Synthesis of 2-[11C]methoxy-3,17β-estradiol to measure the pharmacokinetics of an antitumor drug candidate, 2-methoxy-3,17β-estradiol

Article abstract of DOI:10.1002/jlcr.1131

2-Methoxy-3,17β-estradiol, an endogenous estrogen metabolite, showed cytotoxicity in various cancer cell lines and also has antiangiogenic and proapoptotic activities. Clinical I and II trials of 2-methoxy-3,17β- estradiol for multiple myeloma, advanced solid tumors, metastatic breast and prostate cancer are underway. We prepared 2-[¹¹C]methoxy-3,17β- estradiol to measure the pharmacokinetics and organ distribution of 2-methoxy-3,17β-estradiol in clinical trials. 2-[¹¹C]Methoxy-3, 17β-estradiol was synthesized from a precursor, 2-hydroxy-3,17β-O- bis(methoxymethyl)estradiol, in two steps with over 99% radiochemical purity. The overall reaction time was 45 min and the decay-corrected radiochemical yield was 32.9%. The distribution coefficient (logP_7.4) of 2-[ ¹¹C]methoxy-3,17β-estradiol at pH 7.4 was measured as 2.95. Copyright

Full text of DOI:10.1002/jlcr.1131

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1117–1124.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1131
Research Article
1
1
Synthesis of 2-[ C]methoxy-3,17b-estradiol to measure
the pharmacokinetics of an antitumor drug candidate,
2
-methoxy-3,17b-estradiol
Jiyoung Mun, Ronald J. Voll and Mark M. Goodman*
Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta,
GA 30322, USA
Summary
2
-Methoxy-3,17b-estradiol, an endogenous estrogen metabolite, showed cytotoxicity
in various cancer cell lines and also has antiangiogenic and proapoptotic activities.
Clinical I and II trials of 2-methoxy-3,17b-estradiol for multiple myeloma, advanced
solid tumors, metastatic breast and prostate cancer are underway. We prepared
11
2
-[ C]methoxy-3,17b-estradiol to measure the pharmacokinetics and organ distribu-
1
1
tion of 2-methoxy-3,17b-estradiol in clinical trials. 2-[ C]Methoxy-3,17b-estradiol
was synthesized from a precursor, 2-hydroxy-3,17b-O-bis(methoxymethyl)estradiol, in
two steps with over 99% radiochemical purity. The overall reaction time was 45 min
and the decay-corrected radiochemical yield was 32.9%. The distribution coef-
1
1
ficient (logP7.4) of 2-[ C]methoxy-3,17b-estradiol at pH 7.4 was measured as
.95. Copyright # 2006 John Wiley & Sons, Ltd.
2
Received 18 March 2005; Revised 18 September 2005; Accepted 27 October 2005
Key Words: 2-methoxy-3,17b-estradiol (2ME2,2ME); positron emission tomography
(
PET); carbon-11; pharmacokinetics
Introduction
-Methoxy-3,17b-estradiol (2-methoxyestradiol, 2ME2, 2ME, Figure 1)
is an endogenous estrogen metabolite, which has been reported to
2
1
,2
show cytotoxicity in various cancer cell lines. The colchicine binding site
of tubulin is occupied by 2ME2, which results in destabilization of
3
–5
microtubule.
2ME2 reduced HIF-1, hypoxia inducible factor, activity
*Correspondence to: Mark M. Goodman, Department of Radiology, Division of Radiological Sciences,
Emory University, 1364 Clifton Road, Atlanta, GA30322, USA. E-mail: mgoodma@emory.edu
Copyright # 2006 John Wiley & Sons, Ltd.

