Synthesis of isotopically labelled SGLT inhibitors and their metabolites

Article abstract of DOI:10.1016/j.tet.2009.12.003

Isotopically labelled analogues of two structurally very similar SGLT inhibitors AVE2268 (1a) and AVE8887 (1b) have been synthesized by various routes. The radioactive labelled [¹⁴C]-AVE2268 was prepared in five steps including a Friedel-Crafts acylation as the key step for the ¹⁴C-label introduction. For [¹⁴C]-AVE8887 the same synthetic approach was not successful and therefore an alternative thiophene metallation/Weinreb amide sequence was developed. This pathway was also applied to obtain stable isotopically labelled analogues of both AVE2268 and AVE8887. Finally, the synthesis of two metabolites, sulfate 12 and glucuronide 13 were achieved by applying interesting protecting group and oxidation strategies.

Full text of DOI:10.1016/j.tet.2009.12.003

Tetrahedron 66 (2010) 1472–1482
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of isotopically labelled SGLT inhibitors and their metabolites
*
Volker Derdau , Thorsten Fey, Jens Atzrodt
Sanofi-Aventis Deutschland GmbH, Isotope Chemistry & Metabolite Synthesis, G876, 65926 Frankfurt/Ho¨chst, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Isotopically labelled analogues of two structurally very similar SGLT inhibitors AVE2268 (1a) and
AVE8887 (1b) have been synthesized by various routes. The radioactive labelled [14C]-AVE2268 was
prepared in five steps including a Friedel–Crafts acylation as the key step for the 14C-label introduction.
For [14C]-AVE8887 the same synthetic approach was not successful and therefore an alternative thio-
phene metallation/Weinreb amide sequence was developed. This pathway was also applied to obtain
stable isotopically labelled analogues of both AVE2268 and AVE8887. Finally, the synthesis of two me-
tabolites, sulfate 12 and glucuronide 13 were achieved by applying interesting protecting group and
oxidation strategies.
Received 29 October 2009
Received in revised form
24 November 2009
Accepted 1 December 2009
Available online 21 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
molecules throughout the body and in excreta, even after their
transformation into different metabolites, the administration of
Sodium glucose co-transporters (SGLT-2)1 are membrane pro-
teins. They play an important role in maintaining glucose equilib-
rium in the human body by re-absorption of glucose in the kidney.
It is therefore expected that the inhibition of SGLT-2 transporters
could decrease glucose blood levels by preventing re-absorption
from the urine. The control of these transporters could be an ef-
fective tool in the normalisation of high blood glucose levels that
are associated with diseases such as Type 1 and Type 2 diabetes. In
combination with a negative energy balance, such a potential
profile could be highly attractive for the treatment of diabetes
mellitus and obesity. Other potential therapeutic applications like
treatment of lipid disorders, atherosclerosis, cardiovascular dis-
eases, high blood pressure and metabolic syndrome could also be
relevant.2 Consequently, the development of small-molecule in-
hibitors of SGLT has received widespread attention across the
pharmaceutical industry (Fig. 1).3
Two new candidates are being developed by Sanofi–Aventis as
novel SGLT-2 inhibitors for the treatment of type II diabetes mel-
litus. They are AVE2268 (1a) and AVE8887 (1b).4 In animal models,
both compounds have reduced the intestinal absorption of glucose
and at the same time increased renal excretion of glucose. In the
course of drug development the candidate’s pharmacokinetic (PK)
properties and the absorption, distribution, metabolism and elim-
ination (ADME)5 characteristics have to be evaluated in vitro, then
in animals and finally in humans.6 In order to keep track of the drug
radiolabelled drugs is considered essential.7 Typically, 14C is the
label of choice because it can be introduced into a metabolically and
chemically stable position in the backbone of the compound
without changing the pharmacological profile of the drug
candidate.8
HO
HO
HO
HO
HO
O
O
HO
O
O
OH
OH
F
F
F
S
S
O
O
1a
AVE2268
1b
AVE8887
Figure 1.
In recent years, liquid chromatography coupled with tandem
mass spectroscopy detection (LC–MS/MS) has become the most
powerful bioanalytical tool for the investigation of samples from
these animal and human toxico-, metabolism and pharmacokinetic
studies.9 For a quantitative LC–MS/MS analysis of the new drug
candidates or relevant metabolites in complex matrices (like blood,
urine, bile etc.,), stable isotopically labelled internal standards are
considered essential.10 That means, in conjunction with the plan-
ned development program for AVE2268 1a and AVE8887 1b, both
14C- and stable isotopically labelled versions of these new drug
candidates were required. In addition, two major metabolites were
requested to be synthesised as the toxicological and pharmaco-
logical profile of relevant metabolites also needs to be assessed
during drug development.11
* Corresponding author. Tel.: þ49 6930538756.
E-mail address: Volker.Derdau@sanofi-aventis.com (V. Derdau).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.12.003

V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
1473
2. Results and discussion
started to look for alternative strategies for the synthesis of 5b in
which greater emphasise was taken in minimising decomposition
by increased reactivity of the thiophene 2.
2.1. Synthesis of 14C-labelled AVE2268 1a and AVE8887 1b
O
O
In choosing the labelling position not only the chemical and
metabolic stability have to be considered but also the availability of
suitable 14C-labelled precursors. For all these reasons placing the
label into the benzylic position was preferred. Using thiophene ring
labelling could have had the advantage of a common 14C-building
block for both candidates however it is less accessible for labelling
and therefore much more expensive. Hence, 14C-synthesis of
AVE2268 1a was achieved following the simple five step synthesis
path depicted in Scheme 1.12 Starting from commercially available 3-
methoxythiophene 2 and [14CO]anisoyl chloride [14C]-3a, purchased
from Amersham Biosciences, a completely regioselective Friedel–
Crafts acylation followed by a selective methyl ether cleavage of the
3-methoxythiophene derivative 4a gave the hydroxyl-thiophene
derivative [14C]-5a. Subsequent alkylation with acetobromo-alpha-
glucose under phase transfer conditions, carbonyl reduction with
NaCNBH3/TMSCl13 and final basic acyl deprotection afforded the
desired compound [14C]-1a in a very good overall yield of 50%. The
structure of 1a was confirmed by an X-ray analysis of 8a (Fig. 2).
O
SnCl4, CH2Cl2
32 %
Cl
2
+
S
OCF3
OCF3
3b
4b
O
HO
BBr3 - SMe2
57 %
S
OCF3
5b
Scheme 2. Synthesis of AVE8887 1b building block 5b.
Stimulated by results reported by Miller et al. and Gronowitz
et al.14 we examined the regioselective lithiation of 3-methox-
ythiophene 2 in refluxing ether over 5 min. However, when
quenching the lithiated thiophene 2 either with anisoyl chloride 3a
or 4-trifluoromethoxybenzoyl chloride 3b only traces of the cor-
responding products 4a and 4b, respectively, could be observed.
Besides acid chlorides the Weinreb amide is well known to be re-
active in organometallic reactions and would be readily accessible
also in labelled form.15 Initial tests of 9b in the reaction with the
lithiated thiophene 2 resulted in the formation of 4b in a promising
40% yield (Table 1, entry 1). Consequently, Weinreb amide 9b was
used during further optimisation of the reaction conditions in the
lithiation step (Table 1). Under the described reaction conditions no
regioisomer of 4b was determined (LC–MS).
O
1. SnCl4, CH2Cl2 (4a)
2. BBr3 - DMS
O
O
HO
14C
14C
Cl
+
S
S
O
O
14C]-3a
[
14C]-5a
80 % (2 steps)
HO
[
2
O
O
Br
O
O
O
O
O
O
1.
HO
O
O
Table 1
Optimisation of lithiation conditions
(7a)
6
HO
OH
O
1. 1.3 equiv. n-BuLi
O
14C
H2
2. NaCNBH3, TMSCl (8a)
3. NaOMe, MeOH
40°C, Ether, 5 min
O
S
2
O
O
63 % (3 steps)
S
2.
[
14C]-1a
O
N
OCF3
Scheme 1. Synthesis of [14C]-AVE2268 1a.
OCF3
4b
9b
0°C, 30 min
Entry
2 (equiv)
Solvent
Yield 4ba [%]
1
2
3
4
5
6
7
1.0
1.3
1.5
2.0
1.3
1.3
1.3
Et2O
Et2O
Et2O
Et2O
Toluene
THF
MTBE
40
66
67
74
9
53
62
a
Yields are based on Weinreb amide 9b.
Initially the focus of this optimisation work was to achieve im-
proved reaction conditions for the synthesis of only 14C-labelled
AVE8887, in which handling of refluxing ether is manageable due to
the small scale. To this end the best results were achieved
employing 2 equiv of n-BuLi (entry 4). However, for a multi-gram
synthesis of AVE8887 both a safer solvent and a more convenient
electrophile were preferred. Diethylether could be substituted by
less flammable MTBE (entry 7) and the Weinreb amide 9b by the
less expensive piperidine 9d or morpholine amide 9e. Interestingly,
reaction of the lithiated thiophene with the bulky diisopropylamide
9c gave no conversion (Scheme 3).
Figure 2. X-ray structure of 8a.
For 14C-labelling of AVE8887 1b a very similar pathway was
envisioned employing 4-trifluoromethoxy-[14CO]benzoyl chloride
[
14C]-3b instead of [14C]-3a. Unfortunately we found in our cold
elaboration experiments that more electron deficient benzoyl
chlorides, such as 3b, reacted much slower with thiophene 2 under
Friedel–Crafts acylation conditions. Consequently the acid labile
thiophene 2 started to decompose resulting in low yields for this
reaction step. Even under optimised conditions and a stepwise
addition (2–3 equiv) of 2, the maximum yield achieved was only
32%, see also Scheme 2. In addition, work up and purification were
difficult due to the numerous side products formed. Therefore, we
The 14C-synthesis of [14C]-1b was finally accomplished following
the pathway shown in Scheme 4. Starting from
[
14CO]tri-
fluoromethoxy benzoic acid [14C]-10b the corresponding Weinreb
amide [14C]-9b was formed under Appel conditions.16 Addition of

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V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
the lithiated thiophene to an ice–cold solution of the Weinreb
amide [14C]-9b yielded [14C]-4b in 76%. Luckily the rest of the
synthesis showed no significant deviation from the synthesis of
account of their mass difference, see also Figure 3. If this mass dif-
ference is selected to be large enough to avoid cross signal over-
lapping of the natural isotope pattern, quantitative determination is
possible. Typically, for small molecules without chlorine, bromine or
sulfur-containing functionalities, an incorporation of 3–4 mass units
is sufficient.18 In this case due to the thiophene ring moiety present
in 1a and 1b at least five deuterium atoms were required.
[
14C]-1a reported above. The colourless compound was isolated
after six reaction steps with an overall yield of 45%.
