Synthesis of [2H8]-enterolactone and [2H10]-enterodiol

Article abstract of DOI:10.1002/jlcr.793

Enterolactone and enterodiol are the main mammalian metabolites of dietary butyrolactone type lignans. The study of biological properties and potential health effects of these compounds requires isotopically labelled compounds as standards for quantitativ

Full text of DOI:10.1002/jlcr.793

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 25–30.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.793
Research Article
Synthesis of [2H8]-enterolactone and
[2H10]-enterodiol
. . . . .
Eija Leppala and Kristiina Wahala*
Department of Chemistry, Laboratory of Organic Chemistry, P.O. Box 55,
FIN-00014 University of Helsinki, Finland
Summary
Enterolactone and enterodiol are the main mammalian metabolites of dietary
butyrolactone type lignans. The study of biological properties and potential health
effects of these compounds requires isotopically labelled compounds as standards
for quantitative measurements. An expedient deutero-labelling method for enter-
olactone is to use the D3PO4 ꢀ BF3/D2O complex at room temperature which
will exchange all eight aromatic hydrogens, even from inactivated meta positions,
to form [2,4,5,6,20,40,50,60-2H8]-enterolactone in 74% yield and 99% isotopic purity.
[2,4,5,6,9,9,20,40,50,60-2H10]-Enterodiol was prepared from [2,4,5,6,20,40,50,60-2H8]-
enterolactone by reduction with LiAlD4 which introduces two more deuterium atoms
into the molecule. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: deuterium; enterodiol; enterolactone; labelling; lignan; phytoestrogen;
metabolite
Introduction
In the human diet, good sources for plant lignans – precursors of the
mammalian lignans enterolactone (3,30-dihydroxylignano-9,90-lactone) 1 and
enterodiol (lignane-3,30,9,90-tetraol) – are flaxseed, wholegrain products, cereals
especially rye, berries and vegetables.1,2 The growing interest in these
compounds is based on their possible health effects. Several epidemiological
studies indicate that enterolignans, enterolactone 1 and enterodiol, may protect
against breast, prostate and colon cancers and cardiovascular diseases.3ꢁ5
.
*Corresponding author. K. Wahala, Department of Chemistry, Laboratory of Organic Chemistry, P.O. Box
55, FIN-00014 University of Helsinki, Finland. E-mail: Kristiina.Wahala@Helsinki.fi
. .
Contract/grant sponsor: Finnish Academy
Contract/grant sponsor: University of Helsinki
Contract/grant sponsor: Chancellor of University of Helsinki
Contract/grant sponsor: Graduate School of Bio-organic and Medicinal Chemistry
Copyright # 2003 John Wiley & Sons, Ltd.
Received 4 September 2003
Revised 2 October 2003
Accepted 5 October 2003

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E. LEPPALA AND K. WAHALA
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Dietary plant lignans matairesinol (4,40-dihydroxy-3,30-dimethoxylignano-
9,90-lactone) and secoisolariciresinol (3,30-dimethoxy-lignane-4,40,9,90-tetraol)
are metabolized mainly to enterolactone 1 and enterodiol by human intestinal
microflora.6,7 These metabolites have been identified in human body fluids and
tissues. Additional minor metabolites of plant lignans and new precursors for
enterolactone 1 and enterodiol have been recently found in vivo and in vitro
studies.8,9
Lignan concentrations are low in biological samples and therefore
quantitation methods require good resolution and sensitivity. Commonly
used analytical techniques for lignans are based on HPLC-MS and GC-MS.10
In these chromatographic methods stable isotopically labelled enterolignans
are needed as internal standards.
We now report that the D3PO4 ꢀ BF3/D2O complex, which we have used
previously for the deuteration of flavonoids,11ꢁ14 gives the [2H8]-enterolactone
2 in one step in excellent yield and isotopic purity. Remarkably all eight
aromatic protons of enterolactone 1, even from the non activated C-5 and C-50
sites, are exchanged at room temperature. In the mass spectrum there is only
an M+ peak and no cluster of molecular ions with lesser amounts of
deuterium and no unlabelled molecules. Thus the isotopic purity of
[2,4,5,6,20,40,50,60-2H8]-enterolactone 2 is >99%.
Previously, [9,9-2H2]-enterolactone has been prepared in 49% overall yield15
by Kirk et al. by multistep synthesis, starting from dideuterated butenolide
followed by a tandem Michael alkylation reaction, desulphurization and
debenzylation with Raney nickel. Another route, only useful for preparing
butyrolactone lignans such as enterolactone with identical aromatic sub-
stituents, was introduced by Bambagiotti-Alberti et al.16 Dibenzylidene
succinic acid, prepared by Stobbe condensation, was deuterated (D2) over
a H4Ru4(CO)8(PBu3)4 catalyst to form the g-lactone ring directly.
[7,8,9,9,70,80-2H6]-enterolactone was obtained after debenzylation in 46%
overall yield accompanied by 12% of the cis isomer. In our own earlier work,17
enterolactone was refluxed with
a mixture of PBr3/D2O to give
[2,4,6,20,40,60-2H6]-enterolactone directly in 35% yield in >90% isotopic
purity. In this method aromatic hydrogens ortho or para to the phenolic
hydroxy group are exchanged for deuterium.
Results and discussion
In the optimized procedure, hydrogens of the phenolic hydroxy groups are
first removed from the equilibrium by exchanging them to deuteriums in an
acetone–D2O mixture and evaporating the solvents. The actual deuteration
reagent D3PO4 ꢀ BF3/D2O is prepared by dissolving dry P2O5 (1.5 g) in D2O
(1.5 ml) at 08C. The resulting deuterated phosphoric acid is then saturated with
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 25–30