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J. MUN ET AL.
1
1
Figure 1. Structures of 2-methoxy-3,17b-estradiol and 2-[ C]methoxy-3,17b-
estradiol
and HIF-1a nuclear accumulation, which is related to antiangiogenic
2ME2 is being evaluated in phase I and II
6–11
and proapoptotic activity.
clinical trials for advanced solid tumors, multiple myeloma, prostate
12
and metastatic breast cancer. One hundred and eighty three references
were searched by SciFinder relating to anticancer activity of 2ME2 from
2
004 to 2006.
The purpose of this paper is to develop a reliable labeling method of
-methoxyestradiol with carbon-11 for positron emission tomography (PET)
2
study. PET can be used to obtain pharmacokinetics, organ distribution and
access to a target organ of a drug candidate in human clinical trials, when a
1
3
drug candidate can be labeled without change in chemical structure.
Generally, blood and plasma pharmacokinetic data can be obtained from
conventional methods in human clinical trials, whereas, organ distribution and
tumor/tissue pharmacokinetic data are limited to body secretions and biopsy
samples. PET of a whole body can provide quantified organ distribution as
1
3
well as tumor/tissue pharmacokinetic data. A tracer quantity of a drug
candidate is used in a PET study (nanomolar to micromolar quantity,
depending on a study model) and half life of carbon-11 is only 20.4 min. Thus,
1
4
chemical toxicity from the drug is within allowable limits. The availability of
the drug candidate labeled with a positron emitter itself is usually the limiting
factor for a PET study. 2ME2 has an inherent methoxy group, which can be
1
1
labeled with [ C]CH I from a phenol precursor, which makes it ideal for PET
3
study.
11
A synthesis of 2-[ C]methoxy-3,17b-estradiol was published by Park et al.
with 8–11% decay-corrected radiochemical yield during 38 min to 40 min of
1
5
the total reaction time as a symposium abstract in 1999. We would like to
present a modified synthesis to give a better radiochemical yield and to provide
a formulation for PET study.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1117–1124
DOI: 10.1002/jlcr

PHARMACOKINETICS OF AN ANTITUMOR DRUG CANDIDATE
1119
Results and discussion
Chemistry
-Hydroxy-3,17b-O-bis(methoxymethyl)estradiol was synthesized from 3,17b-
estradiol in two steps in 61% yield (Scheme 1). The synthetic method,
2
1
6,17
published by Paaren Herbert et al. was slightly modified.
was protected with chloromethyl methyl ether to generate 3,17b-O-bis
methoxymethyl)estradiol in 93.7% yield. 2-Hydroxy-3,17b-O-bis(methoxy-
3,17b-Estradiol
(
methyl)estradiol was synthesized by lithiation with sec-butyllithium at À788C,
subsequent addition of trimethyl borate, followed by oxidation with sodium
perborate tetrahydrate at room temperature in 65.2% yield. In the last step,
after addition of trimethyl borate, the temperature of the reaction mixture was
elevated to 08C. It was essential to stir the reaction mixture for more than 1 h
to get a good yield. Temperature fluctuation between 08C and room
temperature did not affect the yield. Product formation was monitored with
TLC (R =0.1, eluent 1:6 =EtOAc: Hexane) after taking out small aliquots of
f
the reaction mixture and quenching them with saturated aqueous ammonium
chloride solution. Oxidation step was performed with saturated aqueous
sodium perborate tetrahydrate solution overnight. NMR spectrometry of 2-
hydroxy-3,17b-O-bis(methoxymethyl)estradiol was verified by the previous
16,17
literature values.
2-Methoxy-3,17b-estradiol was purchased from Sigma.
Radiochemistry
11
2
-[ C]Methoxy-3,17b-estradiol was synthesized from 2-hydroxy-3,17b-O-bis
methoxymethyl)estradiol in two steps by modification of the labeling method,
(
15
published by Park et al. (Scheme 2). 2-Hydroxy-3,17b-O-bis(methoxymethyl)
estradiol in DMF was deprotonated with NaH and then methylated with
1
1
11
C]CH I at 808C for 10 min. The resulting 2-[ C]methoxy-3,17b-O-bis
[
3
Scheme 1. Synthesis of 2-hydroxy-3,17b-O-bis(methoxymethyl)estradiol
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

