Radioiododestannyllation of novel GHB neuroreceptor ligands

Article abstract of DOI:10.1002/jlcr.1767

A series of 4-substituted gamma-hydroxybutyric acid (GHB) analogues have previously been synthesized and characterized for binding to specific high-affinity GHB sites in rat brain cortical membranes.^1,2 Within this series some halogenated analogues displayed unprecedented high affinity and selectivity for GHB sites, and would potentially be valuable as pharmacological tools if radiolabelled. Here we present the iodine-125 labelling of one of these analogues by radioiodo-destannylation. Copyright

Full text of DOI:10.1002/jlcr.1767

Abstracts
Published online 11 May 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1767
10th International Symposium on the
Synthesis and Applications of Isotopes and
Isotopically Labelled Compounds—Wiley
Young Scientist Awards
Session 10, Tuesday, June 16, 2009
SESSION CHAIRS: LARRY JONESa AND RHYS SALTERb
aNovartis Pharmaceuticals, USA
bJohnson & Johnson, USA
Abstract: Addresses by the three of the four Wiley Young Scientist Award winners are highlighted.
Keywords: Anthocyanins; polyphenols; Radioiodo-de-stannylation; Iodine-125; Steroid fatty acid esters; Ionic liquid; Microwave
STUDIES TOWARDS THE SYNTHESIS OF 13C-LABELLED ANTHOCYANINS
LAURA J. MARSHALLa, KARL M. CABLEb AND NIGEL P. BOTTINGa
aSchool of Chemistry, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK
bGSK Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
Summary: The anthocyanins are a class of polyphenols found in nature, which are widely distributed throughout the plant kingdom
and are thought to possess antioxidant properties. Methodology previously developed in our group for the regioselective
placement of 13C-atoms into aromatic rings is being applied to the synthesis of 13C-labelled anthocyanins–namely cyanidin-3-
glucoside and delphinidin-3-glucoside.
Keywords: anthocyanins; cyanidin; delphinidin; polyphenols
Introduction: Over 500 different anthocyanins have been isolated from plants, and are found mainly in flowers, fruits and
vegetables. They are responsible for the colours observed in these species, which can range from red to blue and purple. This
change in colour is due to the difference in structure observed in response to pH changes, as shown in Scheme 1.1 The
anthocyanins are based on the core structure of the flavylium cation, which itself is based on the benzopyrylium cation. They
possess a positive charge, most commonly with ClÀ as the counterion. Chemically, the anthocyanin group consists of the sugar-free
anthocyanidins and the anthocyanin glycosides which are water-soluble.
R1
R1
OH
R2
O
R5
R4
O
R5
R4
O
R2
O
O
R3
sugar
R3
sugar
Under acidic conditions (red) Under basic conditions (blue)
Scheme 1. Changes in anthocyanin structure due to change in pH.
The anthocyanins are powerful antioxidants, protecting the plant from radicals formed either by UV light or during metabolic
processes. Interest in anthocyanin pigments has intensified over recent years due to the possible health benefits to humans. One
crucial factor is that the antioxidant property is retained after consumption, and so fruits and vegetables containing anthocyanins
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Abstracts
are a good source of dietary antioxidants. Consumption of anthocyanin-containing foods may therefore help to prevent illnesses
such as coronary heart disease, cancer and strokes. This backs up a popular theory that the drinking of red wine can result in much
lower rates of coronary heart disease in countries such as France and Italy, where the consumption of red wine is higher than in
North American and North European countries–many of which have significantly higher rates of coronary heart disease.2
Other health benefits have been suggested, however the effects of anthocyanins on human health are still poorly understood.
Studies into the biological effects of natural products such as these require accurate quantification of the compounds in plasma
samples, with LC-MS and GC-MS being the common analytical techniques used. The accuracy and reproducibility of these
techniques is greatly improved by the use of stable isotope labelled standards for calibration, as the standard will have similar
chemical and physical properties to the analyte.3 New synthetic routes to isotopically labelled anthocyanins are therefore required.
