Synthesis of exemestane labelled with 13C

Article abstract of DOI:10.1016/j.steroids.2008.02.010

The synthesis of exemestane (Aromasin), an irreversible steroidal aromatase inhibitor, specifically labelled with ¹³C is reported. The preparation of [¹³C₃]exemestane was achieved according to an eight-step proc

Full text of DOI:10.1016/j.steroids.2008.02.010

steroids 7 3 ( 2 0 0 8 ) 760–764
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Synthesis of exemestane labelled with 13C
Erminia Fontanaa,∗, Alberto Pignattia, Danilo Giribonea, Enrico Di Salleb
a
Isotope Chemistry, Accelera Unit, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano (MI), Italy
Global Medical Oncology, Pfizer Italia S.r.l, Via Lorenteggio 257, 20152 Milano, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of exemestane (Aromasin®), an irreversible steroidal aromatase inhibitor,
specifically labelled with 13C is reported. The preparation of [13C3]exemestane was achieved
according to an eight-step procedure starting from the commercially available testosterone.
© 2008 Elsevier Inc. All rights reserved.
Received 19 December 2007
Received in revised form
15 February 2008
Accepted 24 February 2008
Published on line 4 March 2008
Keywords:
Exemestane
Aromatase inhibitor
Labelling
13C
Internal standard
LC–MS
1.
Introduction
liquid chromatography–mass spectrometry (LC–MS). In this
paper, the approach followed to prepare a suitable stable
Exemestane (Aromasin®, 6-methylenandrosta-1,4-diene-
3,17-dione; molecular weight 296.41) (Fig. 1) is an orally
active irreversible aromatase inhibitor which is clinically
in use for the treatment of postmenopausal women with
early or advanced breast cancer [1–3]. The initial analytical
methods developed for the determination of exemestane in
biological fluids involved the use of high-performance liquid
chromatography (HPLC) combined with ultraviolet detection
or with thermospray mass spectrometry or followed by
specific radioimmunoassay [4–6]. However, a higher samples
throughput as well as lower limits of quantitation were
needed to assay exemestane in human plasma. Therefore,
the preparation of a stable labelled version of the compound
was required to be used as analytical internal standard (IS) to
develop and validate a sensitive, specific and reliable method
for the quantitation of exemestane in biological fluids using
labelled IS of exemestane is described.
2.
Experimental
Chemicals and materials: All solvents and reagents were of
analytical grade and were used without purification unless
otherwise indicated. Testosterone (1) was purchased from
Fluka, with a chemical purity >99%. [13C2]Acetyl chloride (99
atom % 13C) and [13C]formaldehyde (20% aqueous solution; 99
atom % 13C) were obtained from Aldrich Chemical Co.
Instrumentation and equipment: Chemical purities were
determined by HPLC using a series-200 pump (PerkinElmer)
equipped with series-200 solvent degasser (PerkinElmer),
series-200 autosampler (PerkinElmer) and a LC-235 UV diode
array detector (PerkinElmer) connected with Totalchrom
∗
Corresponding author. Tel.: +39 0331 581148; fax: +39 0331 581682.
E-mail address: erminia.fontana@nervianoms.com (E. Fontana).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.02.010

steroids 7 3 ( 2 0 0 8 ) 760–764
761
flask. At the end of the addition (about 2 h) the mixture was
stirred for 2 h at 40 ◦C then cooled to room temperature and
evaporated to dryness. The obtained residue was dissolved
with water (500 ml), transferred into a separating funnel and
extracted with ethyl acetate (EtOAc 2 ml × 200 ml). After sepa-
ration, the combined organic layers were back extracted with
8% NaHCO3 (3 ml × 100 ml). The basic aqueous phases were
combined with the aqueous phase previously obtained, then
6 N HCl was added up to pH 2. After extraction with EtOAc
(4 ml × 250 ml) the obtained organic phases were combined,
extracted with brine (2 ml × 300 ml) and dried over Na2SO4.
The solvent was removed by evaporation, and the obtained
intermediate (3) (11.09 g, 28.9 mmol) was used without further
purification in the next step.
Fig. 1 – Structural formula of exemestane.