O
O
Stable isotopically labelled 1a was synthesised according to the
path shown in Scheme 5 in eight chemical steps with an overall
yield of 25%. Starting from 4-hydroxy benzoyl methyl ester 11 the
hydroxy group was alkylated with deuterated methyliodide fol-
lowed by basic ester hydrolysis to give the corresponding acid [D3]-
10a in 90% yield over two steps. The subsequent steps were
described in the 14C-synthesis above, however sodiumcyanobor-
odeuteride was employed in the carbonyl reduction to introduce
two further deuterium atoms. A co-injection of AVE2268 and [D5]-
AVE2268 revealed the expected mass difference but identical re-
tention time in the LC–MS (Fig. 3).
Li
S
R
4b
F3CO
NiPr2
N
R =
no conversion
52 %
9c
9d
9e
N
74 %
O
Scheme 3. Alternative amides for lithiation coupling step.
Scheme 5. Synthesis of [D5]-AVE2268 1a.
Scheme 4. Synthesis of [14C]-AVE8887 1b.
The synthesis of stable isotopically labelled AVE8887 1b was
achieved applying exactly the same pathway starting from 13C-la-
belled-trifluoromethoxy benzoic acid 10b.
2.2. Synthesis of stable isotopically labelled AVE2268 1a and
AVE8887 1b
In order to avoid matrix effects in mass spectrum experiments,17
such as ion suppression, stable isotopic labelled internal standards
are of particular advantage due to the similarity of the physical and
chemical properties to the investigated substance.10 Both can be
extracted from biological samples to the same extent, have identical
retention time and ionisation behaviour in the LC–MS but differ on
2.3. Synthesis of sulfate and glucoronite metabolites
12 and 13
During preclinical development the synthesis of two metabo-
lites, sulfate 12 and glucuronide 13, became necessary (Fig. 4).
Figure 3. LC–MS-spectra of a 1:1 mixture of AVE2268 and [D5]-AVE2268.

V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
1475
O
O
electrophile. Unfortunately, we obtained a mixture of phenol 12
and the reduced starting material 24 in the ratio 30:70. We couldn’t
prevent the reductive formation of the by-product even by per-
forming the halogen exchange directly in the presence of the
electrophile at ꢁ100 ꢀC. No further optimisation of this reaction
was performed as separating even small amounts of 24 from
compound 12 under reversed phase HPLC conditions proved to be
difficult and time consuming.
HO
HO
HO
O
S
O
O
O
HO
OH
S
S
OH
O
12
13
Figure 4. Structure of AVE2268 metabolites 12 and 13.
O
1. SOCl2
O
Based on our knowledge from the described syntheses above we
choose a benzyl protection of the phenol to avoid the strong acidic
conditions that would be required for demethylation. Starting from
4-benzyloxybenzoyl chloride 14 Friedel–Crafts acylation with 2 and
subsequent methyl ether cleavage with BBr3–DMS complex the 3-
hydroxy thiophene derivative 15 was obtained as stable crystalline
intermediate without benzyl-group removal (Scheme 6).
HO
N
H
2.
N
O
Br
Br
,CH2Cl2
98 %
O
18
19
O
O
1. BBr3 - DMS (21)
2. NaCNBH3, TMSCl
O
Li
S
1. SnCl4
2. BBr3 - DMS,
O
O
HO
S
0°C, 30 min
Et2O
Br
+
2
43 % (2 steps)
HO
Cl
20
OBn
S
OBn
67 % (2 steps)
49 %
14
15
O
S
O
O
HO
HO
HO
O
NaCNBH3,
TMSCl
S
O
SO3 - NEt3
SO3 - NEt3
O
S
S
S
CH2Cl2, 16 h
81 %
Br
OBn
46 %
quant.
Br
16
S
OBn
22
23
HO
S
O
17
O
HO
HO
Pd/C, H2
(10 - 15 % conversion)
O
O
O
S
S
1. n-BuLi/TMEDA
THF, -100°C, 30 min
O
O
O
O
S
OH
+
2. TMSO-OTMS
90% conversion
(LC-MS)
S
S
12
OH
ratio
30 : 70
H
Scheme 6. Synthesis of sulfate metabolite 12, route 1.
12
24
Scheme 7. Synthesis of sulfate metabolite 12, route 2.
Compared to the previous labelling syntheses a higher excess
(10 equiv) of NaBH3CN was necessary for complete reduction of the
carbonyl function. The corresponding product 16 proved to be
highly unstable and completely decomposed after three days at
room temperature. It was therefore immediately used after syn-
thesis in the next step. Sulfate 17 was formed quantitatively using
excess of sulfurtrioxide–triethylamine-complex. However, in the
last reaction step (debenzylation) we only observed 10–15% con-
version under all conditions tested: (a) 0.1 equiv Pd/C 10%, H2
(40 bar), THF, 6 h, rt and 50 ꢀC; (b) 1 equiv Pd/C 10%, H2 (40 bar),
THF; (c) 1 equiv Pd/C 10%, H2 (40 bar), THF, 1 equiv NEt3; (d) 1 equiv
Pd/C 10%, H2 (40 bar), THF, 1 equiv HOAc. Unfortunately, we were
neither successful in further optimisation of the reaction conditions
(temperature, pressure, solvent, etc.,) nor in a semi-preparative
HPLC separation of the small amounts of metabolite 12 formed. In
order to provide the requested amount (around 200 mg) of 12 in
time we started working on two alternative approaches in parallel.
Firstly, we planned to use a bromo-atom as an anchor for a late
hydroxyl-group introduction and secondly, we choose allyl-pro-
tection due to a higher liability than the benzyl-group under re-
ductive conditions.
In parallel to route 2 we followed route 3 (Scheme 8) starting
from 4-hydroxybenzoic acid 25, which was alkylated with allyl-
bromide and converted to the corresponding acylchloride 26.
Friedel–Crafts acylation with thiophene 2 and selective methyl
ether cleavage resulted in ketone 27. The reduction of the carbonyl
group was followed by the successful formation of the allyl-pro-
tected sulfate 28. After initial unsuccessful attempts to remove the
allyl-group we surprisingly succeeded by using combined condi-
tions of Hara et al.19a and Zhu et al.19b and addition of catalytic
amounts of NaBH4. Accordingly 28 was heated in the presence of
NaBH4-activated Pd/C catalyst in MeOH/KOH under microwave
conditions in a closed vial for 80 min. We found that only the
combination of high temperature, strong basic conditions and
NaBH4-activated Pd/C catalyst gave reasonable yields for the
deprotection of 28.
For the synthesis of metabolite 13 we initially envisioned a one
step synthesis approach starting from parent drug AVE2268 1a.
Firstly, we tried to directly oxidise the glucoside moiety to afford
the corresponding glucuronide. However, any attempts using
reported methods like KMnO4, SeO2, PtO2/O2 TEMPO/NaOCl/Oxone,
peroxidases, etc., failed. Secondly, any trials to perform a reacetali-
sation of AVE2268 1a–13 by adding glucuronic acid in the presence
of a weak acid (p-toluenesulfonic acid) were unsuccessful. Applying
an orthogonal protecting group strategy finally allowed the selec-
tive oxidation of the C6-position. However, several problems had to
be considered; (a) any traces of acid had to be avoided to prevent
acetyl migration, (b) in the first oxidation step utilising the Dess–
Martin periodinane, a very weak base had to be used in order to
Route
2 (Scheme 7) started from commercially available
4-bromo benzoic acid 18, which was transformed into the mor-
pholine amide 19 and subsequently reacted with lithiated thio-
phene 2 to give ketone 20 in moderate yields. After methyl ether
cleavage and reduction of the carbonyl function, the unstable thi-
ophenol 22 was immediately reacted with sulfurtrioxide–triethyl-
amine-complex to give the sulfate 23 as a colourless salt. In the last
reaction step a halogen–lithium exchange was performed at
ꢁ100 ꢀC and then bistrimethylsilyl peroxide was added as

1476
V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
prevent double bond formation by elimination of acetic acid, and
(c) the generated aldehyde was unstable and had to be directly
oxidised to the corresponding acid (Scheme 9).
the acetyl groups with sodium methoxide, water and lithium
chloride had to be added to cleave the methyl ester. Performing this
route enabled the metabolite 13 to be synthesised in only three
steps with 44% overall yield.
O
Allylbromid
1.
O
K2CO3, DMF
OAc O
Cl
HO
1.
90 %
AcO
O
O
O
O
O
2. SOCl2, 79 %
OH
O
AcO
26
25
AcO
AcO
Br
HO
O
O
NaCNBH3
TMSCl
2. SO3 - NEt3
1.
O
SnCl4,
87 %
O
O
HO
Ag2CO3
TBAB
S
OAc
S
O
S
65%
S
2.
BBr3 - DMS
41 %
5a
O
O
27
32
HO
O
O
O
HO
O
S
O
Pd/C (10%)
MeOH/10% KOH
cat. NaBH4
O
S
NaOMe
O
AcO
AcO
NaBH3CN
TMSCl
O
O
LiCl, H2O
13
O
92%
74%
S
OAc
microwave
58% (3steps)
S
OH
O
S
12
O
28
33
Scheme 8. Synthesis of sulfate metabolite 12, route 3.
Scheme 10. Synthesis of glucuronide metabolite 13, route 2.
OTBDPS
1. TBDPSCl
DMAP, 99%
AcO
AcO
3. Conclusions
O
AVE2268
O
1a
We have described in detail the challenges of the synthesis of
isotopically labelled compounds of SGLT inhibitors 1a and 1b. Al-
though very similar in their chemical structure, different synthetic
pathways had to be optimised. Furthermore we have shown in the
synthesis of metabolites 12 and 13 unexpected challenges and
possible new strategies to overcome them.
2. Ac2O, Pyridine
94%
OAc
S
O
29
OH
1. Dess-Martin
Pyridine
AcO
O
HF - Pyridine
99%
O
AcO
2. NaClO2, t-BuOH
OAc
4. Experimental section
NaH2PO4(aq)
S
98% (2 steps)
O
4.1. General
30
4.1.1. [14C]-4a (4-Methoxy-phenyl)-(3-methoxy-thiophen-2-yl)-meth-
HO
AcO
O
O
HO
O
O
ane-[14CO]one. In a flask tin-(IV)-tetrachloride (786
mL, 6.72 mmol,)
HO
HO
was dissolved in dichloromethane (54 mL) and at 5–10 ꢀC [14CO]a-
nisoyl chloride [14C]-3a (915 mg, 5.37 mmol, 11,100 MBq) was added.
Then 3-methoxythiophene (768 mg, 6.72 mmol) at 5–10 ꢀC was
slowly added and the reaction mixture was stirred at rt for 4 h. After
complete conversion (LC–MS) ice-cooled water (30 mL) and 30%ige
HCl (2.5 ml) was added. The different layers were separated and the
organic layer was washed with water (20 mL), sodium bicarbonate
solution (8 wt % in water, 20 mL) and water (20 mL). The organic
phase was dried over Na2SO4 and evaporated in vacuo. The crude
product was purified by chromatography using heptane/ethyl ace-
tate 3:1 as mobile phase. The product [14C]-4a (1.06 g, 4.29 mmol,
8880 MBq, 80%, purity 97% (HPLC, 254 nm)) was isolated as a yellow
NaOMe
75%
O
O
AcO
OAc
OH
S
S
13
O
O
31
Scheme 9. Synthesis of glucuronide metabolite 13, route 1.
