Synthesis of urine drug metabolites: glucuronic acid glycosides of phenol intermediates

Article abstract of DOI:10.1016/j.carres.2007.01.014

The investigation of drug metabolism requires substantial amount of metabolites. Isolation from urine is tedious, therefore, the material obtained by synthesis is preferred. Substantial amounts of three tentative drug metabolites, phenolic glucuronides, h

Full text of DOI:10.1016/j.carres.2007.01.014

Carbohydrate Research 342 (2007) 970–974
Note
Synthesis of urine drug metabolites: glucuronic acid
glycosides of phenol intermediates
a
b
b
a,
*
˚
´
Carl Johan Arewang, Martina Lahmann, Stefan Oscarson and Anna-Karin Tiden
aDepartment of Chemistry, AstraZeneca, R&D So¨derta¨lje, S-151 85 So¨derta¨lje, Sweden
bDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 19 December 2005; received in revised form 23 January 2007; accepted 25 January 2007
Available online 31 January 2007
Abstract—The investigation of drug metabolism requires substantial amount of metabolites. Isolation from urine is tedious, there-
fore, the material obtained by synthesis is preferred. Substantial amounts of three tentative drug metabolites, phenolic glucuronides,
have been prepared using easily available glycosyl donors. The final products [3(2-N-methyl-N-isopropylaminoethoxy)phenyl] b-D-
glucopyranosiduronic acid, 4-amino-3,5-dimethylphenyl b-D-glucopyranosiduronic acid and [2(S)-propanoyl-6-O-naphthyl] b-D-
glucopyranuronic acid are useful as, for example, reference material in metabolite investigations.
ꢀ 2007 Published by Elsevier Ltd.
Keywords: Metabolites; Glycosylation; Glucuronic acid donor; Phenolic glucuronide
One of the most common ways to rid the body of lipid
metabolites is by oxidation or hydrolysis producing
additional hydroxyl- or carboxyl-groups, these deriva-
tives1 are subsequently used forming a glucuronic acid
derivative, either the glycoside or the anomeric ester,
both b-linked. To investigate drug metabolism, identifi-
cation of these glucuronic acid derivatives is important,
as is testing of all aspects of their biological activity.
Since often only minor amounts can be extracted from
the urine, identification is much simplified by compari-
son with a synthetic derivative of known structure. Also,
activity studies require substantial amount of metabo-
lites, preferably obtained by synthesis. Although several
glucuronic acid donor/promoter systems have been
developed,2,3 still there is no universal method found
for the synthesis of glucuronides (Scheme 2). Herein
we report the synthesis of three phenolic glucuronides,
all tentative urine drug metabolites: Glucuronide 14 is
a possible metabolite of a novel local anaesthetic and
glucuronide 17 is likely to be a metabolite of lidocaine,
also a local anaesthetic. The third glucuronide 19 is a
tentative metabolite of the anti-inflammatory substance
naproxen.
Since the synthesis of glucuronides has to be elabo-
rated for each individual case,4 a number of glucuronic
acid donors, 1–7 (Chart 1) were tried with acceptor 10,
which was prepared in an acceptable yield from O-2-
hydroxyethylresorcinol (Scheme 1). A silver triflate-pro-
moted coupling using the acetylated donor 15 at 0 ꢁC
COOMe
O
COOMe
O
COOMe
O
RO
RO
BnO
BnO
BnO
SEt
BnO
RO
1 R= Ac
OBz
BzO
Br
Br
3
4
2 R= Bz
COOMe
O
COOMe
O
Si
O
Si
O
O
SEt
O
O
O
OBz
RO
Br
6 R= Bz
7 R= Piv
Si
Si
5
COOMe
O
COOMe
O
AcO
AcO
PivO
PivO
OAc
OAc
PivO
*
Corresponding author. Tel.: +46 855326991; fax: +46 855328892;
e-mail addresses: martinal@chem.gu.se; anna-karin.tiden@
astrazeneca.com
8
9
Br
Chart 1. Glucuronic acid donors.
