Synthesis and radiosynthesis of 17α -[p-(iodophenylethynyl)]estra-3,17β-diols

Article abstract of DOI:10.1515/znb-2003-0813

Estrones have been subjected to an addition reaction with [(4-ethynylphenyl)azo]pyrrolidine to provide 17 α -{4-[(pyrrolidin-1-yl)-azo]phenylethynyl}estra-3,17 β-diols. A subsequent iodination with iodotrimethylsilane, produced in situ from chlorotrimethy

Full text of DOI:10.1515/znb-2003-0813

Synthesis and Radiosynthesis of
17α-[p-(Iodophenylethynyl)]estra-3,17β-diols
Jian Wanga, Masataka Watanabea, Shuntaro Matakab, Thies Thiemannb, Goreti Ribeiro
Moraisc, Fernanda Roleirac, Elisiario Tavares da Silvac, and Cristina Melo e Silvad
a Interdisciplinary Graduate School of Engineering Sciences and
b Institute of Advanced Material Study, Kyushu University, Fukuoka 816-8580, Japan
c Centro de Estudos Farmaceuticos, Faculdade de Farmacia, Universidade Coimbra,
P-3000-295 Coimbra, Portugal
d Instituto Tecnologico e Nuclear, Estrada Nacional 10, P-2685 Sacavem, Portugal.
E-mail: cmelo@itn.mces.pt
Reprint requests to Dr. Th. Thiemann. E-mail: thies@cm.kyushu-u.ac.jp
Z. Naturforsch. 58b, 799 – 804 (2003); received April 9, 2003
Estrones have been subjected to an addition reaction with [(4-ethynylphenyl)azo]pyrrolidine to
provide 17 α-{4-[(pyrrolidin-1-yl)-azo]phenylethynyl}estra-3,17 β-diols. A subsequent iodination
with iodotrimethylsilane, produced in situ from chlorotrimethylsilane and NaI, leads to 17α-(p-
iodophenylethynyl)estra-3,17 β-diols. This reaction can also be carried out with Na125I to give ra-
diolabelled estra-3,17 β-diols, which are potentially useful radiodiagnostic agents for the detection
of estrogen positive breast cancer. The stability of the radiolabelled compounds is exemplified in a
stability study of 3-O-methyl 17 α-(p-[125I]iodophenylethynyl)estra-1,3,5(10),6-tetraene-17 β-ol in
acetonitrile.
Key words: Steroids, Radioiodination, Triazenes
Radiolabelled estrogens and antiestrogens with a radioiodination will be given and exemplary results of
high selective binding affinity to the estrogen receptor studies on the stability of the radiolabelled compounds
ER α have been shown to have potential as radiodiag- will be disclosed.
nostica for the detection of minimal estrogen positive
breast cancer [1]. Although a number of nuclides have
been forwarded as radiolabels in this context, such as
99mTc [2] and 18F [3], 123I [4] and 125I [5] remain
attractive because they are medically well-studied ra-
dioisotopes for radioimaging. Previously, the authors
have investigated the iodovinyl derivatives 1a and
1b [6]. While especially the Z-iodovinyl compound
1b showed a promising biodistribution in mice [6a], it
was evident that the iodovinyl compounds metabolized
too quickly, as a large amount of iodine was found in
the thyroid rather quickly. The stability of the corre-
sponding iodoethynyl derivatives 1c is also low, even
in vitro.
As ultimately compounds 2 are to carry a radiolabel
[
125I], the iodination has to be the last step of the syn-
thetic sequence and the iodo function cannot be intro-
duced with the phenylethynyl moiety, for instance by
addition of the readily available p-iodophenylacetylene
to estrones 7. A critical point in the iodination is the
sensitivity of the C-17 hydroxy group, which easily
undergoes dehydration. Thus, it was not possible to ex-
change other groups at the para position of the phenyl
unit, such as the trimethylsilyl group (e. g., with NCS,
NaI, AcOH, or with ICl, CH2Cl2 [9] or with ben-
zyltrimethylammonium dichloroiodate [BTMAICl2],
ZnCl2, AcOH) or the tributylstannyl function (I2, THF)
to an iodo functionality.