Starting from AVE2268 1a the primary alcohol functionality
could be selectively silylated using TBDPSCl (tert-butyldiphenyl-
chlorosilane). Afterwards the secondary alcohol functionalities
were acetylated to give 29, which was then reacted with HF-pyri-
dine complex to deprotect the primary alcohol again. Compound 30
could be oxidised to the carboxylic acid 31 in two steps applying
Dess–Martin periodinane and sodium chlorite. Final cleavage of the
acetyl groups with sodium methoxide led to 13 after six steps in
68% overall yield.
In parallel a second route was developed (Scheme 10). While the
alkylation of the corresponding des-keto aglycon precursor did not
proceed the keto aglycon 5a allowed the introduction of the pro-
tected glucuronic acid. The reaction succeeded under phase
transfer conditions using an excess of silver carbonate (Koenigs–
Knorr conditions). Subsequent reduction of the benzylic carbonyl
function in 32 with NaBH3CN and TMSCl gave 33. After removing
solid: mp 98–99 ꢀC; 1H NMR (CDCl3):
d
¼8.37 (d, J¼6.3 Hz, 1H), 7.96
(d, J¼6.9 Hz, 2H), 6.96 (d, J¼6.9 Hz, 2H), 6.37 (d, J¼6.3 Hz, 1H), 3.91,
3.88 (s, 6H) ppm.
4.1.2. [14C]-5a (3-Hydroxy-thiophen-2-yl)-(4-methoxy-phenyl)-meth-
ane- [14CO]one. In a 250 mL flask were weighed BBr3–DMS (1.20 g,
3.83 mmol) and the flask was filled with argon over 10 min. The solid
was dissolved in dichloromethane (62 mL) and at rt [14C]-4a
(887 mg, 3.57 mmol, 7389 MBq) in dichloromethane (10 mL) was
added dropwise. The dark solution was stirred for 7 h at rt (TLC-
control) and then satd sodium bicarbonate solution (8 mL) was
added. The layers were separated and the organic was washed with

V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
1477
water (10 mL), dried over Na2SO4 and the solvent evaporated in
vacuo. The crude, slightly wet product was directly used in the next
reaction step.
m/z (%)¼383.2 [MþHþ] (100); HRMS (ESI-LTQ, neg.) C18H22O7S calcd
381.10135; found, 381.10110.
4.1.6. [14C]-9b 4-Hydroxy-N-methoxy-N-methyl-benzamide-[14CO]. Into
a 50 mL flask [14CO]-4-trifluoromethoxy benzoic acid (721 mg,
3.50 mmol, 7400 MBq), N,O-dimethyl-hydroxylamine–HCl (683 mg,
7.00 mmol) and triphenylphosphine (1.83 g, 7.00 mmol) were added
and the flask was filled with argon. The compounds were dissolved in
dry THF (5 mL) and bromotrichloromethane (0.69 mL, 7.00 mmol) and
pyridine (0.567 mL, 7.00 mmol) were added. The reaction mixture was
heated to reflux (bath temperature 80–90 ꢀC) for 5 h (TLC-control).
After cooling to rt water (5 mL) was added, the phases were separated
and the aqueous phase extracted three times with ethyl acetate
(20 mL). The combined organic phases were dried over Na2SO4 and the
solvent evaporated in vacuo. The crude product was purified by chro-
matography on SiO2 using heptane/ethyl acetate 1:1 as eluent to give
4.1.3. [14C]-7a 3-(2-(4-Methoxy-[14CO]benzoyl)-thiophenyl)-ß-
acetyl-glucopyranoside. Into 250 mL flask was filled
D
-4,5,6-
a
[
14C]-5a
(836 mg, 3.57 mmol, 7389 MBq) and dissolved in dichloromethane
(42 mL). At rt tetrabutylammoniumbromide (571 mg, 1.77 mmol),
potassium carbonate (4.49 g, 32.5 mmol) and water (2.1 mL) were
added. Then at rt acetobrom-alpha-glucose 6 (3.07 g, 7.48 mmol) was
added in portions during 1 h. The brown reaction mixture was stirred
at rt over 16 h (TLC-control). The reaction mixture was filtered and the
organic layer washed with water (10 mL) three times. Finally the or-
ganic layer was dried over Na2SO4 and the solvent removed in vacuo.
The crude product was purified by chromatography (eluent: heptane/
ethyl acetate 1:1) to give [14C]-7a (1.70 g, 3.00 mmol, 6600 MBq, 84%
in two steps, purity 98% (HPLC, 254 nm)) as a colourless solid; mp:
1
[14C]-9b (830 mg, 3.33 mmol, 7030 MBq, 95%) as colourless solid. H
149–151 ꢀC; 1H NMR (DMSO-d6):
J¼6.7 Hz, 2H), 7:1 (d, J¼6.7 Hz, 2H), 7.04 (d, J¼5.6 Hz, 1H), 5.62 (d, 1H),
5.34 (dd, 1H), 4.93 (m, 1H), 4.72 (dd, 1H), 4.24 (m, 2H), 4.12 (m, 1H),
3.81 (s, 3H, O–CH3), 2.05, 2.00, 1.90, 1.85 (s, 12H, acetyl-CH3) ppm.
d
¼8.02 (d, J¼5.6 Hz, 1H), 7.71 (d,
NMR (DMSO-d6):
(s, 3H), 3.28 (s, 3H) ppm.
d
¼7.75 (d, J¼8.8 Hz, 2H), 7.45 (d, J¼8.8 Hz, 2H), 3.57
4.1.7. [14C]-4b [2-(4-Trifluoromethoxy-benzoyl)-(3-methoxy-thiophe-
n-2-yl)-methane-[14CO]one. In a 50 mL flask 3-methoxythiophene 2
(0.7 mL, 7.0 mmol) was dissolved in diethylether (15 mL). Then the
flask was filled with argon and at rt n-BuLi (0.80 mL,1.6 M in hexane)
was added via a syringe. The reaction mixture was stirred at 40 ꢀC
bath temperature for 30 min. Then the reaction mixture was added
slowly to an ice-cooled solution of N-methoxy-N-methyl-4-tri-
4.1.4. [14C]-8a 3-(2-(4-Methoxy-[14CH2]benzyl)-thiophenyl)-ß-
D-1,3,4,
5-acetyl-glucopyranoside. In a 100 mL two-necked flask [14C]-7a
(1.70 g, 3.00 mmol, 6600 MBq) was weighed and the flask was filled
with argon. Then the compound was dissolved in dry acetonitrile
(29 mL) and the reaction mixture was cooled to 0–5 ꢀC. NaCNBH3
(1.49 g, 23.7 mmol) was added in portions at max. 5 ꢀC and the
mixture was stirred for 30 min. Then trimethylsilylchloride (2.90 mL,
23.7 mmol) was added dropwise (internal temperature should not
exceed 5 ꢀC) and the mixture was stirred for 4 h at 5 ꢀC (LC–MS
control). Finally satd sodium bicarbonate solution (44 mL) was
added and was stirred vigorously for 5 min. After addition of
dichloromethane (400 mL) the phases were separated and the
aqueous phase was extracted three times by dichloromethane
(20 mL). The combined organic layers were dried over Na2SO4 and
the solvent was evaporated in vacuo. The crude material was purified
by chromatography (SiO2, heptane/ethyl acetate 1:1) to give [14C]-8a
(1.68 g, 3.00 mmol, 6600 MBq, 100%, purity 93% (HPLC, 254 nm)) as
fluoromethoxy-[14CO]benzamide
[
14C]-9b (830 mg, 3.33 mmol,
7030 MBq) in diethylether (10 mL) via syringe. After one hour stir-
ring at rt complete conversion was monitored by LC–MS and water
(5 mL) was added. Then the layers were separated, the aqueous
phase was extracted three times with dichloromethane (10 mL) and
the combined organic layers were dried over Na2SO4 and the solvent
evaporated in vacuo. The crude product was purified by chroma-
tography heptane/ethyl acetate 3:1 to give [14C]-4b (756 mg,
2.50 mmol, 5342 MBq, 76%) as a yellow oil. 1H NMR (DMSO-d6):
d
¼8.04 (d, J¼5.5 Hz,1H), 7.82 (d, J¼8.6 Hz, 2H), 7.45 (d, J¼8.6 Hz, 2H),
7.19 (d, J¼5.5 Hz, 1H), 3.79 (s, 3H) ppm.
a colourless solid. Mp: 116–118 ꢀC; 1H NMR (DMSO-d6):
d¼7.29 (d,
4.1.8. [14C]-5b (3-Hydroxy-thiophen-2-yl)-(4-trifluoromethoxy-phe-
nyl)-methane-[14CO]one. Into a 250 mL flask was weighed BBr3–
DMS (821 mg, 2.63 mmol) and the flask was filled with argon over
10 min. The solid was dissolved in dichloromethane (50 mL) and at
rt (3-methoxy-thiophen-2-yl)-(4-trifluoromethoxy-phenyl)-meth-
J¼5.5 Hz, 1H), 7.09 (d, J¼6.7 Hz, 2H), 6.87 (d, J¼5.5 Hz, 1H), 6.84 (d,
J¼6.7 Hz, 2H), 5.41–5.33 (m, 2H), 5.07–4.97 (m, 2H), 4.21–4.17 (m,
2H), 4.09 (d, J¼9.7 Hz, 1H), 3.91–3.79 (m, 2H), 3.71 (s, 3H), 2.00, 1.99,
1.96, 1.95 (s, 12H, acetyl–CH3) ppm.
anone
[
14C]-4b (756 mg, 0.43 mmol, 5342 Mbq) in dichloro-
4.1.5. [14C]-1a 3-(2-(4-Methoxy-[14CH2]benzyl)-thiophenyl)-ß-
opyranoside. In a 100 mL flask acetyl-protected glucose derivative
14C]-8a (1.68 g, 3.00 mmol, 6600 MBq) was dissolved in methanol
D-gluc-
methane (10 mL) was added dropwise. The dark solution was
stirred for 7 h at rt (TLC-control) and then satd sodium bicarbonate
solution (8 mL) was added. The layers were separated and the or-
ganic phase was washed with water (10 mL), dried over Na2SO4 and
the solvent was evaporated in vacuo. Chromatography (eluent
heptane/ethyl acetate 3:1) of the crude product yielded [14C]-5b
(621 mg, 2.15 mmol, 4595 MBq, 86%) as a yellow solid. Mp: 67–
[
(25 mL). To this solution a sodium methoxide solution (30% in MeOH,
2.90 mL,15.9 mmol) was added at 0 ꢀC and the reaction mixture was
stirred for 2 h. Then the pH was adjusted to pH 6.7–6.9 by the ad-
dition of 2 N HCl in ethanol and subsequently the solvent evaporated
in vacuo. The residue was suspended in water, two times cen-
trifugated and dried in vacuo. This material was purified by chro-
matography using dichloromethane/methanol 10:1 as eluent,
followed by semi-preparative HPLC (column: Luna RP18 column,
eluents: acetonitrile/water 20:80 for 4 min, then up to 80:20 in
6 min, then 80:20 for 4 min and finally in 1 min down again to 20:80
70 ꢀC; 1H NMR (DMSO-d6):
d
¼11.45 (br s, 1H, OH), 7.97 (d, J¼5.4 Hz,
1H), 7.93 (d, J¼8.7 Hz, 2H), 7.51 (d, J¼8.7 Hz, 2H), 6.87 (d, J¼5.4 Hz,
1H) ppm.