0008-6215/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.carres.2007.01.014

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C. J. Arewang et al. / Carbohydrate Research 342 (2007) 970–974
971
of coupling product was observed (MALDI-TOF), we
decided to protect the amino group. Trifluoroacetyla-
tion gave an almost insoluble compound and the phthal-
imido protected acceptor produced only small amounts
of the desired glucuronide under Koenigs-Knorr condi-
tions both with donors 1, 2 and 8.9 The best result (ap-
prox. 40%) with this acceptor was obtained using donor
95 and BF3–etherate, but the product was difficult to
purify. The benzyloxycarbonyl protected acceptor 11
(Scheme 1) did not react with donor 9 under these con-
ditions but gave a b-glucuronide using donor 2. Depend-
ing on reaction conditions, yields between 20% and 50%
were isolated but both orthoester formation and transe-
sterification were frequent side reactions. The major
drawback, however, was the severe competition of the
elimination reaction during the debenzoylation step.
Fortunately, the acetylated donor 15 gave glucuronide
15 in a good yield (57%, Scheme 2). Deprotection affor-
ded target derivative 17 (38%), which was found to be
rather labile and therefore characterised and stored as
the hydrochloride salt of methyl ester 16.
In case of the naproxen derivative 12, which was pre-
pared in two steps from commercial (S)-2-(6-methoxy-2-
naphthyl)propanoic acid (naproxen, Scheme 1), a silver
promoted coupling was not feasible because the accep-
tor is easily oxidised by silver (Ag+). However, using
the 1-O-acetyl derivative 9 as donor together with
BF3–etherate gave 18 in 67% yield. One-pot conven-
tional deprotection then afforded the third target struc-
ture 19 in 70% yield.
i,ii
OH
N
HO
HO
HO
HO
O
O
10
62%
iii
96%
NH2
NHCbz
11
12
COOH
COOMe
iv
quant.
HO
HO
Scheme 1. Synthesis of the acceptor molecules. Reagents and condi-
tions: (i) (1) MsCl, Et3N, t-BuOMe; (2) i-PrMeNH; (ii) NaOH, t-
BuHSO4, 1,4-dioxane; (iii) CbzCl, NaOAc/water; (iv) TMSCl, MeOH.
gave an a/b-mixture (1:3) in 40% yield, whereas the
benzoylated donor 26 produced a mixture of b-glycoside
13 and the corresponding orthoester in similar yield.
Exclusive formation of b-glycoside 13, albeit still only in
a moderate yield (40%), was obtained when the coupling
between donor 2 and acceptor 10 was carried out at
ambient temperature. The use of more elaborated do-
nors7,8 (4–7) did not improve the yield. Further it was
found that the use of thioglycoside donors 3 and 5 in
combination with thiophilic promoters, that is, DMTST
and NIS/TfOH, was not compatible with acceptor 10.
DMTST as promoter produced a number of unidenti-
fied products, while NIS/TfOH gave iodinated phenyl
derivatives (as identified by MS), products of possible
side reactions with the activated aromatic acceptor.
However, taking into account the accessibility of donor
2 and acceptor 10, the yield in the glycosylation was
sufficient to synthesise enough material of the desired
glucuronide 13 (Scheme 2). Conventional deprotection
afforded in two steps target compound 14 (51%).
In conclusion, the synthesis of three possible drug
metabolite phenolic glucuronides has been accom-
plished. The synthetic pathways utilise easily available
glycosyl donors and allow preparation of substantial
amounts of the target glucuronides. The use of more
complex donors developed for oligosaccharide synthe-
sis7,8 was not an advantage with these phenolic aglycons.
Furthermore, activated phenol aglycons were not com-
patible with thiophilic promoters, especially iodonium
reagents.