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J. MUN ET AL.
(
methoxymethyl)estradiol was deprotected by 6 N aqueous HCl at 1258C
1
for 5 min. After neutralization with 6 N aqueous NaOH, crude 2-[ C]
1
methoxy-3,17b-estradiol, was purified by means of semi-preparative HPLC.
11
2
-[ C]Methoxy-3,17b-estradiol was concentrated in 1 ml ethanol via a solid
phase extraction procedure based upon the method described by Lemaire et al.
18
1
1
The distribution coefficient (log P ) of 2-[ C]methoxy-3,17b-estradiol,
7.4
measured in 1-octanol and 0.02 M phosphate buffer at pH 7.4, was 2.95.
11
11
-[ C]Methoxy-3,17 -estradiol was formulated to dissolve in 10% ethanolic,
2
4
5% aqueous 3-hydoxypropyl g-cyclodextrin solution. 3-Hydoxypropyl
1
1
g-cyclodextrin was used as a delivery vehicle of 2-[ C]methoxy-3,17b-estradiol
to increase solubility in serum. The overall reaction time was 45 Æ 12.5 min
(
n=5) and the decay-corrected radiochemical yield was 32.9 Æ 8.3% (n=5)
1
1
11
based on the production of [ C]CH I. The identity of 2-[ C]methoxy-3,
3
1
7b-estradiol was confirmed by comparing the retention time on an analytical
HPLC system with a co-injected authentic sample of 2-methoxy-3,
1
1
1
7b-estradiol. 2-[ C]Methoxy-3,17b-estradiol was synthesized with more than
9
9% radiochemical purity based on the radiometric detection.
11
Scheme 2. Synthesis of 2-[ C]methoxy-3,17b-estradiol
Experimental
General
All chemicals were analytical grade, purchased from Aldrich Chemical Co.
(Milwaukee, WI, USA) and used without further purification. 2-Methoxyes-
tradiol was purchased from Sigma. Glassware was flame-dried under argon
gas and reactions were performed under argon gas. Two hundred and fifty
micrometer layers of F-254 silica gel absorbed on aluminum plates, purchased
from Whatman Ltd. (Clifton, NJ, USA), was utilized for thin-layer
chromatography (TLC) analyses. Silica gel 60 from EMD was used for
1
column chromatography. H NMR spectra were recorded on a Varian
spectrometer at 400 MHz or 300 MHz and referenced to the NMR solvent
1
chemical shifts in ppm values, J-values in Hz). No-carrier-added [ C]CO
1
(
2
14
16
was produced from the bombardment of N gas containing 2% O by a
2
2
Siemens 11 MeV RDS 112 negative-ion cyclotron at Emory University
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1117–1124
DOI: 10.1002/jlcr

