Synthesis and bioactivity of N-glycosylated 3-(2-oxo-2-arylethylidene)-indolin-2-ones

Article abstract of DOI:10.1039/c4ra14301a

N-Glycosyl-3-alkylideneoxindoles, N-glycosylated 3-(2-oxo-2-arylethylidene)indolin-2-ones, were prepared by reaction of isatin-N-glycosides with substituted acetophenones. The biological activities of these new compounds revealed significant antitumor act

Full text of DOI:10.1039/c4ra14301a

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1
Synthesis and Bioactivity of N-glycosylated 3-(2-Oxo-2- arylethylidene)-
indolin-2-ones
a
a
b
c
Dennis Kleeblatt, Martin Becker, Michael Plötz, Madeleine Schönherr, Alexander
a
a
b
c
a,d
a,e
Villinger, Martin Hein, Jürgen Eberle, Manfred Kunz, Qamar Rahman, Peter Langer*
a
Institut für Chemie, Universität Rostock, AlbertꢀEinsteinꢀStr. 3a, 18059 Rostock, Germany
Charité Centrum 12 für Innere Medizin und Dermatologie, Hauttumorzentrum, Charité –
b
Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
c
Klinik für Dermatologie, Venerologie und Allergologie, Universitätsklinik Leipzig, Philippꢀ
RosenthalꢀStr. 23ꢀ25, 04103 Leipzig, Germany
d
Amity University, Lucknow Campus, Viraj Khandꢀ5, Gomti Nagar, Lucknow – 226010,
India
e
LeibnizꢀInstitut für Katalyse e. V. an der Universität Rostock, AlbertꢀEinsteinꢀStr. 29a,
18059 Rostock, Germany
Fax: +49 (381)4986412, Eꢀmail: peter.langer@uniꢀrostock.de
Abstract:
NꢀGlycosylꢀ3ꢀalkylideneoxindoles,
Nꢀglycosylated
3ꢀ(2ꢀoxoꢀ2ꢀ
arylethylidene)indolinꢀ2ꢀones, were prepared by reaction of isatinꢀNꢀglycosides with
substituted acetophenones. The biological activities of these new compounds revealed
significant antitumor activity in melanoma human cell lines. Inhibition of cell
proliferation and loss of cell viability were strongly enhanced by the combination with
the death ligand TRAIL (TNFꢀrelated apoptosisꢀinducing ligand). The antitumor effects
were related to the inhibition of the survival pathway of cꢀJun and JNK2 (Jun Nꢀterminal
kinase).
Keywords: isatines, indirubin analogues, carbohydrates, heterocycles

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2
O
OH
O
N
O
OAc
HO
OH
O
OAc
HO
OH
AcO
OAc
OAc
O
AcO
N
O
AcO
OAc
O
R
Graphical abstract

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Introduction
The natural product indirubin (1) is an isomer of the blue dye indigo, which has, in contrast to
indigo, a violet colour (Figure 1). During the isolation of indigo from natural sources, as well
as during its industrial synthesis, indirubin is formed as a byꢀproduct and is responsible for the
reddish shine. In addition, indirubin is the pharmacologically active substance of the
traditional chinese medicinal recipe Danggui Longhui Wan, which has been used for the
1
treatment of myelocytic leukemia. The biological activity of indirubin has been related to the
2
inhibition of cyclin dependent kinases (CDKs). During the last years, inhibition of CDKs is
becoming an attractive research field for combating cancer and other deseases, which are
3
related to hyperproliferation. Indirubin and its substituted derivatives are not only potent
2
inhibitors of cyclinꢀdependent kinases (CDKs) , but also of several other kinases, such as
4
glycogen synthase kinaseꢀ3 (GSKꢀ3). For example, 5ꢀsubstituted indirubins displayed high
2,4
inhibitory potency toward various CDKs and GSKꢀ3β. Also several Oꢀ and Nꢀglycosylated
5
indigoid dyes have been synthesized in recent years. Many of the compounds studied so far
exhibit higher antiproliferative activity toward human cancer cells in vitro than their nonꢀ
glycosylated analogues. Notably, both deprotected and protected Nꢀglycosides may be of
6
pharmacological relevance. For example, the biological activity of so called ‘Natura’, i. e.
acetylꢀprotected βꢀDꢀxylopyranosylꢀNꢀisoindigo, was reported to be higher than that of its
6a
deprotected analogue.
7
In 2006, our group published the first synthesis of Nꢀglycosylated indirubins. We also
reported significant activities of several derivatives against various human cancer cell lines.
Based on these results, other glycosylated and nonꢀglycosylated derivatives and
8
heteroanalogues of indirubin and isoindigo have been synthesized. Some of them, especially
the thiaꢀanalogous indirubinꢀNꢀglycosides, have shown substantial activity against different
8
c
human cancer cell lines. In all these studies, we have found that the presence of a
carbohydrate moiety located at the nitrogen atom of the heterocycle resulted in a significant
increase of the antiꢀcancer activity.
In the course of our program related to the development of new, biologically active indirubin
derivatives, we turned our attention to 3ꢀalkylideneoxindoles, such as 2, which can be

