Chemical synthesis of allyl-[13C6]-glucuronate

Article abstract of DOI:10.1002/jlcr.1875

The synthesis of allyl-[¹³C₆]-glucuronate from α-D-[¹³C₆]-glucose in five steps is described. Selective protection of the primary alcohol in the glucose with the bulky trityl group followed by acetylation in the same pot gave the fully protected sugar. Removal of the trityl group followed by oxidation of the primary alcohol to the acid and removal of the acetate groups using catalytic amounts of sodium methoxide gave the glucuronic acid. Reaction with allyl bromide using resin-bound fluoride gave the title compound. Copyright 2011 John Wiley & Sons, Ltd. The synthesis of allyl-[¹³C₆]-glucuronate from α-D-[¹³C₆]-glucose in five steps is described. Copyright

Full text of DOI:10.1002/jlcr.1875

Research Article
Received 22 September 2010,
Revised 27 December 2010,
Accepted 3 January 2011
Published online 19 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1875
Chemical synthesis of allyl-[13C6]-glucuronate
Ã
Bachir Latli, Matt Hrapchak, Ravikumar Seetharama,
Dhileepkumar Krishnamurthy, and Chris H. Senanayake
The synthesis of allyl-[13C6]-glucuronate from a-D-[13C6]-glucose in five steps is described. Selective protection of the
primary alcohol in the glucose with the bulky trityl group followed by acetylation in the same pot gave the fully protected
sugar. Removal of the trityl group followed by oxidation of the primary alcohol to the acid and removal of the acetate
groups using catalytic amounts of sodium methoxide gave the glucuronic acid. Reaction with allyl bromide using resin-
bound fluoride gave the title compound.
Keywords: carbon-13; allyl-[13C6]-glucuronate; glucuronic acid; metabolism
coupling it in the second step to the glucuronic acid moiety.
The only labeled glucuronide derivatives we found in the
Introduction
Drug candidates or their phase I metabolites subject to
glucuronidation include compounds that contain alcohol,
phenol, amine, sulfhydryl, and carboxylic acid functional
groups.1 The preparation of these glucuronide derivatives either
by chemical or enzymatic means is required to further assess
their biological activities, especially if the glucuronidation in vivo
is extensive.2,3 Glucuronidation is usually a phase II metabolic
process that was initially considered to be a detoxification and
clearance pathway for drugs and xenobiotics. However, mount-
ing evidence has suggested that these glucuronides may have
potent pharmacological and/or adverse effects. For example,
1-admantylamino-N-glucuronide was reported to have anti-
Parkinson activity,4 glucuronidation of morphine leads to
pharmacologically active conjugates that are more potent than
morpholine itself,5,6 the glucuronide derivatives of budesonide
and retinoyl were suggested for the treatment of ulceratic colitis
and acne, respectively,7,8 the glucuronide derivatives or
modifications on the sugar moiety of the cholesterol absorption
inhibitor Zetia have actually enhanced the inhibition of
cholesterol absorption,9 and recently the acyl glucuronide
derivative of dabigatran was shown to be a pharmacologically
active metabolite and contribute to the overall anti-coagulant
activity of dabigatran.10
literature were the preparation of the glucuronide derivative of
Ezetimibe (Zetia), which was prepared enzymatically,30 and
the transformation of 18O-labeled UDP-glucuronic acid to the
glucuronic acid derivative of p-nitrophenol.33
Herein, we report the synthesis of allyl-[13C6]-glucuronate
from [13C6]-a-D-glucose.
Experimental
General
[13C6]-a-D-Glucose was purchased from Sigma-Aldrich (Saint
Louis, MO). Mass spectra were acquired with a Hewlett-Packard
auto sampler Series 1150 connected to a Micromass Platform
LCZ in ES mode. NMR spectra were recorded with a Bruker
400 MHz DPXB spectrometer using deuterated chloroform,
DMSO, or methanol as solvent and tetramethyl silane as the
internal standard. The analytical HPLC purity was determined on
a Agilent 1200, using a Zorbax SB-C18 column, particle size
5 mm, 4.6 ꢀ 150 mm, and eluting with 10–100% water in
acetonitrile (both solvents contain 10 mM TFA) in 20 min. Flow
rate 1 mL/min UV detection was at 254 nm.