4.1.9. [14C]-7b 3-(2-(4-Trifluoromethoxy-[14CO]benzoyl)-thiophenyl)-
ß-
[
D-1,3,4,5-acetyl-glucopyranoside. Into a 50 mL flask was filled
for 1 min, 14 mL flow) to give
[
14C]-1a (860 mg, 2.25 mmol,
14C]-5b (625 mg, 2.15 mmol, 4595 MBq) and was dissolved in
4950 MBq, 75%, purity 99.4% (HPLC, 254 nm)) as a colourless solid.
dichloromethane (20 mL). At rt tetrabutylammoniumbromide
(346 mg, 1.07 mmol), potassium carbonate (2.30 g, 9.67 mmol) and
water (0.2 mL) were added. Then at rt acetobromo-alpha-glucose 6
(1.50 g, 3.65 mmol) was added in portions during 1 h. The brown
reaction mixture was stirred at rt over 16 h (TLC-control) then fil-
tered and the organic layer washed three times with water (10 mL).
Finally the organic layer was dried over Na2SO4 and the solvent
1
Mp: 154–155 ꢀC; H NMR (DMSO-d6):
d
¼7.16–7.14 (m, 3H), 6.91 (d,
J¼5.5 Hz, 1H), 6.80 (d, J¼8.6 Hz, 2H), 5.35 (s, 1H), 5.05 (s, 1H), 4.99 (s,
1H), 4.63–4.53 (m, 2H), 4.01–3.97 (m, 2H), 3.71 (s, 3H), 3.66 (s, 1H),
3.49–3.44 (m, 1H), 3.32–3.05 (m, 4H); 13C NMR (DMSO-d6):
d¼157.6,
150.7, 132.7, 129.5, 123.8, 121.3, 120.2, 113.7, 103.4, 77.1, 76.5, 73.4,
20
69.8, 60.8, 54.9, 30.0 ppm; [
a
]
ꢁ34.3 (c 5, methanol); MS (ESI, pos.)
D

1478
V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
removed in vacuo. The crude product was purified by chromatog-
raphy (eluent: heptane/ethyl acetate 1:1) to give [14C]-7b (1.20 g,
1.94 mmol, 4135 MBq, 90%) as a colourless solid. Mp: 90–93 ꢀC; 1H
with 2 N HCl (10 mL). The combined organic phases were dried over
Na2SO4 and subsequently the solvent evaporated to dryness and im-
mediately used in the next reaction step.
NMR (DMSO-d6):
d
¼8.09 (d, J¼5.5 Hz, 1H), 7.78 (d, J¼6.7 Hz, 2H),
(b) To the ester was dissolved in 3 N NaOH (40 mL) and eth-
anol (10 mL) to obtain a clear solution. This mixture was heated to
reflux for 2 h. Under ice cooling a pH of 1 was obtained by the
addition of 2 N HCl. The precipitated colourless solid was filtered,
washed with water (3 mL) and dried in vacuo to give [D3]-10a
(3.49 g, 22.5 mmol, 90% in two steps). 1H NMR (DMSO-d6):
7.43 (d, J¼6.7 Hz, 2H), 7.13 (d, J¼5.5 Hz, 1H), 5.60 (d, J¼7.9 Hz, 1H),
5.27 (dd, J¼9.5/9.5 Hz, 1H), 4.94–4.90 (m, 1H), 4.63 (dd, J¼9.6/
9.5 Hz, 1H), 4.21–4.17 (m, 2H), 4.06–4.04 (m, 1H), 2.02, 1.99, 1.90,
1.84 (s, 12H, acetyl-CH3) ppm.
4.1.10. [14C]-8b 3-(2-(4-Trifluorormethoxy-[14CH2]benzyl)-thiophenyl)-
d
¼12.55 (br s, 1H), 7.88 (d, J¼8.6 Hz, 2H), 7.03 (d, J¼8.6 Hz, 2H);
ß-D
-1,3,4,5-acetyl-glucopyranoside. In a 250 mL flask ketone [14C]-7b
2H NMR (DMSO):
d
¼3.78 (s, 3D) ppm; MS (ESI, neg.) m/z (%)¼154
(1.20 g, 1.94 mmol, 4135 MBq) was weighed and the flask was filled
with argon. Then the compound was dissolved in dry acetonitrile
(50 mL) and the reaction mixture was cooled to 0–5 ꢀC. NaCNBH3
(2.51 g, 40.0 mmol) was added in portions at max. 5 ꢀC and the
mixture was stirred for 30 min. Then trimethylsilylchloride (5.11 mL,
40.0 mmol) was added dropwise (internal temperature should not
exceed 5 ꢀC) and the mixture was stirred for 4 h at 5 ꢀC (LC–MS
control). Finally satd sodium bicarbonate solution (10 mL) was added
and the reaction mixture was stirred vigorously for 5 min. After ad-
dition of dichloromethane (400 mL) the phases were separated and
the aqueous phase was extracted three times by dichloromethane
(20 mL). The combined organic layers were dried over Na2SO4 and the
solvent was evaporated in vacuo. The crude material was purified by
chromatography (SiO2, heptane/ethyl acetate 1:1) to give [14C]-8b
(1.11 g, 1.84 mmol, 3928 MBq, 95%) as a colourless solid. Mp: 113–
[MꢁH]þ (100).
4.1.13. [D3]-9a 4-[CD3]Methoxy-N-methoxy-N-methyl-benzamide. Into
a 250 mL flask benzoic acid [D3]-11a (3.20 g, 20.6 mmol), N,O-di-
methyl-hydroxylamine–HCl (4.02 g, 41.2 mmol) and triphenylphos-
phine (10.8 g, 41.2 mmol) were added and the flask filled with argon.
The compounds were dissolved in dry THF (30 mL) and bromotri-
chloromethane (3.97 mL, 41.2 mmol) and pyridine (3.33 mL,
41.2 mmol) were added. The reaction mixture was heated to reflux
(bath temperature 80–90 ꢀC) for 5 h (TLC-control). After cooling to rt
water (30 mL) was added, the phases were separated and the aqueous
phase extracted three times with ethyl acetate. The combined organic
phases were dried over Na2SO4 and the solvent was evaporated in
vacuo. The crude product was purified by chromatography on SiO2
using heptane/ethyl acetate 1:1 as eluent to give [D3]-9a (3.60 g,
18.2 mmol, 88%, purity 97 % (HPLC, 254 nm)) as a colourless solid. 1H
114 ꢀC; 1H NMR (DMSO-d6):
d¼7.47 (d, J¼8.1 Hz, 2H), 7.40 (d, J¼5.5 Hz,
1H), 7.30 (d, J¼8.1 Hz, 2H), 6.86 (d, J¼5.5 Hz, 1H), 5.89 (d, J¼3.6 Hz,
1H), 5.45 (dd, J¼9.8/9.3 Hz, 1H), 5.38 (d, J¼8.0 Hz, 1H), 5.11 (dd, J¼8.0/
9.8 Hz, 1H), 5.04 (dd, J¼9.3/9.3 Hz, 1H), 4.21–4.17 (m, 2H), 4.10 (dd,
J¼5.0/9.8 Hz, 1H), 3.33 (s, 2H), 2.09, 2.01, 2.00, 1.99 (s, 12H, acetyl-
NMR (DMSO-d6):
d
¼7.64 (d, J¼8.9 Hz, 2H), 6.99 (d, J¼8.9 Hz, 2H), 3.55
2
(s, 3H), 3.24 (s, 3H); H NMR (DMSO):
d
¼3.78 (s, 3D) ppm; MS (ESI,
pos.) m/z (%)¼199 [MþH]þ (100).
CH3); 13C NMR (DMSO-d6):
d¼170.0, 169.6, 169.3, 169.3, 148.9, 147.2,
4.1.14. [D3]-4a (4-[CD3]Methoxy-phenyl)-(3-methoxy-thiophen-2-yl)-
methan-one. (a) In a 250 mL flask 3-methoxythiophene 2 (3.10 mL,
31.0 mmol) was dissolved in diethylether (50 mL). Then the flask was
filled with argon and at rt n-BuLi (20.0 mL, 31.0 mmol, 1.6 M in hex-
ane) was added. The reaction mixture was stirred at 35 ꢀC bath
temperature for 30 min. Then the reaction mixture was added to an
ice-cooled solution of amide [D3]-9a (3.00 g, 15.1 mmol) in dieth-
ylether (30 mL) via syringe. After one hour stirring at rt complete
conversion was monitored by LC–MS and water (30 mL) was added.
Then the layers were separated, the aqueous phase was extracted
three times with dichloromethane (30 mL) and the combined organic
layers were dried over Na2SO4 and the solvent was evaporated in
vacuo. The crude product was purified by chromatography heptane/
ethyl acetate 1:1 to give [D3]-4a (2.50 g, 9.90 mmol, 65%), which was
used for the next step without further analysis.
144.1, 129.5, 127.4, 123.8, 120.7, 118.9, 99.6, 71.8, 70.9, 70.8, 68.1, 66.1,
61.7, 20.4, 20.4, 20.3, 20.3 ppm; HRMS (ESI-LTQ, pos.) C26H27O11F3NaS
calcd 627.11293; found, 627.11285.