The preparation of glucuronide 17 (Scheme 2) was
first tried with the unprotected aminophenol as acceptor
under various conditions. Since no significant formation
COOMe
COOH
ii, iii
O
2, i
O
BzO
BzO
N
HO
N
O
O
O
N
O
HO
40%
51%
HO
HO
O
OBz
OH
10
11
13
14
COOMe
O
COOR
ii, iv
1, i
AcO
AcO
O
O
NHCbz
NHCbz
HO
HO
O
NH2
57%
70%
OAc
OH
15
16 R = Me
17 R = H
v
COOMe
COOH
COOMe
COOMe
O
COOH
O
ii, vii
70%
9, vi
AcO
AcO
HO
HO
O
O
67%
HO
OAc
OH
12
18
19
Scheme 2. Synthesis of phenyl glucuronides. (i) AgOTf, CH2Cl2; (ii) NaOMe, MeOH; (iii) NaOH, MeOH; (iv) H2, Pd/C, MeOH; (v) LiOH, MeOH;
(vi) BF3–Et2O, CH2Cl2; (vii) LiOH, water.

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972
1. Experimental
cinol (7.5 g, 26.1 mmol) was dissolved in dioxane
(100 mL) and saturated sodium hydroxide (45 mL) and
tert-butyl hydrogensulfate (0.8 g, 2.4 mmol) was added.
Then the reaction mixture was heated to 105 ꢁC over-
night and left at room temperature for two days
(TLC: 9:1 MeCN–Et3N). The reaction mixture was di-
luted with CH2Cl2, and the water phase (pH ꢀ 12), con-
taining most of the desired product, collected. The water
phase was adjusted to pH = 8 with HCl (2 M) and then
extracted with CH2Cl2 twice. The combined organic
phases were dried with sodium sulfate, filtered and evap-
orated to give 10 (4.1 g, 19.5 mmol, 75%). NMR
(CD3OD, Ref. d 3.31): 1H, d 1.14 (d, 6H, J 6.6 Hz,
CH–Me), 2.45 (s, 3H, NMe), 2.98 (dd, 3H, J 5.6 Hz,
NCH2), 3.08 (m, 1H, NCH), 4.09 (dd, 2H, J 5.6 Hz,
OCH2), 6.41 (m, 3H, PhH), 7.06 (t, 3H, PhH); 13C d
17.7 (2 · CHMe), 38.3 (NCH3), 53.0 (NCH2), 56.4
(NCH), 66.4 (OCH2), 103.1, 106.8, 109.4, 131.1, 159.9,
161.3 (Ph). HRMS: Calcd for C12H19NO2 210.1494
[M+H]+. Found: 210.1485 [M+H]+.
1.1. General methods
All organic solvents were distilled before use, except
Et2O, which was stored over Na. Organic solutions were
dried over MgSO4 before concentration, which was
performed under diminished pressure at <40 ꢁC (bath
temperature). NMR spectra were recorded at 300 or
400 MHz (Varian/JEOL) (1H) or at 75 or 100 MHz
(13C), respectively, in CDCl3, D2O or CD3OD. Except
for D2O (d 4.80), TMS was used as internal standard
1
(d 0) for H spectra. 13C Spectra were referred to the
CHCl3 signal (d 77.17) or MeOH signal (d 49.15). Silica
Gel E. Merck 60 (0.040–0.063) was used for flash chro-
matography. TLC was performed on Silica Gel 60 (E.
Merck) glass plates with detection by UV-light and/or
charring with 8% sulfuric acid. MALDI-TOF spectra
were recorded on a Bruker Biflex III using PEG 600,
PEG 1000 and PEG 1500 as calibration reference and
20,40,60-trihydroxy-acetophenone monohydrate (THAP)
as matrix. High resolutions mass spectra were recorded
on a Micromass Q-TOF Micro (ESI) with 10 mM
NH4OOCH at pH 3.2 as a buffer.