Client/Server (PENelson) as integrator via link 600 inter-
face (PerkinElmer). Preparative-HPLC was performed at 25 ◦C
using a PrepStar HPLC system (Varian). 1H NMR data were
recorded on Varian INOVA 400 spectrometer, equipped with
a 5 mm 1H{15N-31P} z-axis pulse field gradient (PFG) indi-
rect detection probe. 13C NMR data were obtained by direct
detection on a varian INOVA 500 spectrometer, equipped
with a 3 mm broadband 15N-31P{1H-19F} probe optimized
for 13C sensitivity and by heteronuclear correlation spec-
tra (H-C) recorded on Varian Unity INOVA 500 spectrometer,
equipped with triple resonance 1H{13C, 15N} z-axis pulse
field gradient (PFG) indirect detection Cold-Probe. Chem-
ical shifts were referenced with respect to the residual
solvent signals (DMSO-d6: 2.50 ppm for 1H and 39.5 ppm
for 13C).
2.3.
3-Oxo-4-oxa-5-androsten-17ˇ-yl benzoate (4)
The intermediate (3) (11.09 g, 28.9 mmol) was dissolved in
dry EtOAc (480 ml) under nitrogen then freshly pre-
a
pared solution of acetic anhydride (94 ml) and 65% HClO4
(0.24 ml) in dry EtOAc (480 ml) was introduced into the reac-
tion flask. After stirring at room temperature for exactly
10 min, 8% NaHCO3 (740 ml) was added, the mixture was
stirred for 30 min then transferred into a separating fun-
nel. After separation of the aqueous layer, the organic
phase was extracted with 8% NaHCO3 (3 ml × 300 ml), water
(2 ml × 300 ml), brine (2 ml × 300 ml) and dried over Na2SO4.
After solvent evaporation, the oily yellow residue was sub-
mitted to 3 crystallizations from mixtures of CH3OH:H2O 3:1
by volume. After drying under high vacuum at 40 ◦C, the
intermediate (4) was recovered as a white powder (5.79 g,
14.7 mmol) with a chemical purity of 90% (determined by
HPLC; see analytical method; Rt = 11.7 min). MS (ESI-MS): m/z
395 ([MH]+).
Analytical method: HPLC: X-Terra Waters RP18 column (mm
4.6 × 100, 5 ␮m) eluting with H2O:CH3CN:HCOOH 90:10:0.1 by
volume (A) and H2O:CH3CN:HCOOH 10:90:0.1 by volume (B):
from 100% A to 0% A in 10 min; 5 min at 0% A; from 100% B to
100% A in 1 min; 4 min at 100% A. Flow rate: 1 ml/min. Column
temperature: 25 ◦C. Analytical wavelength: 215 nm.
2.4.
2-[1ꢀ,2ꢀ-13C2]Acetyl-3-oxo-4-oxa-5-androsten-
2.1.
17ˇ–Benzoyloxy-testosterone (2)
17ˇ-yl benzoate (5)
A solution of benzoyl chloride (BzCl; 49 ml, 42 mmol) in
dry toluene (60 ml) was slowly dripped into a stirred and
cooled (0 ◦C) solution of testosterone (1) (10.08 g, 34.9 mmol)
in dry toluene (100 ml). After addition of dry pyridine (14 ml,
173 mmol), the reaction mixture was stirred at reflux for
20 h then cooled to room temperature. An aqueous solu-
tion of 0.5 N HCl (60 ml) was added and the mixture was
stirred for 1 h. After separation, the organic layer was treated
with 0.5 N HCl (4 ml × 100 ml), 8% NaHCO3 (3 ml × 100 ml),
brine (2 ml × 100 ml) and dried over Na2SO4. The solvent was
evaporated to dryness giving the intermediate (2) (14.03 g,
35.7 mmol), with a chemical purity of 91% (determined by
HPLC; see analytical method; Rt = 14.0 min).