PHARMACOKINETICS OF AN ANTITUMOR DRUG CANDIDATE 1121
Hospital through the N[p,a] C reaction. A GE MicroLab methyl iodide
14
11
1
1
11
system was utilized to convert [ C]CO to [ C]CH I. C-18 SepPaks were
2
3
purchased from Waters Inc. (Milford, MA, USA).
Chemistry
Synthesis of 3,17b-O-bis(methoxymethyl)estradiol (3). Diisopropylethyla-
mine (14.7 ml, 84.8 mmol) and chloromethyl methyl ether (6 ml, 79.0 mmol)
were added to a solution of 3,17b-estradiol (2) (3.82 g, 14.0 mmol) in
tetrahydrofuran (24 ml) at room temperature, to form a white suspension.
The reaction mixture was stirred between 508C and 558C for 24 h to form a
yellowish solution with white solid on the wall of the reaction flask. After
cooling, 20% aqueous NH Cl (10 ml) was added to the reaction mixture,
4
which was extracted with diethyl ether (20 ml  3). The organic layer was
washed with 20% aqueous NH Cl (10 ml  2) and brine (20 ml). Then, the
4
organic layer was dried with MgSO and concentrated in vacuo. The crude
4
product was purified by chromatography on silica (R =0.3, eluent 1:5 =
f
EtOAc:Hexane) to give 3,17b-O-bis(methoxymethyl)estradiol (3) (4.74 g,
1
9
3.7%) as clear highly viscous oil. H NMR (CDCl ) d=0.82 (s, 1 H);
3
1
.18–2.33 (m, 13 H); 2.84–2.88 (m, 2 H); 3.39 (s, 3 H); 3.48 (s, 3 H); 3.63 (t, 1 H,
J=8); 4.67 (dd, 2 H, J=6.6, J=0.95); 5.16 (s, 2 H); 6.78 (d, 1 H, J=2.8); 6.825
dd, 1 H, J=8.5, J=2.8); 7.22 (d, 1 H, J=8.5).
(
Synthesis of 2-hydroxy-3,17b-O-bis(methoxymethyl)estradiol (4). sec-Butyl-
lithium (7.7 ml, 10.8 mmol, 1.4 M in cyclohexane) was added to the solution of
3
,17b-O-bis(methoxymethyl)estradiol (0.945 g, 2.62 mmol) in tetrahydrofuran
14 ml) at À788C slowly, so that the temperature of the solution did not exceed
À688C. The reaction mixture was stirred for 1 h at À788C and trimethylborate
1.19 ml, 10.67 mmol) was added to the reaction mixture at a rate such that the
(
(
temperature of the solution did not elevate more than 108C. The reaction
mixture was stirred for 30 min at À788C. The temperature of the reaction
mixture was allowed to increase to 08C and stirred for at least 1 h. Saturated
aqueous ammonium chloride (3 ml) was added to the reaction mixture, which
was stirred to form a white emulsion. Water (10 ml) and ethyl acetate (20 ml)
was added to the reaction mixture, followed by extraction and then the
aqueous layer was extracted two more times (10 ml  2) with ethyl acetate.
Saturated aqueous sodium perborate tetrahydrate (50 ml) was added to the
combined organic layer (60 ml). The mixture was stirred vigorously overnight
and extracted with ethyl acetate (50 ml  3). The organic layer was washed
with brine (70 ml) and dried with MgSO and concentrated in vacuo. The crude
4
product was purified by chromatography on silica (R =0.3, eluent 1:4 =
f
EtOAc:Hexane) to give 2-hydroxy-3,17b-O-bis(methoxymethyl)estradiol (4)
1
(
0.643 g, 65.2%) as clear highly viscous oil. H NMR (CDCl ) d=0.82(s, 3 H);
3
Copyright # 2006 John Wiley & Sons, Ltd.
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1
.18–2.27 (m, 13 H); 2.76–2.81(m, 2 H); 3.38(s, 3 H); 3.53 (s, 3 H); 3.62 (t, 1 H,
J=8.5); 4.67 (dd, 2 H, J=6.6, J=0.95); 5.17 (s, 2 H); 5.75 (bs, 1 H); 6.80
s, 1 H); 6.90 (s, 1 H).
(
Radiochemistry
Radiosynthesis of [ C]CH I. [ C]CH I was produced from [ C]CO by a
11
11
11
3
3
2
1
1
11
GE MicroLab methyl iodide system. [ C]CO was reduced to [ C]CH by
2
4
11
NiH and then [ C]CH was iodinated by I at 7208C. The preparation time
2
4
2
was approximately 15 min.
1
1
Preparation of 2-[ C]methoxy-3,17b-estradiol. 2-Hydroxy-3,17b-O-bis(methoxy-
methyl)estradiol (2.0 mg) was dissolved in anhydrous N,N-dimethylformamide
(
DMF, 200 ml) in a 2 ml septum-sealed v-vial. Slow stream (approximately
11
3
0ml/min) of [ C]CH I was added to the reaction mixture with a vent at 08C.
3
The reaction mixture was heated at 808C for 10 min and cooled down for
min in a cold water bath. Aqueous hydrochloric acid (6 N, 100 ml) was added
to the reaction vial and then the reaction mixture was heated at 1258C for
min. Aqueous sodium hydroxide (6 N, 100 ml) was added to the reaction
1
5
mixture, after it was cooled down for 1 min in a cold water bath. The reac-
tion mixture was diluted with HPLC eluent (0.5 ml, 50:50:0.1=CH CN:
3
water: NaH PO ) and injected onto a semi-preparative HPLC column
2
4
(
X-Terra Prep RP , 5mm, 19 Â 100 mm, Waters, Inc.). The HPLC eluate was
18
monitored using a radioactivity probe (SRI Instruments Peak Simple Chromato-
graphy Data System Model 202, Torrance, CA, USA; Carrol-Ramsey Model
101-S-DC-P single channel detector with preamplifier and standard probe, Carrol-
Ramsey Associates, Berkley, CA, USA) and each 1 min fraction was collected with
1
1
9
ml/min elution rate. 2-[ C]Methoxy-3,17b-estradiol was eluted in the 8-min
fraction.
11
Dose formulation of 2-[ C]methoxy-3,17b-estradiol. The fraction, containing
11
2
-[ C]methoxy-3,17b-estradiol, was concentrated via a solid phase extraction
procedure based upon the method described by Lemaire et al. All transfers
1
8
of liquid were performed using vacuum suction. The fraction of
11
2
-[ C]methoxy-3,17b-estradiol (9 ml) was diluted with sterile water (18 ml)
and drawn to a C-18 SepPak (pre-activated with 10 ml of ethanol, followed by
0 ml of water), which was then washed with saline (20 ml) and ethanol
1
1
1
(0.5 ml). 2-[ C]Methoxy-3,17b-estradiol was eluted from the C-18 SepPak
1
1
with 1 ml of ethanol. The ethanolic solution (1 ml) of 2-[ C]methoxy-3,17b-
estradiol was diluted with 9 ml of 45% aqueous 3-hydroxypropyl
g-cyclodextrin solution. The reaction time was 45 Æ 12.5 min (n = 5) and
the decay-corrected radiochemical yield was 32.9 Æ 8.3% (n =5) based on
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1117–1124
DOI: 10.1002/jlcr