Additionally, although described for a number of
It is known that a pyrrolidinylazo group as a tri-
compounds [7] in our hands, the radioiodination of azene moiety can be exchanged effectively to an iodo
a terminal acetylene did not proceed to satisfaction. function. Thus, an introduction of the iodo function in
Recently, the preparation of 17 α-(p-iodophenyl)estra- 17 α-{4-[(pyrrolidin-1-yl)-azo]phenylethynyl}estra-3,
3,17 β-diols has been reported [8]. In the following, 17 β-diols of type 8 seemed viable. The starting ma-
the synthesis of 17α-(p-iodophenylethynyl)estra-3,17 terial should be easily accessible via addition of the
β-diols 2 will be described, representative examples of [(4-ethynylphenyl)azo]pyrrolidine 6 to estrones 7. [(4-
c
0932–0776 / 03 / 0800–0799 $ 06.00 ꢀ 2003 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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J. Wang et al. · Synthesis and Radiosynthesis of 17α-[p-(Iodophenylethynyl)]estra-3,17β-diols
Fig. 1. Iodinated estradiols as potential di-
agnostic agents for estrogen positive breast
cancer.
3-O-methyl-equiline (7c) and with 3-O-methyl-estra-
1,3,5(10),6-tetraen-17-one (7d). Especially derivatives
of 7b and 7c have been used in combination with
other substances in hormone replacement therapy and
are known to bind to the estrogen receptor. The
iodophenylethynyl derivatives of 7d are analogs to the
iodovinyl compounds 1a and 1b, which have been
studied by the authors in in vivo biodistribution exper-
iments [6a].
Iodination of the triazenes is carried out with in situ
produced iodotrimethylsilane (Scheme 3). It is not an
advantage to use an excess of reagent as then the 17
β-hydroxy group will be silylated. Most likely, the
mechanism of the iodination involves an initial attack
of the iodotrimethylsilane on the lone electron pair of
the pyrrolidine nitrogen followed by a nucleophilic dis-
placement reaction by the iodide (Scheme 4).
Scheme 1. Synthesis of 6, reaction conditions: i. NaNO2,
HCl; ii. pyrrolidine, KOH (64%); iii. (Ph3P)2PdCl2, Et3N,
48 h (91%); iv. KOH, MeOH, 5 h (58%).
The 3-hydroxy group of equilinine can be protected
as a silyloxy group without affecting the addition of 6.
After the addition and subsequent iodination, the sily-
loxy group is cleaved under fluoride catalysis in the
usual way (Scheme 5).
Ethynylphenyl)azo]pyrrolidine 6 itself can be synthe-
sized from p-bromoaniline (3) in a number of ways in-
volving the transformation of the aniline function to
the triazene, a Sonogashira coupling and a desilylation
of the ethynyl terminus. While the order of the Sono-
gashira coupling and the triazene formation can be
changed, i. e., the triazene formation can be performed
as the second or even as the final step, the best yield of
6 is achieved by initial reaction of the aniline 3 to the
triazene 4, subsequent ethynylation to 5 and desilyla-
tion. The yield of the Sonogashira coupling could be
increased from 67% [12] to 91% by changing the pub-
lished reaction conditions [(Ph3P)2PdCl2, CuI, Et2NH,
rt] to (Ph3P)2PdCl2, Et3N, CuI, 70 ◦C, 2 d (Scheme 1).
The radioiodination [14] can be carried out in a sim-
ilar way as the cold iodination (Scheme 6).