A solution of the intermediate (4) (1.51 g, 3.83 mmol) in
dry tetrahydrofuran (THF; 34.4 ml) was slowly added under
nitrogen to a cooled (−78 ◦C) and stirred 1 M solution of
lithium bis(trimethylsilyl)amide (7.66 ml; 7.66 mmol) in dry
THF. After stirring under nitrogen at −78 ◦C for 1 h, a solu-
tion of [13C2]acetyl chloride (308 mg, 3.83 mmol) in dry THF
(2.7 ml) was added. The mixture of reaction was stirred at
−78 ◦C for 90 min, then 1 N HCl (4.8 ml) and water (4.8 ml)
were added and the mixture was allowed to reach room tem-
perature then diluted with EtOAc (50 ml). After transferring
into a separating funnel and separation, the organic layer
was washed with brine (4 ml × 200 ml), dried over Na2SO4
and evaporated to dryness. The obtained crude intermedi-
ate (5) was purified by flash chromatography on silica gel
using a mixture n-hexane:EtOAc 2:1 by volume as the elut-
ing solvent system. The collected fractions were combined
as appropriate and after solvent evaporation to dryness,
the intermediate (5) (446 mg; 1.02 mmol) was recovered as
a white solid with a chemical purity >95% (determined
by HPLC; see analytical method; Rt = 13.8 min) and an iso-
2.2.
17ˇ-Benzoyloxy-5-oxo-3,5-seco-4-norandrostan-
3-oic acid (3)
An aqueous solution of 2M K2CO3 (32.6 ml) was added to a sus-
pension of (2) (13.00 g, 33.2 mmol) in tert-butanol (325 ml). The
reaction mixture was heated at 40 ◦C under stirring and two
separate solutions of 0.13 M KMnO4 (42.25 ml) and 0.75 M KIO4
(419 ml) were contemporaneously introduced into the reaction
topic purity of 99 atom
([MH]+).
%
13C. MS (ESI-MS): m/z 439

762
steroids 7 3 ( 2 0 0 8 ) 760–764
[3,4-13C2]Testosterone (6)
after solvent evaporation to dryness, the intermediate (8)
2.5.
(101 mg, 0.335 mmol) was recovered as a white solid with a
chemical purity >89% (determined by HPLC; see analytical
method; Rt = 9.8 min) and an isotopic purity of 99 atom % 13C.
MS (ESI-MS): m/z 302 ([MH]+).
The intermediate (5) (446 mg; 1.02 mmol) was dissolved in a
solution of KOH (500 mg) in a mixture of CH3OH:H2O 75:25
by volume (50 ml). The mixture of reaction was refluxed for
about 20 h. After cooling to room temperature, 1 N HCl was
added up to pH 4 and methanol was removed by evapora-
tion. The aqueous residue was transferred into a separating
funnel and extracted with EtOAc (3 ml × 25 ml). The com-
bined organic phases were washed with 1 N HCl (3 ml × 20 ml),
4% NaHCO3 (4 ml × 20 ml), brine (2 ml × 20 ml) and dried over
Na2SO4. After solvent evaporation to dryness, the intermedi-
ate (6) was recovered as a yellow solid (270 mg, 0.93 mmol) with
a chemical purity >77% (determined by HPLC; see analytical
method; Rt = 6.2 min) and an isotopic purity of 99 atom % 13C.
MS (ESI-MS): m/z 291 ([MH]+).
2.8.
[
13C3]Exemestane
The intermediate (8) (0.101 mg, 0.335 mmol) was dissolved
under nitrogen in dry 1,4-dioxane (7 ml) then p-TsOH
(128.7 mg, 0.674 mmol) and 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ; 133.6 mg, 0.588 mmol) were added. The
reaction mixture was stirred under reflux for about 1 h then
cooled to room temperature. After solvent evaporation, the
residue was dissolved in EtOAc (25 ml), transferred into a
separating funnel and washed with water (3 ml × 25 ml), 4%
NaHCO3 (3 ml × 25 ml), brine (2 ml × 25 ml) and dried over
Na2SO4. The crude [13C3]exemestane was purified on a silica
gel column eluting with a mixture n-hexane:EtOAc 1:1 by
volume. The obtained fractions were combined as appropriate
and, after solvent evaporation to dryness, [13C3]exemestane
(74.5 mg; 0.25 mmol) was recovered 84% chemically pure
(determined by HPLC; see analytical method). The obtained
2.6.
