoma cancer, imaging agents
Triphenylacetamides (TPAs) are small molecules that
have recently been developed as potential anti-melanoma
agents by Dothager et al. Some of these TPAs were found
to potently induce apoptosis in melanoma cells through
G1 cell cycle arrest and dramatically reduce the level of
active nuclear factor k-B (NFkB) in the cell.1 We are in-
terested in the development of new cancer imaging
agents. TPAs labeled with carbon-11 may enable non-in-
vasive monitoring of cancer proliferation and apoptosis in
melanoma cells and NFkB, and melanoma cancer re-
sponse to chemotherapy using positron emission tomog-
raphy (PET) imaging technique. To further develop
potential therapeutic drugs as diagnostic agents, we de-
signed and synthesized carbon-11 labeled TPAs as novel
potential PET melanoma cancer imaging agents.
The synthesis of reference standards and desmethylated
precursors,
N-(4-methoxyphenyl)-2,2,2-triphenylacet-
Compounds 2a and 2b are new TPAs, synthesized for the
first time in this laboratory. Compounds 1a and 1b have
been reported to be potent anti-melanoma agents of TPAs,
and the reported IC50 values in melanoma cell lines (nor-
mal human bone marrow) UACC-62, B16-F10, SK-
MEL-5, and bone marrow for 1a are 0.69 mM, 0.60 mM,
0.83 mM, and 4.77 mM, respectively, while those for 1b
are 0.62 mM, 0.80 mM, 0.57 mM, and 7.00 mM, respective-
ly.1 To better understand the structure of the new PET
tracers, the structure of compound 1a was determined by
X-ray crystallography. Compound 1a was obtained as air-
stable, colorless crystals by slow evaporation from a solu-
tion of 1a in acetone. We report the first single crystal
structure of compound 1a, and the perspective view of the
asymmetric unit 1a with one solvent molecule (acetone) at
amide (MTA, 1a), 3-phenyl-(R)-2-(2,2,2-triphenylacetyl-
amino)propionic acid methyl ester (PAME, 1b), N-(4-
hydroxyphenyl)-2,2,2-triphenylacetamide (2a), and 3-
phenyl-(R)-2-(2,2,2-triphenylacetylamino)propionic acid
(2b), was performed using a modification of the literature
procedure.1 The synthetic approach is outlined in
Scheme 1. Commercially available starting material,
triphenylacetyl acid, was reacted with thionyl chloride to
afford triphenylacetyl chloride (3), which was reacted
with corresponding amines p-anisidine (a) and D-phenyl-
alanine methyl ester (b) to give reference standards TPAs,
SYNTHESIS 2006, No. 14, pp 2301–2304
x
x.
x
x
.
2
0
0
6
Advanced online publication: 28.06.2006
DOI: 10.1055/s-2006-942442; Art ID: M00806SS
© Georg Thieme Verlag Stuttgart · New York

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M. Gao et al.
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O
O
SOCl2, DMF
reflux
amine a or b, Et3N
CH2Cl2
OH
Cl
3
O
O
AlCl3, EtSH
CH2Cl2
HN
OCH3
HN
OH
1a
2a
KOH, MeOH
HCl
O
O
N
H
HN
OCH3
OH
O
O
1b
2b
Scheme 1 Synthesis of triphenylacetamides.