1.3. 4-Benzyloxyamido-3,5-dimethylphenol (11)
Benzyl chloroformate (8.5 mL, 4 equiv) was added
in two portions to a slurry of 4-amino-3,5-dimethyl-
phenol10,11 (2.0 g, 14.6 mmol) and NaOAc (50 mL, 2 M
in water). After 10 min a mixture of EtOAc and toluene
(50 mL, 1:1, v/v) was added and the stirring continued
for further 30 min. The organic layer was then sepa-
rated, dried and concentrated. The crude product was
crystallised from toluene to obtain 11 (3.8 g, 14.0 mmol,
96%). Mp: 87–88 ꢁC. NMR (CD3OD): 1H, d 2.13 (s, 6H,
PhMe), 5.22 (s, 2H, Cbz), 6.30 (s, 2H, PhH), 7.36–7.42
(m, 5H, Cbz); 13C, d 18.4 (PhMe), 67.6 (Cbz), 115.4
(PhC2, PhC4), 125.2 (PhC3, PhC5), 128.3, 128.5, 128.8
(Cbz), 136.3 (PhC4), 137.4 (Cbz), 155.4, 156.0 (Cbz,
PhC6).
1.2. 3-(2-N-Methyl-N-isopropylaminoethoxy)phenol (10)
O-2-Hydroxyethyl resorcinol (5 g, 32.43 mmol) was dis-
solved in tert-butyl methyl ether (250 mL), flushed with
nitrogen and cooled to 0 ꢁC. Triethylamine (10.9 mL,
77.8 mmol) was added and then mesyl chloride
(5.6 mL, 71.3 mmol) during a period of 10 min. The
ice bath was removed after 1 h and stirring was contin-
ued for another hour, when TLC (4:1 toluene–EtOAc)
showed complete reaction. The reaction mixture was di-
luted with Et2O, washed with saturated sodium hydro-
gen carbonate twice, concentrated and co-evaporated
with toluene. The residue was dissolved in 1:4 EtOAc–
toluene (250 mL), then N-methyl-N-isopropylamine
(60 mL, 575 mmol) was added and the soln was heated
and left at 70 ꢁC overnight. Another 1.5 mL (14 mmol)
of N-methyl-N-isopropylamine was added and the reac-
tion was complete within 4 h. The reaction mixture was
diluted with toluene, washed with water three times,
dried, filtered and concentrated. The residue was puri-
fied by flash-chromatography (10:1:0.1 EtOAc–
MeOH–Et3N) to produce 3-(2-N-methyl-N-isopropyl-
aminoethoxy)-1-mesyl resorcinol (7.6 g, 26.3 mmol,
82%). NMR (CD3OD): 1H, d 1.05 (d, 6H, J 6.6 Hz,
CH–Me), 2.34 (s, 3H, NMe), 2.80 (dd, 2H, J 6.2 Hz,
NCH2), 2.90 (m, 1H, NCH), 3.13 (s, 3H, SCH3), 4.05
(dd, 2H, J 6.2 Hz, OCH2), 6.86 (3H, m, PhH), 7.30 (t,
1H, PhH); 13C, d 18.0 (2 · CHMe), 37.4, 38.5 (SCH3
and NCH3), 51.7, 54.4 (NCH and NCH2), 67.3
(OCH2), 108.8, 113.8, 114.0, 130.4, 150.2, 160.2 (Ph).
3-(2-N-Methyl-N-isopropylaminoethoxy)-1-mesyl resor-
Anal. Calcd for C16H17NO3: C, 70.8; H, 6.3; N, 5.2;
O, 17.7. Found: C, 70.8; H, 6.3; N, 5.2.
1.4. Methyl 2(S)-(6-hydroxy-2-naphthyl)propionate (12)
TMSCl (0.6 mL, 4.6 mmol) was added to a soln of
2(S)-(6-hydroxy-2-naphthyl)propionic acid12 (0.50 g,
2.3 mmol) in MeOH (20 mL). TLC (45:4:1 CHCl3–
MeOH–AcOH) showed complete conversion after 2 h.