For the isotope chemist, these new synthetic routes must also be applicable to the incorporation of isotopically-labelled atoms
through commercially available compounds. Isotopically-labelled small molecules are more widely available and cheaper than
uniformly-labelled aromatic rings, and their use can allow the regioselective incorporation of labelled atoms into the structure, as
previously demonstrated in work by our group.3
An efficient route towards the synthesis of two anthocyanins, namely cyanidin-3-glucoside 1 (found in strawberries) and
delphinidin-3-glucoside 2 (found in grapes), (Scheme 2) is being examined, with the unlabelled synthesis being carried out initially in
order to optimise the conditions. The synthetic route builds on the strategy developed in the synthesis of [1,3,5–13C3]gallic acid,3
with regioselective incorporation of 13C-atoms in the B-ring using either [1,3–13C2]acetone or triethyl [13C]orthoformate, as desired.
The third 13C-atom will be introduced using [13C]methyl iodide during the synthesis of the A-ring.
O H
C
O H
C
13
13
O H
O H
C l
Cl
13
13
HO
O
C
H O
O
CH
O H
O H
O H
O H
13
13
C
C
O
O
O
O
O H
O H
O H
O H
O H
H O
HO
1
2
Scheme 2. Final target compounds.
Results and Discussion: Our aim was to apply the synthetic route and labelling strategy used in the synthesis of [1,3,5–13C3]gallic
acid to the synthesis of our two target anthocyanins,3 the retrosynthetic analysis of which is shown in Scheme 3. Using this approach
the 13C-labels are situated in the A and B rings, rather than the central heterocyclic, C-ring. This means that such derivatives would
also have utility in studies for the investigation of the metabolism and pharmacokinetics of anthocyanins. The A and B rings usually
remain intact during metabolism, whereas the C-ring is broken down, so that 13C-labels in the C-ring are often lost.
OH
OAc
*
*
OH
O
H
Cl
AcO
X
HO
O
HO
OH
*
*
+
* X
OAc
OH
*
OH
O
O
O
O
OAc
OH
OH
O
OAc
OH
4
AcO
HO
1 or 2
6
*
HO
OH
O
*
OAc
OH
O
OEt
O
+
OAc
OAc
+
13
*
*
EtO OEt
*
OAc
6
5
RO
X
Br
OR
3
18
O
O
*
(* = 13C)
I CH3
Cl
OMe
Scheme 3. Retrosynthetic analysis of compounds 1 and 2.
Scheme 4 shows the preparation of the initial component 3 starting with synthesis of the 4H-pyran-4-one 4 from triethyl
orthoformate 5 and acetone 6 via the intermediate enone 7. The proposed 13C-labelled synthesis will make use of [1,3–13C2]acetone to
introduce the two 13C-atoms into the ring.3 Using our previously optimised procedure, the required phenol 4-hydroxyacetophenone 8
was produced from the reaction of the pyranone with the pronucleophile acetylacetone under basic conditions, followed by acidic
hydrolysis to yield the aromatic product in 95% yield.3 Bromination was achieved by one of two methods, which gave either mono- or
di-brominated 9 as required, with acetic acid/sodium acetate/bromine giving di-bromination in quantitative yield and
dichloromethane/bromine giving mono-bromination in quantitative yield.3 Subsequent substitution of bromine with hydroxyl groups
was carried out in aqueous sodium hydroxide in the presence of copper sulfate to give 10.3 Protection of the hydroxyl groups as the
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J. Label Compd. Radiopharm 2010, 53 315–321

Abstracts
benzyl ethers 11, produced the first requirement for purification during the synthesis of component 3.4 Bromination on the methyl
group using tetrabutylammonium tribromide and subsequent substitution of the bromine with a hydroxyl group followed by
purification gave component 3 in either the tri- or di-benzyloxy form as desired (29% tri-benzyloxy, 37% di-benzyloxy overall).5 This is
now being repeated with the use of 13C-labelled starting materials to give the labelling pattern as indicated in Scheme 4.
The synthesis of component 4 (Scheme 5) was then carried out, which began with the preparation of 13C-labelled resorcinol 13 as
previously developed in our group.6 The use of [13C]methyl iodide in the first stage of this synthesis gives the 13C-label at the 2 position
of the resorcinol. MOM protection7 of the two hydroxyl groups was then achieved without any problems to give 14 in 67% yield.