4.1.11. [14C]-1b 3-(2-(4-Trifluoromethoxy-[14CH2]benzyl)-thiophenyl)-ß-
D-glucopyranoside. In a 250 mL flask acetyl-protected glucose de-
rivative [14C]-8b (1.11 g, 1.84 mmol, 3928 MBq) was dissolved in
methanol (30 mL). To this solution sodium methoxide solution
(1.14 mL, 6.00 mmol, 30% in MeOH) was added at 0 ꢀC and the
reaction mixture stirred for 3 h. Then the pH was adjusted to
a value of 6.7–6.9 using 2 N HCl in ethanol and the solvent was
evaporated in vacuo to a volume of 2–3 mL. The crude material
was purified by chromatography using dichloromethane/metha-
nol 8:1 as eluent, followed by semi-preparative HPLC (column:
Luna RP18 column, eluents: acetonitrile/water 20:80 for 4 min,
then up to 80:20 in 6 min, then 80:20 for 4 min and finally in
1 min down again to 20:80 for 1 min, 14 mL flow) to give [14C]-1b
(576 mg, 1.32 mmol, 2828 MBq, 72%, purity 99.2% (HPLC,
254 nm)) as a colourless solid. Mp: 144–145 ꢀC; 1H NMR (DMSO-
4.1.15. [D3]-5a (3-Hydroxy-thiophen-2-yl)-(4-[CD3]methoxy-phenyl)-
methanone. Into a 250 mL flask were weighed BBr3–DMS (3.25 g,
10.4 mmol) and the flask filled with argon over 10 min. The solid was
dissolved in dichloromethane (40 mL) and as solution of thiophene
[D3]-4a (2.50 g 9.97 mmol) in dichloromethane (10 mL) was added
dropwise at rt. The dark solution was stirred for 3 h at rt (LC–MS
control) and then satd sodium bicarbonate solution (20 mL) was
added. The layers were separated and the organic layer was washed
with water (10 mL), dried over Na2SO4 and the solvent was evapo-
rated in vacuo. Chromatography (eluent heptane/ethyl acetate 4:1)
of the crude product yielded [D3]-5a (1.79 g, 7.54 mmol, 79%, purity
98% (HPLC, 254 nm)) as a colourless solid. 1H NMR (DMSO-d6):
d6):
d
¼7.41 (d, J¼8.5 Hz, 2H), 7.27 (d, J¼8.5 Hz, 2H), 7.24 (d,
J¼5.5 Hz, 1H), 6.97 (d, J¼5.5 Hz, 1H), 5.37 (d, J¼4.9 Hz, 1H), 5.05
(d, J¼4.5 Hz, 1H), 4.98 (d, J¼5.3 Hz, 1H), 4.64 (d, J¼7.3 Hz, 1H),
4.56 (dd, J¼5.7/5.7 Hz, 1H), 4.12–4.04 (m, 2H), 3.72–3.68 (m, 1H),
3.51–3.47 (m, 1H), 3.32–3.12 (m, 4H); 19F NMR (DMSO-d6):
d
¼56.8 ppm; HRMS (ESI-LTQ, pos.) C18H21O7F3S calcd 437.08763;
found, 437.08752.
4.1.12. [D3]-10a [CD3]anisic acid. (a) Into a 250 mL flask were weighed
methyl-4-hydroxy benzoate 11a (3.80 g, 25.0 mmol) and potassium
carbonate (6.90 g, 50.0 mmol) and the flask was filled with argon.
Then the compounds were suspended in dry DMF (50 mL) and
[D3]methyliodide (5.0 g, 35 mmol) was added dropwise via syringe.
The flask was closed and heated to 80 ꢀC over 10 h. Then ethyl acetate
(50 mL) was added and the organic phase was washed three times
d
¼11.85 (br s, 1H, OH), 7.96 (d, J¼5.4 Hz, 1H), 7.89 (d, J¼8.9 Hz, 2H),
7.10 (d, J¼8.9 Hz, 2H), 6.93 (d, J¼5.4 Hz, 1H); 2H NMR (DMSO):
d
¼3.82 (s, 3D) ppm; MS (ESI, pos.) m/z (%)¼238 [MþH]þ (100).
4.1.16. [D5]-1a 3-(2-(4-[CD3]Methoxy-[CD2]benzyl)-thiophenyl)-ß-
glucopyranoside. (a) In a 250 mL flask [D3]-5a (1.73 g, 7.30 mmol)
was dissolved in dichloromethane (90 mL). At rt
D-

V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
1479
-(4-benzyloxy-phenyl)-methanone
(1.90 g,
5.86 mmol)
in
tetrabutylammoniumbromide (1.18 g, 3.65 mmol), potassium car-
bonate (9.06 g, 65.7 mmol) and water (5 mL) were added. Then at rt
acetobrom-alpha-glucose 6 (6.00 g, 14.6 mmol) was added in por-
tions. The brown reaction mixture was stirred at rt over 16 h (TLC-
control) and then water (20 mL) was added. The phases were
separated, the aqueous phase was extracted three times with
dichloromethane (20 mL), the combined organic phases were dried
over Na2SO4 and the solvent removed in vacuo. The crude product
was purified by chromatography (eluent: heptane/ethyl acetate
1:1) to give [D3]-7a (3.53 g, 6.22 mmol, 85%) as a colourless solid.
(b) In a 250 mL flask ketone [D3]-7a (2.00 g, 3.50 mmol) was
weighed and the flask filled with argon. Then the compound
was dissolved in dry acetonitrile (60 mL) and the reaction mixture
was cooled to 0–5 ꢀC. NaCNBD3 (1.84 g, 28.0 mmol) was added in
portions at max. 5 ꢀC and the mixture was stirred for 30 min. Then
trimethylsilylchloride (3.55 mL, 28.0 mmol) was added dropwise
(internal temperature should not exceed 5 ꢀC) and the mixture was
stirred for 4 h at 5 ꢀC (LC–MS control). Finally satd sodium bi-
carbonate solution (30 mL, in D2O) was added and the solution was
stirred vigorously over 5 min. After addition of dichloromethane
(60 mL) the phases were separated and the aqueous phase was
extracted three times with dichloromethane (20 mL). The com-
bined organic layers were dried over Na2SO4 and the solvent was
evaporated in vacuo. This crude material (2 g, >100%) was directly
used in the next reaction step without further purification.
dichloromethane (10 mL) was added dropwise at rt. The dark so-
lution was stirred for 3 h at rt (TLC-control) and then satd sodium
bicarbonate solution (30 mL) was added. The layers were separated
and the organic phase was washed with water (30 mL), dried over
Na2SO4 and the solvent was evaporated in vacuo. Chromatography
(eluent heptane/ethyl acetate 2:1) of the crude product yielded 15
(1.40 g, 4.51 mmol, 77%, purity 95% (HPLC, 254 nm)) as yellow oil,
LC–MS (ESI, pos.) m/z (%)¼311 [MþH]þ (100).
4.1.18. Compound
16
2-(4-benzyloxy-benzyl)-thiophen-3-ol. In
a 250 mL flask ketone 15 (1.90 g, 6.12 mmol) was weighed and the
flask filled with argon. Then the compound was dissolved in dry
acetonitrile (53 mL) and the reaction mixture was cooled to 0–5 ꢀC.
NaCNBH3 (2.50 g, 37.8 mmol) was added in portions at max. 5 ꢀC
and the mixture was stirred for 30 min. Then trimethylsilylchloride
(6.00 mL, 46.5 mmol) was added dropwise (internal temperature
should not exceed 5 ꢀC) and the mixture was stirred for 4 h at 5 ꢀC
(LC–MS control). Finally satd sodium bicarbonate solution (45 mL)
was added and the mixture was stirred vigorously over 5 min. After
addition of dichloromethane (400 mL) the phases were separated
and the aqueous phase was extracted three times by dichloro-
methane (20 mL). The combined organic layers were dried over
Na2SO4 and the solvent was evaporated in vacuo. This material was
directly used in the next reaction step without further purification.
(c) In a 100 mL flask acetyl-protected glucose derivative [D5]-8a
(1.00 g, 1.80 mmol) was dissolved in methanol (15 mL). To this so-
lution sodium methoxide solution (0.343 mL, 1.80 mmol, 30% in
MeOH) was added at 0 ꢀC and the reaction mixture was stirred for
2 h. Then with 2 N HCl in ethanol a pH of 6.7–6.9 was adjusted and
water (100 mL) was added. 1/3 of the solvent was evaporated in
vacuo and the resulting solid was isolated by centrifugation. The
solid was washed twice with water and dried in vacuo. This ma-
terial was further purified by chromatography using dichloro-
methane/methanol 6:1 as eluent, followed by semi-preparative
HPLC (column: Luna RP18 column, eluents: acetonitrile/water
20:80 for 4 min, then up to 80:20 in 6 min, then 80:20 for 4 min and
finally in 1 min down again to 20:80 for 1 min, 14 mL flow) to give
[D5]-1a (503 mg, 1.29 mmol, 72% in two steps, purity 99.6% (HPLC,
254 nm)) as a colourless solid. Mp: 151–152 ꢀC; 1H NMR (DMSO-
4.1.19. Compound 17 sulfuric acid mono-[2-(4-benzyloxy-benzyl)-
thiophen-3-yl] ester. Thiophene 16 (1.81 g, 6.11 mmol) was dis-
solved in dichloromethane (20 mL) and triethylamine (TEA,
4.29 mL, 30.6 mmol). Then sulfurtrioxide–triethylamine-complex
(2.21 g, 12.2 mmol) was added in one portion and the reaction
mixture was stirred for 16 h at rt. Water (10 mL) was added and
with 1 N HCl a pH of 1 was obtained. The phases were separated
and the aqueous phase was extracted three times by dichloro-
methane (20 mL). The combined organic layers were dried over
Na2SO4 and the solvent was evaporated in vacuo to give sulfate 17
(1.50 g, 3.14 mmol, 46%). This material was used in the next reaction
step without further purification. 1H NMR (DMSO-d6):
d¼7.54–7.33
(m, 5H), 7.22 (d, J¼8.9 Hz, 2H), 7.15 (d, J¼5.5 Hz, 1H), 6.96 (d,
J¼5.5 Hz, 1H), 6.92 (d, J¼8.9 Hz, 2H), 5.05 (s, 2H), 3.98 (s, 2H) 3.09
(q, 6H, TEA), 1.19 (t, 9H, TEA) ppm.
d6):
d
¼7.16–7.14 (m, 3H), 6.91 (d, J¼5.5 Hz, 1H), 6.80 (d, J¼8.6 Hz,
2H), 5.35 (s, 1H), 5.05 (s, 1H), 4.99 (s, 1H), 4.63–4.53 (m, 2H), 3.66 (s,
1H), 3.49–3.44 (m, 1H), 3.32–3.05 (m, 4H) ppm; MS (ESI, pos.) m/z
(%)¼408 [MþNaþ3D] (3), 409 [MþNaþ4D] (22), 410, [MþNaþ5D]
(53), 411 [MþNaþ6D] (17), 412, [MþNaþ7D] (5).
4.1.20. Compound 20 [2-(4-bromo-phenyl)-(3-methoxy-thiophen-2-
yl)-methanone. In a 250 mL flask 3-methoxythiophene 2 (1.30 mL,
13.1 mmol) was dissolved in diethylether (30 mL). Then the flask
was filled with argon and n-BuLi (7.00 mL, 17.5 mmol, 2.5 M in
hexane) added at rt. The reaction mixture was stirred at 35 ꢀC bath
temperature for 10 min. Then the reaction mixture was slowly
dropped into an ice-cooled solution of amide 19 (3.50 g, 13.0 mmol)
in diethylether (50 mL) via syringe. After two hours stirring at rt
complete conversion was monitored by LC–MS and water (25 mL)
was added. Then the layers were separated, the aqueous phase was
extracted three times with dichloromethane (30 mL) and the
combined organic layers were dried over Na2SO4 and the solvent
was evaporated in vacuo. The crude product was purified by
chromatography heptane/ethyl acetate 4:1 to give 20 (1.90 g,
4.1.17. Compound 15 (3-hydroxy-thiophen-2-yl)-(4-benzyloxy-phe-
nyl)-methanone. (a) In
a 500 mL flask 3-methoxythiophene 2
(2.75 mL, 27.5 mmol) and 4-benzyloxy benzoic acid chloride (5.40 g,
21.9 mmol) were dissolved in dichloromethane (200 mL) and at 0–
5 ꢀC tin-(IV)-tetrachloride (5.52 mL, 46.8 mmol) was added. The re-
action mixture was stirred at 5 ꢀC for 2 h and a further 2 h at rt. After
complete conversion (LC–MS) ice-cooled water (20 mL) was added.
