Synthesis of carbon-11-labeled casimiroin analogues as new potential PET agents for imaging of quinone reductase 2 and aromatase expression in breast cancer

Article abstract of DOI:10.1016/j.steroids.2010.06.004

Carbon-11-labeled casimiroin analogues were first designed and synthesized as new potential PET agents for imaging of quinone reductase (QR) 2 and aromatase expression in breast cancer. [¹¹C]casimiroin (6-[ ¹¹C]methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one, [ ¹¹C]11) and its carbon-11-labeled analogues 5,6,8-trimethoxy-1-[ ¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]17), 8-methoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([ ¹¹C]21a), 6,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin- 2(1H)-one ([¹¹C]21b), and 5,8-dimethoxy-1-[¹¹C]methyl-4- methylquinolin-2(1H)-one ([¹¹C]21c), were prepared from their corresponding precursors with [¹¹C]methyl triflate ([ ¹¹C]CH₃OTf) under basic conditions (NaH) through either O- or N-[¹¹C]methylation and isolated by semi-preparative HPLC method in 40-50% radiochemical yields decay corrected to end of bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).

Full text of DOI:10.1016/j.steroids.2010.06.004

Steroids 75 (2010) 967–973
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of carbon-11-labeled casimiroin analogues as new potential PET agents
for imaging of quinone reductase 2 and aromatase expression in breast cancer
Min Wanga, Mingzhang Gaoa, Kathy D. Millerb, George W. Sledgeb, Gary D. Hutchinsa, Qi-Huang Zhenga,∗
a Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, L3-208, 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:
Carbon-11-labeled casimiroin analogues were first designed and synthesized as new potential
PET agents for imaging of quinone reductase (QR) 2 and aromatase expression in breast cancer.
[
Received 16 April 2010
Received in revised form 27 May 2010
Accepted 7 June 2010
11C]casimiroin (6-[11C]methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one, 11C]11) and its
[
carbon-11-labeled analogues 5,6,8-trimethoxy-1-[11C]methyl-4-methylquinolin-2(1H)-one ([11C]17),
8-methoxy-1-[11C]methyl-4-methylquinolin-2(1H)-one ([11C]21a), 6,8-dimethoxy-1-[11C]methyl-4-
methylquinolin-2(1H)-one ([11C]21b), and 5,8-dimethoxy-1-[11C]methyl-4-methylquinolin-2(1H)-one
([11C]21c), were prepared from their corresponding precursors with [11C]methyl triflate ([11C]CH3OTf)
under basic conditions (NaH) through either O- or N-[11C]methylation and isolated by semi-preparative
HPLC method in 40–50% radiochemical yields decay corrected to end of bombardment (EOB), based on
Available online 15 June 2010
Keywords:
Positron emission tomography (PET)
Quinone reductase (QR) 2
Aromatase
Casimiroin analogues
Radiotracers
[
11C]CO2, and 111–185 GBq/␮mol specific activity at the end of synthesis (EOS).
© 2010 Elsevier Inc. All rights reserved.
Cancer imaging
1. Introduction
certain quinone substrates into more highly reactive species
that are capable of causing more cellular damage in cancer, and
cancer is characterized by the uncontrolled growth and spread of
abnormal cells [7–13]. The enzyme aromatase, a key cytochrome
P450 enzyme, is responsible for a key step in the biosynthesis of
estrogens and catalyzes the rate-limiting aromatization step in the
conversion of androgens to estrogens [2,7]. The enzymes QR and
aromatase are key enzymes in sex steroid production related to ER,
and provide the attractive molecular targets for the development
of anticancer drugs for breast cancer. The inhibition of estrogen
synthesis by aromatase inhibitors (AIs) and inhibition of estrogen
action by compounds interacting with ERs (antiestrogens) are two
strategies for the design of drugs that can be used to ameliorate
the growth effects of estrogens on ER positive (ER+) tumor cells
[14]. AIs have been extensively studied as effective hormonal
therapeutic drugs in the treatment of ER+ breast cancer. They
are equivalent or superior to antiestrogen tamoxifen in women
with metastatic diseases. In addition, AIs provide an effective
treatment in some patients relapsing from tamoxifen [14–16]. The
up-regulation of QR may contribute to the beneficial effects of
tamoxifen and other antiestrogens in breast cancer prevention and
treatment [4]. A next logical step to improve cancer therapeutic
benefit would be to develop dual inhibitors to target both enzymes
simultaneously [17]. Recently a new series of casimiroin ana-
logues has been developed as new QR2 and aromatase inhibitors,
potential chemopreventive or chemotherapeutic agents [7]. The
Breast cancer is the most common cancer diagnosed in women
and is the second leading cause of death in women after lung can-
cer in the United States [1]. Approximately 75% of breast cancers
test positive for estrogen receptor (ER), the progesterone receptor
(PgR), or both, and estrogen stimulation of these receptors is a
significant factor in the development and growth of breast cancer
[2,3]. The enzyme quinone reductase (QR), a detoxifying phase
II antioxidant enzyme, is a flavoprotein that reversibly catalyzes
the oxidation of the reduced form of nicotinamide adenine dinu-
cleotide or nicotinamide adenine dinucleotide phosphate (NADH
or NADPH) by various quinones and oxidation-reduction dyes [4].
There are two main QRs reported in the literature, QR1 and QR2.