O
O
O
2x
b)
O
c)
H
2x
OEt
a)
d,e)
C
EtO OEt
EtO
OEt
*
*
EtO OEt
*
*
*
*
-
EtOBF3
6
O
5
*
*
7
4
OH
8
O
O
*
O
O
O
i)
f or g)
j)
OH
X
h)
k)
Br
*
*
*
*
*
*
*
*
*
BnO
X
BnO
Br
X
BnO
X
HO
X
OBn
OBn
OH
9
OBn
12
OH
10
3
11
Scheme 4. Preparation of component 3- indicates proposed point of 13C introduction and labelled positions. Reagents and Conditions: (a) BF3 Á OEt2, DCM; (b) iPr2Net,
acetone (or [1,3-13C2]acetone), quant; (c) Aq. HCl, EtOH, efflux, 24 h, quant; (d) Acetylacetone, KOtBu, tBuOH, reflux, 20 h; (e) 1 M HCl, reflux, 1 h (95%); (f ) AcOH, NaOAc, Br2
1 h (99%, X = Br); (g) DCM, Br2; (h) CuSO4 Á 5H2O, Aq. NaOH, reflux, 18 h (88% X = OH), 82% X = H); (i) BnBr, 18-crown-6, K2CO3, acetone, reflux, 6 h (69% X = OBn, 73% X = H);
( j) Tetrabutylammonium tribromide, DCM, MeOH, 3 h (99% X = OBn or H); (k) Sodium formate, EtOH, reflux, 18 h (52% X = OBn, 66% X = H).
Ã
*
O
O
O
O
a)
O
O
b,c)
*
Cl
d)
OMe
H3C
OMe
*
*
HO
OH
*
MOMO
OMOM
MOMO
OMOM
e)
f,g)
13
14
15
OH
O
H
O
H
*
*
*
MOMO
OMOM
MOMO
OMOM
HO
OH
h)
OTMS
OTMS
OH
17
16
4
Ã
Scheme 5. Studies towards the preparation of component 4 - indicates proposed point of 13C introduction and labelled positions. Reagents and Conditions: (a) CH3I (or
13CH3l), Li, Cul, Et2O (55%); (b) THF, KOtBu, reflux, 6 h; (c) Aq. HCl (74%); (d) 10% Pd/C, xylene, reflux, 3 h (20%); (e) MOMCl, DMF, Et2O, NaH (67%); (f) B2 Pin2, dtbpy,
[lr(OMe)(COD)]2, iso-hexane, reflux, 18 h; (g) Aq. oxone, acetone (61%); (h) TMSCI, pyridine, DMAP (54%).
The key step was the introduction of a third hydroxyl group meta to both MOM ethers (15), using a procedure originally reported
by Maleczka,8 and developed further in our group for this purpose. TMS protection to yield 16 was also straightforward. It is now
proposed that regioselective formylation of the TMS-protected compound (17) followed by global deprotection will yield
component 4, where the 13C-label will be present adjacent to the formyl group as indicated by the asterisk in Scheme 5.
O
OBn
OAc
OH
X
BnO
X
a)
O
*
*
OAc
OAc
OAc
*
*
OAc
BnO
O
Br
O
OAc
OAc
OBn
O
18
AcO
3
19
OH
OAc
HO
X
AcO
X
*
*
*
*
OAc
OAc
c)
b)
O
O
O
O
OAc
OAc
OAc
OAc
O
O
AcO
AcO
20
6
Scheme 6. Coupling of components 3 and 5 to give component 18- indicates proposed point of introduction of 13C label and labelled positions. Reagents and
Ã
˚
Conditions: (a) Hg(CN)2, 4 A mol. sieves, Na2SO4, toluene, reflux, 2 h (34% X = OBn, 47% X = H); (b) H2(g), 10% Pd/C, THF, 18 h (95% X = OH, 47%, X = H); (c) Acetic anhydride,
pyridine, 18 h (55% X = OAc, 10% X = H).