SYNTHESIS OF [2H8]-ENTEROLACTONE
27
BF3 gas at room temperature. The reagent is added to the pre-deuterated
enterolactone and the mixture stirred at room temperature.12
In previous methods only two to six hydrogens of enterolactone undergo
exchange whereas the D3PO4 ꢀ BF3/D2O complex is acidic enough to exchange
all eight aromatic hydrogens of enterolactone, even from the non-active
aromatic sites C-5 and C-50 (Scheme 1). [2,4,5,6,9,9,20,40,50,60-2H10]-Enterodiol
3 was obtained by the reduction of [2,4,5,6,20,40,50,60-2H8]-enterolactone 2 with
LiAlD4.
Scheme 1. Synthesis of [2H8]-enterolactone and [2H10]-enterodiol
Since the deuterated enterolactone will be used in analytical procedures as
the TMS derivative, the deuterium content of [2H8]-enterolactone was
determined by comparing the EI mass spectra of TMS-derivatised labelled
and unlabelled compounds. Also, it was important to demonstrate that the
newly generated D labels are stable under the silylation conditions. The ion
patterns are practically superimposable in the M+ region (Figure 1) and
calculated isotopic purity for the TMS-derivatised [2H8]-enterolactone was
>99%. EI mass spectroscopy cannot be used for the calculation of
deuteration level of TMS-derivatised [2H10]-enterodiol as no molecular ion
peak can be seen. However an APCI negative mode mass spectrum gives a
strong M+-1 peak, both for deuterated and undeuterated enterodiols,
allowing the estimation of isotopic purity of [2H10]-enterodiol as >95%.
1
The H and 13C NMR data also confirm the exchange of all aromatic
protons with excellent deuteration levels.
Experimental
All compounds, homogeneous by TLC, were characterized by H NMR, 13C
1
NMR, LRMS and HRMS. Melting points were determined in open capillary
.
tubes with a Buchi B-545 melting point apparatus and are uncorrected. NMR
data were recorded on a Varian GEMINI 2000 or Bruker Avance 500
spectrometer. Chemical shifts are given in ppm and SiMe4 was used as an
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 25–30

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E. LEPPALA AND K. WAHALA
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Figure 1. Part of the MS spectra for TMS derivatised [2H8]-enterolactone (a)
and enterolactone (b)
internal standard. In the 13C NMR spectra the shifts given for the C-D triplets
or C-D2 multiplet are those corresponding to the centre of the pattern and are
marked ‘D’. EIMS and HRMS were obtained using a JEOL JMS SX 102
spectrometer and the samples were introduced by a direct inlet probe. The
deuterium content of TMS derivatised [2H8]-enterolactone was determined
with a GC/MS Varian Saturn CP-3800. The deuterium content of [2H10]-
enterodiol was determined with a Micromass Quattro II LC/MS using APCI
in the negative mode. TLC was conducted with Merck silica gel 60 F254 plates
and [2H8]-enterolactone was purified with Sephadex LH-20 (Sigma). D2O
(99.9%) was obtained from Aldrich and LiAlD4 (99%) from Fluka. THF was
dried by distilling over sodium and enterolactone was synthesized by a tandem
Michael addition alkylation according to our published method.18
[2,4,5,6,20,40,50,60-2H8]-Enterolactone 2
A mixture of D2O-treated enterolactone (0.10 g, 0.34 mmol) and D3PO4 ꢀ BF3/
D2O (4 ml) reagent12 was stirred at room temperature for 20 h. The reaction
mixture was poured into ice water and the product extracted with EtOAc. The
extract was washed with water, dried over anhydrous Na2SO4 and evaporated.
The crude product was re-treated with D3PO4 ꢀ BF3/D2O if the deuteration
level as ascertained by NMR was unsatisfactory. The crude product was
purified with Sephadex LH-20 (column: 0.5 cm ꢂ 17 cm, eluent: first 5 ml 20:1
(v/v) CH2Cl2–MeOH then 10 ml 40:3 (v/v) CH2Cl2–MeOH) and recrystallized
from CH2Cl2–EtOAc as off white crystals (0.74 g, 74%), isotopic purity
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 25–30