QR1, a FAD (flavin adenine dinucleotide)-binding protein that
forms homodimers and reduces quinones to hydroquinones, has
its activity up regulated by antiestrogens in an ER-dependent man-
ner in breast cancer cells [5,6]. QR2, a cytosolic FAD-dependent
flavoenzyme that catalyzes metabolic reduction of quinones
with unexpected cosubstrate specificity, may actually transform
∗
Corresponding author at: Department of Radiology and Imaging Sciences, Indi-
ana University School of Medicine, 1345 West 16th Street, L3-208, Indianapolis, IN
46202-2111, USA. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.06.004

968
M. Wang et al. / Steroids 75 (2010) 967–973
enzymes QR and aromatase also provide the attractive targets for
the development of enzyme-based breast cancer imaging agents
for use in the biomedical imaging technique positron emission
tomography (PET). Casimiroin analogues labeled with a positron-
emitting radionuclide such as carbon-11 may enable non-invasive
monitoring QR and aromatase expression in breast cancer and
breast cancer response to QR and aromatase inhibitor therapy
using PET imaging technique. Significant efforts in the field of brain
aromatase imaging with PET using carbon-11-labeled aromatase
inhibitors such as [11C]vorozole and [11C-cyano]letrozole have
been reported [18–21], and we have previously published the syn-
thesis and preliminary biological evaluation of carbon-11-labeled
aromatase inhibitors, carbon-11-labeled sulfonanilide analogues
as new PET breast cancer aromatase imaging agents [3]. In cancer
molecular imaging, different molecular targets, enzyme-based
imaging agents, radionuclides, and imaging modalities have their
own advantages and disadvantages, and cancer is a complicated
group of diseases involving multiple targets, and thus there is great
interest in development of dual cancer targets, dual enzyme-based
imaging agents or dual radiolabeled ligands and dual imaging
modalities [1,17,22,23]. These simultaneously ‘mixed’ agents or
modalities would be advantageous over single agent or modality.
We are interested in the development of radiolabeled dual enzyme
inhibitors as enzyme-based PET breast cancer imaging agents.
We focus on the development of carbon-11-labeled non-steroidal
analogues that are inhibitors of steroid biosynthetic enzymes or
ligands for steroid hormone receptors. In our previous work, we
have developed carbon-11-labeled dual aromatase-steroid sulfa-
tase inhibitors [1] based on their unlabeled compounds [24]. In
this ongoing study, to further develop therapeutic agent for diag-
nostic use, we have first designed and synthesized [11C]casimiroin
(6-[11C]methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-
4.6 mm × 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 mm × 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 Corpora-
tion, Bedford, MA.
2.2. Synthesis of casimiroin and its analogues
2.2.1. Methyl 2,3-dihydroxybenzoate (2)
To a solution of 2,3-dihydroxybenzoic acid 1 (18.0 g, 117 mmol)
in MeOH (150 mL) was added thionyl chloride (12.8 mL, 175 mol)
dropwise at 0 ◦C. After the reaction mixture was heated to reflux
for 16 h, the solvent and excess thionyl chloride was evaporated in
vacuo. The residue was dissolved in EtOAc, and washed with sat-
urated NaHCO3. The organic layer was washed with brine, dried
over Na2SO4, and filtered. The solvent was evaporated in vacuo,
and the crude product was purified over a short pad of silica gel
using CH2Cl2 as eluent with to afford 2 (19.5 g, 99%) as a white
solid, m.p. 70–72 ◦C. 1H NMR (CDCl3) ı: 10.89 (s, 1H, OH), 7.37 (dd,
J = 1.5, 8.5 Hz, 1H, Ph-H), 7.12–7.10 (m, 1H, Ph-H), 6.80 (t, J = 8.0 Hz,
1H, Ph-H), 5.66 (s, 1H, OH), 3.96 (s, 3H, OCH3).
2.2.2. Methyl 2,3-methylenedioxybenzoate (3)
A
mixture of compound 2 (12.0 g, 71 mmo), KF (20.6 g,
355 mmol) in DMF (150 mL) was stirred at room temperature for
30 min. CH2Cl2 (5.94 mL, 85.2 mmol) was added and the reaction
mixture was heated at 120 ◦C for 3 h. After cooling to room tem-
perature, the mixture was poured into water and extracted with
Et2O. The combined organic layer was washed with brine, dried
over Na2SO4, and filtered. The solvent was evaporated in vacuo, and
the crude product was purified by column chromatography, eluting
with hexanes/EtOAc (2:1) to afford 3 (9.95 g, 77%) as a white solid,
m.p. 63–64 ◦C. 1H NMR (CDCl3) ı: 7.42 (dd, J = 1.5, 8.5 Hz, 1H, Ph-H),
6.97 (dd, J = 1.0, 8.0 Hz, 1H, Ph-H), 6.87 (t, J = 8.0 Hz, 1H, Ph-H), 6.10
(s, 2H, OCH2O), 3.93 (s, 3H, OCH3).
one,
[
11C]11) and its carbon-11-labeled analogues 5,6,8-
trimethoxy-1-[11C]methyl-4-methylquinolin-2(1H)-one ([11C]17),
8-methoxy-1-[11C]methyl-4-methylquinolin-2(1H)-one
([11C]
21a), 6,8-dimethoxy-1-[11C]methyl-4-methylquinolin-2(1H)-one
([11C]21b), and 5,8-dimethoxy-1-[11C]methyl-4-methylquinolin-
2(1H)-one ([11C]21c), as new potential PET agents for imaging of
QR 2 and aromatase expression in breast cancer.
2.2.3. 2,3-Methylenedioxybenzoic acid (4)
2. Experimental
A mixture of compound 3 (9.6 g, 53 mmol), 2N KOH (53 mL,
106 mmol) in MeOH (150 mL) was stirred at room temperature for
3 h. After evaporating MeOH in vacuo, the solution was cooled in an
ice bath and acidified to pH 3.5 with 2N HCl. The white precipitate
was collected by filtration, washed with water and Et2O, dried in
vacuo to afford 4 (8.08 g, 91%) as a white solid, m.p. 234–235 ◦C.