The stability of the iodinated compounds in ace-
tonitrile is good. This has also been determined for
the radiolabelled compounds by RP-HPLC analysis of
2a−125 I in acetonitrile 6 days and 21 days after la-
belling (Fig. 2). Stability tests in PBS, PBS/PEG, in
vitro binding affinity assays to ER α and in vivo biodis-
tribution studies of the compounds are underway.
The addition of 6 to ketones 7 proceeds in fair yields
(Scheme 2). The above mentioned reactions were car-
ried out with methoxyestra-1,3,5(10)-triene 7a, but
also with estranes with further insaturations within the
Experimental Section
General remarks: Synthesis. Melting points were mea-
sured on a Yanaco microscopic hotstage and are uncorrected.
B-ring such as with 3-O-methyl-equilinine (7b), with Infrared spectra were measured with JASCO IR-700 and
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J. Wang et al. · Synthesis and Radiosynthesis of 17α-[p-(Iodophenylethynyl)]estra-3,17β-diols
801
Scheme 2. Synthesis of 8.
Fig. 2. Stability of 2a−125 I,
acetonitrile, shown by reversed
phase HPLC analysis (see Exp.
Section for details).
Nippon Denshi JIR-AQ2OM instruments. 1H and 13C NMR tion (KOH, DMSO, MeI) [14] of commercial material.
spectra were recorded with a JEOL EX-270 spectrometer. 3-O-Methylequilinine (7c) has been obtained from
The chemical shifts are relative to TMS (solvent CDCl3, un- 3-O-methylestrone (7a) via 3-O-methyl-9-hydroxy-
less otherwise noted). Mass spectra were measured with a 6-ketoestra-1,3,5(10)-trien-17-one
JMS-01-SG-2 spectrometer (EI, 70 eV). Column chromatog- dimethyl-1’,3’-dioxane]) and
17,17-(2’-[5’,5’-
3-O-methyl-6-ketoestra-
raphy was carried out on Wakogel 300. All experiments were 1,3,5(10),9(11)-tetraen-17-one 17,17-(2’-[5’,5’-dimethyl-
purged with argon at the start.
1’,3’-dioxane]) in a four-step procedure (i. KMnO4,
Adogen, benzene, reflux; ii. p-TsOH, neopentylglycol,
benzene, reflux; iii. NaBH4, MeOH; iv. p-TsOH, benzene,
reflux) [15].
All chemical reagents were of reagent grade. THF
was dried over sodium ketyl. 3-O-Methylestrone (7a)
and 3-O-methylequiline (7b) were obtained by methyla-
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802
J. Wang et al. · Synthesis and Radiosynthesis of 17α-[p-(Iodophenylethynyl)]estra-3,17β-diols
Scheme 3. Synthesis of 2, reaction conditions: i. TMSCl, NaI, CH CN, 1 h.
3
Scheme 4. Mechanism of the iodination.
Radiosynthesis: Acetonitrile was dried over P2O5 under washed with water and brine and subsequently dried over
N2. The radioiodinated product was purified by reversed anhydrous MgSO4. Concentration of the solution in vacuo
phase HPLC on a C18 Nucleosil column (250/4 10 µm) and column chromatography (hexane/ether 1:1) gave 5
with a flow of 1 ml/min using a gradient of acetonitrile-water [see also lit. 12] (972 mg, 91%) as colorless crystals; IR
(70 : 30 to 100 : 0). The compounds were detected by their (KBr) ν = 2146, 1650, 1596, 904, 759 cm−1. – 1H NMR
UV absorption at 254 nm with an UV detector SPD-10AV (67.8 MHz, CDCl3) δ = 0.24 (s, 9H, 3 CH3), 2.00 (m, 4H),
3
from Shimadzu and by their γ-radiation using a γ-Radioflow 3.78 (bs, 4H, 2 CH2N), 7.32 (d, 2H, J = 8.6 Hz), 7.39 (d,
detector LB 509 from Berthold. The commercially available 2H, 3J = 8.6 Hz). – 13C NMR (67.8 MHz, CDCl3, DEPT 90,
[
125I] in NaOH solution (pH = 7.0 – 11) was used as pur- DEPT 135) δ = 0.07 (3C, SiMe3), 23.72 (2C, CH2), 49.15
chased from Amersham.