Synthesis
However, some glucuronides, like the acyl glucuronides of
NSAIDS, containing carboxylic acid groups have been implicated
in adverse effects.11,12 These acyl glucuronides may covalently ½13C6ꢁ-(3R,4S,5R,6R)-6-(trityloxymethyl)tetrahydro-2H-pyran-
2,3,4,5-tetrayl tetraacetate (1)
bind to macromolecules like proteins and become immuno-
genic.13–22
A mixture of [13C6]-a-D-glucose (1.0 g, 5.4 mmol) and trityl
chloride (1.5 g, 5.4 mmol) in pyridine (5 mL) was stirred at
ambient temperature overnight. HPLC analysis revealed 10% of
The preparation of glucuronide derivatives used to be a very
tedious procedure involving low yielding multi-step syntheses
and very often researchers turned to enzymatic processes
to prepare a few milligrams of the desired glucuronide
derivatives.23–34
Detailed metabolism studies, including identification, charac-
terization, and even toxicology studies, are required by
regulatory bodies when the glucuronidation is extensive.2,3
Usually, labeled glucuronide derivatives are prepared by first
introducing the isotopes on the compound of interest and then
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900
Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA
*Correspondence to: Bachir Latli, Chemical Development, Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-
0368, USA.
E-mail: bachir.latli@boehringer-ingelheim.com
J. Label Compd. Radiopharm 2011, 54 337–339
Copyright r 2011 John Wiley & Sons, Ltd.

B. Latli et al.
trityl chloride remaining. Additional labeled glucose (0.1 g) was (1H, m), 3.59 (1H, m), 2.29 (1H, m), 2.12 (3H, s), 2.08 (3H, s), 2.05
added and the mixture was warmed to 651C for 1 h. Then, acetic (3H, s), 2.03 (3H, s). 13C NMR (100 MHz, CDCl3) d: 170.3, 170.1,
anhydride (3.0 mL) was added and the mixture was allowed to 169.3, 169.0, 91.7 (d, J = 48 Hz, b-anomer), 89.1 (d, J = 45 Hz,
cool to ambient temperature and was stirred for 1 h. HPLC a-anomer), 74.9 (t, J = 42 Hz), 72.6 (t, J = 42 Hz), 70.4 (dd, J = 42,
analysis showed that the intermediate product was consumed. 48 Hz), 68.0 (t, J = 42 Hz), 61.4 (d, J = 46 Hz, a-anomer), 60.8
The reaction mixture was then poured into ice water (300 mL) (d, J = 42 Hz, b-anomer), 20.8, 20.6.
and stirred vigorously for 0.5 h. The solid was collected by
vacuum filtration, washed with water, and air dried to give a ½13C6ꢁ-(2S,3S,4S,5R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-
carboxylic acid (3)
white solid. The material was recrystallized from MeOH (30 mL)
to give [13C6]-(3R,4S,5R,6R)-6-(trityloxymethyl)tetrahydro-2H-
pyran-2,3,4,5-tetrayl tetraacetate b-anomer (1.6 g, 49 %) as a
white solid after vacuum filtration. The mother liquor was
concentrated and purified by CombiFlash Companion (120 g,
20–50% EtOAc/Hexane). The desired fractions were combined
and concentrated to give [13C6]-(3R,4S,5R,6R)-6-(trityloxymethyl)
tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate a/b-anomers
(0.9 g, 28%) as a white solid. HPLC: 18.1 min (b-anomer),
NaIO4 (2.5 g, 11.8 mmol) and RuCl3 ꢂ H2O (77 mg, 0.3 mmol) were
added to (2) (1.0 g, 2.96 mmol) in CCl4 (7.4 mL), MeCN (7.4 mL)
and H2O (11.8 mL) at ambient temperature. The mixture was
stirred for 3 h, diluted with H2O, and extracted with CH2Cl2 ( ꢀ 3).
The combined extracts were dried over Na2SO4 and concen-
trated to give a dark brown solid residue, which was diluted with
Et2O, and filtered through Celite. The filtrate was concentrated
in vacuo to give [13C6]-(2S,3S,4S,5R)-3,4,5,6-tetraacetoxytetra-
hydro-2H-pyran-2-carboxylic acid (950 mg, 89%) as a colorless
oil. 1:1.6 ratio of a/b. LCMS m/z: 391; 1H NMR (400 MHz, CDCl3) d:
6.41 (1H, bd, J = 179 Hz, a-anomer), 5.81 (1H, dd, J = 7.5, 169 Hz),
5.33 (2H, m), 5.16 (1H, m), 4.45 (1H, bd, J = 150 Hz, a-anomer),
4.27 (1H, m), 2.14 (3H, s), 2.07 (3H, s), 2.06 (3H, s), 2.05 (3H, s).