J. Label Compd. Radiopharm 2010, 53 315–321
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Abstracts
Scheme 6 shows the coupling of component 3 with sugar component 18–obtained from a-D-glucose and hydrogen bromide in
acetic acid. The optimal conditions for this coupling were found to require the use of mercuric cyanide in toluene under rigorously
anhydrous conditions9 to give 19 in 34% yield (X = OBn) and 47% yield (X = H). Deprotection of the benzyl groups using hydrogen in
the presence of palladium on carbon (20) followed by subsequent acetyl protection gave component 6. Once again, either the tri- or
di-acetoxy compounds could be obtained depending on the route followed in Scheme 1.
The final stage in the synthesis will involve the coupling of components 4 and 6, and subsequent cyclisation to construct the
anthocyanin ring system. Final deprotection of all the hydroxyl groups, on the benzene rings and the sugar, gives the target
anthocyanins 1 and 2 as shown in Scheme 7 below.9 Currently this has yet to be achieved unlabelled, but the synthesis will follow
good literature precedent.9 When completed in 13C-labelled form, the target compounds will possess three 13C-labels–introduced by
the use of [1,3–13C2]acetone and [13C]methyl iodide–one in the A-ring and two in the B-ring.
OH
*
OAc
OH
O
H
Cl
AcO
X
HO
O
*
HO
OH
*
*
X
OH
*
+
OAc
*
O
O
O
OAc
OH
OH
O
O
OAc
OH
OH
AcO
HO
(X=H)
2 (X=OH)
4
1
6
Scheme 7. Proposed coupling of components 6 and 4 to give target compounds 1 and 2. (Ã indicates proposed position of labelling).
Conclusion: Studies towards the synthesis of 13C-labelled anthocyanins cyanidin-3-glucoside 1 and delphinidin-3-glucoside 2 are
underway, with the synthesis of the unlabelled target compounds nearing completion. Optimisation of the route has led to high
yields in the majority of steps, with very little purification being required in the synthesis of component 3. Both of these factors are
beneficial to syntheses employing 13C-labelled materials as it ensures the minimum amount of losses of material. The application of
our previous methodology3 to this synthesis will allow the regioselective placement of 13C-atoms into the anthocyanin core, thus
producing multiply-labelled anthocyanins for use in biological testing involving GC-MS and LC-MS analysis.
Acknowledgements: Financial support from the BBSRC and GlaxoSmithKline is gratefully acknowledged.
References
[1] J. A. Joule, G. F. Smith, Heterocyclic Chemistry, 2nd Edition ed.; Van Nostrand Reinhold (UK): Wokingham, 1987.
[2] S. Renaud, The Lancet 1992, 355, 48.
[3] L. J. Marshall, K. M. Cable, N. P. Botting, Org Biomol Chem 2009, 7, 785–788.
[4] Q. Zhang, N. P. Botting, Tetrahedron 2004, 60, 12211–12216.
[5] C. Ma, W. M. Kwok, W. S. Chan, Y. Du, J. T. W. Kan, P. H. Toy, D. L. Phillips, J Am Chem Soc 2006, 128, 2558–2570.
[6] M. F. Oldfield, L. Chen, N. P. Botting, Tetrahedron 2004, 60, 1887–1893.
[7] R. C. Ronald, M. R. Winkle, Tetrahedron 1983, 39, 2031–2042.
[8] R. E. Maleczka, F. Shi, D. Holmes, M. R. Smith, J Am Chem Soc 2003, 125, 7792–7793.
[9] O. Dangles, H. Elhajji, Helv Chim Acta 1994, 77, 1595–1610.
RADIOIODODESTANNYLLATION OF NOVEL GHB NEURORECEPTOR LIGANDS
MARTIN PEDERSENb, SIGNE HØGa, PETRINE WELLENDORPHa AND LARS MARTINYb
aDepartment of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
bThe Hevesy Laboratory, Radiation Research Division, Risø, The Technical University of Denmark, 399 Frederiksborgvej, DK-4000 Roskilde, Denmark.
Abstract: A series of 4-substituted gamma-hydroxybutyric acid (GHB) analogues have previously been synthesized and characterized
for binding to specific high-affinity GHB sites in rat brain cortical membranes.1,2 Within this series some halogenated analogues
displayed unprecedented high affinity and selectivity for GHB sites, and would potentially be valuable as pharmacological tools if
radiolabelled. Here we present the iodine-125 labelling of one of these analogues by radioiodo-destannylation.