(2C, NCH2, very broad signal), 93.56 (C≡), 105.68 (C≡),
117.68 (Cquat), 120.09 (CH), 132.68 (CH), 151.19 (Cquat). –
MS (EI, 70 eV): m/z (%) = 271 (6), 233 (100), 173 (20), 121
(7), 77(3).
Typical procedures: [(4-Trimethylsilylethynylphenyl)-
azo]pyrrolidine (5): To a solution of [(4-bromophenyl)-
azo]pyrrolidine 4 (1.0 g, 3.94 mmol) in anhydrous Et3N
(10 ml) was added trimethylsilylacetylene (463 mg,
3-O-Methyl-17α-{4-[(pyrrolidin-1-yl)-azo]phenylethy-
4.73 mmol) and (PPh3)2PdCl2 (55 mg, 0.08 mmol) and nyl}estra-1,3,5(10)-trien-3,17β-diol (8a): To a solution of
CuI (15 mg, 0.08 mmol) under an inert atmosphere. The N-[p-ethynylphenylazo]pyrrolidine (6) (88 mg, 0.44 mmol)
◦
medium was heated at 70 C for 2 d. Then, the solution in dry THF (2 ml) was added LDA (2M LDA in a mixture
was cooled and poured into water (50 ml) and extracted of heptane / THF / ethylbenzene, 0.24 ml, 0.48 mmol) at
with dichloromethane (3 × 50 ml). The organic layer was −78 ◦C under an Ar atmosphere. The resulting solution
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J. Wang et al. · Synthesis and Radiosynthesis of 17α-[p-(Iodophenylethynyl)]estra-3,17β-diols
803
Scheme 5. Synthesis of 2d, reaction conditions: i. LDA, THF (6); ii. TMSCl, NaI, Et N, 1 h (32%); iii. n-Bu4NF, THF,
3
0 ◦C, 15 min (84%).
Scheme 6. Radiosynthe-
sis of 2a−125 I, reac-
tion conditions: i. TM-
SCl, Na125I, CH3CN, ∆,
3 h.
was stirred at −78 ◦C for 1 h. Then, 3-O-methylestrone 8d (50 mg, 0.10 mmol) in acetonitrile (3.3 ml) was added
(7a) (114 mg, 0.4 mmol) was added to the solution. The and the mixture was stirred for 1 h. After hydrolysis (sat.
reaction mixture was warmed slowly to r.t. and stirring NaHCO3, 10 ml) and evaporation of the acetonitrile, the
was continued for 24 h. Thereafter, the mixture was poured residue was extracted with dichloromethane (2 × 15 ml).