1H NMR (DMSO-d6) ı: 12.97 (s, 1H, OH), 7.27 (dd, J = 1.5, 8.0 Hz, 1H,
Ph-H), 7.11 (d, J = 7.5 Hz, 1H, Ph-H), 6.89 (t, J = 8.0 Hz, 1H, Ph-H), 6.11
(s, 2H, OCH2O).
2.1. General
All commercial reagents and solvents from Sigma–Aldrich
and Fisher Scientific were used without further purification.
[
11C]Methyl triflate ([11C]CH3OTf) was prepared according to a
literature procedure [25]. Melting points were 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 stan-
dard. Chemical shift data for the proton resonances were reported
in parts per million (ppm, ı scale) relative to internal standard
TMS (ı 0.0), and coupling constants (J) were reported in hertz (Hz).
Thin-layer chromatography (TLC) was run using Analtech silica gel
GF uniplates (5 × 10 cm2). Plates were visualized under UV light.
Preparative TLC was run using Analtech silica gel UV 254 plates
(20 × 20 cm2). Normal phase flash column chromatography was
carried outonEMScience silicagel60(230–400mesh)with aforced
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.
2.2.4. Benzyl benzo[d][1,3]dioxol-4-ylcarbamate (5)
A mixture of compound 4 (7.78 g, 47 mmol), diphenylphos-
phonyl azide (DPPA) (11.2 mL, 52 mmol), triethylamine (7.25 mL,
52 mmol) in benzene (100 mL) was heated to reflux for 3 h. Benzyl
alcohol (10 mL) was added, and the reaction mixture was heated at
reflux for another 5 h. After cooling to room temperature, the mix-
ture was washed successively with cold 1N HCl, saturated NaHCO3,
and brine. The organic layer was dried over Na2SO4, and filtered.
The solvent was evaporated in vacuo, and the residue was puri-
fied by column chromatography, eluting with hexanes/EtOAc (2:1)
to afford 5 (9.36 g, 74%) as a white solid, m.p. 92–94 ◦C. 1H NMR
(CDCl3) ı: 7.49 (br s, 1H, NH), 7.46–7.32 (m, 5H, Ph-H), 6.81(t,
J = 8.0 Hz, 1H, Ph-H), 6.63 (s, 1H, Ph-H), 6.59 (dd, J = 1.0, 8.0 Hz, 1H,
Ph-H), 5.94 (s, 2H, OCH2O), 5.21 (s, 2H, CH2Ph).
Analytical high performance liquid chromatography (HPLC) was
performed using a Prodigy (Phenomenex) 5 ␮m C-18 column,

M. Wang et al. / Steroids 75 (2010) 967–973
969
2.2.5. Benzyl benzo[d][1,3]dioxol-4-yl(methyl)carbamate (6)
NaH (60% dispersion in mineral oil, 1.86 g, 46.5 mmol) was
added to a solution of compound 5 (8.44 g, 31 mmol) in DMF
(40 mL). After the mixture was stirred at room temperature for 1 h,
CH3I (5.8 mL, 93 mmol) was added. The resulting mixture was con-
tinued to stir at room temperature for 4 h, then it was poured into
ice water and extracted with CH2Cl2. The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The sol-
vent was evaporated in vacuo, and the crude product was purified
by column chromatography, eluting with hexanes/CH2Cl2 (1:2) to
afford 6 (8.44 g, 95%) as colorless oil. 1H NMR (CDCl3) ı: 7.32–7.30
(m, 5H, Ph-H), 6.81–6.71 (m, 3H, Ph-H), 5.90 (s, 2H, OCH2O), 5.17
(s, 2H, CH2, CH2Ph), 3.27 (s, 3H, NCH3).
0 ◦C, and the mixture was stirred for 30 min. Anhydrous MeOH
(15 mL) was added dropwise, and the reaction mixture was stirred
for 6 h. Aqueous acetic acid (0.5 mL) was added to decompose the
excess TMS-diazomethane. The solvent was evaporated in vacuo,
and the residue was purified by column chromatography hex-
anes/acetone (1:1) to afford 11 (117 mg, 37%) as an orange solid,
m.p. 197–198 ◦C (lit. 199–201 ◦C [7]). 1H NMR (CDCl3) ı: 7.55
(d, J = 8.5 Hz, 1H, Ph-H), 6.80 (d, J = 8.0 Hz, 1H, Ph-H), 6.05 (s, 2H,
OCH2O), 6.01 (s, 1H, C CH), 3.92 (s, 3H, OCH3), 3.86 (s, 3H, NCH3).
2.2.11. Benzyl 2,4,5-trimethoxyphenylcarbamate (13)
A mixture of 2,4,5-trimethoxybenzoic acid 12 (8.0 g, 37.7 mmol),
DPPA (8.96 mL, 41.5 mmol), triethylamine (5.78 mL, 41.5 mmol) in
benzene (100 mL) was heated to reflux for 2 h. Benzyl alcohol
(10 mL) was added, and the reaction mixture was heated at reflux
for another 5 h. After cooling to room temperature, the mixture
was washed successively with cold 1N HCl, saturated NaHCO3,
and brine. The organic layer was dried over Na2SO4, and filtered.
The solvent was evaporated in vacuo, and the crude product was
purified by column chromatography, eluting with hexanes/EtOAc
(1:1) to afford 13 (9.67 g, 81%) as a white solid, m.p. 98–99 ◦C (lit.