The different layers were separated and the organic layer was
washed with water (10 mL), sodium bicarbonate solution (10 mL, 8
wt % in water) and brine (10 mL). The organic phase was dried over
Na2SO4 and was evaporated in vacuo. The crude product was puri-
fied by chromatography using heptane/ethyl acetate 3:1 as mobile
phase. The product (6.20 g, 19.1 mmol, 87%, purity 95% (HPLC,
6.39 mmol, 49%) as a yellow solid. 1H NMR (DMSO-d6):
d
¼8.06 (d,
J¼5.3 Hz, 1H), 7.71 (d, J¼9.0 Hz, 2H), 7.67 (d, J¼9.0 Hz, 2H), 7.19 (d,
J¼5.3 Hz, 1H), 3.80 (s, 3H) ppm; LC–MS (ESI, pos.) m/z (%)¼297
[MþH]þ (95), 299 [MþH]þ (100).
254 nm)) was isolated as a yellow oil. 1H NMR (CDCl3):
d¼7.87 (d,
J¼8.9 Hz, 2H), 7.58 (d, J¼5.5 Hz, 1H), 7.48–7.35 (m, 5H), 7.14 (d,
J¼8.9 Hz, 2H), 6.93 (d, J¼5.5 Hz, 1H), 5.17 (s, 2H), 3.86 (s, 3H) ppm;
LC–MS (ESI, pos.) m/z (%)¼325 [MþH]þ (100), 347 [MþH]þ (35).
b) Into a 250 mL flask was weighed BBr3–DMS (1.99 g, 6.29 mmol)
and the flask filled with argon over 10 min. The solid was dissolved
in dichloromethane (130 mL) and (3-methoxy-thiophen-2-yl)
4.1.21. Compound 21 [2-(4-bromo-phenyl)-(3-hydroxy-thiophen-2-
yl)-methanone. Into a 250 mL flask were weighed BBr3–DMS
(2.79 g, 8.92 mmol) and the flask filled with argon over 10 min. The
solid was dissolved in dichloromethane (100 mL) and a solution of
ketone 20 (2.50 g, 8.41 mmol) in dichloromethane (10 mL) was

1480
V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
added dropwise at rt. The dark solution was stirred for 3 h at rt (LC–
MS control) and then satd sodium bicarbonate solution (30 mL)
was added. The layers were separated and the organic phase was
washed with water (10 mL), dried over Na2SO4 and the solvent was
evaporated in vacuo. Chromatography (eluent heptane/ethyl ace-
tate 2:1) of the crude product yielded 21 (1.40 g, 4.96 mmol, 59%) as
120.1 (arom. CH), 45.3 (TEA, CH2), 30.9 (CH2), 8.3 (TEA, CH3); MS
(ESI, neg.) m/z (%)¼269 [MþH]þ (100).
4.1.25. Compound 26 4-allyloxy-benzoyl chloride. (a) 4-hydroxyben-
zoic acid 25 (8.10 g, 58.6 mmol), allylbromide (10.7 mL, 123 mmol)
and potassium carbonate (20.0 g, 145 mmol) were suspended in
DMF (60 mL) and stirred for 48 h. Then water (20 mL) and
dichloromethane (50 mL) were added and the layers were sepa-
rated. The aqueous layer was extracted three times by dichloro-
methane (30 mL). The combined organic layers were dried over
Na2SO4 and the solvent was evaporated in vacuo. To the residue 2 N
NaOH (100 mL) and ethanol (20 mL) were given and the solution
was heated to reflux for 5 h (LC–MS control). A pH of 1 was
obtained by conc HCl and the precipitated solid was filtered,
washed with ice-cooled water-methanol 5:1 and dried in vacuo
over CaCl2 to give the allyl ether (9.40 g, 52.7 mmol, 90%).
(b) 4-allyloxy benzoic acid (6.40 g, 35.9 mmol) was suspended
in thionyl chloride (25 mL) and heated to reflux for 8 h. Thionyl
chloride was removed in vacuo. Toluene (20 mL) was added and
again the solvent was removed in vacuo. This procedure was re-
peated twice. The residue 26 (5.60 g, 28.5 mmol, 79%) was directly
used in the next reaction step.
a yellow solid. 1H NMR (DMSO-d6):
d
¼11.45 (s, 1H, OH), 7.98 (d,
J¼5.3 Hz, 1H), 7.79–7.70 (m, 4H), 7.87 (d, J¼5.3 Hz, 1H); LC–MS (ESI,
pos.) m/z (%)¼283 [MþH]þ (95), 285 [MþH]þ (100).
4.1.22. Compound 22 2-(4-bromo-benzyl)-thiophen-3-ol. In a 250 mL
flask ketone 21 (1.00 g, 3.53 mmol) was weighed and the flask filled
with argon. Then the compound was dissolved in dry acetonitrile
(15 mL) and the reaction mixture was cooled to 0–5 ꢀC. NaCNBH3
(1.71 g, 25.9 mmol) was added in portions at max. 5 ꢀC and the mix-
ture stirred for 30 min. Then trimethylsilylchloride (4.11 mL,
31.9 mmol) was added dropwise (internal temperature should not
exceed 5 ꢀC) and the mixture was stirred for 4 h at 5 ꢀC (LC–MS
control). Finally satd sodium bicarbonate solution (30 mL) was added
and the reaction mixture was stirred vigorously for 5 min. After ad-
dition of dichloromethane (40 mL) the phases were separated and the
aqueous phase was extracted three times with dichloromethane
(20 mL). The combined organic layers were dried over Na2SO4 and the
solvent was evaporated in vacuo. This crude material (684 mg,
2.54 mmol, 72%, purity 94% (HPLC, 254 nm)) was directly used in the
next reaction step without further purification.
4.1.26. Compound 27 (4-allyloxy-phenyl)-(3-hydroxy-thiophen-2-
yl)-methanone. (a) In a 500 mL flask 3-methoxy-thiophene 2
(3.61 mL, 36.0 mmol) and 4-allyloxy benzoic chloride 26 (5.60 g,
28.5 mmol) were dissolved in dichloromethane (200 mL) and tin-
(IV)-tetrachloride (5.52 mL, 46.8 mmol) added at 0–5 ꢀC. The re-
action mixture was stirred for 2 h at 5 ꢀC and further 2 h at rt. After
complete conversion (LC–MS) ice-cooled water (20 mL) was added.
The different layers were separated and the organic layer was
washed with water (10 mL), sodium bicarbonate solution (10 mL, 8
wt % in water) and brine (10 mL). The organic phase was dried over
Na2SO4 and was evaporated in vacuo. The crude product was pu-
rified by chromatography using heptane/ethyl acetate 3:1 as mobile
phase. The product (6.80 g, 24.8 mmol, 87%) was isolated as a yel-
low solid.
(b) Into a 250 mL flask were weighed BBr3–DMS (22.5 g,
72.0 mmol) and the flask filled with argon over 10 min. The solid
was dissolved in dichloromethane (200 mL) and a solution of (3-
methoxy-thiophen-2-yl)-(4-allyloxy-phenyl)-methanone (9.88 g,
36.0 mmol) in dichloromethane (10 mL) added dropwise at rt. The
dark solution was stirred for 3 h at rt (TLC-control) and then satd
sodium bicarbonate solution (30 mL) was added. The layers were
separated and the organic layer was washed with water (30 mL),
dried over Na2SO4 and the solvent was evaporated in vacuo.
Chromatography (eluent heptane/ethyl acetate 3:1) of the crude
product yielded 27 (3.80 g, 14.6 mmol, 41%; purity 93% (HPLC,
4.1.23. Compound 23 sulfuric acid mono-[2-(4-bromo-benzyl)-thio-
phen-3-yl] ester. Thiophene 22 (950 mg, 3.53 mmol) was dissolved
in dichloromethane (10 mL) and triethylamine (2.49 mL,
5.01 mmol). Then sulfurtrioxide–triethylamine-complex (1.28 g,
7.06 mmol) was added in one portion and the reaction mixture was
stirred for 16 h at rt. Water (10 mL) was added and with 1 N HCl
a pH of 1 was obtained. The phases were separated and the aqueous
phase was extracted three times by dichloromethane (20 mL). The
combined organic layers were dried over Na2SO4 and the solvent
was evaporated in vacuo. The crude product was purified by
chromatography using dichloromethane/EtOH/TEA 90:5:5 as sol-
vent to give sulfate 23 (1.10 g, 2.44 mmol, 69%, purity 97% (HPLC,
254 nm)) as a slightly brown solid. 1H NMR (DMSO-d6):
s, 1H, SO3H), 7.48 (d, J¼9.0 Hz, 2H), 7.23 (d, J¼9.0 Hz, 2H), 7.15 (d,
J¼5.4 Hz, 1H), 6.96 (d, J¼5.4 Hz, 1H), 4.00 (s, 2H), 3.15–3.09 (m, 6H,
TEA), 1.23–1.11 (m, 9H, TEA) ppm.
d¼8.91 (br
4.1.24. Compound 24 sulfuric acid mono-[2-benzyl-thiophen-3-yl]
ester. Bromide 23 (400 mg, 0.89 mmol) was weighed into a 100 mL
flask and the flask was filled with argon. Then 23 was dissolved in
THF (20 mL) and TMEDA (0.3 mL, 2.0 mmol) was added. The solu-
tion was cooled to ꢁ100 ꢀC in a dichloromethane/dry ice-cooling
bath and n-BuLi (1.0 mL, 2.5 mmol, 2.5 M in hexane) was added
dropwise via syringe. The mixture was stirred for 15 min at ꢁ100 ꢀC
and then bis(trimethylsilyl)peroxide (474 mg, 2.66 mmol) in THF
(5 mL) was added via syringe. The resulting mixture was allowed to
warm to rt during 3 h. (LC–MS control showed 90% conversion of
starting material). Satd NH4Cl solution (10 mL) was added and the
layers were separated. The aqueous phase was extracted five times
with dichloromethane/1-butanol 4:1 (20 mL). The two main
products 24 and 12 were analysed by 1H NMR and MS (for detailed
analytical information of 12 see below). Attempts to separate both
compounds by HPLC and RP columns on a preparative scale failed.