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4
regarded as structurally simplified analogues of indirubin (Figure 1). We assumed that these
structurally more flexible structures might better interact with the reactive center of an
enzyme, which should lead to an enhanced biological activity. WOODARD et al. studied the
9a
activity of several nonꢀglycosylated 3ꢀalkylideneoxindoles as CDK inhibitors. They reported
a very high inhibition in case of derivative 3b containing a halogen substituent. In contrast,
only a weak inhibition was observed for unsubstituted derivative 3a (Figure 2). BELENKAYA
9b
et al. reported antiꢀmalaria activity of nitro substituted compound 3f.
Herein, we report what is, to the best of our knowledge, the first synthesis of Nꢀglycosylated
ꢀalkylideneoxindoles and their antitumor activity in human melanoma cell lines. During the
3
optimization of the biological activity, the most promising substitution patterns were selected
9
based on known nonꢀglycosylated derivatives.
O
O
NH
O
O
N
H
N
H
1
2
Figure 1. Indirubin (1) and 3ꢀalkylideneoxindole 2
R''
3
3
3
3
3
a: R' = H, R'' = H
b: R' = H, R'' = Br
c: R' = Me, R'' = Br
d: R' = Br, R'' = H
e: R' = F, R'' = Br
O
R'
HN
O
3f: R' = NO , R'' = H
2
Figure 2. Known biologically active nonꢀglycosylated 3ꢀalkylideneoxindoles

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Results and Discussion
8
IsatinꢀNꢀglycoside 7a was prepared, following our previously reported procedure, by reaction
of ꢀrhamnose with aniline, acetylation and subsequent cyclization with oxalyl chloride
Scheme 1). Likewise, derivatives 7b, 7c and 7d were prepared from ꢀmannose, ꢀglucose
The condensation of isatinꢀNꢀglycoside 7a with
L
(
D
D
6b,7b,10
and
Dꢀgalactose, respectively.
acetophenone afforded Nꢀglycosylated 3ꢀalkylideneoxindole 9a in up to 71% yield (Scheme
2). The reaction was optimized based on our previous experience with indirubinꢀNꢀglycosides
8
and their heteroanalogous compounds, and based on known chemistry of nonꢀglycosylated 3ꢀ
9
alkylideneoxindoles. We tried several conditions for the condensation reaction and found that
a twoꢀstep procedure worked best in our hands. The first step was the aldol reaction of the
acetophenone with the carbonyl group (Cꢀ3) of the isatinꢀNꢀglycoside (Scheme 2). For this
reaction step we used triethylamine as the base and ethanol as the solvent at a temperature of
20 °C. Only in case of parent acetophenone no additional solvent was used. In two cases we
could isolate and characterize the aldol addition products, which were formed as
diastereomeric mixtures, because of the new stereocenter at Cꢀ3. The ratio of the diasteromers
was found to be about 2:1, but we did not investigate the configuration of the main/ minor
isomer at Cꢀ3. In general, it was sufficient to isolate the crude products, by simple distillation
of the solvent from the reaction mixture, to be used as the starting materials in the following
step. The purified or the crude products were reacted with mesyl chloride, in the presence of
triethylamine (and in some cases in the additional presence of DMAP), to give the desired
dehydrated final products. After workup and purification, the products could be isolated in
71% yield.
The preparative scope was next studied (Table 1). The condensation of isatinꢀNꢀglycoside 7a-
d with various substituted acetophenones afforded Nꢀglycosylꢀ3ꢀalkylideneoxindoles 9a-m in
45 ꢀ 78% yields (based on 7).

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6
Me
HO
Me
HO
NH2
O
O
i
HO
OH
+
NHPh
OH
HO
OH
4
a
5a
ii
Me
Me
O
O
iii
AcO
AcO
AcO
NHPh
OAc
O
AcO
OAc O
7
a
6a
Scheme 1. Synthesis of 1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀβꢀ
L
ꢀrhamnopyranosyl)isatin (7a). Reagents and
O, pyridine, 0 °C, 12 h; (iii) oxalyl
3
(1 equiv.), 55°C, 1.5 h, then chromatographic separation of the
conditions: (i) PhNH , EtOH (abs.), 20°C, 24 h; (ii) Ac
2
2
chloride (10 equiv.), AlCl
anomers.

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7
4
5
O
3
a
3
6
2
7
a
O
7
N
1
i
+
O
1
'
OAc
'
2
OH
O
N
O
3
'
O
6
'
Me
O
5
'
Me
4'
OAc
OAc
OAc
AcO
7
a
OAc
8
a
ii
O
O
OSO CH
3
N
O
N
2
O
O
Me
O
Me
AcO
OAc
OAc
AcO
OAc
OAc
9
a
Scheme 2. Synthesis of 9a. Reagents and conditions: (i) NEt (1 eq.), EtOH 20 °C, 6ꢀ10 h. (ii)
3
3 2 2
MsCl, NEt , (DMAP), CH Cl , 0ꢀ20 °C, 2 h, 20°C, 6ꢀ8 h.
For the overall reaction, using crude intermediates 8 for the final dehydratisation step, we
observed a trend to higher yields for acetophenones containing electronꢀwithdrawing
substituents. The highest yields were observed for the nitroꢀsubstituted derivative, while the
lowest yield was observed for the methoxyꢀsubstituted derivatives. This result is surprising,
because of the lower nucleophilicity of the acceptorꢀsubstituted acetophenones.
Table 1. Synthesis and biological activity of acetyl protected Nꢀglycosylꢀ3ꢀ(2ꢀ
oxoꢀ2ꢀarylethylidene)indolinꢀ2ꢀones 9a-m (IC50 values for the tests of selected