99–100 ◦C [7]). 1H NMR (CDCl3) ı: 7.85 (br s, 1H, NH), 7.43–7.32 (m,
5H, Ph-H), 7.14 (s, 1H, Ph-H), 6.54 (s, 1H, Ph-H), 5.20 (s, 2H, CH2Ph),
3.86 (s, 6H, OCH3), 3.82 (s, 3H, OCH3).
2.2.6. N-Methylbenzo[d][1,3]dioxol-4-amine (7)
A solution of compound 6 (8.4 g, 29.4 mmol) in EtOH (150 mL)
was hydrogenated at a pressure of 45 psi in the presence of 10%
Pd/C (840 mg) for 6 h. The catalyst was filtered off through Celite.
The solvent was evaporated in vacuo, and the residue was purified
by column chromatography with hexanes/CH2Cl2 (1:1.5) to afford
7 (4.45 g, 92%) as a colorless oil. 1H NMR (CDCl3) ı: 6.76 (t, J = 8.0 Hz,
1H, Ph-H), 6.34 (dd, J = 1.0, 8.0 Hz, 1H, Ph-H), 6.32 (dd, J = 1.0, 8.0 Hz,
1H, Ph-H), 5.90 (s, 2H, OCH2O), 2.89 (s, 3H, NCH3).
2.2.7. Ethyl 3-(benzo[d][1,3]dioxol-4-yl(methyl)amino)-3-
oxopropanoate (8)
2.2.12. 2,4,5-Trimethoxyaniline (14)
A mixture of compound 7 (4.0 g, 26.5 mmol) and excess diethyl
malonate (40 mL) was heated and stirred at 110 ◦C for 3 h under
a nitrogen atmosphere. The excess diethyl malonate was removed
in vacuo, and the residue was purified by column chromatography
A solution of compound 13 (6.0 g, 18.9 mmol) in THF (50 mL)
and MeOH (15 mL) was hydrogenated at a pressure of 40 psi in
the presence of 10% Pd/C (60 mg) for 7 h. The catalyst was fil-
tered off through Celite. The solvent was evaporated in vacuo, and
the residue was purified by column chromatography with hex-
anes/EtOAc (1:1) to afford 14 (3.15 g, 91%) as a white solid, m.p.
83–84 ◦C (lit. 84–86 ◦C [7]). 1H NMR (CDCl3) ı: 6.54 (s, 1H, Ph-H),
6.41 (s, 1H, Ph-H), 3.83 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3).
with hexanes/EtOAc (2:1) to afford 8 (7.02 g, 70%) as a yellow oil. 1
H
NMR (CDCl3) ı: 6.86 (t, J = 8.0 Hz, 1H, Ph-H), 6.82 (dd, J = 1.5, 7.5 Hz,
1H, Ph-H), 6.72 (dd, J = 1.5, 8.0 Hz, 1H, Ph-H), 6.03 (s, 2H, OCH2O),
4.12 (q, J = 7.0 Hz, 2H, CH2CH3), 3.30 (s, 2H, C(O)CH2C(O)), 3.27(s,
3H, NCH3), 1.23 (t, J = 7.0 Hz, 3H, CH2CH3).
2.2.8. 3-(Benzo[d][1,3]dioxol-4-yl(methyl)amino)-3-
oxopropanoic acid (9)
2.2.13. 3-oxo-N-(2,4,5-Trimethoxyphenyl)butanamide (15)
A mixture of compound 14 (3.0 g, 16.4 mmol) and methyl ace-
toacetate (1.77 mL, 16.4 mmol) in toluene (50 mL) was stirred and
refluxed using a Dean-Stark apparatus for 24 h. The solvent was
evaporated in vacuo, and the crude product was purified by column
chromatography, eluting with CH2Cl2/MeOH (100:3) to afford 15
(3.25 g, 74%) as a tan solid, m.p. 88–89 ◦C (lit. 90–92 ◦C [7]). 1H NMR
(CDCl3) ı: 9.10 (br s, 1H, NH), 8.07 (s, 1H, Ph-H), 6.56 (s, 1H, Ph-H),
3.89 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.59 (s,
2H, C(O)CH2C(O)), 2.34 (s, 3H, CH3).
A mixture of compound 8 (3.0 g, 11.3 mmol) and 1N NaOH
(40 mL) was heated and stirred at 70 ◦C for 4 h. The solution was
cooled in an ice bath and acidified to pH 3.0 with 2N HCl. The mix-
ture was extracted with CH2Cl2. The combined organic layer was
washed with brine, dried over Na2SO4, and filtered. The solvent
was evaporated in vacuo, and the residue was triturated under hex-
anes/EtOAc (2:1) to afford 9 (2.68 g, 84%) as a pale brown solid,
m.p. 112–114 ◦C (lit. 115–117 ◦C [7]). 1H NMR (CDCl3) ı: 6.92 (t,
J = 8.0 Hz, 1H, Ph-H), 6.88 (dd, J = 1.0, 8.0 Hz, 1H, Ph-H), 6.67 (dd,
J = 1.5, 8.0 Hz, 1H, Ph-H), 6.06 (s, 2H, OCH2O), 3.33 (s, 3H, NCH3),
3.26 (s, 2H, CH2, C(O)CH2C(O)).