Dry toluene (5 mL) was added and the mixture concen-
trated and coevaporated with dry toluene to give methyl
2(S)-(6-hydroxy-2-naphthyl)propionate
12
(0.53 g,
1
2.3 mmol, quant.) as a white solid. NMR (CDCl3): H,
d 1.59 (d, J 6.4 Hz, Me), 3.70 (s, 3H, OMe), 3.87 (m,
1H, CH), 7.05 (m, 2H, PhH), 7.37 (d, 1H, PhH), 7.65
(m, 3H, PhH); 13C (CD3OD), d 18.6 (PhMe), 45.5
(CH), 52.4 (OMe), 109.4, 118.2, 126.1, 126.4, 127.0,
128.9, 129.8, 133.8, 153.7 (Ph), 175.0 (CO).

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973
˚
1.5. [3(2-N-Methyl-N-isopropylaminoethoxy)phenyl] b-D-
glucopyranosiduronic acid (14)
(0.81 g, 3.0 mmol) in CH2Cl2 (50 mL) containing 4 A
molecular sieves. After 3 h, Et3N (1 mL) was added
and the stirring was continued for 15 min. The mixture
was diluted with CH2Cl2 and filtered through Celite,
concentrated, and the residue was purified by silica gel
chromatography (toluene ! 6:1 toluene–EtOAc) to give
15 (15 g, 1.7 mmol, 57%). [a]D ꢁ14.8 (c 1.0, CH2Cl2);
13C NMR (CDCl3): d 18.7 (PhMe), 20.6, 20.7 (COCH3),
53.1 (OMe), 67.2 (Cbz), 69.3, 71.2, 72.0, 72.8 (C2, C3,
C4, C5), 99.2 (C1), 116.6 (PhC2, PhC4), 125.4 (PhC3,
PhC5), 128.3, 128.7, 129.2 (Cbz), 137.8, 138.0 (PhC4,
Cbz), 155.3, 155.8 (Cbz, PhC6), 167.0 (COOMe),
169.4, 169.5, 170.2 (CO). A soln of 15 (900 mg,
1.53 mmol) in MeOH (100 mL) was treated with a cata-
lytic amount of sodium in MeOH. After 30 min an addi-
tional amount of sodium in MeOH was added. The
stirring was continued for 2 h. Although some starting
material was left according to TLC (5:1 CHCl3–MeOH),
the reaction was quenched by addition of Dowex 50
(H+) ion exchange resin, because the formation of elim-
ination product started to occur. After filtration and
concentration, the crude product was purified by flash
chromatography (CHCl3–MeOH 20:1 ! CHCl3–MeOH
10:1) giving methyl [(4-benzyloxyamido-3,5-dimethyl-
phenyl) b-D-glucopyranosid]uronate (500 mg, 1.07
mmol, 71%). [a]D ꢁ62.0 (c 1.03, MeOH); 1H NMR
(MeOD): d 2.20 (s, 6H, PhMe), 3.48 (m, 2H), 3.62
(t, J 9.7 Hz, 1H), 3.76 (s, 3H, OMe), 4.03 (d, J
9.7 Hz, H-1), 4.95 (d, J 7.0 Hz, 1H), 5.18 (s, 2H, Cbz),
6.80 (s, 2H, PhH), 7.36 (m, 5H, Cbz). 13C NMR
(MeOD): d 18.7 (PhMe), 53.0 (OMe), 67.8 (Cbz), 73.1,
74.7, 76.8, 77.2 (C2–C5), 102.6 (C1), 117.4 (PhC2,
PhC4), 126.0 (PhC3, PhC5), 128.9, 129.2, 129.6 (Cbz),
138.4, 138.9 (PhC4, Cbz), 156.0, 157.3 (PhC6, Cbz),
171.8 (CO). The above obtained compound (856 mg,
1.85 mmol) was dissolved in MeOH (100 mL) and 10%
Pd/C (50 mg) was added. Hydrogenolysis was carried
out under H2 at atmospheric pressure overnight. The
suspension was filtered through a plug of Celite and
RP-C18 gel, which was washed with MeOH (100 mL).