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8
compounds 9 in the melanoma cell line SKꢀMel 147). The intermediately
formed Nꢀglycosylated 3ꢀhydroxyꢀ3ꢀ(2ꢀoxoꢀ2ꢀarylethyl)indolinꢀ2ꢀones 8 are
also given (when isolated).
IC50 (µM)
7
a
8 Yield
9 Yield
(mean +/ꢀ
SD)
O
O
AcO
OAc
O
OH
O
N
AcO
N
AcO
N
AcO
AcO
a
O
a
O
ꢀ
O
Me
O
AcO
AcO
O
Me
b
7
1%
OAc
Me
93%
OMe
O
O
AcO
AcO
OAc
O
O
OH
O
N
N
4
.4
O
a
AcO
AcO
b
N
b
O
AcO
Me
O
+/ꢀ 0.5
OMe
O
Me
a
b
AcO
4
5% (80% )
OAc
Me
AcO
95%
O
AcO
AcO
OAc
O
O
N
N
4
.3
O
a
a
a
b
AcO
AcO
c
O
Me
O
+/ꢀ 0.7
NO
2
Me
a
a
a
OAc
78%
O
AcO
AcO
OAc
O
O
N
N
2
.8
O
AcO
AcO
d
O
Me
O
+/ꢀ 0.6
Cl
Me
4
9%
5%
OAc
O
AcO
AcO
OAc
O
O
N
N
4
.2
O
O
AcO
AcO
e
O
Me
O
+/ꢀ 0.4
F
Me
6
OAc
O
AcO
AcO
O
O
N
N
6
.0
OAc
f
O
AcO
OAc O
+
/ꢀ 0.6
NO
2
OAc
a
7
0%
OAc OAc

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9
O
AcO
O
O
O
N
AcO
N
b
b
b
c
OAc
g
ꢀ
O
AcO
OAc O
Cl
OAc
a
a
a
a
5
2%
3%
OAc OAc
O
AcO
AcO
O
O
O
N
N
7
.0
OAc
h
i
O
AcO
OAc
O
+/ꢀ 0.7
F
OAc
6
OAc OAc
O
AcO
AcO
O
O
O
O
O
N
N
5
.9
OAc
O
AcO
OAc O
+/ꢀ 0.5
CF
3
OAc
6
9%
1%
7%
4%
OAc OAc
O
AcO
AcO
O
O
N
N
OAc
j
ꢀ
ꢀ
ꢀ
ꢀ
O
AcO
OAc O
NO
2
OAc
7
OAc OAc
O
AcO
AcO
O
O
N
N
c
OAc
k
l
O
AcO
OAc O
CF
3
OAc
a
a
6
OAc OAc
O
AcO
AcO
O
O
O
N
N
d
OAc
O
AcO
OAc
O
F
OAc
6
OAc OAc
O
AcO
AcO
O
O
O
N
N
d
OAc
m
O
AcO
OAc O
CF
3
OAc
a
6
6%
OAc OAc
a
Yields of isolated products 9 based on 7 using crude intermediate 8 for the dehydratisation
step
b
Yields of isolated products 9 based on purified intermediate 8 used for the dehydratisation
step

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1
0
1
1
For the structure elucidation of compounds 9 two dimensional NMR experiments ( H, Hꢀ
1
1
1
1
COSY, HSQC and H, HꢀNOESY) were carried out. In the H, HꢀNOESY spectra, the
missing correlation between Hꢀ8 and Hꢀ4 (Figure 3) was a first, but unreliable indication of
the Eꢀconfiguration of the double bond between Cꢀ3 and Cꢀ8. Furthermore, the βꢀ
configuration of the carbohydrate substituents could be clearly shown by the correlation
1
1
signals of Hꢀ1’, Hꢀ3’ and Hꢀ5’ in the H, HꢀNOESY spectra (only βꢀconfigured starting
isatinꢀNꢀglycosides were applied for the reactions). These correlations can appear in case of
1
βꢀL
ꢀrhamno configured substituents, when they have a C ꢀ (or similar) conformation, as well
4
4
as in case of the other investigated carbohydrate substituents, when they have a C
1
ꢀ
conformation. The above mentioned correlations were observed for all studied examples.
Additionally, the structure of 9c was independently confirmed by Xꢀray crystal structure
11
analysis (Figure 4). Here, the Eꢀconfiguration of the double bond as well as the anomeric βꢀ
1
configuration and C
4
ꢀconformation of the sugar moiety was clearly proven. Because of
comparable NMR data of all the 3ꢀ(2ꢀoxoꢀ2ꢀarylethylidene)indolinꢀ2ꢀones, we assumed that
all these compounds possess an Eꢀconfigured double bond.
5
6
o
m
4
a
O
p
i
7
NO2
3
H
H
9
7a
3
o
m
6
'
H
8
N
1
H C
5'
1'
3
O
2
3
'
AcO
H
O
4'
2'
AcO
H
OAc
1
1
Figure 3. H, HꢀNOESY correlations for compound 9c

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1
Figure 4. Xꢀray structure image of compound 9c (ORTEP plot with displacement ellipsoids at
the 50 % probability level).
We also tried to remove the acetyl protecting groups from the carbohydrate substituents of
compounds 9. Therefore, typical methods under basic (e.g. 1% methanolic sodium
1
2
methanolate) as well as acidic conditions (hydrogen chloride in methanol) were applied.
However, it was not possible to isolate the deprotected products from the reaction mixture.
The result can be explained by a retroꢀaldol reaction as the formation of the corresponding
acetophenone and of the isatinꢀNꢀglycoside was detected. However, as mentioned in the
introduction, acetylꢀprotected glycosylated isoindigo and indirubin derivatives proved, in
6
a
some cases even more active than their deprotected analogues. Therefore, we studied the
pharmacological activity of our acetylated products and, in fact, interesting biological effects
were observed which are outlined below.
In previous studies, we had investigated the biological activity of compound 9b in melanoma
cell lines SKꢀMelꢀ19, SKꢀMelꢀ29, SKꢀMelꢀ103 and SKꢀMelꢀ147 and observed only moderate
8
c
direct activities in these cells. In the present work, we studied the growth inhibitory
activities (IC50) of selected examples of various derivatives of derivatives 9 (Table 1). Here,
we focussed on cell line SKꢀMelꢀ147 in order to put an emphasis on the differences between
the individual substances. This cell line is characterized by a very high proliferative activity,