2.2.14. 5,6,8-Trimethoxy-4-methylquinolin-2(1H)-one (16)
To a preheated PPA (20 g) was added compound 15 (1.5 g,
5.6 mmol) at 100 ◦C. After stirring at the same temperature for 2 h,
the reaction mixture was poured into ice water and neutralized
with 5N NaOH. The mixture was extracted with CH2Cl2. The com-
bined organic layer was washed with brine, dried over Na2SO4,
and filtered. The solvent was evaporated in vacuo, and the crude
product was purified by column chromatography, eluting with
CH2Cl2/MeOH (100:3) to afford 16 (290 mg, 21%) as a purple solid,
m.p. 149–150 ◦C (lit. 149–151 ◦C [7]). 1H NMR (CDCl3) ı: 9.40 (br s,
1H, NH), 6.78 (s, 1H, Ph-H), 6.49(d, J = 0.5 Hz, 1H, C CH), 3.97 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 2.68 (d, J = 1.0 Hz, 3H,
CH3).
2.2.9. 6-Hydroxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-
8(9H)-one (10)
To a preheated polyphosphoric acid (PPA) (20 g) was added
compound 9 (2.0 g, 8.4 mmol) at 100 ◦C. After stirring at the same
temperature for 2 h, the reaction mixture was poured into ice
water. After PPA had dissolved, the pale yellow precipitate was col-
lected by filtration, washed with water and Et2O, dried in vacuo to
afford 10 (1.65 g, 89%) as a pale yellow solid, m.p. 309 ◦C (dec.) (lit.
310 ◦C (dec.) [7]). 1H NMR (DMSO-d6) ı: 11.30 (br s, 1H, OH), 7.46
(d, J = 8.5 Hz, 1H, Ph-H), 6.89 (d, J = 8.5 Hz, 1H, Ph-H), 6.10 (s, 2H,
OCH2O), 5.72 (s, 1H, C CH), 3.66 (s, 3H, NCH3).
2.2.15. 5,6,8-Trimethoxy-1,4-dimethylquinolin-2(1H)-one (17)
NaH (60% dispersion in mineral oil, 20 mg, 0.50 mmol) was
added to a suspension of compound 16 (100 mg, 0.4 mmol) in DMF
(3 mL). After the mixture was heated and stirred at 40 ◦C for 1 h,
it was cooled to room temperature, and CH3I (0.1 mL, 1.60 mmol)
2.2.10. Casimiroin (6-methoxy-9-methyl-[1,3]dioxolo[4,5-
h]quinolin-8(9H)-one, 11)
To a suspension of compound 10 (300 mg, 1.37 mmol) in Et2O
(1 mL) was added TMS-diazomethane (2 M in hexanes, 15 mL) at

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M. Wang et al. / Steroids 75 (2010) 967–973
was added. The reaction mixture was continued to stir at room
temperature for 0.5 h, and then heated at 80 ◦C overnight. The
mixture was poured into ice water and extracted with CH2Cl2.
The combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated in vacuo, and the
crude product was purified by preparative TLC plate, eluting with
CH2Cl2/MeOH (100:3) to afford 17 (50 mg, 47%) as a pale brown
solid, m.p. 129–130 ◦C (lit. 128–130 ◦C [7]). 1H NMR (CDCl3) ı: 6.83
(s, 1H, Ph-H), 6.57 (s, 1H, C CH), 3.96 (s, 3H, OCH3), 3.93 (s, 3H,
OCH3), 3.87 (s, 3H, OCH3), 3.82 (d, J = 2.0 Hz, 3H, NCH3), 2.63 (s, 3H,
CH3).
2.2.21. 5,8-Dimethoxy-4-methylquinolin-2(1H)-one (20c)
According to the procedure for preparation of 20a, compound
20c (640 mg, 46%) was prepared from PPA (20 g) and 19c (1.5 g,
6.3 mmol) as a pale yellow solid, m.p. 186–187 ◦C (lit. 187–189 ◦C
[7]). 1H NMR (CDCl3) ı: 9.38 (br s, 1H, NH), 6.89 (d, J = 8.5 Hz, 1H,
Ph-H), 6.53 (d, J = 9.0 Hz, 1H, Ph-H), 6.42 (s, 1H, C CH), 3.92 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 2.65 (d, J = 1.0 Hz, 3H, CH3).
2.2.22. 8-Methoxy-1,4-dimethylquinolin-2(1H)-one (21a)
To a suspension of compound 20a (120 mg, 0.63 mmol) in DMF
(2 mL) was added NaH (60% dispersion in mineral oil, 31.6 mg,
0.79 mmol). The reaction mixture was stirred and heated at 40 ◦C
for 1 h. The mixture was cooled to room temperature, and CH3I
(0.16 mL, 2.52 mmol) was added. After stirring at room temperature
for 0.5 h and at 60 ◦C overnight, the reaction mixture was poured
into ice water and extracted with CH2Cl2. The combined organic
layer was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated in vacuo, and the crude product was puri-
fied by preparative TLC plate, eluting with hexanes/EtOAc (1:1) to
afford 21a (65 mg, 50%) asayellow solid, m.p. 71–72 ◦C (lit. 72–74 ◦C
[7]). 1H NMR (CDCl3) ı: 7.30 (dd, J = 1.5, 8.0 Hz, 1H, Ph-H), 7.18 (t,
J = 8.0 Hz, 1H, Ph-H), 7.09 (dd, J = 1.5, 8.0 Hz, 1H, Ph-H), 6.62 (s, 1H,
2.2.16. N-(2-Methoxyphenyl)-3-oxobutanamide (19a)
A mixture of o-anisidine 18a (3.0 g, 24.4 mmol) and methyl ace-
toacetate (2.63 mL, 24.4 mmol) in toluene (50 mL) was stirred and
refluxed using a Dean-Stark apparatus for 24 h. The solvent was
evaporated in vacuo, and the crude product was purified by col-
umn chromatography, eluting with hexanes/EtOAc (2.5:1) to afford
19a (1.3 g, 26%) as a yellow solid, m.p. 82–83 ◦C (lit. 82–84 ◦C [7]).
