A versatile approach to the regioselective synthesis of diverse (-)-epicatechin-β-d-glucuronides

Article abstract of DOI:10.1016/j.tetlet.2012.01.054

A versatile new approach is reported for the total synthesis of five glucuronide metabolites of epicatechin, using selective protection/deprotection techniques.

Full text of DOI:10.1016/j.tetlet.2012.01.054

Tetrahedron Letters 53 (2012) 1501–1503
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A versatile approach to the regioselective synthesis of diverse
(ꢀ)-epicatechin-b-D-glucuronides
Eric S. Mull a, , Michael Van Zandt a, Adam Golebiowski a, R. Paul Beckett a, Pradeep K. Sharma b,
⇑
Hagen Schroeter c
a Institute of Bioanalytics, 23 Business Park Drive, Branford, CT 06405, USA
b Johnson Matthey Pharma Services, 25 Patton Road, Devens, MA 01434, USA
c Mars, Incorporated, 6885 Elm Street, McLean, VA 22101, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A versatile new approach is reported for the total synthesis of five glucuronide metabolites of epicatechin,
using selective protection/deprotection techniques.
Received 15 December 2011
Revised 12 January 2012
Accepted 13 January 2012
Available online 21 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Flavanols
Epicatechin
Metabolites
O-glucuronide
Asymmetric cis-dihydroxylation
Introduction
R1O
OH
O
R1O
OH
O
a
The health benefits of flavanols have been widely investigated in
recent years.1 Specifically, the consumption of flavanol-rich foods
such as cocoa is known to be associated with improved cardiovascu-
lar health.2 Recent work has shown that many of these effects are
mediated by the natural product (ꢀ)-epicatechin.3 (ꢀ)-Epicatechin
is readily metabolized in humans to give various O-methylated,
O-glucuronidated, and O-sulfated metabolites.4
Major human metabolites of (ꢀ)-epicatechin have been isolated
from plasma and urine, and then structurally characterized by
NMR.3a,4a It is known that (ꢀ)-epicatechin is rapidly metabolized
after ingestion and that one or more metabolites are likely to be
biologically active.5 Isolation of these species is tedious and leads
only to trace quantities of metabolite. It is therefore desirable to
develop a synthetic strategy for generating these metabolites in
sufficient quantities for further study of their biological activities.
We recently reported the synthesis of optically active 13C-labeled
(ꢀ)-epicatechin.6 As part of an ongoing program to synthesize
putative metabolites of (ꢀ)-epicatechin in a stereoselective man-
ner, we report here the first synthesis of five epicatechin glucuron-
ides via a selective protection/deprotection methodology.
OR2
OR2
R1=H, R2=Bn
R1=Bn, R2=H
1a
1b
R1=MOM, R2=Bn
R1=Bn, R2=MOM
2a
2b
Scheme 1. Reagents and conditions: (a) methoxymethylbromide, Hünigs base,
CH2Cl2, 0 °C, 1 h.
Results and discussion
Syntheses of acetophenones 1a,7 and 1b8 were previously
described in the literature. By utilizing the hydrogen bonding be-
tween ketone and the adjacent phenol, orthogonally bis-protected
acetophenones 2a and 2b were synthesized by the reaction of
acetophenones 1a and 1b with methoxymethyl bromide in dichlo-
romethane as the solvent (Scheme 1). Condensation of acetophe-
nones 2a–c (2c commercially available) and benzaldehydes 3a–d
with sodium hydride in dimethylformamide gave chalcones 4a–e
(Scheme 2). Reduction of chalcones 4a–e with sodium borohydride
and cerium chloride resulted in the decarbonylated product, which
was purified by column chromatography. TLC analysis indicated
that disappearance of the chalcone was rapid (the reaction appears
⇑
Corresponding author. Tel.: +1 203 677 5791.