Keywords: Radioiododestannyllation; GHB; iodine-125; chloramines-T
Introduction: Gamma-hydroxybutyric acid (GHB) is a neuroreceptor ligand with weak affinity for gamma-aminobutyric acid
subtype B (GABAB) receptors and high affinity for specific GHB sites which to date still have unclear pharmacological and physiological
importance. GHB is
The compound is listed as an illicit drug because of its drug-abuse potential (so-called: Fantasy) and examples of uses as a date
a
metabolite of GABA and is found in micromolar concentrations in the mammalian brain.3
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Abstracts
rape drug.4 GHB is also a registered drug for the treatment of excessive daytime sleepiness and cataplexy associated with the disorder
narcolepsy,5,6 formulated as the sodium salt (Xyrems).7 Because of the public concern relating to the misuse of GHB along with the
pharmaceutical use and its natural occurrence in the mammalian brain, investigation of its exact mechanism of action is needed.
Developing a suitable radioligand with binding affinity to GHB sites is an important tool for the further understanding of the GHB
action and mechanism and we therefore set out to develop a radioligand with high affinity for GHB receptors. Several cold
analogues were tested and the iodine-127 version of 2 (Scheme 1) showed superior binding to GHB receptor rich regions of the rat
brain. The iodine-125 compound was prepared according to the following.
Radioiododestannyllation has proved to be a widely applicable method for labelling organic molecules with radioiodine.8 Electrophilic
iodine can be created (as Id1-CldÀ) in situ by the reaction of NaI with the oxidant Chloramine-T.9 In this example we have used a
palladium coupling reaction for the synthesis of the organostannate using hexa-n-butylditin and tetrakistriphenylphosphine-palladium.
Results and Discussion: Iodine-125 labelled compound 2 was readily prepared and is currently undergoing pharmacological
evaluation. The tributyl-stannate precursor in its lactone form 1 was reacted with electrophilic iodine-125 (generated from carrier
free Na125I in the presence of chloramine-T). Efficient labelling was achieved within 30 min, and subsequent deprotection was
achieved by lactone hydrolysis (Scheme 1). RP-HPLC purification and subsequent reformulation using SEP PAK C18 cartridges
provided the labelled compound 2 in excellent radiochemical yield (RCY 83%) and high radiochemical purity (RCP 97%).
Scheme 1. (i) Chloramine-T in a mixture of 94% EtOAc, 4% DMF, 1% AcOH and 1% water. Room temperature, 30 min.
Experimental: 4-hydroxy-4-[4-(2-[125-iodo]benzyloxy)phenyl]butanoic acid. (2) Chloramine-T trihydrate (40.8 mg, 145 mmole)
was suspended in a mixture of EtOAc (4.75 mL), DMF (0.25 mL), acetic acid (50 mL) and a small amount of water was added (50 mL) to
dissolve the compound. This reagent solution (1000 mL) was added to the stannyl precursor 1 (1.19 mg, 2.1 mmole) in a small HPLC
vial, the vial was capped and shaken to dissolve the compounds. I-125 NaI (30 ml, 3 mCi) was added to the vial which was quickly
reclosed. The vial was shaken and the contents left to react for 30 min.
Sodium metabisulfite (9.70 mg) in water (350 mL) was added to quench excess Chloramine-T and the reaction mixture was washed
with purified water (3 Â 300 mL). The organic layer was evaporated to dryness under an argon flow.
Subsequently, the reaction mixture was dissolved in acetonitrile (500mL) and NaOH (20 mL, 4M) the latter to hydrolyse the lactone.
The sample was shaken and left over night at 51C, neutralized with HCl (80 mL, 1 M) and immediately subjected to HPLC purification.
The desired product 2 was collected at 24.7 min, and analysed to have a radiochemical purity of 97% and a total activity of
2.48 mCi (83%). HPLC conditions: Buffer A (4.9% MeCN, 95% Water, 0.1% TFA), Buffer B (99.9% MeCN, 0.1% TFA). Gradient: 0 min to
15 min (30% B); 15 min to 35 min (gradient to 100% B); 35 min to 45 min (100% B); 45 min to 46 min (gradient to 30% B).