1H NMR (CDCl3) ı: 9.23 (br s, 1H, NH), 8.32 (dd, J = 1.5, 8.0 Hz, 1H,
Ph-H), 7.06 (dt, J = 1.5, 8.0 Hz, 1H, Ph-H), 6.95 (dt, J = 1.0, 8.0 Hz, 1H,
Ph-H), 6.89 (dd, J = 1.0, 8.5 Hz, 1H, Ph-H), 3.91 (s, 3H, OCH3), 3.60 (s,
2H, C(O)CH2C(O)), 2.33 (s, 3H, CH3).
C
CH), 3.93 (s, 3H, OCH3), 3.90 (s, 3H, NCH3), 2.43 (s, 3H, CH3).
2.2.23. 6,8-Dimethoxy-1,4-dimethylquinolin-2(1H)-one (21b)
According to the procedure for preparation of 21a, compound
21b (139 mg, 65%) was prepared from 20b (200 mg, 0.91 mmol),
NaH (60% dispersion in mineral oil, 45.6 mg, 1.14 mmol), and
CH3I (0.23 mL, 3.64 mmol) as a yellow solid, m.p. 158–160 ◦C (lit.
157–159 ◦C [7]). 1H NMR (CDCl3) ı: 6.71 (d, J = 3.0 Hz, 1H, Ph-H),
6.68 (d, J = 3.0 Hz, 1H, Ph-H), 6.66 (s, 1H, C CH), 3.91 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 3.87 (s, 3H, NCH3), 2.41 (d, J = 1.0 Hz, 3H, CH3).
2.2.17. N-(2,4-Dimethoxyphenyl)-3-oxobutanamide (19b)
According to the procedure for preparation of 19a, compound
19b (9.2 g, 59%) was prepared from 2,4-dimethoxyaniline 18b
(10.0 g, 65.0 mmol) and methyl acetoacetate (7.05 mL, 65.0 mmol)
as a beige solid, m.p. 91–92 ◦C (lit. 92–44 ◦C [7]). 1H NMR (CDCl3)
ı: 9.02 (br s, 1H, NH), 8.18 (d, J = 9.0 Hz, 1H, Ph-H), 6.49–6.45 (m,
2H, Ph-H), 3.88 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.58 (s, 2H,
C(O)CH2C(O)), 2.33 (s, 3H, CH3).
2.2.24. 5,8-Dimethoxy-1,4-dimethylquinolin-2(1H)-one (21c)
According to the procedure for preparation of 21a, compound
21c (121 mg, 57%) was prepared from 20c (200 mg, 0.91 mmol),
NaH (60% dispersion in mineral oil, 45.6 mg, 1.14 mmol), and
CH3I (0.23 mL, 3.64 mmol) as a yellow solid, m.p. 186–187 ◦C (lit.
187–189 ◦C [7]). 1H NMR (CDCl3) ı: 7.03 (d, J = 9.0 Hz, 1H, Ph-H),
6.63 (d, J = 9.0 Hz, 1H, Ph-H), 6.50 (s, 1H, C CH), 3.84 (s, 6H, OCH3),
3.82 (s, 3H, NCH3), 2.60 (d, J = 0.5 Hz, 3H, CH3).
2.2.18. N-(2,5-Dimethoxyphenyl)-3-oxobutanamide (19c)
According to the procedure for preparation of 19a, compound
19c (11.7 g, 75%) was prepared from 2,5-dimethoxyaniline 18c
(10.0 g, 65.0 mmol) and methyl acetoacetate (7.05 mL, 65.0 mmol)
as a pale yellow solid, m.p. 68–70 ◦C (lit. 70–72 ◦C [7]). 1H NMR
(CDCl3) ı: 9.25 (br s, 1H, NH), 8.06 (d, J = 3.0 Hz, 1H, Ph-H), 6.80 (d,
J = 8.5 Hz, 1H, Ph-H), 6.59 (dd, J = 3.0, 9.0 Hz, 1H, Ph-H), 3.87 (s, 3H,
OCH3), 3.78 (s, 3H, OCH3), 3.59 (s, 2H, CH2, C(O)CH2C(O)), 2.33 (s,
3H, CH3).
2.3. Synthesis of [11C]casimiroin ([11C]11) and its
carbon-11-labeled analogues [11C]17 and [11C]21a–c
2.2.19. 8-Methoxy-4-methylquinolin-2(1H)-one (20a)
[
11C]CO2 was produced by the 14N(p,␣)11C nuclear reaction in
To a preheated PPA (10 g) was added compound 19a (800 mg,
3.9 mmol) at 100 ◦C. After stirring at the same temperature for 2 h,
the reaction mixture was poured into ice water and neutralized
with 5N NaOH. The mixture was extracted with CH2Cl2. The com-
bined organic layer was washed with brine, dried over Na2SO4,
and filtered. The solvent was evaporated in vacuo, and the crude
product was purified by column chromatography, eluting with
CH2Cl2/MeOH (100:2.5) to afford 20a (200 mg, 27%) as a white solid,
m.p. 189–190 ◦C (lit. 188–190 ◦C [7]). 1H NMR (CDCl3) ı: 9.33 (br s,
1H, NH), 7.28 (d, J = 1.0, 1H, Ph-H), 7.17 (t, J = 8.0 Hz, 1H, Ph-H), 7.00
(dd, J = 0.5, 8.0 Hz, 1H, Ph-H), 6.56 (d, J = 1.0 Hz, 1H, C CH), 3.98 (s,
3H, OCH3), 2.49 (d, J = 1.5 Hz, 3H, CH3).
small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV
proton cyclotron on research purity nitrogen (+1% O2) in a Siemens
radionuclide delivery system (Eclipse RDS-111). The precursor
10, 16, 20a, 20b, or 20c (0.1–0.3 mg) was dissolved in CH3CN
(300 ␮L). To this solution was added NaH (0.5–1 mg). The mixture
was transferred to a small reaction vial. No-carrier-added (high
specific activity) [11C]CH3OTf that was produced by the gas phase
production method [25] from [11C]CO2 through [11C]CH4 and
[
11C]CH3Br with silver triflate (AgOTf) column was passed into the
reaction vial until radioactivity reached a maximum (∼2 min), and
then the reaction vial was isolated and heated at 80 ◦C for 3 min.