The combined solutions were titrated with HCl (1 N,
ꢀ1.8 mL) and concentrated to approx. 5 mL. The resid-
ual soln was diluted with water (50 mL) and washed
with Et2O. After freeze-drying, 16 (665 mg, 1.83 mmol,
99%) was obtained. [a]D ꢁ78.0 (c 2.0, MeOH); 1H
NMR (MeOD): d 2.14 (s, 6H, PhMe), 3.45 (m, 2H),
3.61 (t, J 9.4 Hz, 1H), 3.77 (s, 3H, OMe), 3.94 (d,
J 9.8 Hz, H-1), 4.78 (d, J 7.6 Hz, 1H), 6.68 (s,
2H, PhH). 13C NMR (CD3OD): d 18.1 (PhMe),
53.0 (OMe), 73.2, 74.8, 76.8, 77.3 (C2–C5), 104.1
(C1), 118.6 (PhC2, PhC4), 124.9 (PhC3, PhC5), 139.6
(PhC4), 151.3 (PhC6), 171.2 (CO). Q-TOF HRMS:
Calcd for C15H22NO7 328.1396 [M+H]+, 350.1216
[M+Na]+. Found: 328.1391 [M+H]+, 350.1216
[M+Na]+. The free amino compound 16 (42 mg,
128 lmol) was dissolved in a methanolic LiOH soln
To a stirred soln of 10 (69 mg, 0.33 mmol) and 2
(307 mg, 0.53 mmol) in dry CH2Cl2 (3 mL) containing
˚
4 A molecular sieves (0.5 g), silver triflate (203 mg,
0.79 mmol) dissolved in dry toluene (1.5 mL) was added
in small portions. After 4 h at room temperature under
darkness the mixture was diluted with CH2Cl2 and fil-
tered through Celite, concentrated, and the residue
was purified by silica gel chromatography (24:1:0.025
CH2Cl2–MeOH–TEA) to give 13 (94 mg, 0.13 mmol,
1
40%). H NMR (CDCl3): d 1.08 (dd, 6H, CH(Me)2),
2.32 (s, 3H, NMe), 2.76 (t, 2H, NCH2), 2.88 (m, 1H,
NCH), 3.65 (s, 3H, OMe), 3.97 (t, 2H, OCH2), 4.50
(d, J4,5 9.0 Hz, 1H, H5), 5.45 (d, J1,2 7.0 Hz, 1H, H1),
5.74 (dd, J1,2 7.4 Hz, J2,3 9.2 Hz, 1H, H2), 5.82 (t, J3,4
9.2 Hz, J4,5 9.2 Hz, 1H, H4), 5.96 (t, J2,3 9.2 Hz, J3,4
9.2 Hz, 1H, H3), 6.60 (m, 3H), 7.17 (t, 1H), 7.30–7.40
(m, 7H), 7.44–7.52 (m, 2H), 7.90–7.98 (m, 6H). Thermo-
spray mass spectra from a Finnigan SSQ 7000 showed
[M+H] = 712.