E-mail address: eric.mull@bms.com (E.S. Mull).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.01.054

1502
E. S. Mull et al. / Tetrahedron Letters 53 (2012) 1501–1503
R1O
OTBS
OR4
OR3
R1O
OH
OR4
OR3
OR4
OR3
R1O
OH
b, c
a
+
O
OR2
5a-e
OR2
O
O
OR2
R1
R2
R3
Bn
Bn
R4
Me
Me
→ 4a
4b
2a + 3a
2b + 3a →
2a R1=MOM, R2=Bn
2b
3a R3=Bn, R4=Me
3b
MOM Bn
Bn
Bn
MOM
Bn
R1=Bn, R2=MOM
2c R1=Bn, R2=Bn
R3=MOM, R4=Me
3c R3=Me, R4=Bn
3d
2c + 3b → 4c
2a + 3c → 4d
MOM Me
MOM Bn
MOM Bn
Me
Bn
Bn
Bn
R3=Bn, R4=Bn
d, e
4e
2a + 3d →
OR4
OR3
OR4
OR3
R1O
O
R1O
O
R1O
OH
OH
OR4
OR3
f
g, h
O
O
OR2
7a-e
OR2
8a-e
O
OR2
OH
6a-e
i, j
OR4
OR3
OR4
OR3
R1O
O
R1O
O
HO2C
HO
O
l, m, n
= GluA
OBn
OH
OR2
OH
OR2
OH
R
x = MOM
9a-e
10a-e
R1
R2
H
R3
H
H
GluA
Me
H
R4
k
x
R
= H
11a
11b
11c
11d
11e
GluA
H
Me
Me
Me
H
GluA
H
H
GluA
GluA
H
H
H
Scheme 2. Reagents and conditions: (a) NaH, DMF, 0 °C to RT, 1 h; (b) CeCl3ꢃ7H20, 1:4 EtOH/THF, then NaBH4, ꢀ40 to 0 °C, 5 h; (c) TBSCl, imidazole, DMF, 16 h, RT; (d) AD-mix
, MeSO2NH2, 1:1 t-BuOH/water, 0 °C to RT, 16 h; (e) TBAF, AcOH, 0 °C, 1 h; (f) EtC(OEt)3, PPTS, 1,2-dichloroethane, 65 °C, 2 h; (g) 7 N NH3 in MeOH, 48 h; (h) DMP(wet),
CH2Cl2, 2 h; (i) Al(OiPr)3, 2:1 toluene/IPA, 100 °C, 1 h; (j) NaH, BnBr, DMF, 2 h; (k) 4 N HCl in dioxane, 1:1 MeOH/CH2Cl2, 1 h, (l) 2,3,4-tri-O-acetyl- -glucuronic acid methyl
a
a-D
ester trichloroacetimidate, 4 Å mol. sieves, CH2Cl2, 1 h at RT then cooled to ꢀ40 °C, then BF3ꢃEt2O, ꢀ40 to 0 °C, 2 h; (m) 1 N NaOH, 0 °C to RT, 2 h; (m) H2 (g), Pd(OH)2, MeOH,
2 h.
complete after 1 h), however high yield requires continuous stirring
for an additional 5 h.6 The remaining free phenol was protected as
the tert-butyldimethylsilyl (TBS) ether 5a–e before performing an
protected O-glucuronide in moderate yield after silica gel chroma-
tography.12 The O-acyl protected glucuronic acid methyl ester was
deprotected with 1 N sodium hydroxide at 0 °C then subjected to
hydrogenation conditions with Pearlman’s catalyst to provide
crude 11a–e (purity ꢁ85% by HPLC).
asymmetric Sharpless cis-dihydroxylation using AD-mix-a 9
, and
then deprotecting the TBS ether with a 1:1 molar mixture of tetra-
butylammonium fluoride:acetic acid to give 6a–e.6 It is important
to use an equimolar amount of glacial acetic acid to avoid epimer-
ization of the product. Cyclization of compounds 6a–e in the
presence of triethyl orthopropionate and pyridinium p-toluenesul-
fonate in 1,2-dichloroethane gave 7a–e in good yield after
chromatography.6
After room temperature deprotection of the ester with 7 N
ammonia in methanol, the protected catechin was converted to
protected epicatechin by oxidizing the hydroxyl group to the
ketone with Dess–Martin periodinane (DMP) in wet dichlorometh-
ane 8a–e10 and then stereoselectively reducing the ketone with
aluminum triisopropoxide and isopropyl alcohol under Meerw-
ein–Ponndorf–Verley reduction conditions.11 Benzyl protection of
the 3-hydroxyl with benzyl bromide and sodium hydride 9a–e,
followed by deprotection of the methoxymethyl (MOM) group
with hydrochloric acid, provided the free phenol at the desired po-
sition while all other hydroxy groups were protected as the benzyl
ethers 10a–e. Coupling of phenols 10a–e with commercially avail-
Pure (>95% by HPLC) 11a–e14 was obtained by reverse phase
semi-preparative HPLC (Column: Sunfire prep C18 OBO 5 lM,
30 ꢂ 75 mm by Waters Corp.) using a gradient of 0% to 20% ACN/
Water with 0.1% TFA modifier.