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2
which may mirror the situation of highly aggressive metastatic cells in the human system. We
selected the derivatives for biotests only those compounds which are stable in water for an
extended period of time. Compounds which tend to be slightly unstable in aqueous solution
were not tested in order to avoid misleading biological results. Those derivatives containing a
βꢀLꢀrhamnosyl substituent revealed slightly higher activities than the mannosylated
compounds. The highest activity was found for the chloroꢀsubstituted derivative 9d. The
higher growth inhibitory activity of derivative 9b in our current experiments (IC50 value: 4.4
8
c
+
/ꢀ 0.5), as compared to our earlier results (IC50 value: 27.2 +/ꢀ 1.3), may be explained by i)
culture conditions, ii) the different incubation times applied (48 h in current vs. 24 h in
previous experiments) as well as by iii) two different assays, used (WSTꢀ1 in previous vs.
XTT assay in current experiments). In addition, human benign dermal fibroblasts were used
here as controls, and the IC50 values of indirubin derivatives were significantly higher in
fibroblasts than in melanoma cells (9b: 8.8 +/ꢀ1.1 ꢁM; 9c: 32.9 +/ꢀ 0.9 ꢁM; 9d: 9.1 +/ꢀ 0.7
ꢁM; 9e: 9.5 +/ꢀ 0.3 ꢁM; 9f: 11 +/ꢀ1.5 ꢁM; 9h: 19.3 +/ꢀ 0.9 ꢁM; 9i: 9.6 +/ꢀ2.4 ꢁM; cf. Table 1).
Further biological effects of the Nꢀglycosylated 3ꢀ(2ꢀoxoꢀ2ꢀarylethylidene)indolinꢀ2ꢀone
derivatives 9c, 9d, 9e, 9f, 9i, 9j, 9k and 9m were exemplary investigated in the frequently
1
3
investigated human melanoma cell line Aꢀ375 , which is well characterized for apoptosis
regulation. In particular, apoptosis regulation by the cytokine and death ligand TRAIL (TNFꢀ
14,15
related apoptosisꢀinducing ligand) is well described in this cell line.
Cell viability was
monitored by calcein staining and flow cytometry. The applied concentrations of 10 µM of
the indirubin derivatives resulted in a significant reduction of cell viability after 24 h (20% ꢀ
30%; Figure 5a). We had previously reported that indirubin derivatives, as also their
heteroanalogous compounds, can strongly enhance apoptosis induced by death ligand
8
,14
TRAIL.
Thus, combinations with TRAIL were also tested for the new series of Nꢀ
glycosylated 3ꢀ(2ꢀoxoꢀ2ꢀarylethylidene)indolinꢀ2ꢀones. Whereas TRAIL (20 ng/ml) alone
showed only moderate effects on Aꢀ375 cell viability (20% reduction), the combinations
dramatically reduced the numbers of viable cells (50% ꢀ 90% reduction; Figure 5a).
Decreased cell viability also exerted a strong impact on cell proliferation, monitored in Aꢀ375
cells here by WSTꢀ1 assay. Thus, inhibition of cell proliferation upon combination treatment
with TRAIL ranged between 70% and 90% (Figure 5b). Direct cytotoxicity on the other hand
remained unaffected at this time, as determined by LDH release in cell culture supernatants
(data not shown). Cell proliferation was also determined in real time. In fact, Aꢀ375 cells were
still proliferative when treated with TRAIL (20 ng/ml) or several indirubin derivatives alone,

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3
e.g. 9c, 9f at 1.25 µM and 9e, 9i, 9m, 9k at 2.5 µM. Inhibition by these indirubin derivatives
alone ranged between 40% and 80% at a time point of 100 h (75 h after treatment). At this
time, inhibition by TRAIL alone was at 60%. However upon combination treatments, cell
proliferation and cell attachment was almost completely abolished for all tested indirubins.
For 9d and 9j, an enhanced effect by combination treatment could not be clearly shown, as in
this assay the effect of 9d and 9j alone was already very strong (Figure 5c).
Figure 5. Effects of Nꢀglycosylated 3ꢀ(2ꢀoxoꢀ2ꢀarylethylidene)indolinꢀ2ꢀones on melanoma
cells: (a) Cell viability of Aꢀ375 cells was determined after 24 h by calcein staining. Cells
were treated with TRAIL (20 ng/ml), compounds of the series 9 (10 ꢁM) or with
combinations. (b) Cell proliferation of Aꢀ375 cells expressed as percent of nonꢀtreated
controls was determined after 48 h by WSTꢀ1 assay. Cells were treated with 1.25 ꢁM of
compounds of series 9 and/or 20 ng/ml TRAIL. (c) Realꢀtime cell analysis (RTCA) of Aꢀ375
cells treated with 1.25 µM / 2.5 µM of 9c, 9d, 9e, 9f, 9i, 9m, 9j and 9k +/- 20 ng/ml TRAIL.
Seeding density was 1,250 cells per microtiter well. The cell index was normalized at 24 h,