The product was diluted five times with ultra purified water and loaded onto a pre-activated C-18 Sep-PAK (160 mg). The loaded
cartridge was washed with ultra purified water (5 mL), eluted with EtOH (5 mL) and subsequently diluted to a concentration of
approximately 5 MBq/ml with a dilute NaOH solution (0.001 M).
Radiochemical analysis (Figure 1) was performed on the same HPLC system as the preparation with another HPLC method.
A Carroll Ramsey S105 equipped with a silicon diode was used as radio detector and UV-detection confirmed a chemically pure product.
Figure 1. HPLC Radiogram for RCP of 2.
J. Label Compd. Radiopharm 2010, 53 315–321
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Abstracts
Material and Methods: Chemicals were used as arrived from the supplier. Carrier free sodium[I–125]iodine was purchased from
Perkin Elmer with a radioactive concentration of 10 mCi/100 mL.
Acknowledgements: The work on design and synthesis of GHB ligands has been supported by The Danish Medical Research
Council, Beckett-fonden and ‘Carl og Ellen Hertz Legat for Dansk Natur- og Lægevidenskab’ and is an ongoing collaboration with
¨
Drs. Hans Brauner-Osborne, Rasmus P. Clausen and Bente Frølund (Department of Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, University of Copenhagen) who are gratefully acknowledged. Radiolabelling experiments were performed at the Hevesy
Laboratory, Radiation Research Division, Risø-DTU, The Technical University of Denmark, who are gratefully thanked for the supply of
radioisotopes and instruments necessary for the labelling.
References
¨
[1] S. Høg, P. Wellendorph, B. Nielsen, K. Frydenvang, I. F. Dahl, H. Brauner-Osborne, L. Brehm, B. Frølund, R. P. Clausen, Journal of
Medicinal Chemistry 2008, 51, 8088–8095.
[2] P. Wellendorph, S. Høg, J. R. Greenwood, A. de Lichtenberg, B. Nielsen, B. Frølund, L. Brehm, R. P. Clausen, H. Brauner-Osborne,
¨
Journal of Pharmacology and Experimental Therapeutics 2005, 315, 346–351.
[3] C. G. T. Wong, K. M. Gibson, O. C. Snead, Trends in Pharmacological Sciences 2004, 25, 29–34.
[4] A. L. Girard, C. Y. Senn, Journal of Interpersonal Violence 2008, 23, 3–20.
[5] D. E. Fuller, C. S. Hornfeldt, Pharmacotherapy 2003, 23, 1205–1209.
[6] D. M. Robinson, G. M. Keating, Cns Drugs 2007, 21, 337–354.
[7] L. P. Carter, D. Pardi, J. Gorsline, R. R. Griffiths, Drug and Alcohol Dependence 2009, 104, 1–10.
[8] R. N. Hanson, G. L. Tonnesen, W. H. McLaughlin, W. D. Bloomer, D. E. Seitz, Journal of Labelled Compounds &
Radiopharmaceuticals 1981, 18, 128–129.
[9] D. S. Wilbur, Bioconjugate Chemistry 1992, 3, 433–470.
SYNTHESIS OF [3H] STEROID FATTY ACID ESTERS IN IONIC LIQUIDS UNDER MICROWAVE
IRRADIATION
a
SOMDATTA DEB AND KRISTIINA WAHALA
a,b
¨
¨ ¨
aLaboratory of Organic Chemistry, University of Helsinki, A.I.Virtasen aukio 1, PL 55, Helsinki, 00014, Finland
bQUILL, The Queen’s University of Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK
Abstract: Fatty acid esters of steroid hormones are naturally occurring compounds but their physiological role is not fully
understood. These esters have potential therapeutic uses. To study their properties a reference compound was required.
Radiolabelled analogues of these compounds can serve as a standard. In this work we report the synthesis of long chain fatty acid
ester derivatives of tritiated steroids by microwave irradiation in an ionic liquid.