13C NMR (100 MHz, CDCl3) d: 169.7 (d, J = 65 Hz, b-anomer), 169.3
(d, J = 62 Hz, a-anomer), 91.3 (d, J = 49 Hz, b-anomer), 89.0
(d, J = 42 Hz, a-anomer), 72.4 (dd, J = 40, 65 Hz), 71.8
(t, J = 40 Hz), 70.0 (dd, J = 40, 49 Hz), 68.5 (t, J = 40 Hz), 61.6
(t, J = 42 Hz, a-anomer), 20.8, 20.5.
1
18.6 min (a-anomer) 1:1.5 ratio of a/b; LCMS m/z: 619; H NMR
(400 MHz, CDCl3) d: 7.44 (6H, m), 7.30 (6H, m), 7.24 (3H, m), 6.46
(1H, bd, J = 179 Hz, a-anomer), 5.74 (1H, dd, J = 7.5, 167 Hz), 5.38
(1H, m), 5.28 (1H, m), 5.00 (1H, m), 3.71 (1H, m), 3.35 (1H, m), 3.08
(1H, m), 2.17 (3H, s), 2.06 (3H, s), 2.02 (3H, s), 1.76 (3H, s). 13C NMR
(100 MHz, CDCl3) d: 170.3, 168.9, 169.4, 169.0, 143.5, 128.7, 127.0,
92.0 (d, J = 49 Hz, b-anomer), 89.4 (d, J = 45 Hz, a-anomer), 74.0
(t, J = 43 Hz), 73.2 (t, J = 40 Hz), 71.3 (t, J = 44 Hz), 70.5 (dd, J = 40,
49 Hz), 69.4 (t, J = 42 Hz), 68.3 (t, J = 43 Hz), 61.7 (d, J = 44 Hz,
b-anomer), 61.2 (d, J = 44 Hz, a-anomer), 20.9, 20.6, 20.4.
½13C6ꢁ-(2S,3S,4S,5R)-allyl-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-
2-carboxylate (4)
½13C6ꢁ-(3R,4S,5R,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-
2,3,4,5-tetrayl tetraacetate (2)
NaOMe (6.2 mL, 3.10 mmol, 0.5 M in MeOH) was added to a
solution of (3) (0.95 g, 2.58 mmol) in MeOH (15 mL) at ambient
temperature. The mixture was stirred for 2 h (a precipitate
formed after 5 min). The reaction was quenched by the addition
of H2O (100 mL) and then concentrated to give an off-white solid.
The solid was diluted with DMF (8 mL), then 3 N HCl (1.0 mL,
3 mmol) was added to convert the salt to the acid and then
added to a flask containing the Fluoride bound resin (1.3 g).
The mixture was stirred for 2 h, then allyl bromide (0.3 mL,
2.89 mmol) was added and the mixture was stirred at 401C
overnight. The mixture was filtered and concentrated to give
brown oil. The crude product was purified by CombiFlash
Companion (40 g, EtOAc: IPA: H2O 5:3:1) and the desired
Hydrogen bromide in acetic acid (1.02 g, 4.2 mmol) was added
to a solution of (1) (2.5 g, 4.2 mmol) in AcOH (20 mL) at ambient
temperature. The mixture was stirred for 30 min, filtered, and the
filtrate was diluted with H2O and CH2Cl2. The layers were
separated and the organic layer was washed with H2O ( ꢀ 3),
dried over Na2SO4, and concentrated to give a colorless oil.
The crude product was diluted with MTBE and then hexane was
added. The liquid was decanted off and the solid residue was
dried under vacuum to give [13C6]-(3R,4S,5R,6R)-6-(hydroxy-
methyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (1.03 g,
70%) as a sticky solid. 1:1.7 ratio of a/b. LCMS m/z: 377; 1H NMR
(400 MHz, CDCl3) d: 6.36 (1H, bd, J = 179 Hz, a-anomer), 5.74 (1H,
dd, J = 7.5, 167 Hz), 5.32 (1H, m), 5.11 (2H, m), 3.78 (1H, m), 3.67
Ph
Ph
O
OH
OH
O
OAc
OAc
O
OAc
OAc
1) TrCl, Pyr
HBr
HO
HO
HO
AcO
Ph
O
2) Ac2O
77%
AcOH
70%
AcO
OH
OAc
OAc
13C6
13C6
13C 6
(1)
(2)
.