A soln of 13 (70 mg, 0.10 mmol) in MeOH (4 mL) was
treated with sodium methoxide (4.4 mL, 0.1 M,
0.44 mmol) and stirred for 3 h. pH was adjusted to 6
with Dowex H+ ion exchange resin, filtered and concen-
trated to give crude 14 (28 mg, 0.07 mmol, 70%). No
further purification was performed. Thermospray
mass spectra from a Finnigan SSQ 7000 showed
[M+H] = 400. The crude product (10 mg, 0.025 mmol)
was dissolved in MeOH (0.5 mL) before NaOH
(40 lL, 0.038 mmol, 1 M) was added. After 4 h the mix-
ture was neutralised with H+-ion exchange resin, filtered
and concentrated. The residue was purified by gel filtra-
tion on a Bio-Gelꢂ P-2 (Fine) column and then freeze-
dried to give 14 (7 mg, 0.018 mmol, 73%). NMR
1
(CD3OD): H, d 1.34 (dd, 6H, CH(Me)2), 2.81 (s, 3H,
NCH3), 3.41 (m, 2H, NCH2), 3.51 (m, 3H, H2–H5),
3.63 (m, 1H, NCH), 3.76 (m, 1H, H2–H5), 4.26 (m,
2H, OCH2), 4.90 (d, J1,2 7.2 Hz, 1H, H1), 6.64 (dd,
J4,5 8.4 Hz, J2,4 2.3 Hz, 1H, PhH4), 6.75 (dd, J5,6
8.1 Hz, J2,6 1.8 Hz, 1H, PhH6), 6.85 (t, J2,4 2.3 Hz, J2,6
2.2 Hz, 1H, PhH2), 7.20 (t, J4,5 8.2 Hz, J5,6 8.2 Hz,
1H, PhH5). 13C, d 16.6–16.7 (CH(Me)2), 37.1 (NCH3),
52.9 (NCH2), 59.1 (NCH), 63.8 (OCH2), 73.7, 74.8,
76.7, 78.0 (C2–C5), 102.5 (C1), 105.0 (PhC2), 109.9
(PhC4), 112.0 (PhC6), 131.2 (PhC5), 160.2, 160.4
(PhC1, PhC3), 176.4 (COOH). Q-TOF HRMS: Calcd
for C18H28NO8 386.1815 [M+H]+. Found: 386.1810
[M+H]+.
1.6. 4-Amino-3,5-dimethylphenyl b-D-glucopyranosid-
uronic acid (17)
Silver triflate (1.0 g, 3.9 mmol) was added at room tem-
perature to a stirred soln of 1 (1.6 g, 4.0 mmol) and 11

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974
(0.5 mL, 0.1 M). The mixture was stirred at room tem-
perature overnight. The TLC (12:3:3:1 EtOAc–AcOH–
MeOH–water, Rf ꢀ 0.5) showed complete conversion
to the uronic acid. Excess of base was removed by addi-
tion of Dowex 50 (H+) ion exchange resin. Crude 17
(40 mg) was obtained after concentration. 1H NMR
(D2O): d 2.26 (s, 6H, PhMe), 3.52 (m, 3H), 3.81 (d, J
7 Hz, 1H), 4.92 (d, J 9 Hz, 1H), 6.71 (s, 2H, PhH).
MALDI-TOF MS: Calcd for C14H19NO7 313.13 [M];
Found 335.98 [M+Na]+, 352.00 [M+K]+.
(5 mL). After 2 h the mixture was neutralised with H+
ion exchange resin, filtered and concentrated. The crude
residue was dissolved in water (2 mL), filtered through a
short RP C18 column (Sep-pack). Freeze drying gave 19
1
(600 mg, 1.50 mmol, quant.). NMR (D2O): H, d 1.46
(d, J 7.2 Hz, Me), 3.63 (m, 3H), 3.75 (m, 1H), 3.92 (d,
J 8.2 Hz, 1H), 5.19 (d, J 5.2 Hz, 1H), 7.28 (m, 2H,
PhH), 7.44 (m, 2H, PhH), 7.71 (s, 1H, PhH), 7.79 (m,
2H, PhH); 13C, d 18.7 (PhMe), 48.7 (CH), 72.2, 73.6,
76.1, 77.0 (C2–C5), 100.9 (C1), 111.3, 119.3, 126.0,
127.4, 128.0, 130.1, 130.2, 133.2, 139.9, 154.9 (Ph),
176.0, 183.9 (COOH). Q-TOF HRMS: Calcd for
C19H20O9 410.1451 [M+NH4]+, 415.1005 [M+Na]+.
Found: 410.1450 [M+NH4]+, 415.1007 [M+Na]+.