Compounds 12 and 24 furthermore decomposed under acidic
254 nm)) as yellow oil. 1H NMR (DMSO-d6):
d
¼11.85 (s, 1H, OH),
7.95 (d, J¼5.4 Hz, 1H), 7.88 (d, J¼9.0 Hz, 2H), 7.12 (d, J¼9.0 Hz, 2H),
6.93 (d, J¼5.4 Hz, 1H), 6.11–6.03 (m, 1H, allyl-H), 5.45 (dd, J¼1.6 Hz,
J¼17.2 Hz, 1H, allyl-H), 5.32 (dd, J¼1.6 Hz, J¼17.2 Hz, 1H, allyl-H),
4.68–4.66 (m, 2H) ppm; LC–MS (ESI, pos.) m/z (%)¼261 [MþH]þ
(100).
4.1.27. Compound 28 sulfuric acid mono-[2-(4-allyloxy-benzyl)-thi-
ophen-3-yl] ester. (a) In
a 250 mL flask ketone 27 (3.80 g,
14.6 mmol) was weighed and the flask filled with argon. Then the
compound was dissolved in dry acetonitrile (176 mL) and the re-
action mixture was cooled to 0–5 ꢀC. NaCNBH3 (5.52 g, 87.8 mmol)
was added in portions at max. 5 ꢀC and the mixture was stirred for
30 min. Then trimethylsilylchloride (14.1 mL, 111 mmol) was added
dropwise (internal temperature should not exceed 5 ꢀC) and the
mixture stirred for 4 h at 5 ꢀC (LC–MS control). Finally satd sodium
bicarbonate solution (9 mL) was added and the reaction mixture
stirred vigorously over 5 min. After addition of dichloromethane
conditions. Compound 24: 1H NMR (DMSO-d6):
d
¼9.6 (br s, 1H,
SO3H), 7.29–7.23 (m, 4H), 7.19–7.16 (m, 1H), 7.13 (d, J¼5.4 Hz, 1H),
6.98 (d, J¼5.4 Hz, 1H), 4.02 (s, 2H), 3.10–3.04 (m, 6H, TEA), 1.20 (t,
J¼7.3 Hz, 9H, TEA); 13C NMR dept135
¼128.4, 128.2, 125.8, 122.7,
d

V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
1481
(400 mL) the phases were separated and the aqueous phase was
extracted three times by dichloromethane (20 mL). The combined
organic layers were dried over Na2SO4 and the solvent was evap-
orated in vacuo. This material was directly used in the next reaction
step without further purification.
was purified by column chromatography (heptane/ethyl acetate
3:1) to give 29 (1.76 g, 2.35 mmol, 94%) as a white foam. 1H NMR
(DMSO-d6):
d
¼7.64–7.60 (m, 4H), 7.45–7.39 (m, 4H), 7.34 (t,
J¼7.5 Hz, 2H), 7.23 (d, J¼5,5 Hz, 1H), 7.09 (d, J¼8.6 Hz, 2H), 6.95 (d,
J¼5.5 Hz, 1H), 6.82 (d, J¼8.6 Hz, 2H), 5.41–5.38 (m, 2H), 5.17–5.14
(m, 1H), 5.08–5.03 (m, 1H), 4.09–4.04 (m, 1H), 3.93–3.83 (m, 2H),
3.73 (m, 2H), 3.70(s, 3H), 1.97 (s, 6H), 1.93 (s, 3H), 0.99 ppm (s, 9H);
LC–MS (ESI, pos.) m/z (%)¼769 MþNa]þ (100).
(b) Thiophene from step (a) (3.60 g, 14.6 mmol) was dissolved
in dichloromethane (20 mL) and triethylamine (8.12 mL,
58.4 mmol). Then sulfurtrioxide–triethylamine-complex (7.95 g,
43.9 mmol) was added in one portion and the reaction mixture
was stirred for 16 h at rt. Water (10 mL) was added and with 1 N
HCl a pH of 1 was adjusted. The phases were separated and the
aqueous phase was extracted three times by dichloromethane
(20 mL). The combined organic layers were dried over Na2SO4 and
the solvent was evaporated in vacuo. The crude product was pu-
rified by chromatography using dichloromethane/MeOH/TEA
90:5:5 as solvent to give crude sulfate 28 (7.8 g, >100%) as brown
oil (with excess TEA). This material was used in the next reaction
step without further purification; MS (ESI, neg.) m/z (%)¼325
[MꢁH]þ (100).
4.1.30. Compound 30 3-(2-(4-methoxy-benzyl)-thiophenyl)-ß-D-
3,4,5-acetyl-glucopyranoside. To a solution of 29 (1.42 g, 1.90 mmol)
in dry THF (20 mL) and dry pyridine (20 mL) hydrofluoric acid–
pyridine-complex (3.42 mL, 24.7 mmol) was added slowly at 0 ꢀC.
After stirring for 30 min at 0 ꢀC, the reaction mixture was allowed
to come to rt. After one hour further HF-complex (1.31 mL,
9.50 mmol) was added to the mixture at 0 ꢀC and stirring was
continued for another hour at rt. Then ethyl acetate (60 mL) and
water (60 mL) were added. The layers were separated and the
aqueous layer was extracted twice with ethyl acetate (50 mL). The
combined organic layers were dried over Na2CO3. The crude
product was purified by column chromatography (heptane/ethyl
acetate 3:2) to give 30 (956 mg,1.88 mmol, 99%) as a white foam. 1H
4.1.28. Compound 12 sulfuric acid mono-[2-(4-hydroxy-benzyl)-thi-
ophen-3-yl] ester. In a 20 mL microwave vial sulfate 28 (700 mg,
1.64 mmol) and 200 mg Pd/C (10% Pd, water free, Fa. Heraeus) were
suspended in KOH (6 mL, 10% in MeOH). The vial was filled with
argon and NaBH4 (20 mg) was added in one portion. The reaction
mixture was stirred for 3 min, and then the vial was closed and
heated at 110 ꢀC for 80 min (biotage Initiator microwave; absorp-
tion level d normal; pressure 10 atm). Caution: Overpressure might
be possible after reaction and must be removed carefully. The reaction
mixture was filtered, the filter cake washed twice with methanol
(5 mL) and the filtrate concentrated in vacuo to give an orange oil.
The crude product was purified by chromatography using
dichloromethane/MeOH/TEA 90:5:5 as solvent, followed by semi-
preparative HPLC (YMC-Basic RP8 column, 10 mL flow; acetonitrile/
water/TEA gradient program) to give sulfate 12 d TEA (370 mg,
0.95 mmol, 58%, purity 99.6% (HPLC)) as colourless needles. 1H
NMR (DMSO-d6):
d
¼7.27 (d, J¼5.5 Hz, 1H), 7.10 (d, J¼8.6 Hz, 2H),
6.95 (d, J¼5.5 Hz, 1H), 6.84 (d, J¼8.6 Hz, 2H), 5.37–5.30 (m, 2H),
5.04–4.95 (m, 2H), 4.89 (s, br s, OH), 3.91–3.86 (m, 1H), 3.84 (d,
J¼7.8 Hz, 2H), 3.71 (s, 3H), 3.57–3.53 (m, 1H), 3.46–3.42 (m, 1H),
2.00 (s, 3H), 1.95 (s, 3H), 1.93 ppm (s, 3H); LC–MS (ESI, pos.) m/z
(%)¼531 [MþNa]þ (100).
4.1.31. Compound 31 3-(2-(4-methoxy-benzyl)-thiophenyl)-ß-
3,4,5-acetyl-glucuronide. Dry pyridine (37.6 L, 0.46 mmol) and
Dess–Martin periodinane (779 L, 0.30 mmol, 12% in dichloro-
D-
m
m
methane) were added to a solution of 30 (102 mg, 0.20 mmol) in
dry dichloromethane (2 mL) under an argon atmosphere. After
stirring for 2 h at rt the starting material had disappeared (LC–MS
control). Due to the low stability of the formed aldehyde (unstable
in LC–MS) the solvent was just evaporated and the residue directly
redissolved in tert-butanol (2 mL) and satd NaH2PO4-solution
NMR (DMSO-d6):
d
¼9.18 (s, 1H, OH), 8.85 (br s, 1H, SO3H), 7.09 (d,
J¼5.5 Hz, 1H), 7.04 (d, J¼8.4 Hz, 2H), 6.96 (d, J¼5.5 Hz, 1H), 6.66 04
(d, J¼8.4 Hz, 2H), 3.88 (s, 2H) 3.11–3.04 (m, 6H, TEA), 1.20–1.17 (m,
(1.4 mL). Then 2-methyl-2-butene (224 mL, 2.00 mmol) and sodium
9H, TEA); 13C NMR (DMSO-d6):
d¼155.6, 146.0, 130.9, 129.4, 127.8,
chlorite (NaClO2, 27.1 mg, 0.24 mmol) were added and the reaction
mixture was stirred overnight. For work up water (3 mL) was
added, the aqueous phase was extracted three times with ethyl
acetate (20 mL) and the combined organic layers were dried over
Na2SO4. The residue was purified by column chromatography
(dichloromethane/methanol 9:1/4:1) to give 31 (103 mg,
122.9, 120.1, 115.0, 45.7, 30.4, 8.6 ppm; (MS (m/z) neg. ESI¼285
(100); HPLC (UV, 230 nm)¼99.5%); HRMS (ESI-LTQ, neg.) C11H9O5S2
calcd 284.98969; found, 284.98990.
4.1.29. Compound 29 3-(2-(4-methoxy-benzyl)-thiophenyl)-ß-
D-1-
tertbutyldiphenylsilyl,3,4,5-acetyl-glucopyranoside. Dry dichloro-
0.20 mmol, 98%) as a pale yellow solid. 1H NMR (DMSO-d6):
d¼13.4
methane (140 mL), TEA (3.72 mL, 26.4 mmol) and 4-dimethylami-
nopyridine (DMAP, 296 mg, 2.40 mmol) were added to a solution of
AVE2268 1a (4.59 g, 12.0 mmol) in dry DMF (16 mL). Then tert-
butyldiphenylchlorosilane (TBDPSCl, 6.67 mL, 25.2 mmol) in
dichloromethane (10 mL) was added dropwise at 0 ꢀC during
10 min and the reaction mixture was stirred overnight without
further cooling. Afterwards water (120 mL) was added, the organic
layer was separated and the aqueous layer was extracted twice with
ethyl acetate. The combined organic layers were washed with brine
and dried over Na2SO4. The crude product was purified by column
chromatography (heptane/ethyl acetate 1:2/1:4) to give TBDPS-
protected AVE2268 (7.40 g, 11.9 mmol, 99%) as a white foam. LC–MS
(ESI, pos.) m/z (%)¼643 [MþNa]þ (100).
(s, br s, 1H), 7.28 (d, J¼5.5 Hz, 1H), 7.10 (d, J¼8.6 Hz, 2H), 6.87 (d,
J¼5.5 Hz, 1H), 6.83 (d, J¼8.6 Hz, 2H), 5.43–5.39 (m, 2H), 5.13–5.07
(m, 2H), 4.50 (d, J¼10.0 Hz, 1H), 3.84 (s, 2H), 3.71 (s, 3H), 2.00 (s,
3H), 1.98 (s, 3H), 1.97 ppm (s, 3H); LC–MS (ESI, pos.) m/z (%)¼545
[MþNa]þ (100).