[3,4-13C2]Androstenedione (7)
Pyridinium chlorochromate (PCC; 599.7 mg, 2.78 mmol) was
added under nitrogen to a stirred solution of the interme-
diate (6) (270 mg, 0.93 mmol) in dry dichloromethane (10 ml).
After 4 h stirring at room temperature, the conversion was
completed (determined by TLC on silica gel; n-hexane:EtOAc
1:1 by volume) and diethyl ether (18 ml), silica gel and celite
were introduced into the reaction flask. After stirring at room
temperature for about 1 h, the suspention was filtered and
washed with diethyl ether (20 ml). The filtrate was evapo-
rated to dryness giving the intermediate (7) as a yellowish
solid (235 mg, 0.82 mmol) with a chemical purity >90% (deter-
mined by HPLC; see analytical method; Rt = 9.1 min) and an
isotopic purity of 99 atom % 13C. MS (ESI-MS): m/z 289
([MH]+).
[
13C3]exemestane was submitted to a further purification
by preparative HPLC (XTerra RP18 column, mm 100 × 50 I.D.,
5 ␮m, eluting with H2O (A) and CH3CN (B): from 90%A to 20%A
in 20 min; from 20%A to 0%A in 3 min; from 0% A to 90% A in
5 min; Flow rate: 120 ml/min. Column temperature: ambient;
Analytical wavelength: 215 nm]. After solvent evaporation to
dryness, [13C3]exemestane (52 mg; 0.038 mmol) was recovered
as a white solid with a chemical purity >98% (determined
by HPLC; see analytical method; Rt = 7.9 min) and an isotopic
purity of 99 atom % 13C. MS (ESI-MS): m/z 300 ([MH]+). 1H
NMR (400 MHz, DMSO-d6): ı ppm 0.86 (s, 3 H, CH3-18) 1.12 (s,
3 H, CH3-19). 1.17–1.33 (m, 2H, H12 ax, H9), 1.39 (m, 1H, H14),
1.51–1.73 (m, 3H, H11 ax, H12 eq, H15 ␤), 1.80–1.94 (m, 4H, H11
eq, H7 ax, H8, H15 ␣), 2.02 (ddd, J = 19.08, 9.21, 9.02 Hz, 1 H,
H16 ␣) 2.42 (dd, J = 19.14, 8.41 Hz, 1 H, H16 ␤) 2.62 (dd, J = 9.08,
5.18 Hz, 1 H, H7 eq) 5.04 (d, JH–13C = 159.1 Hz, 2 H, CH2-20) 5.99
(d, JH–13C = 162.1 Hz, 1 H, H4) 6.16 (m, 1 H, H2) 7.25 (t, J = 10.06 Hz,
JH–13C = 10.2 Hz 1 H, H1). 13C NMR (125 MHz, DMSO-d6): ı ppm
12.8 (C18), 19.1 (C19), 20.7 (C15), 21.3 (C11), 30.5 (C12), 34.2 (C8),
34.8 (C16), 37.9 (C7), 43.9 (C10), 47.5 (C13), 48.7 (C9), 49.6 (C14),
112.1 (d, J = 3.6) (C20), 121.7 (dt, J = 53.3, 3.6) (C4), 127.4 (C2),
145.9 (C6), 155.3 (C1), 167.6 (C5), 185.1 (d, J = 53.3) (C3), 219.4
(C17).
2.7.