Keywords: fatty acid ester; oleate; linoleate; steroid; estradiol; dehydroepiandrosterone; testosterone; tritium label; microwave; ionic
liquid
Introduction: Fatty acid esters (FAE) of steroid hormones (SH) occur naturally. Since the hydroxyl group is protected as an ester they
are metabolically stable, act as a hormone reservoir and can release active hormone to the target tissues as required by the action of
esterase.1,2 SH-FAE are found in different tissues, but their biological significance is not known completely. In vitro studies suggest
that in blood estradiol (E2)-FAE increases the anti-oxidant potential of lipoprotein.3 Ovarian follicular fluid contains a mixture of
different E2-FAE, comprising predominantly of unsaturated FAE. Since estradiol has action on ovaries, it is expected that E2-FAE may
also have an important role in the female reproductive system.4,5 Concentration of dehydroepiandrosterone fatty acid esters (DHEA-
FAE) in the adrenal gland is three times higher than that of free DHEA.1 In blood DHEA-FAE are produced in high density lipoprotein
(HDL) by lecithin-cholesterol acyltransferase (LCAT) and then transferred to low density lipoprotein (LDL) and very low density
lipoprotein (VLDL).6,7 In vitro studies show that DHEA-FAE is formed in the ZR-75-1 breast cancer cells and they may act as a
prehormone for other steroids.8,9 These findings are significant for estrogen responsive breast cancer. Testosterone esters occur in
the fat and testes of male rat.10 In rat testosterone is esterified in the brain also and esterification may play an important role in the
development of rat brain.11
All these studies indicate that SH-FAE may have some unique actions. A better understanding of their physiological role may be
therapeutically helpful. Studies were actively carried out to reveal their biological effects requiring a standard compound.
Radiolabelled analogue of these compounds can serve as a standard. We have synthesized FAE of [3H] labeled SH for use as standard
in biological studies.12
Results and discussion: [3H] labelled estradiol 17b-oleate (1), DHEA oleate (2), DHEA linoleate (3) and testosterone linoleate (4)
were synthesized in mg scale from the [3H] labelled steroid in an ionic liquid solvent under microwave irradiation (MW) in high yield.
All the starting materials were converted to the corresponding esters in a very short time.
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Abstracts
Figure 1. Tritum-labeled fatty acid eters of Stroud synthesized in Mw/IL.
Selective esterification at C-17 of estradiol was done in one pot. After preparation of the diester, the product was not isolated.
KOH was added to the same vial and the mixture irradiated again to give selectively the C-3 monoester. Since esterification and
selective saponification were both done in the same reaction vessel no significant loss of the costly[3H] labelled materials occurs.
This methodology is very effective for synthesis with isotopically labeled materials, where cost of the materials compels one to run
the reactions on a small scale.
Experimental: We synthesized tritium-labelled fatty acid esters of steroids from [1,2,6,7-3H]-DHEA (94.5 Ci/mmol), [2,4,6,7-3H]-
estradiol (70 Ci/mmol) obtained from PerkinElmer, Boston and [1,2,6,7-3H]-testosterone (74 Ci/mmol) obtained from Amersham.
General procedure for synthesis of fatty acid esters of steroids
A mixture of 7.1 Â 10À6 mmol of steroid, catalytic amount 4-(dimethylamino)pyridine, 1-butyl-3-methylimidazolium chloride[B-
mim]Cl and excess of fatty acid chloride in dry pyridine was MW irradiated at 401C with 20 W power for 15 min. After the reaction,
the mixture was acidified with 1N HCl. The aqueous phase was extracted three times with EtOAc. The combined organic phase was
washed successively with a NaHCO3 solution and brine and dried over Na2SO4. The crude product was purified on a short silica gel
column eluting with a mixture of 8:1 or 3:1 n-hexane/EtOAc depending on the procduct.
For the synthesis of estradiol-17b-oleate, after irradiation at 401C, an excess of KOH and toluene was added to the vial and
irradiated at 801C for a further 5 min. The product was then isolated by the procedure described above.
The resulting[3H] steroid esters gave a single radioactive spot coincident with authentic cold steroid esters on thin layer
chromatography.
Conclusion: Tritium labeled steroid fatty acid esters were synthesized with the assistance of IL and MW. The method is rapid and
high yielding.
Acknowledgements: KW is grateful to Finnish Academy (the grant number 126431) for support.
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