All commercial reagents and solvents were used without further pu-
rification unless otherwise specified. The 11CH3OTf was synthe-
sized according to a literature procedure.3 1H NMR spectra were
recorded on a Bruker QE 300 NMR spectrometer using TMS as an
internal standard. LRMS were obtained using a Bruker Biflex III
MALDI-TOF mass spectrometer, and HRMS were obtained using
a Kratos MS80 mass spectrometer, in the Department of Chemistry
at Indiana University. TLCs were run using Analtech silica gel GF
uniplates (5 × 10 cm2). Plates were visualized by UV light. Normal
phase flash 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-sensitive
reactions were performed under a positive pressure of nitrogen
maintained by a direct line from a nitrogen source. Analytical
HPLC was performed using a Prodigy (Phenomenex) 5 mm C-18
column, 4.6 × 250 mm; MeCN–MeOH– KHPO4– (20 mM, pH 6.7;
buffer solution), 3:1:3 mobile phase, flow rate 1.5 mL/min, and UV
(254 nm) and g-ray (NaI) flow detectors. Semi-preparative HPLC
was performed using a Prodigy (Phenomenex) 5 mm C-18 column,
10 × 250 mm; MeCN–MeOH–KHPO4– (20 mM, pH 6.7) 3:1:3 mo-
bile phase, 5.0 mL/min flow rate, UV (254 nm) and g-ray (NaI) flow
detectors. Semi-prep C-18 guard cartridge column (1 × 1 cm) was
obtained from E. S. Industries, Berlin, NJ, with part number
300121-C18-BD 10m. Sterile vented Millex-GS 0.22 mm filter unit
was obtained from Millipore Corporation, Bedford, MA.
O
11CH3OTf, 6 N NaOH
O
11CH3
2a
HN
[
11C]1a
O
11CH3OTf, 6 N NaOH
2b
HN
O
11CH3
O
[
11C]1b
Scheme 2 Synthesis of carbon-11 labeled triphenylacetamides.
ellipsoid of 30% (anisotropic picture) is shown in
Figure 1.
In summary, the synthetic procedures that provided both
the phenol and carboxylic acid precursors, reference stan-
dards MTA and PAME, and target tracers [11C]TMA and
[11C]PAME have been well developed. We have reported
the first single crystal structure of the potent compound
MTA. The results obtained for in vitro data warrant fur-
ther in vivo evaluation of the new tracers [11C]MTA and
[11C]PAME as potential PET melanoma cancer imaging
agents.
Triphenylacetyl Chloride (3)
Triphenylacetyl acid (4.5 g, 0.016 mol) was mixed with SOCl2 (30
mL, excess) and DMF (two drops) was added, the resulting mixture
was heated at reflux for 3 h. Excess SOCl2 was removed by evapo-
ration under reduced pressure. Residual SOCl2 was removed by co-
evaporation with anhyd benzene (40 mL) to afford compound 4 as
a white solid in ca. 100% yield; mp 125–127 °C.
1H NMR (300 MHz, CDCl3): d = 7.28–7.37 (m, 15 H, Ph).
Synthesis 2006, No. 14, 2301–2304 © Thieme Stuttgart · New York

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Synthesis of Carbon-11 Labeled Triphenylacetamides
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Figure 1 Molecular structure of compound 1a.
N-(4-Methoxyphenyl)-2,2,2-triphenylacetamide (1a) and
3-Phenyl-(R)-2-(2,2,2-triphenylacetylamino)propionic Acid
Methyl Ester (1b); General Procedure
To a cold solution (–5 to 0 °C) of p-anisidine (a) or D-phenylalanine
methyl ester (b) (5.4 mmol) and Et3N (2.5 mL) in CH2Cl2 (50 mL)
was added a solution of compound 3 (5 mmol) in CH2Cl2 (6 mL)
over 30 min. The reaction mixture was stirred at 0 °C for 2 h and
then at r.t. for 3 h. The mixture was washed with H2O (30 mL) and
dried over MgSO4. The crude product was purified by column chro-
matography (EtOAc–hexanes, 2:3).
3-Phenyl-(R)-2-(2,2,2-triphenylacetylamino)propionic Acid
(2b)
A mixture of compound 1b (0.45 g, 1.0 mmol), KOH (0.82 g, 14.6
mmol), and MeOH (25 mL) was stirred at r.t. for 16 h. The pH of
the mixture was adjusted to 6 by the addition of aq HCl (2 N). The
mixture was concentrated, extracted with CH2Cl2 (2 × 50 mL), and
washed with brine (50 mL). The organic phase was dried over
Na2SO4 and concentrated under reduced pressure to afford com-
pound 2b as a white solid in 95% yield; mp 76–78 °C; Rf = 0.62
(MeOH–CH2Cl2, 1:9).