6-[13C]Methylenandrost-[3,4-13C2]4-ene-
3,17-dione (8)
Anhydrous triethyl orthoformate (0.24 ml, 1.44 mmol) and
p-toluenesulfonic acid monohydrate (p-TsOH; 9.1 mg,
0.047 mmol) were added under nitrogen to a solution of
the intermediate (7) (235 mg, 0.82 mmol) in dry THF (2.8 ml)
and absolute ethanol (0.25 ml) and the reaction mixture
was stirred at 40 ◦C for about 1 h. After cooling to room
temperature, N-methylaniline (97 ␮l, 0.9 mmol) and a 20%
[
13C]formaldehyde aqueous solution (0.225 ml, 1.47 mmol)
were introduced into the reaction flask and the mixture was
stirred at 40 ◦C for about 2 h. As soon as the conversion was
completed (determined by TLC on silica gel; n-hexane:EtOAc
1:1 by volume), the mixture was cooled to room temperature,
37% HCl was added (0.65 ml) and the stirring was continued
for further 4 h. The obtained brown solution was transferred
into a separating funnel with water (40 ml) and the mix-
ture was extracted with EtOAc (3 ml × 30 ml). The combined
organic phases were washed with 0.1 N HCl (3 ml × 20 ml),
brine (2 ml × 20 ml) and dried over Na2SO4. After solvent
evaporation, the oily crude intermediate (8) was purified
by flash chromatography on silica gel using a mixture n-
hexane:EtOAc 4:1 by volume as the eluting solvent system.
The collected fractions were combined as appropriate and
3.
Results and discussion
Standard requirements of an acceptable IS usually include
a molecular weight at least three mass unit higher than
that of the non-labelled material and stability of the labels
during sample preparation and analyses. In case of exemes-
tane, deuterium and 18O were discarded due to possible
exchange reactions during sample preparation as well as
possible formation of ions interfering with the quantita-
tion of the analyte during the ionization processes in the
mass spectrometer. Therefore, the introduction of three 13C
was considered. In the literature several preparations of

steroids 7 3 ( 2 0 0 8 ) 760–764
763
Fig. 2 – Synthesis of carbon-13 labelled exemestane. Reagents and conditions: (a) BzCl, Pyridine, toluene, reflux, 20 h; (b)
KMnO4, KIO4, K2CO3, tBuOH, 40 ◦C, 5 h; (c) HClO4, Ac2O, EtOAc, rt, 1 h; (d) 13CH313COCl ((CH3)3Si)2N-Li, THF, −78 ◦C; (e) KOH,
MeOH, H2O, reflux, 20 h; (f) PCC, CH2Cl2, rt, 5 h; (g) 1. Triethylorthoformate, THF, EtOH, p-TsOH, 40 ◦C 1 h 2. N-methylaniline,
H13CHO, 40 ◦C, 2 h; (h) DDQ, p-TsOH, dioxane, reflux, 1 h.
carbon-13 labelled testosterone [7–10] are reported involv-
ing the partial degradation of the ring A of the non-labelled
testosterone followed by reconstruction of the ring using
suitable carbon-13 labelled synthons. According to these pro-
cedures up to five carbon-13 atoms can be introduced in
the ring A of testosterone. However, when considering the
preparation of [13C3]exemestane, the commercial availabil-
ity at reasonable prices of both [13C2]acetyl chloride and
tion of the 17-hydroxyl group of testosterone (1) by reaction
with benzoylchloride, the A-ring of (2) was opened by oxida-
tion with KMnO4/KIO4. The obtained ketoacid (3) was treated
with HClO4 giving the lactone (4). The reaction of (4) with
lithium bis(trimethylsilyl)amide at −78 ◦C followed by treat-
ment with [13C2]acetyl chloride, afforded the intermediate (5).
The rearrangement of (5) to the desired [13C2]testosterone (6)
was obtained at reflux in methanol/water in the presence of
KOH. The conversion of (6) to the labelled androstenedione
(7) was achieved by treatment with pyridinium chlorochro-
mate in methylene chloride. The reaction of (7) with the
commercially available [13C]formaldehyde in the presence of
N-methylaniline afforded the intermediate (8). The treatment
of (8) with 2,6-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
in dioxane in the presence of p-toluenesulfonic acid gave
[
13C]formaldehyde and the simplicity of synthetic meth-
ods indicated the synthesis of [13C2]testosterone followed
by the introduction of the required third carbon-13 in the
6-methylene group as the most convenient approach. Accord-
ing to this procedure, the preparation of [13C3]exemestane
was accomplished in eight steps starting from testosterone
(1). The synthetic pathway is shown in Fig. 2. After protec-

764
steroids 7 3 ( 2 0 0 8 ) 760–764
the crude [13C3]exemestane. After purification by preparative
HPLC, [13C3]exemestane was obtained >98% chemically pure
and with an isotopic purity of 99 atom % 13C. The overall chem-
ical yield was approximately 10% from (1). The compound
obtained was suitable for use as internal standard to develop a
convenient LC–MS assay [11], which was then used to measure
exemestane levels in a number of clinical studies [12–15].
new irreversible aromatase inhibitor, using liquid
chromatography–thermospray mass spectrometry. J Mass
Spectrom 1995;30:693–7.