4.1.32. Compound 13 3-(2-(4-methoxy-benzyl)-thiophenyl)-ß-
curonide. To a solution of 31 (310 mg, 0.59 mmol) in methanol
(40 mL) sodium methylate (452 L, 2.37 mmol, 30% in methanol)
D-glu-
m
was added dropwise. After stirring for 1 h at rt the starting material
had disappeared (LC–MS control) and the reaction mixture was
adjusted to a pH value of 6.5 with hydrochloric acid (1.25 M in
ethanol). The solvent was evaporated and the residue was purified
first by column chromatography (dichloromethane/methanol
3:1/2:1/1:1) and afterwards by semi-preparative HPLC (col-
umn: YMC Pack Pro C18, eluents: acetonitrile/water 10:90 for
4 min, then up to 80:20 in 12 min, then 80:20 for 4 min and finally
in 1 min down again to 10:90 for 1 min, flow: 15 mL/min, retention
time: 10.5 min). Final freeze-drying of the product containing
fractions yielded 13 (176 mg, 0.44 mmol, 75%, purity 99.6% (HPLC,
TBDPS-protected AVE2268 (1.55 g, 2.50 mmol) was dissolved in
dry pyridine (130 mL) and reacted with acetic acid anhydride
(35.8 mL, 375 mmol). The mixture was stirred for 4 h before
methanol (60 mL) was added. After one hour water (130 mL) was
added and the mixture was stirred overnight. Afterwards the
aqueous layer was extracted twice with ethyl acetate (50 mL) and
the combined organic layers were dried over Na2SO4. The residue

1482
V. Derdau et al. / Tetrahedron 66 (2010) 1472–1482
230 nm)) as a white solid. Mp: 185 ꢀC (decomp.); 1H NMR (DMSO-
d6):
2H), 5.38 (s, br s, 1H), 5.08 (s, br s, 1H), 4.67 (d, J¼7.1 Hz, 1H), 3.98 (s,
2H), 3.71 (s, 3H), 3.49–3.47 (m, 2H), 3.26–3.23 ppm (m, 3H); LC–MS
(ESI, pos.) m/z (%)¼419, [MþNa]þ (100); HRMS (ESI-LTQ, neg.)
C18H19O8S calcd 395.08061; found, 395.08072.
2.20 mmol, 74%, purity 98.3% (HPLC, 230 nm)) as a white solid.
(Analytical data: see above).
d
¼7.21–7.18 (m, 3H), 6.92 (d, J¼5.5 Hz, 1H), 6.83 (d, J¼8.6 Hz,
Acknowledgements
We thank Dr. Lars Bierer for his support during development of
the synthesis and Dr. Norbert Nagel and Winfried Heyse for the
X-ray structure of 8a. Our special thanks to Dr. Jens Riedel, Gerald
Scholz, Claudia Loewe and Dr. Martin Sandvoss for analytical sup-
port. Furthermore we thank Dr. John Allen and Dr. Tom Gregson for
helpful suggestions during the preparation of the manuscript.
4.1.33. Compound 32 3-(2-(4-methoxy-benzoyl)-thiophenyl)-ß-D-
3,4,5-acetyl-glucuronic methyl ester. Potassium carbonate (3.70 g,
26.5 mmol) and tetrabutylammoniumbromide (TBAB, 863 mg,
2.65 mmol) dissolved in water (20 mL) were added to keto aglycon
5a (1.24 g, 5.30 mmol) and triaceto-bromo-a-D-glucuronic acid
methyl ester (4.62 g, 11.1 mmol) dissolved in dichloromethane
(80 mL) and the reaction mixture was stirred for 24 h at rt. Then
silver carbonate (1.11 g, 3.98 mmol) and TBAB (863 mg, 2.65 mmol)
in water (15 mL) were added. After 3.5 h the addition of silver
carbonate (1.11 g, 3.98 mmol) and TBAB (863 mg, 2.65 mmol) in
water (15 mL) was repeated. After stirring over two nights water
was added and the reaction mixture was extracted three times with
ethyl acetate (30 mL). The combined organic layers were washed
with brine and then dried over Na2SO4. The crude product was
purified by column chromatography (heptane/ethyl acetate 3:2) to
give 32 (1.89 g, 3.44 mmol, 65%) as a pale yellow foam. 1H NMR
Supplementary data
1H and/or 13C NMR spectra of 1a,1b, 4a, 4b, 5a, 5b, 7a, 7b, 8a, 8b,
9b, 9e, 12, 13, 15, 17, 20, 23, 24, 27, 28, 29, 30, 31, 32 and 33. Sup-
plementary data associated with this article can be found in the
online version, at doi:10.1016/j.tet.2009.12.003.
References and notes
1. (a) Zhao, F.-Q.; Keating, A. F. Curr. Genomics 2007, 8, 113–128; (b) Wood, S. I.;
Trayhurn, P. Br. J. Nutr. 2003, 89, 3–9.
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Curr. Opin. Invest. Drugs 2007, 8, 285–292.
(DMSO-d6):
d
¼7.96 (d, J¼5.5 Hz, 1H), 7.68 (d, J¼6.9 Hz, 2H), 7.10 (d,
J¼5.5 Hz, 1H), 6.98 (d, J¼6.9 Hz; 2H), 5.66 (d, J¼7.7 Hz, 1H), 5.31 (t,
J¼9.6 Hz, 1H), 4.98 (t, J¼9.6 Hz, 1H), 4.73–4.62 (m, 2H), 3.84 (s, 3H),
3.61 (s, 3H), 1.98 (s, 3H), 1.91 (s, 3H), 1.86 ppm (s, 3H); LC–MS (ESI,
pos.) m/z (%)¼573 [MþNa]þ (100).
4. Glombik, H.; Frick, W.; Heuer, H.; Kramer, W.; Brummerhop, H.; Plettenburg, O.
WO 2004007517.
5. (a) Caldwell, J.; Gardner, I.; Swales, N. Toxicol. Pathol.1995, 23,102–112; (b) Roffey,
S. J.; Obach, R. S.; Gedge, J. I.; Smith, D. A. Drug Metab. Rev. 2007, 39, 17–43.
6. Vogel, H. G. In Drug Discovery and Evaluation: Safety and Pharmacokinetic Assays;
Hock, F. J., Maas, J., Mayer, D., Eds.; Springer: New York, NY, 2006.
7. (a) Marathe, P. H.; Shyu, W. C.; Humphreys, W. G. Curr. Pharm. Des. 2004, 10,
2991–3008; (b) Dalvie, D. Curr. Pharm. Des. 2000, 6, 1009–1028.
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14; Wiley-Blackwell (an imprint of John Wiley & Sons Ltd): Chichester, 2009.
9. (a) Guidance for Industry: Bioanalytical Method validation; US Administration,
CDER, Rockwill, MD, USA, 2001; (b) Stovkis, E.; Rosing, H.; Beijnen, J. H. Rapid
Commun. Mass Spectrom. 2005, 19, 401–407; (c) Jemal, M.; Xia, Y. Q. Curr. Drug.
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Appl. 1996, 676, 53–59; (b) Kao, C.-Y.; Giese, R. Chem. Res. Toxicol. 2005, 18, 70–
75; (c) Wang, H.; Hussain, A. A.; Pyrek, J. S.; Goodman, J.; Wedlund, P. J. J. Pharm.
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Dunn, J. A.; Grosse, C. M. J. Chromatography, B: Biomed. Appl. 1999, 732, 383–393.
11. (a) Guidance for Industry: Testing of Drug Metabolites; US Administration, CDER,
Rockwill, M.D, USA, 2008; (b) Humphreys, W. G.; Unger, S. E. Chem. Res. Toxicol.
2006, 19, 1564–1569; (c) Smith, D. A.; Obach, R. S. Chem. Res. Toxicol. 2006, 19,
1570–1579.
12. Derdau, V.; Bierer, L.; Kossenjans, M. DE102004063099. Derdau, V.; Bierer, L.;
Kossenjans, M. USPTO 20080207882.
13. Reaction conditions have been reported earlier Box, V. G. S.; Meleties, P. Tet-
rahedron Lett. 1998, 39, 7059–7062.
14. (a) Miller, L. L.; Yu, Y. J. Org. Chem. 1995, 60, 6813–6819; (b) Gronowitz, S.;
Cederlund, B.; Ho¨rnfeldt, A.-B. Chem. Scr. 1974, 5, 217–226.
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bramaniam, S.; Aidhen, I. S. Synthesis 2008, 23, 3707–3738.
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7911; Angew. Chem., Int. Ed. 2007, 46, 7744–7765.
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4.1.34. Compound 33 3-(2-(4-methoxy-benzyl)-thiophenyl)-ß-D-glu-
curonic methyl ester. To a solution of 32 (1.85 g, 3.36 mmol) in dry
acetonitrile (90 mL) Na(BH3)CN (1.78 g, 26.9 mmol) was added at
0 ꢀC and the reaction stirred for 30 min under cooling. Afterwards
chlorotrimethylsilane (3.50 mL, 26.9 mmol) was dropped slowly
into the reaction mixture (caution: development of gases). After
stirring for further 3 h at 0–4 ꢀC satd NaHCO3-solution was added
and the reaction mixture was extracted with dichloromethane. The
combined organic layers were washed once with water and dried
over Na2SO4. The crude product was purified by column chroma-
tography (heptane/ethyl acetate 3:2) to give 33 (1.65 g, 3.09 mmol,
92%) as a white solid. 1H NMR (DMSO-d6):
d
¼7.27 (d, J¼5.5 Hz, 1H),
7.08 (d, J¼8.6 Hz, 2H), 6.85–6.81 (m, 3H), 5.48–5.42 (m, 2H), 5.12–
5.02 (m, 2H), 4.63 (d, J¼9.9 Hz, 1H), 3.83 (s, 2H), 3.70 (s, 3H), 3.63 (s,
3H), 1.99 (s, 3H), 1.98 (s, 3H), 1.96 ppm (s, 3H); LC–MS (ESI, pos.) m/z
(%)¼559 [MþNa]þ (100).
4.1.35. Compound 13 3-(2-(4-methoxy-benzyl)-thiophenyl)-ß-D-glu-
curonide. Sodium methylate (2.54 mL, 13.4 mmol, 30% in metha-
nol) was dropped into a suspension of 33 (1.60 g, 2.98 mmol) in
methanol (200 mL). After 2 h stirring at rt lithium chloride
(644 mg, 14.9 mmol) and water (70 mL) were added to the yellow
solution and the reaction mixture was stirred overnight. Then the
reaction mixture was adjusted to a pH value of 5.5 with hydro-
chloric acid (1.25 M in methanol). The solvent was evaporated in
vacuo and the residue was purified firstly by column chromatog-
raphy (dichloromethane/methanol 3:1/2:1/1:1) and secondly
by semi-preparative HPLC (information: see above). Final freeze-
drying of the product containing fractions yielded 13 (872 mg,