1H NMR (300 MHz, CDCl3): d = 2.90 (dd, J = 13.97, 8.45 Hz, 1 H,
CHH), 3.19 (dd, J = 13.97, 4.78 Hz, 1 H, CHH), 4.89 (dt, J = 8.45,
4.78 Hz, 1 H, CH), 6.17 (d, J = 6.62 Hz, 1 H, NH), 6.90 (dd,
J = 6.98, 1.35 Hz, 2 H, Ph), 7.10 (dd, J = 6.20, 3.30 Hz, 2 H, Ph),
7.17–7.25 (m, 16 H, Ph).
Compound 1a
Yield: 92%; white solid; mp 121–123 °C; Rf = 0.82 (EtOAc–hex-
anes, 1:1).
1H NMR (300 MHz, CDCl3): d = 3.74 (s, 3 H, CH3), 6.79 (d,
J = 8.82 Hz, 2 H, Ph), 7.22–7.32 (m, 18 H, Ph, NH).
LRMS (CI): m/z (%) = 436 (M+ + H, 88), 243 (100).
HRMS (CI): m/z calcd for C29H26NO3: 436.1907; found: 436.1901.
Compound 1b
Yield: 90%; colorless solid; low mp; Rf = 0.82 (EtOAc–hexanes
1:1).
Tracer [11C]1a
Phenol precursor 2a (0.3 mg) was dissolved in MeCN (400 mL). To
this solution was added a 6 N aq solution of NaOH (2 mL). The mix-
ture was transferred to a small volume, three-neck reaction tube.
[11C]CH3OTf, produced from bubbling 11CO2 through 11CH4, and
11CH3Br were passed into the air-cooled reaction tube at –15 °C to
–20 °C, which was generated by a Venturi cooling device powered
with 100 psi of compressed air, until the radioactivity in solution
reached a maximum (ca. 3 min); then the reaction tube was heated
at 70–80 °C for 2 min. The contents of the reaction tube were dilut-
1H NMR (300 MHz, CDCl3): d = 2.92 (dd, J = 7.88, 13.97 Hz, 1 H,
CHH), 3.11 (dd, 5.15, 13.97 Hz, 1 H, CHH), 3.70 (s, 3 H, CH3), 4.94
(dt, J = 5.15, 7.88 Hz, 1 H, CH), 6.15 (d, J = 7.35 Hz, 1 H, NH),
6.88 (dd, J = 1.35, 7.16 Hz, 2 H, Ph), 7.12–7.24 (m, 18 H, Ph).
N-(4-Hydroxyphenyl)-2,2,2-triphenylacetamide (2a)
To a stirred solution of compound 1a (393 mg, 1.0 mmol, 1 equiv)
in EtSH (1 mL) and CH2Cl2 (10 mL) cooled in an ice water bath was
added AlCl3 (0.53 g, 4.0 mmol, 4 equiv). The reaction was stirred
for 2 h. Then the mixture was quenched by the addition of 1 N HCl
(2 mL), extracted with CH2Cl2 (2 × 10 mL), washed with brine (15
mL), dried over Na2SO4, filtered, and evaporated to give the crude
product, which was purified by column chromatography (silica gel;
EtOAc–hexanes, 1:4) to afford compound 2a as a white solid in
90% yield; mp 194–196 °C; Rf = 0.75 (MeOH–CH2Cl2, 1:9).
–
ed with 0.1 M NaHCO3 (0.8 mL) and MeCN–MeOH–KHPO4
(3:1:3, 1 mL), and injected onto the preparative HPLC column. The
product fraction was collected and the solvent was removed by ro-
tary evaporation under vacuum. The final product [11C]1a was for-
mulated in saline, sterile-filtered through a sterile vented Millex-GS
0.22 mm cellulose acetate membrane, and collected in a sterile vial.