SYNTHESIS OF [2H8]-ENTEROLACTONE
29
>99%, mp 145–1468C (Reference 19, 141–1438C D0-enterolactone); 1H NMR
(200 MHz, D6-acetone): d 2.48–2.70 (4H, m, 70, 8, 80), 2.89 (1H, m, 7b), 2.96
(1H, m, 7a), 3.88 (1H, m, 90a), 4.07 (1H, m, 90b), 8.26 and 8.30 (s, 2 ꢂ OH);
13C-NMR (50 MHz, D6-acetone): d 35.1 (C-7), 38.6 (C-70), 42.1(C-80), 46.8 (C-
8), 71.4 (C-90), 114.1 (t, C-4, C-40)D, 116.2 (t, C-2, C-20)D, 120.6 (t, C-6, C-60)D,
129.8 (C-5, C-50)D, 140.6 (C-1), 141.1 (C-10), 158.3 (C-3, C-30), 178.7(C-9);
EIMS (70 eV) m/z: M+ 306 (39%), 196 (24%), 149 (16%), 137 (33%), 111
(100%), 80 (25%), 43 (46%); HRMS (m/z): M+ calcd for C18H10D8O4,
306.1707, found, 306.1718.
[2,4,5,6,20,40,50,60-2H8]-3,30-Di-O-TMS-enterolactone
[2H8]-Enterolactone (1 mg) was stirred in the pyridine/HDMS/TMCS 9:3:1 (v/
v) reagent8 for 2 h. Volatiles were evaporated under a flow of Ar and the
product was dissolved in hexane. The filtered hexane solution was used as such
for GC/MS analysis. EIMS (70 eV) m/z: M+ 450 (100%), 185 (52%).
[2,4,5,6,9,9,20,40,50,60-2H10]-Enterodiol 3
[2H8]-Enterolactone (0.054 g, 0.18 mmol) was treated with LiAlD4 (0.04 g, 0.95
mmol) in dry THF (6 ml) at room temperature. After 1h the reaction mixture
was poured into a cold, saturated NH4Cl (aq) solution. The solution was
neutralised with 2M H2SO4 and extracted with EtOAc. The extract was
washed with brine, dried over Na2SO4 and evaporated. Crude [2H10]-
enterodiol was recrystallized from CHCl3–CH3COCH3 as off white crystals
(0.041 g, 75%), isotopic purity >95%, mp 174–1758C (Reference 20, 175–
1768C for D0-enterodiol).
1H NMR (500 MHz, D6-acetone): d 1.95 (2H, m, 8, 80), 2.70 (4H, m, 7,70),
3.51 (1H, m, 90a), 3.68 (1H, m, 90b), 8.08 (s, arom. OH); 13C-NMR (126 MHz,
D6-acetone): d 36.1 (C-7, C-70), 44.8 and 44.9 (C-8, C-80), 61.1 (weak m, C-9D
and s, C-90), 113.1 (C-4, C-40)D, 116.6 (C-2, C-20)D, 120.7 (C-6, C-60)D, 129.4
(C-5, C-50)D, 143.9 (C-1, C-10), 158.1 (C-3, C-30); EIMS (70 eV) m/z: M+ 312
(3%), 294 (33%), 182 (36%), 164 (62%), 150 (50%), 137 (54%), 111 (100%),
80 (60%), 44 (59%).
Conclusion
All eight aromatic hydrogens, even at the nonactive C-5 and C-50 sites, of
enterolactone can be exchanged for deuterium under strongly acidic
conditions using the D3PO4 ꢀ BF3/D2O complex as deuteration reagent, in
good yield and excellent isotopic purity. [2,4,5,6,20,40,50,60-2H8]-Enterolactone
2 and its reduction product [2,4,5,6,9,9,20,40,50,60 -2H10]-enterodiol 3 are
stable molecules and can be used as reference compounds in quantitative
analyses.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 25–30

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E. LEPPALA AND K. WAHALA
30
Acknowledgements
.
We thank Dr Jorma Matikainen and Mr Heikki Bjork for running the mass
spectra. Financial support from The Finnish Academy, University of Helsinki,
Chancellor of University of Helsinki and the Graduate School of Bio-organic
and Medicinal Chemistry is gratefully acknowledged.
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