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when treatment was started. Cell indices indicate cell adhesion. (a ꢀ c) Each two independent
experiments with triplicate determinations revealed highly comparable results.
As reported previously, treatment of melanoma cells with glycosylated indirubin analoga
8c
resulted in an inhibition of the phosphorylation of the transcription factor cꢀJun. This may be
due to a direct inhibition of its upstream kinases cꢀJun Nꢀterminal kinases 1/2 (JNK1/2). To
further address this question, SKꢀMelꢀ147 cells were treated with derivative 9b and cꢀJun
phosphorylation was determined by immunoblotting. As treatment with 10 µM of 9b was
associated with significant induction of cell death, lower concentrations were used (5 µM, 6.5
µM and 7.5 µM). Inhibited cꢀJun phosphorylation was seen for all three concentrations, and
strongest effects were obtained with 6.5 ꢁM (Figure 6). The three concentrations also induced
upregulation of p53 levels, which may support apoptosis induction in melanoma cells via p53ꢀ
dependent pathways.
Figure 6. Derivative 9b suppresses cꢀJun phosphorylation in melanoma cells. SKꢀMelꢀ147
melanoma cells were treated for indicated times with 6.5 ꢁM of 9b. Total protein lysates of
cells were subjected to immunoblotting using specific antibodies for phosphorylated cꢀJun
(Ser63/73) and total p53. Staining for GAPDH was used as loading control.
To test whether inhibition of cꢀJun phosphorylation was due to a direct inhibitory effect on the
upstream kinases JNK1/2, ATPꢀcompetitive kinase binding assays were performed in vitro
using recombinant JNK2 kinase. The small molecule inhibitor sorafenib, a wellꢀknown multiꢀ

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5
kinase inhibitor which strongly inhibits JNK1/2 kinases, was used as positive control. The
IC50 of sorafenib for the inhibition of JNK2 was determined at 1.6 µM, whereas the IC50 of
7
9
b was at 1.06 ꢁM. As negative control, the indirubin derivative 10 (Figure 7) was used,
which did not inhibit the activity of JNK2 (figure 8).
H C
3
O
O
HO
N
HO
OH
O
HN
Figure 7. Structure of the NꢀβꢀLꢀrhamnopyranosylindirubin 10 used as control substance.
Thus, it could be demonstrated that derivative 9b exerted a strong inhibitory effect on JNK2,
which explains its inhibitory activity on cꢀJun phosphorylation. Suppression of JNK2 and cꢀ
Jun may significantly contribute to the decreased proliferation and enhanced apoptosis
sensitivity of melanoma cells after treatment with derivative 9b, as cꢀJun plays a decisive role
in melanoma cell proliferation and survival.

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6
Figure 8. Derivative 9b strongly inhibits JNK2 activity in melanoma cells. Kinase ATPꢀ
binding assays were performed with recombinant JNK2 using different concentrations of the
positive control inhibitor sorafenib as well as of derivatives 9b and 10. Experiments were
performed three times each in triplicates. IC50 values were calculated after analysis of all
experiments. Graphs correspond to the means of one representative experiment.
Conclusions
In conclusion, the first synthesis of acetyl protected Nꢀglycosylated 3ꢀ(2ꢀoxoꢀ2ꢀ
arylethylidene)indolinꢀ2ꢀones was reported. The synthesis relies on the aldol condensation of
readily available isatinꢀNꢀglycosides with substituted acetophenones. The preparation of the
corresponding deprotected derivatives failed in most of the cases, probably due to the
preferred formation of products of the retroaldol reaction. However, the protected derivatives
showed interesting pharmacological activities, such as a significant decrease of cell viability
and inhibition of cell proliferation. The effects were stronger in combination with the
proapoptotic death ligand TRAIL (TNFꢀrelated apoptosisꢀinducing ligand). A reduction of the

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7
cell viability by up to 90% and complete abrogation of cell proliferation are strongly
suggestive for testing therapeutic strategies. With regard to our investigations related to the
mode of antitumor activity of indirubin derivatives in the cell, our present study suggests that
the investigated derivatives affected the prosurvival pathway of JNK and cꢀJun.
Experimental section
Material and methods for the syntheses and characterisation of the new compounds
All solvents were anhydrous and commercially available and all reactions were performed
under argon atmosphere. The synthesis of Nꢀglycosylated isatin derivatives has been
6b,7b,10
described in the literature.
The substituted acetophenones were commercially available.
1
Yields refer to isolated products. H NMR spectra (250.13 MHz, 300.13 MHz and 500.13
1
3
MHz) and C NMR spectra (62.9 MHz, 75.5 MHz and 125.8 MHz) were recorded on Bruker
spectrometers AV 250, AV 300 and AV 500 in CDCl
3
and DMSOꢀd
6
as solvents. The
1
13
calibration of spectra was carried out on solvent signals (CDCl
3
: δ ( H) = 7.25, δ ( C) = 77.0;
1
13
acetoneꢀd : δ ( H) = 2.05, δ ( C) = 29.83). For the NMR signals of the compounds 8a,b and
6
9a-m the atom labelling given in Figure 3 was used. Mass spectrometric data (MS) were
obtained by electron ionization (EI, 70 eV), chemical ionization (CI, isobutane) or
electrospray ionization (ESI). The mass spectra and highꢀresolution mass spectra (HRMS)
were recorded on the following MS instruments: TimeꢀofꢀFlight (LC / MS 6210 (Agilent
Technologies) for ESI and Finnigan MAT 95 XP (Thermo Electron Corporation) for EI / CI.
Elemental analyses were performed on a C,H,N,S analyser (Thermo Quest Flash EA 1112).
The melting points were measured with a polarizing microscope (Leitz Laborlux 12 PolꢀS)
equipped with a hot stage (Mettler FP 90). Thin layer chromatography was performed on
silicaꢀgel foil (Merck DCꢀfoil, silica gel 60, F254) and silica gel plates (Merck HPTLC
precoated plates, silica gel 60, F254) pursues. Detection was carried out via UV absorbance at
2
54 nm or 366 nm and/or development with 10% methanolic sulfuric acid and subsequent
heat treatment. Column chromatography was performed on MachereyꢀNagel silica gel 60
particle size 63ꢀ200 nm, 70ꢀ230 mesh).
(
General procedure for the synthesis of the intermediate addition products 8 and
aldolcondensation products 9