The optical purity of the final compounds was not determined,
although a high degree of stereoselectivity is inferred, based upon
similar methodology used for the synthesis of 13C-labled (ꢀ)-epi-
catechin,6 as well as the synthesis of (+)-myristinin A,13 in which
the absolute stereochemistry at C-2 was determined to be S. The cis
-configuration at C-2 and C-3 after stereoselective reduction of the
ketone was supported by the fact that the resultant C-2 proton
exists as a singlet in the 1H NMR spectrum (J2,3 <1 Hz), as reported
previously.6,11 Some loss of stereochemical purity, (possibly due to
diminished asymmetric induction in the Sharpless cis-dihydroxyla-
tion step), was revealed after the optically pure glucuronide was
attached to the epicatechin core, thus forming diastereomers,
which were separable by reverse phase HPLC. Analysis of the minor
isomers by NMR showed nearly identical spectra, with the C-2
proton appearing as a singlet. The anomeric proton was deter-
mined to be b based upon the coupling constant (J = ꢁ7.7 Hz).12
able 2,3,4-tri-O-acetyl-
a-D-glucuronic acid methylester trichloro-
acetimidate and boron trifluoride etherate provided the fully

E. S. Mull et al. / Tetrahedron Letters 53 (2012) 1501–1503
1503
Mullen, W.; Steiling, H.; Williamson, G.; Lean, M. E.; Crozier, A. Mol. Nutr. Food
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5. Terao, J. J. Med. Invest. 1999, 46, 159–168.
6. Sharma, P. K.; He, M.; Romanczyk, L.; Schroeter, H. J. Labelled Comp. Rad. 2010,
53, 605–612.
Synthesis of optically pure glucuronide metabolites of epicatechin
as well as its in-depth analytical and stereochemical evaluation
will be the subject of a future publication.
7. Kumazawa, T.; Minatogawa, T.; Matsuba, S.; Sato, S.; Onodera, J. Carbohydr. Res.
2000, 329, 507–513.
Conclusion
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Wakimoto, T.; Kan, T. Org. Lett. 2009, 11, 2233–2236.
9. van Rensburg, H.; van Heerden, P. S.; Bezuidenhoudt, B. C. B.; Derreira, D.
Tetrahedron Lett. 1997, 38, 3089–3092.
10. Schreiber, S.; Meyer, S. J. Org. Chem. 1994, 59, 7549–7552.
11. Sharma, P. K.; Kolchinski, A.; Shea, H. A.; Nair, J. J.; Gou, Y.; Romanczyk, L. J., Jr;
Schmitz, H. H. Org. Proc. Res. Dev. 2007, 11, 422–430.
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G.; Kiefel, M. J.; Ferro, V.; von Itzstein, M. Carbohydr. Res. 2005, 340, 2077–
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13. Maloney, D. J.; Deng, J.; Starck, S. R.; Gao, Z.; Hecht, S. M. J. Am. Chem. Soc. 2005,
127, 4140–4141.
We have developed efficient methods to prepare epicate-
chin-b-D-glucuronides in a stereo- and enantio-specific manner.
These methods have been utilized in the first total syn-
theses
of
40-O-methyl-(ꢀ)-epicatechin-7-b-
D-glucuronide
D-glucuronide (11b),
(11a), 40-O-methyl-(ꢀ)-epicate chin-5-b-
40-O-methyl-(ꢀ)-epicatechin-30-b-
D
-glucuronide (11c), 30-O-
methyl-(ꢀ)-epicatechin-7-b- -glucuronide (11d), and (ꢀ)-epi-
D
catechin-7-b-D-glucuronide (11e).
14. Analytical data for 11a: MS m/z = 481.3 [M++1]. 1H NMR (500 MHz, d6-
acetone): d 7.07 (d, 1H, J = 2.0 Hz), 6.95 (dd, 1H, J = 8.4, 2.0 Hz), 6.91 (d, 1H,
J = 8.4 Hz), 6.22 (d, 1H, J = 2.4 Hz), 6.18 (d, 1H, J = 2.4 Hz), 5.02 (d, 1H,
J = 7.7 Hz), 4.94 (s, 1H), 4.27–4.23 (m, 1H), 4.07 (d, 1H, J = 9.4 Hz), 3.83 (s,
3H), 3.70 (dd, 1H, J = 9.4, 9.1 Hz), 3.60 (t, 1H, J = 9.1 Hz), 3.49 (dd, 1H, J = 9.1,
7.7 Hz), 2.90 (dd, 1H, J = 16.9, 4.5 Hz), 2.77 (dd, 1H, J = 16.9, 3.0 Hz). 13C NMR
(125 MHz, d6-acetone): d 170.3, 157.9, 157.4, 157.0, 147.7, 147.0, 133.4, 119.0,
115.1, 111.9, 102.8, 101.9, 97.5, 96.8, 79.5, 77.1, 76.0, 74.3, 72.6, 66.7, 56.3,
29.1.
Acknowledgment
This work was funded by the Mars, Incorporated.