[6] Persiani S, Broutin F, Cicioni P, Stefanini P, Strolin Benedetti
M. Determination of the new aromatase inhibitor
exemestane in biological fluids by automated
high-performance liquid chromatography followed by
radioimmunoassay. Eur J Pharm Sci 1996;4:331–40.
[7] Yuan S. Synthesis of 3,4-13C2 steroids. Steroids
1982;39:279–90.
[8] Zomer G, Wyberg H, Drayer NM. [1,2,3,4-13C] Testosterone
and [1,2,3,4-13C] estradiol. Steroids 1984;44:283–92.
[9] Joubert C, Beney C, Marsura A, Cuong LJ. Synhesis of labelled
Acknowledgement
The authors wish to thank Dr. Nicoletta Colombo, Analytical
Chemistry Unit at Nerviano Medical Sciences (Nerviano, Italy),
for NMR spectroscopy.
[
13C6] testosterone and [13C5] 19-nortestosterone. Labelled
Comp Radiopharm 1995;36:745–54.
[10] Kockert K, Vierhapper FW. Novel partial synthetic
approaches to replace carbons 2,3,4 of steroids. A
methodology to label testosterone and progesterone with
13C in the steroid A ring. Part 2. Tetrahedron 2000;56:
9967–74.
r e f e r e n c e s
[11] Cenacchi V, Baratte` S, Cicioni P, Frigerio E, Long J, James C. J
Pharm Biomed Anal 2000;22:451–60.
[12] Mauras N, Lima J, Patel D, Rini A, di Salle E, Kwok A, et al.
Pharmacokinetics and dose finding of a potent aromatase
inhibitor, Aromasin (exemestane), in young males. J Clin
Endocrinol Metab 2003;88:5951–6.
[13] Rivera E, Valero V, Francis D, Asnis AG, Schaaf LJ, Duncan B,
et al. Pilot study evaluating the pharmacokinetics,
pharmacodynamics, and safety of the combination of
exemestane and tamoxifen. Clin Cancer Res 2004;10:1943–8.
[14] Hutson PR, Love RR, Havighurst TC, Rogers E, Cleary JF. Effect
of exemestane on tamoxifen pharmacokinetics in
postmenopausal women treated for breast cancer. Clin
Cancer Res 2005;11:8722–7.
[15] Traiana TA, Poggesi I, Robson M, Asnis A, Duncan BA, Heerdt
A, et al. Pharmacokinetics and tolerability of exemestane in
combination with raloxifene in postmenopausal women
with a history of breast cancer. Breast Cancer Res Treat 2007
[Epub ahead of print].
[1] Coombes RC, Kilburn LS, Snowdon CF, Paridaens R, Coleman
RE, Jones SE, et al. Survival and safety of exemestane versus
tamoxifen after 2–3 years’ tamoxifen treatment (Intergroup
Exemestane Study): a randomised controlled trial. Lancet
2007;369:559–70.
[2] Kaufmann M, Bajetta E, Dirix LY, Fein LE, Jones SE, Zilembo
N, et al. Exemestane is superior to megestrol acetate after
tamoxifen failure in postmenopausal women with advanced
breast cancer: results of a phase III randomized double-blind
trial. J Clin Oncol 2000;18:1399–411.
[3] Goss P, von Eichel L. Summary of aromatase inhibitor trials:
the past and future. J Steroid Biochem Mol Biol
2007;106:40–8.
[4] Breda M, Pianezzola E, Strolin Benedetti M. Determination of
exemestane, a new aromatase inhibitor, in plasma by
high-performance liquid chromatography with ultraviolet
detection. J Chromatogr 1993;620:225–31.
[5] Allievi C, Zugnoni P, Strolin Benedetti M, Dostert P.
Determination of plasma levels of exemestane (FCE 24304) a