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8
1
Eq. of the Nꢀglycosylisatin and 2 eq. of the substituted acetophenone are dissolved in abs.
ethanol (4ꢀ8 ml per mmol Nꢀglycosylisatin) (in the case of the unsubstituted acetophenone as
reactant, this was used as the solvent in excess and no ethanol was used) . To this solution, 1ꢀ2
eq. of triethylamine are added and the mixture is stirred for 6ꢀ12 hours at room temperature.
After complete conversion of the starting material (TLC control), the reaction mixture is
diluted with toluene and then evaporated under reduced pressure. The crude aldol addition
products 8 can be directly used for the next step or (if containing larger amounts of byꢀ
products) can be purified by column chromatography (heptane : ethyl acetate, v:v =
3:1→2:1).
The crude or purified aldol addition product 8 (1 eq.) and 4ꢀ(dimethylamino)pyridine (1.1
eq.)* are dissolved in dry dichloromethane and the solution is cooled to 0 °C. Then
triethylamine (1.1 eq.) (*in the cases were no DMAP was used, 2.2 eq. of NEt were added)
3
and methanesulfonyl chloride (1.1 eq.) are carefully added (syringe). After that, the cooling
bath is removed and the mixture is allowed to warm to room temperature during 2 hours.
After additional 6ꢀ8 hours stirring at room temperature (TLC control), the solution is diluted
3 4
with dichloromethane and washed twice with saturated NaHCO solution, 1M NaHSO
solution and brine, respectively. The organic layer is then dried over sodium sulfate, filtered
and concentrated under diminished pressure. The residue is purified by column
chromatography (heptane : ethyl acetate, v:v = 2:1) to give product 9.
3ꢀHydroxyꢀ3ꢀ(2ꢀoxoꢀ2ꢀphenylethyl)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀβꢀLꢀrhamnopyranosyl)indolinꢀ2ꢀone
(8a)
According to the general procedure 7a (200 mg, 0.48 mmol) was dissolved in about 1.5 ml
acetophenone and triethylamine (67 µL, 0.48 mmol) was added to start the reaction. After
work up 8a could be isolated after column chromatography (heptane/ ethyl acetate v:v = 6:1
→
3:1) as colourless amorphous solid (yield: 238 mg, 93 %) which consists of a
diastereomeric mixture (diasteromer 1: diasteromer 2 = 4:1) due to the new formed chiral
f
center at Cꢀ3. R 0.15 (heptane/ ethyl acetate, v:v = 3:1).
1
3
H NMR (250 MHz, CDCl ): diasteromer 1: δ = 7.87 (d, J = 7.9 Hz, 2H, 2Hortho), 7.54
3
o,m
3
3
3
3
(
“t”, Jm,p = 7.6 Hz, 1H, Hpara), 7.53 (d, J7,6 = 7.5 Hz, 1H, Hꢀ7), 7.41 (“t”, Jo,m ≈ Jm,p ≈ 7.8
3
3
3
Hz, 2H, 2Hmeta), 7.32 (d, J = 7.5 Hz, 1H, Hꢀ4), 7.23 (“t”, J ≈ J ≈ 7.6 Hz, 1H, Hꢀ6),
4,5
6,7
6,5