into water (15 ml) and extracted with ether (3 × 20 ml). The organic phase was washed with brine and dried over
The organic phase was dried over anhydrous MgSO4 and anhydrous MgSO4. Concentration of the solution in vacuo
concentrated in vacuo. The resulting residue was subjected and column chromatography on silica gel (hexane/ether 1:1)
to chromatography on silica gel (hexane/ether 2:1) to give gave 2c (28 mg, 53%) as a colorless solid; IR (KBr): ν =
8a (102 mg, 53%) as colorless needles, m.p. 237 – 238 ◦C. – 3434, 3026, 2356, 1603, 1147, 1037, 1005, 818 cm−1. – 1H
IR (KBr): ν = 3436, 2928, 2866, 1610, 1498, 1423, 1398, NMR (270 MHz, CDCl3): δ = 0.93 (s, 3H, CH3), 1.32 – 2.45
3
1340, 1315, 843, 754 cm−1. – 1H NMR (270 MHz, CDCl3): (m, 12H), 3.80 (s, 3H, OCH3), 6.00 (dd, 1H, J = 9.6 Hz,
δ = 0.93 (s, 3H, CH3), 1.35 – 2.45 (m, 14H), 2.02 – 2.05 4J = 1.7 Hz, C6), 6.46 (d, 1H, 3J = 9.6 Hz, 3J = 2.6 Hz, C7),
(m, 4H), 2.87 (m, 2H), 3.78 (s, 3H, OCH3), 3.70 – 3.93 (bs, 6.65 (d, 1H, 4J = 2.6 Hz, C4), 6.75 (dd, 1H, 4J = 2.6 Hz,
4
3
3
3
4H), 6.63 (d, 1H, J = 2.6 Hz), 6.71 (dd, 1H, J = 8.9 Hz, 3J = 8.6 Hz), 7.15 (d, 2H, J = 8.6 Hz), 7.18 (d, 1H, J =
4J = 2.6 Hz), 7.23 (d, 1H, J = 8.9 Hz), 7.35 (d, 2H, J = 8.6 Hz), 7.63 (d, 2H, 3J = 8.6 Hz). – 13C NMR (67.8 MHz,
3
3
8.6 Hz), 7.41 (d, 2H, J= 8.6 Hz). – 13C NMR (67.8 MHz, CDCl3): δ = 12.63, 22.79, 24.24, 32.60, 38.90, 39.32, 41.78,
3
CDCl3): δ = 12.90, 22.93, 23.77, 26.50, 27.24, 29.87, 30.96, 48.07, 48.16, 55.31, 80.18, 85.22, 94.12, 111.79, 122.32,
33.06, 39.06, 39.51, 43.61, 47.60, 49.68, 55.18, 80.39, 124.29, 127.96, 128.28, 131.30, 131.62, 132.70, 133.15 (2C),
86.40, 92.34, 111.46, 113.75, 119.12, 120.21 (2C), 126.39, 135.34, 137.45 (2C), 158.13. – MS (FAB, 3-nitrobenzyl al-
132.38 (2C), 132.61, 138.00, 151.17, 157.37. – MS (FAB, cohol): m/z (%) = 510 (30), 329 (37), 309 (27), 273 (29), 242
3-nitrobenzyl alcohol): m/z (%) = 484 (2.1) [MH+]. – HRMS (26), 178 (18). – HRMS (FAB) Found: 510.1056. Calcd. for
(Found) 484.2963. Calcd. for C31H38O2N3: 484.2964. – C17H27IO2: 510.1056.
3-O-Methyl 17α-(p-[125I]iodophenylethynyl)estra-1,3,5
C31H3702N3 (483.6): calcd. C 76.98; H 7.71; N 8.69; found:
C 77.09; H 7.84; N 8.29.
(10),6-tetraene-17β-ol (2a−125I): To a solution of 8d
3-O-Methyl 17α-(p-iodophenylethynyl)estra-1,3,5(10),6- (120 µg) in dry acetonitrile (50 µl) were added 50 µl of
tetraene-3,17 β-diol (2c): Under an argon atmosphere, NaI a trimethylsilyl chloride solution (5 µl of dry trimethylsi-
(70 mg, 0.47 mmol) was dissolved in dry acetonitrile lyl chloride in 5 ml of acetonitrile) and 15 µl (ꢀ 600 µCi)
(0.6 ml). TMSCl (25 mg, 0.23 mmol) was added and the so- of sodium iodide (125I). The reaction mixture was diluted in
lution was stirred at r.t. for several minutes. A solution of 70 µl of acetonitrile and the capped vial was stirred at 80 –
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804
J. Wang et al. · Synthesis and Radiosynthesis of 17α-[p-(Iodophenylethynyl)]estra-3,17β-diols
◦
90 C for 2.5 h in an oil bath. The mixture was purified by radioiodinated compound were eluted in a volume of ∼ 2 ml.
reversed phase HPLC and the fractions corresponding to the The radiochemical yield was 40%.
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