Total radioactivity was assayed and the total volume noted. The
overall synthesis, purification, and formulation time was ca. 30 min
from EOB.
1H NMR (300 MHz, CDCl3): d = 5.42 (s, 1 H, OH), 6.67 (d, J = 8.82
Hz, 2 H, Ph), 7.18 (d, J = 8.82 Hz, 2 H, Ph), 7.25–7.36 (m, 16 H, Ph,
NH).
Analytical HPLC: precursor 2a tR 2.36 min, MTA 1a tR 3.75 min,
[11C]1a tR 3.75 min.
LRMS (CI): m/z (%) = 380 (M+ + H, 100).
HRMS (CI): m/z calcd for C26H22NO2: 380.1645; found: 380.1649.
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M. Gao et al.
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Preparative HPLC: precursor 2a tR 2.85 min, MTA 1a tR 5.32 min,
[11C]1a tR 5.32 min.
ited with the Cambridge Crystallographic Data Centre (CCDC) for
small molecules and the deposition number is CCDC 294956.
Tracer [11C]1b
Acknowledgment
Carboxylic acid precursor 2b (0.3 mg) was dissolved in MeCN (400
mL). To this solution was added 6 N NaOH (2–3 mL). The mixture
was transferred to a small volume, three-neck reaction tube.
11CH3OTf was passed into the air-cooled reaction tube at –15 to –
20 °C, which was generated by a Venturi cooling device powered
with 100 psi compressed air, until the radioactivity reached a max-
imum (ca. 3 min); then the reaction tube was heated at 70–80 °C for
3 min. The contents of the reaction tube were diluted with 0.1 M
NaHCO3 (1 mL). This solution was passed onto a C-18 cartridge by
gas pressure. The cartridge was washed with H2O (2 × 3 mL), and
the aqueous washing was discarded. The product was eluted from
the column with EtOH (2 × 3 mL), and the eluent was concentrated
on a rotary evaporator at high vacuum. The labeled product [11C]1b
was formulated with 50 mM NaH2PO4, whose volume was depen-
dent upon the use of the labeled product [11C]1b in tissue biodistri-
bution studies (ca. 6 mL, 3 × 2 mL) or in micro-PET imaging
studies (1–3 mL), sterile-filtered through a sterile vented Millex-GS
0.22 mm cellulose acetate membrane, and collected in a sterile vial.
Total radioactivity was assayed and total volume noted. The overall
synthesis time was ca. 20 min.
This work was partially supported by the Susan G. Komen Breast
Cancer Foundation, Breast Cancer Research Foundation, and India-
na Genomics Initiative (INGEN) of Indiana University, which is
supported in part by Lilly Endowment Inc. A. Clearfield acknow-
ledges the financial support from the Department of Energy, Basic
Sciences Division through grant number DE-FG03-00ER 15806.
The authors would like to thank Dr. Bruce H. Mock, Barbara E.
Glick-Wilson, and Michael L. Sullivan for their assistance in pro-
duction of 11CH3OTf. The referees’ criticisms and editor’s com-
ments for the revision of the manuscript are greatly appreciated.
References
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Analytical HPLC: precursor 2b tR 2.18 min, PAME 1b tR 4.04 min,
and [11C]1b tR 4.04 min.
X-ray Crystallography
The crystallographic measurements were carried out on a Siemens
P4 diffractometer with graphite-monochromated Mo-Ka radiation
(l = 0.71073 Å) and 12 kW rotating generator. The data were col-
lected at 110 K. The structure was solved and refined using the pro-
grams SHELXS-97 (Sheldrick, 1997) and SHELXL (Sheldrick
1997). The program X-Seed (Barbour, 1999) was used as an inter-
face to the SHELX programs. X-ray coordinates have been depos-
Synthesis 2006, No. 14, 2301–2304 © Thieme Stuttgart · New York