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9
3
3
3
3
6
.98 (“t”, J5,4 ≈ J5,6 ≈ 7.6 Hz, 1H, Hꢀ5), 5.85 (d, J1’,2’ = 1.5 Hz, Hꢀ1’), 5.59 (dd, J2’,1’ = 1.5
3
Hz, J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.26ꢀ5.21 (m, 2H, Hꢀ3’, Hꢀ4’), 3.79 (s, 1H, OH), 3.78ꢀ3.66 (m,
2
2
1
1
7
1
H, Hꢀ5’), 3.71 (d, J8a,8b = 17.2 Hz, 1H, Hꢀ8a), 3.42 (d, J8a,8b = 17.2 Hz, 1H, Hꢀ8b), 2.08,
3
.98, 1.77 (3s, 9H, 3CH
3
CO), 1.35 (d, J5’,6’ = 6.2 Hz, 3H, 3Hꢀ6’). diasteromer 2 (minor): δ =
3
3
3
.87 (d, Jo,m = 7.9 Hz, 2H, 2Hortho), 7.54 (“t”, Jm,p = 7.6 Hz, 1H, Hpara), 7.53 (d, J7,6 = 7.5 Hz,
3
3
3
H, Hꢀ7), 7.41 (“t”, Jo,m ≈ Jm,p ≈ 7.8 Hz, 2H, 2Hmeta), 7.32 (d, J4,5 = 7.5 Hz, 1H, Hꢀ4), 7.23
3
3
3
3
3
(
“t”, J6 ≈ J ≈ 7.6 Hz, 1H, Hꢀ6), 6.98 (“t”, J ≈ J ≈ 7.6 Hz, 1H, Hꢀ5), 5.87 (d, J
=
,7
6,5
5,4
5,6
1’,2’
3
3
1
.4 Hz, Hꢀ1’), 5.59 (dd, J1’,2’ = 1.4 Hz, J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.26ꢀ5.21 (m, 2H, Hꢀ3’, Hꢀ
2
2
4
’), 4.12 (d, J8a,8b = 14.1 Hz, 1H, Hꢀ8a), 4.08 (d, J8a,8b = 14.1 Hz, 1H, Hꢀ8b), 3.79 (s, 1H,
3
OH), 3.78ꢀ3.66 (m, 1H, Hꢀ5’), 2.08, 1.98, 1.68 (3s, 9H, 3CH CO), 1.40 (d, J
= 6.2 Hz,
5’,6’
3
1
3
3
1
1
7
H, 3Hꢀ6’). C NMR (75 MHz, CDCl
3
): diasteromer 1: δ = 198.5 (Cꢀ9), 175.3 (Cꢀ2), 170.1,
70.0, 169.7 (3CH CO), 141.0 (Cꢀ7a), 136.3 (Cipso), 133.8 (Cpara), 129.9 (Cꢀ3a), 129.2 (Cꢀ6),
3
28.7 (2Cmeta), 128.3 (2Cortho), 123.5 (Cꢀ4), 123.1 (Cꢀ5), 114.2 (Cꢀ7), 80.7 (Cꢀ1’), 74.3 (Cꢀ3),
3.9 (Cꢀ5’), 70.5 (Cꢀ2’), 70.4 (Cꢀ3’), 70.3 (Cꢀ4’), 44.2 (Cꢀ8), 20.9, 20.8, 20.6 (3CH CO), 17.7
3
(
Cꢀ6’). diasteromer 2 (minor): δ = 196.8 (Cꢀ9), 175.7 (Cꢀ2), 171.1, 170.0, 169.6 (3CH
3
CO),
141.5 (Cꢀ7a), 136.1 (Cipso), 133.6 (Cpara), 130.1 (Cꢀ3a), 129.4 (Cꢀ6), 128.6 (2Cmeta), 128.1
(2Cortho), 123.3 (Cꢀ4), 123.1 (Cꢀ5), 114.0 (Cꢀ7), 81.2 (Cꢀ1’), 74.0 (Cꢀ3), 73.6 (Cꢀ5’), 70.4 (Cꢀ
3
2’), 70.3 (Cꢀ3’), 70.1 (Cꢀ4’), 44.8 (Cꢀ8), 20.9, 20.8, 20.6 (3CH CO), 17.7 (Cꢀ6’). HRMS
+
(ESI): calc. for C28
H
30NO10 ([M+H] ) 540.18642, found 540.18695.
3ꢀHydroxyꢀ3ꢀ(2ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀoxoethyl)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀβꢀLꢀ
rhamnopyranosyl)indolinꢀ2ꢀone (8b)
According to the general procedure 7a (200 mg, 0.48 mmol) and 4ꢀmethoxyacetophenone
144 mg, 0.96 mmol) were dissolved in about 1.5 ml of ethanol. Then triethylamine (67 µL,
.48 mmol) was added to start the reaction. After work up 8b could be isolated after column
chromatography (heptane/ ethyl acetate v:v = 6:1 → 3:1) as colourless amorphous solid
yield: 260 mg, 95 %) which consists of a diastereomeric mixture (diasteromer 1: diasteromer
(
0
(
2
= 3:1) due to the new formed chiral center at Cꢀ3. R 0.22 (heptane/ ethyl acetate, v:v = 3:1).
f
1
3
H-NMR (250 MHz, CDCl
3
): diasteromer 1: δ = 7.91ꢀ7.83 (m, Jo,m = 7.9 Hz, 2H, 2Hortho),
3
3
4
7
.50 (d, J7,6 = 7.9 Hz, 1H, Hꢀ7), 7.32 (d, J4,5 = 7.6 Hz, 1H, Hꢀ4), 7.22 (“dt”, J6,4 = 1.2 Hz,
3
3
4
3
3
J
6,7 ≈ J6,5 ≈ 7.8 Hz, 1H, Hꢀ6), 6.98 (“dt”, J5,7 = 1.3 Hz, J5,4 ≈ J5,6 ≈ 7.6 Hz, 1H, Hꢀ5), 6.88
3
3
3
(
“d”, J = 7.8 Hz, 2H, 2Hmeta), 5.83 (d, J
= 1.4 Hz, Hꢀ1’), 5.58 (dd, J
= 1.5 Hz,
o,m
1’,2’
1’,2’