Supplementary data
Analytical data for 11b: MS m/z = 481.3 [M++1]. 1H NMR (500 MHz, d6-
acetone): d 7.06 (d, 1H, J = 1.9 Hz), 6.94 (dd, 1H, J = 8.4, 1.9 Hz), 6.91 (d, 1H,
J = 8.4 Hz), 6.30 (d, 1H, J = 2.2 Hz), 6.09 (d, 1H, J = 2.2 Hz), 5.01 (d, 1H,
J = 7.4 Hz), 4.91 (s, 1H), 4.23–4.20 (m, 1H), 4.07 (d, 1H, J = 9.6 Hz), 3.83 (s,
3H), 3.73 (dd, 1H, J = 9.6, 9.0 Hz), 3.61 (t, 1H, J = 9.0 Hz), 3.57 (dd, 1H, J = 9.0,
7.4 Hz), 2.91 (dd, 1H, J = 17.1, 3.1 Hz), 2.85 (dd, 1H, J = 17.1, 4.4 Hz). 13C NMR
(125 MHz, d6-acetone): d 170.3, 157.9, 157.6, 156.7, 147.7, 147.0, 133.4, 118.9,
115.0, 112.0, 102.4, 102.0, 98.2, 96.5, 79.4, 77.2, 76.1, 74.4, 72.6, 66.7, 56.3,
29.0.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2012.01.054. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
Analytical data for 11c: MS m/z = 481.3 [M++1]. 1H NMR (500 MHz, D2O): d 7.18
(d, 1H, J = 1.7 Hz), 7.07 (dd, 1H, J = 8.6,1.7 Hz), 7.00 (d, 1H, J = 8.6 Hz), 6.06 (m,
0.79H, partial deuterium exchange), 6.02 (m, 0.42H, partial deuterium
exchange), 5.08 (d, 1H, J = 7.7 Hz), 4.3 (m, 1H), 4.03 (d, 1H, J = 9.6 Hz), 3.83
(s, 3H), 3.69–3.56 (m, 3H), 2.81 (dd, 1H, J = 16.9, 4.5 Hz), 2.66 (dd, 1H, J = 16.9,
2.5 Hz). 13C NMR (125 MHz): d 155.6, 155.2, 155.1, 148.3, 145.0, 131.2, 121.9,
114.4, 112.5, 112.5, 100.4, 99.6, 96.0, 95.4, 77.7, 75.2, 72.6, 71.3, 65.5, 55.8,
27.3.
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Analytical data for 11d: MS m/z = 481.3 [M++1]. 1H NMR (500 MHz, d6-
acetone): d 7.19 (d, J = 1.3 Hz, 1H), 6.97 (dd, 1H, J = 8.5, 1.3 Hz), 6.81 (dd, 1H,
J = 8.5, 1.3 Hz), 6.24 (d, J = 1.9 Hz, 1H), 6.17 (d, J = 1.9 Hz, 1H), 4.99 (d, 1H,
J = 7.7 Hz), 4.96 (s, 1H), 4.27–4.22 (m, 1H), 4.07 (d, 1H, J = 9.3 Hz), 3.84 (s, 3H),
3.70 (t, 1H, J = 9.3 Hz), 5.58 (t, 1H, J = 9.3 Hz), 3.48 (dd, 1H, J = 9.3, 7.7 Hz), 2.91
(dd, 1H, J = 16.9, 4.5 Hz), 2.80 (dd, 1H, J = 16.7, 2.2 Hz). 13C NMR (125 MHz, d6-
acetone): d 170.3, 158.0, 157.6, 157.1, 148.0, 147.0, 132.0, 120.7, 115.2, 112.0,
102.9, 102.1, 97.6, 97.1, 79.8, 77.2, 76.1, 74.4, 72.7, 66.8, 56.3, 29.3.
Analytical data for 11e: MS m/z = 467.3 [M++1]. 1H NMR (500 MHz, d6-acetone):
d 7.06 (d, 1H, J = 1.6 Hz), 6.84 (dd, 1H, J = 8.1, 1.6 Hz), 6.79 (d, 1H, J = 8.1 Hz),
6.22 (d, 1H, J = 2.4 Hz), 6.16 (d, 1H, J = 2.4 Hz), 5.01 (d, 1H, J = 7.7 Hz), 4.90 (s,
1H), 4.25–4.21 (m, 1H), 4.07 (d, 1H, J = 9.7 Hz), 3.71 (t, 1H, J = 9.0 Hz), 3.60 (t,
1H, J = 9.0 Hz), 3.49 (dd, 1H, J = 9.0, 7.7 Hz), 2.90 (dd, 1H, J = 16.7, 4.6 Hz), 2.77
(dd, 1H, J = 16.7, 3.0 Hz). 13C NMR (125 MHz, d6-acetone): d 170.2, 158.0,
157.5, 157.1, 145.4, 145.4, 132.1, 119.5, 115.5, 115.4, 102.9, 102.0, 97.5, 97.0,
79.6, 77.2, 76.1, 74.4, 72.7, 66.8, 29.1.