The contents of the reaction vial were diluted with NaHCO3 (0.1 M,
1 mL), 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]11, [11C]17, [11C]21a, [11C]21b, or [11C]21c, was for-
mulated in saline, sterile-filtered through a sterile vented Millex-GS
0.22 ␮m 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
2.2.20. 6,8-Dimethoxy-4-methylquinolin-2(1H)-one (20b)
According to the procedure for preparation of 20a, compound
20b (320 mg, 23%) was prepared from PPA (20 g) and 19b (1.5 g,
6.3 mmol) as a yellow solid, m.p. 230–231 ◦C (lit. 232–234 ◦C [7]).
1H NMR (DMSO-d6) ı: 10.48 (br s, 1H, NH), 6.80 (d, J = 2.5 Hz, 1H,
Ph-H), 6.72 (d, J = 2.5 Hz, 1H, Ph-H), 6.42 (d, J = 1.0 Hz, 1H, C CH),
3.88 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 2.40 (d, J = 1.0 Hz, 3H, CH3).

M. Wang et al. / Steroids 75 (2010) 967–973
971
Table 1
active compound (21b versus QR2 and 17 versus aromatase) has
resulted from previous structural modification of parent natural
product casimiroin [7]. The relative affinity of casimiroin analogues
for aromatase versus QR2 is calculated and also listed in Table 1.
These in vitro data indicate some interesting information about
the structure–activity relationship (SAR). Overall, [1,3]dioxolo
ring-opened analogues 17, 21a–c displayed superior inhibitory
potency for both QR2 and aromatase compared with the parent
compound 11, the replacement of the 6-methoxy group of com-
pound 11 by a methyl group increased the inhibitory activity, and
the trimethoxy compound 17 is most selective aromatase inhibitor
(aromatase/QR2 ratio 108) in comparison with the dimethoxy
or methoxy compounds 21a–c. These compounds possess an
O-methyl and/or N-methyl position amenable to labeling with a
positron-emitting radioisotope such as carbon-11 as PET agents.
Therefore, these compounds can be proposed as tools for PET
experiments. Previous work [7] indicated N-methylated analogues
showed better potency than non-N-methylated quinolinones in
inhibiting QR2. This combined with other SAR information and
the impact of the structural modification of casimiroin analogues
suggested that the N-methyl position is the better isotope incorpo-
ration site than the O-methyl position. Therefore, we designed and
synthesized carbon-11-labeled casimiroin analogues by labeling
casimiroin 11 at the O-methyl position and labeling its analogues
17 and 21a–c at the N-methyl position.
Inhibition (IC50) of QR2 and aromatase by casimiroin and its analogues [7].
Compound
QR2 IC50 (␮M)
Aromatase
IC50 (␮M)
Aromatase/
QR2 ratio
Casimiroin (11)
17
21a
21b
21c
54.1 6.7
10.8 1.3
5.8 0.9
1.9 0.2
4.1 0.6
3.92 0.67
0.10 0.01
2.02 0.1
0.96 0.02
0.96 0.01
13.8
108
2.87
1.98
4.27
formulation time was 25–30 min from end of bombardment (EOB).
The radiochemical yields decay corrected to EOB, from [11C]CO2,
were 40–50%. Retention times in the analytical HPLC system
were: tR 10 = 2.52 min, tR 11 = 3.47 min, tR
[
11C]11 = 3.47 min;
11C]17 = 3.31 min; tR
11C]21a = 2.95 min; tR
11C]21b = 3.15 min; and tR
11C]21c = 3.17 min. Retention
times in the semi-preparative HPLC system were: tR 10 = 5.11 min,
tR 11 = 7.23 min, tR
11C]11 = 7.23 min; tR 16 = 4.58 min, tR
11C]17 = 6.76 min; tR 20a = 4.23 min, tR
11C]21a = 6.48 min; tR 20b = 4.65 min, tR
11C]21b = 6.91 min; and tR 20c = 4.47 min,
11C]21c = 6.88 min.
tR 16 = 2.27 min, tR 17 = 3.31 min, tR
20a = 2.02 min, tR 21a = 2.95 min, tR
[
[
20b = 2.23 min, tR 21b = 3.15 min, tR
20c = 2.11 min, tR 21c = 3.17 min, tR
[
[
[
17 = 6.76 min, tR
21a = 6.48 min, tR
21b = 6.91 min, tR
tR 21c = 6.88 min, tR
[
[
[
[
3. Results and discussion
3.2. Chemistry
3.1. In vitro data and radiotracer design
The precursors 10, 16, 20a-c for radiolabeling and their stan-
dards 11, 17, 21a–c were synthesized based on the literature
method with modification [7,26]. The improvements included
modified synthetic approaches with moderate to excellent chem-
ical yields, more complete experimental procedures and detailed
spectral data.