PHARMACOKINETICS OF AN ANTITUMOR DRUG CANDIDATE
1123
1
[
1
C]CH I production. The specific activity was 0.9 Ci/mmol at the end of
3
synthesis.
11
Quality control of 2-[ C]methoxy-3,17b-estradiol dose. The identity of
11
2
-[ C]methoxy-3,17b-estradiol was examined by comparing retention time
1
1
with standard 2-methoxy-3,17b-estradiol, purchased from Sigma. 2-[ C]
Methoxy-3,17b-estradiol and standard 2-methoxy-3,17b-estradiol were co-
injected onto analytical HPLC (C18, 5 mm, 3.9 Â 150 mm, Waters Inc.). Both
UV absorbance (254 nm) and radiometric detection (IN/US Gamma RAM
radiometric detector; IN/US Systems Inc., FL, USA) were monitored at the
11
same time. Both of 2-[ C]methoxy-3,17b-estradiol and 2-methoxy-3,17b-
estradiol were eluted at the same retention time (R =3.5 min, eluent
t
5
0:50:0.1=CH CN: water: NaH PO , rate=1 ml/min). The radiochemical
3 2 4
1
1
purity was determined by the percentage of the peak area of 2-[ C]methoxy-
1
1
3,17b-estradiol with the radiometric detection. 2-[ C]Methoxy-3,17b-estradiol
in a dose vial showed over 99% radiochemical purity.
18;19
Measurement of distribution coefficient (logP ).
7
1-Octanol (10 ml) and
.4
phosphate buffer (5 ml, 0.02 M, pH 7.4) were pre-saturated with each other in
11
a separatory funnel (30 ml) and 2-[ C]methoxy-3,17b-estradiol (40 mCi) in
ethanol (200 mL) was added to 1-octanol layer. The layers were mixed for 3 min
and the bottom layer was discarded. Aliquots (2 ml) of the 1-octanol layer
were added to four test tubes containing 2 ml phosphate buffer (0.02 M, pH
7
5
.4). The test tubes were mechanically shaken for 10 min and centrifuged for
min at 1000 g. 0.5 ml of the organic layer was transferred to a test tube and
the rest of the organic layer was discarded for each test tube. 0.5 ml of the
aqueous layer was transferred to a test tube from each test tube. The activity of
each layer was measured with a Packard Cobra automated gamma-counter for
four samples. The distribution coefficient was calculated by the ratio of activity
in 1-octanol layer to aqueous layer. The average distribution coefficient of
1
1
2
-[ C]methoxy-3,17b-estradiol (logP ) was 2.95 Æ 0.3 (n =4).
7.4
Conclusion
11
2
-[ C]Methoxy-3,17b-estradiol was synthesized in two steps with 32.9 Æ 8.3%
(
(
n =5) of the decay-corrected radiochemical yield during 45 Æ 12.5 min
1
n = 5) of the total reaction time, based on [ C]CH I production.
1
3
The precursor of the labeling, 2-hydroxy-3, 17b-O-bis(methoxymethyl)
estradiol, was prepared in two steps from 3,17b-estradiol in 61% of the
11
isolated yield. 2-[ C]Methoxy-3,17b-estradiol was formulated in 10%
ethanolic, 45% aqueous 3-hydroxypropyl g-cyclodextrin solution with over
99% radiochemical purity and 0.9 Ci/mmol of specific activity at the end of
synthesis.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1117–1124
DOI: 10.1002/jlcr

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1
1
2
-[ C]Methoxy-3,17b-estradiol can be used to measure the pharmacoki-
netics and organ distribution of 2-methoxy-3,17b-estradiol in clinical trials.
Acknowledgements
We thank the cyclotron staff at Emory University for their collaboration in
this work.
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DOI: 10.1002/jlcr