O
O
RuCl3 H2O
NaIO4
O
OAc
OAc
O
OH
OH
1) NaOMe, MeOH
HO
O
CCl4, MeCN
H2O
2) PS-F, Allyl Bromide
DMF
AcO
13C6
HO
OAc
OH
37%
89%
13C6
(4)
(3)
Scheme 1. Synthesis of allyl-[13C6]-glucuronate.
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 337–339

B. Latli et al.
fractions were combined to give 230 mg of [13C6]-(2S,3S,4S,5R)-
allyl 3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylate in
37% yield as a light brown solid. 1:1.6 ratio of a/b. LCMS m/z:
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263. H NMR (400 MHz d6-DMSO): 6.82 (1H, m, a-anomer), 6.56
(1H, m), 5.91 (1H, m), 5.33 (1H, dddd, J = 17, 1.5, 1.5, 1.5 Hz), 5.21
(1H, dddd, J = 10.5, 1.5, 1.5, 1.5 Hz), 5.15 (0.3H, m), 5.00 (1H, m),
4.84 (1H, m), 4.73 (0.3H, m), 4.64 (1H, m), 4.58 (2H, m), 4.27 (0.3H,
m), 4.16 (0.3H, bd, J = 8 Hz), 3.90 (0.6H, m), 3.50 (0.6H, m),
3.35 (0.6H, m), 3.22 (0.3H, m), 3.14 (0.6H, m), 3.00 (0.3H, m).
13C NMR (100 MHz d6-DMSO): 169.5 (d, J = 61 Hz, b-anomer),
168.7 (d, J = 69 Hz, a-anomer), 97.5 (d, J = 45 Hz, a-anomer), 93.0
(d, J = 44 Hz, b-anomer), 132.3, 118.0, 64.7, 75.5 (m), 74.4 (m),
72.3(m), 71.9 (m). 1H NMR (400 MHz d4-MeOH): 5.93 (1H, m), 5.34
(1H, m), 5.21 (1H, m), 4.89 (0.4H, m), 4.66 (0.4H, m), 4.65 (2H, m),
4.50 (0.4H, m), 4.28 (0.3H, m), 3.98–4.16 (0.6H, m), 3.78–3.93
(0.7H, m), 3.45 (1H, m), 3.29-3.43 (0.6H, m), 2.93–3.27 (0.6H, m).
Results and discussion
Commercially available [13C6]-a-D-glucose was protected at the
C6 primary alcohol using trityl chloride in pyridine followed by
acetyl protection of the other hydroxyl groups using acetic
anhydride in the same reaction pot in 77% overall yield.35
Removal of the trityl group followed by oxidation of the primary
alcohol to the carboxylic acid in a one-pot procedure gave the
labeled tetraacetoxytetrahydropyran-2-carboxylic acid in 89%
yield. The oxidation was carried out with ruthenium trichloride
and sodium periodate in carbon tetrachloride. In our hands this
gave better yields than Jones reagent.35 The acid thus produced
can be converted to the methyl ester and then treated with
hydrobromide or titanium tetrabromide to give [13C6]-labeled
methyl acetobromo-[13C6]-a-D-glucuronate36 (not described).
Alternatively, the acetate groups can be removed using catalytic
amounts of sodium methoxide in methanol and then treated
with polymer supported fluoride and allyl bromide in DMF to
give allyl-[13C6]-glucuronate in 37% yield.37
´
´
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Drug. Metab. Dispos. 1997, 25, 1389–1394.
This labeled allyl-[13C6]-glucuronate has been used in our
laboratory to access 13C-labeled glucuronide conjugates of drug
candidates or their phase I metabolites according to the
literature.38–42 Scheme 1.
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Summary
´
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A five-step synthesis starting from the commercially available
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in 18% overall yield. This versatile intermediate can be used to
prepare stable labeled glucuronide derivatives of drug candi-
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Acknowledgements
We thank our colleague Dr Heewon Lee for analytical help.
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www.jlcr.org