Asshown in Scheme 1, 2,3-dihydroxybenzoic acid(1)wasesteri-
fied using MeOH in the presence of thionyl chloride to afford methyl
ester 2 in 99% yield. The yield via this method was much higher
Five title compounds casimiroin (11) and its analogues 5,6,8-
trimethoxy-1,4-dimethylquinolin-2(1H)-one (17), 8-methoxy-
1,4-dimethylquinolin-2(1H)-one
(21a),
6,8-dimethoxy-1,4-
dimethylquinolin-2(1H)-one (21b), and 5,8-dimethoxy-1,4-
dimethylquinolin-2(1H)-one (21c) were served as the reference
standards and selected for radiolabeling. These casimiroin ana-
logues are potent QR2 and aromatase inhibitors, and their in vitro
data from previous report are summarized in Table 1, [7]. The most
Scheme 1. Synthesis of casimiroin (11) and its precursor (10).

972
M. Wang et al. / Steroids 75 (2010) 967–973
Scheme 2. Synthesis of a casimiroin analogue (17) and its precursor (16).
than that obtained using catalystic amount of sulfuric acid, which
yield was 40%. The cyclization of catechol 2 was accomplished with
dibromomethane to give benzodioxole 3 in 77% yield, which was
saponified with 2N KOH in MeOH to provide dioxole benzoic acid
4 in 91% yield. Direct transformation of carboxylic acid 4 into car-
bamate 5 was accomplished through Curtius rearrangement using
DPPA and Et3N in benzene, followed by benzyl alcohol, in 74% yield.
N-Methylation of 5 using NaH and CH3I in DMF provided 6 in 95%
yield, which was hydrogenated using Pd/C as catalyst to give the
secondary amine 7 in 92% yield. Acylation of amine 7 with diethyl
malonate gave ␤-keto ester 8 in 70% yield, followed by saponifi-
cation with 1N NaOH to provide the acid 9 in 84% yield. Precursor
10 was obtained by the cyclization of 9 with PPA in 89% yield. O-
Methylation of 10 with TMS-diazomethane involving the addition
of MeOH as a cosolvent afforded standard 11 in 37% yield [27].
As depicted in Scheme 2, Curtius rearrangement of commer-
cially available 2,4,5-trimethoxyl benzoic acid (12) gave carbamate
13 in 81% yield. Hydrogenation of 13 yielded amine 14 in 91%
yield. Acylation of 14 using methyl acetoacetate provided ␤-keto
amide 15 in 74% yield [28]. Cyclization of 15 with PPA afforded
precursor 16 in 21% yield. N-Methylation of 16 using NaH and
CH3I in DMF generated its corresponding standard 17 in 47%
yield.
Similarly, as indicated in Scheme 3, acylation of commercially
available free amines 18a–c provided ␤-keto amides 19a–c in
26–75% yield [28], which were cyclized with PPA to afford pre-
cursors 20a–c in 23–46% yield. N-Methylation of 20a–c gave their
corresponding standards 21a–c in 50–65% yield.
Scheme 4. Synthesis of [11C]casimiroin ([11C]11) and its carbon-11-labeled ana-
logues [11C]17 and [11C]21a–c.
Scheme 3. Synthesis of casimiroin analogues (21a–c) and their precursors (20a–c).

M. Wang et al. / Steroids 75 (2010) 967–973
973
3.3. Radiochemistry
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Synthesis of [11C]casimiroin ([11C]11) and its carbon-11-labeled
analogues,
[ [
11C]17 and 11C]21a–c is outlined in Scheme 4.
The precursors 10, 16 and 20a–c were labeled by [11C]CH3OTf
[25,29] through either O- or N-[11C]methylation and isolated by
reversed-phase HPLC method [1,3] to produce the correspond-
ing pure radiolabeled compounds [11C]11, [11C]17 and [11C]21a–c
in 40–50% radiochemical yields, based on [11C]CO2, decay cor-
rected to EOB. Overall synthesis time was 25–30 min from EOB. The
radiosynthesis was performed in an automated multi-purpose 11C-
radiosynthesis module, allowing measurement of specific activity
during synthesis [30,31]. The specific activity was in a range of
222–370 GBq/␮mol at EOB measured by the on-the-fly technique
[31] and 111–185 GBq/␮mol at the end of synthesis (EOS) mea-
sured by analytical HPLC [32], respectively. Chemical purity and
radiochemical purity were determined by analytical HPLC [32].
The chemical purity of the precursors and reference standards
was >96%. The radiochemical purity of the target tracers was >99%
determined by radio-HPLC through ␥-ray (PIN diode) flow detector,
and the chemical purity of the target tracers was >95% determined
by reversed-phase HPLC through UV flow detector.
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In conclusion, an efficient and convenient synthesis of
[
11C]casimiroin and its carbon-11-labeled analogues has been
developed. The synthetic methodology employed classical organic
chemistry such as esterification, cyclization, saponification, Cur-
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to produce precursor and standard compounds. Carbon-11-
labeled casimiroin analogues were prepared by either O- or
N-[11C]methylation of their corresponding precursors using a reac-
tive [11C]methylating agent, [11C]CH3OTf, and isolated by HPLC
purification procedure in high radiochemical yields, short overall
synthesis time, and high specific activity. These chemistry results
combined with the reported in vitro biological data [7] encour-
age further in vivo biological evaluation of carbon-11-labeled
casimiroin analogues as new potential PET agents for imaging of
QR2 and aromatase expression in breast cancer.
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Acknowledgments
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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]CH3OTf. 1H NMR
spectra were recorded on a Bruker Avance II 500 MHz NMR spec-
trometer in the Department of Chemistry and Chemical Biology at
Indiana University Purdue University Indianapolis (IUPUI), which
is supported by a NSF-MRI Grant CHE-0619254. The referees’ criti-
cisms and editors’ comments for the revision of the manuscript are
greatly appreciated.
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