1.7. (2(S)-Propanoyl-6-O-naphthyl) b-D-glucopyranuronic
acid (19)
A stirred soln of 9 (750 mg, 2.0 mmol) and 12 (460 mg,
2.0 mmol) in dry CH2Cl2 (15 mL) was cooled (0 ꢁC)
when BF3ÆEt2O (650 lL, 5 mmol) was added. The reac-
tion mixture was allowed to attain room temperature
and after additional 12 h, it was poured onto a slurry
of ice water (100 mL). The organic phase was separated,
diluted with CH2Cl2, washed with NaHCO3 and brine
and concentrated. The residue was applied onto a silica
gel column and eluted (toluene ! toluene–EtOAc 10:1)
to give 18 (730 g, 1.34 mmol, 67%). [a]D +19.5 (c 1.9,
CH2Cl2); 1H NMR (CDCl3): d 1.59 (d, J 7.2 Hz,
CHMe), 2.02, 2.04, 2.05 (s, 9H, COMe), 3.66 (s, 3H,
OMe), 3.73 (s, 3H, OMe), 3.87 (q, 1H, CHMe), 4.28
(d, J 9.5 Hz, 1H), 5.27–5.38 (m, 4H), 7.16 (dd, 1H, J
2.4 Hz, J 8.8 Hz, PhH), 7.31 (d, 1H, J 2.2 Hz, PhH),
7.41 (dd, 1H, J 1.0 Hz, J 8.5 Hz, PhH), 7.65 (m, 3H,
PhH). 13C NMR (CDCl3): d 18.7 (PhMe), 20.7, 20.8
(COMe), 45.5 (CH), 52.2, 53.2 (OMe), 69.3, 71.3, 72.0,
72.9 (C2–C5), 99.4 (C1), 111.7, 119.2, 126.1, 126.6,
127.8, 129.7, 130.4, 133.3, 137.1, 154.6 (Ph), 167.1,
169.4, 169.5, 170.2, 175.1 (CO, COMe). NaOMe (1M,
approx. 15 drops) was slowly added to a soln of 18
(1.2 g, 2.2 mmol) in MeOH (50 mL) until the colour
stopped changing (from bright yellow to orange-red).
The reaction mixture was left overnight at room temper-
ature to obtain complete removal of the acetate groups
(TLC: 6:1 CH2Cl2–MeOH), neutralised with H+ ion ex-
change resin, filtered and concentrated. The residue was
purified by flash-chromatography (CH2Cl2 ! CH2Cl2–
MeOH 10:1) to produce methyl [2(S)-(methylpropan-
oyl)-6-O-naphthyl]-b-D-glucopyranosid]uronate (655 mg,
1.55 mmol, 70%). [a]D ꢁ40.0 (c 1.0, MeOH); NMR
(CDCl3): 1H, d 1.56 (d, J 7.2 Hz, Me), 3.66 (s, 3H,
OMe), 3.70 (s, 3H, OMe), 3.80–4.05 (m, 5H), 4.52 (sb,
1OH), 4.59 (sb, 1OH) 5.08 (d, J 7.2 Hz, 1H), 5.14 (sb,
1OH), 7.16 (dd, 1H, PhH), 7.33 (m, 2H, PhH), 7.59
(m, 3H, PhH); 13C, d 18.7 (PhMe), 45.5 (CH), 52.2,
53.1 (OMe), 71.4, 73.0, 74.6, 75.6 (C2–C5), 101.2 (C1),
111.8, 119.3, 126.0, 126.4, 127.8, 129.6, 130.2, 133.3,
136.8, 154.7 (Ph), 169.7, 175.1 (COMe). LiOHÆH2O
(130 mg, 3.1 mmol) was added to a soln of the obtained
methyl ester derivate (630 mg, 1.5 mmol) in water
Acknowledgements
We thank Dr. Benita Forngren, Analytical Chemistry,
Process R&D, AstraZeneca, R&D So¨derta¨lje for record-
ing the HRMS on compounds 14, 16 and 19. We also
thank Dr. Kristina Lycknert and Dr. Rune Sandberg
(former colleagues at AZ) for their contributions in
the synthesis of compound 10.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.01.014.
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