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2
0
3
J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.25ꢀ5.20 (m, 2H, Hꢀ3’, Hꢀ4’), 5.00 (s, 1H, OH), 3.83 (s, 3H,
2
2
OCH
3
), 3.77ꢀ3.69 (m, 1H, Hꢀ5’), 3.59 (d, J8a,8b = 16.8 Hz, 1H, Hꢀ8a), 3.29 (d, J8a,8b = 16.8
3
Hz, 1H, Hꢀ8b), 2.08, 1.98, 1.76 (3s, 9H, 3CH
3
CO), 1.35 (d, J5’,6’ = 6.2 Hz, 3H, 3Hꢀ6’).
3
3
diasteromer 2 (minor): δ = 7.91ꢀ7.83 (m, Jo,m = 7.9 Hz, 2H, 2Hortho), 7.50 (d, J7,6 = 8.0 Hz,
3
4
3
3
1
H, Hꢀ7), 7.32 (d, J4,5 = 7.6 Hz, 1H, Hꢀ4), 7.22 (“dt”, J6,4 = 1.2 Hz, J6,7 ≈ J6,5 ≈ 7.8 Hz, 1H,
4
3
3
3
Hꢀ6), 6.98 (“dt”, J5,7 = 1.3 Hz, J5,4 ≈ J5,6 ≈ 7.6 Hz, 1H, Hꢀ5), 6.88 (“d”, Jo,m = 7.8 Hz, 2H,
3
3
3
2
5
1
Hmeta), 5.86 (d, J
= 1.4 Hz, Hꢀ1’), 5.58 (dd, J
= 1.5 Hz, J = 3.0 Hz, 1H, Hꢀ2’),
2’,3’
1’,2’
1’,2’
2
2
.25ꢀ5.20 (m, 2H, Hꢀ3’, Hꢀ4’), 4.12 (d, J8a,8b = 14.4 Hz, 1H, Hꢀ8a), 4.09 (d, J8a,8b = 14.4 Hz,
H, Hꢀ8b), 4.03 (s, 1H, OH), 3.83 (s, 3H, OCH
3
), 3.77ꢀ3.69 (m, 1H, Hꢀ5’), 2.08, 1.97, 1.69
3
13
(
3s, 9H, 3CH CO), 1.39 (d, J
= 6.2 Hz, 3H, 3Hꢀ6’). C-NMR (75 MHz, CDCl ):
3
5’,6’
3
diasteromer 1: δ = 197.4 (Cꢀ9), 175.3 (Cꢀ2), 170.2, 170.0, 169.6 (3CH
3
CO), 164.1 (Cpara),
1
5
7
40.8 (Cꢀ7a), 130.8 (2Cmeta), 130.1 (Cipso), 129.5 (Cꢀ3a), 129.1 (Cꢀ6), 123.6 (Cꢀ4), 123.1 (Cꢀ
), 113.9 (2Cortho), 113.8 (Cꢀ7), 80.6 (Cꢀ1’), 74.5 (Cꢀ3), 73.9 (Cꢀ5’), 70.5 (Cꢀ2’), 70.4 (Cꢀ3’),
0.3 (Cꢀ4’), 55.5 (OCH ), 43.2 (Cꢀ8), 20.8, 20.7, 20.5 (3CH CO), 17.7 (Cꢀ6’). diasteromer 2
3
3
(
minor): δ = 195.5 (Cꢀ9), 175.7 (Cꢀ2), 170.0, 170.0, 169.6 (3CH
a), 130.6 (2Cmeta), 130.3 (Cipso), 129.3 (Cꢀ3a), 129.3 (Cꢀ6), 123.3 (Cꢀ4), 123.1 (Cꢀ5), 113.8
2Cortho), 113.6 (Cꢀ7), 81.1 (Cꢀ1’), 73.9 (Cꢀ5’), 73.8 (Cꢀ3), 70.4 (Cꢀ2’), 70.4 (Cꢀ3’), 70.1 (Cꢀ
3
CO), 163.9 (Cpara), 141.4 (Cꢀ
7
(
3 3
4’), 55.5 (OCH ), 44.2 (Cꢀ8), 20.7, 20.6, 20.5 (3CH CO), 17.7 (Cꢀ6’). MS (EI, 70eV): m/z
+
(
%) = 569 (M , 1), 551 (4), 273 (45), 171 (15), 153 (53). HRMS (ESI): calc. for C29
H32NO11
+
(
[M+H] ) 570.19699, found 570.19621.
(E)ꢀ3ꢀ(2ꢀOxoꢀ2ꢀphenylethylidene)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀβꢀLꢀrhamnopyranosyl)indolinꢀ2ꢀone
(9a)
According to the general procedure purified 8a (200 mg, 0.37 mmol) and 4ꢀ
dimethylamino)pyridine (50 mg, 0.41 mmol) were dissolved in about 2 ml of
dichloromethane. Then triethylamine (57 µl, 0.41 mmol) and methanesulfonyl chloride (32 µl,
.41 mmol) were added carefully to the reaction mixture. After work up 9a could be isolated
after column chromatography (heptane/ ethyl acetate v:v = 6:1 → 3:1) as orange solid (yield:
(
0
137 mg, 71 %). mp 74ꢀ76 °C (heptane/ ethyl acetate).
1
3
HꢀNMR (300 MHz, CDCl ): δ = 8.22 (d, J = 7.9 Hz, 1H, Hꢀ4), 8.08ꢀ8.05 (m, 2H, 2Hortho),
3
4,5
3
3
7
7
1
.83 (s, 1H, Hꢀ8), 7.61 (“t”, J = 7.8 Hz, 2H, 2Hmeta), 7.53ꢀ7.47 (m, 1H, Hpara), 7.48 (d, J7,6
=
=
4
3
3
4
.6 Hz, 1H, Hꢀ7), 7.30 (“dt”, J6 = 1.2 Hz, J ≈ J ≈ 7.6 Hz, 1H, Hꢀ6), 6.98 (“dt”, J
5,7
,4
6,7
6,5
3
3
3
3
.2 Hz, J5,4 ≈ J5,6 ≈ 7.8 Hz, 1H, Hꢀ5), 5.94 (d, J1’,2’ = 1.4 Hz, Hꢀ1’), 5.59 (dd, J1’,2’ = 1.5

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2
1
3
3
3
Hz, J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.29ꢀ5.17 (m, J2’,3’ = 3.0 Hz, J3’,4’ = 10.1 Hz, 2H, Hꢀ3’, Hꢀ4’),
3
3
3
1
1
1
3
2
.80ꢀ3.71 (m, J5’,6’ = 6.1 Hz, J4’,5’ = 9.4 Hz, 1H, Hꢀ5’), 2.09, 1.97, 1.84 (3s, 9H, 3CH
3
CO),
3
13
.35 (d, J5’,6’ = 6.2 Hz, 3H, 3Hꢀ6’). CꢀNMR (75 MHz, CDCl
3
): δ = 191.1 (Cꢀ9), 169.9,
69.7, 169.6 (3CH CO), 166.9 (Cꢀ2), 143.2 (Cꢀ3), 137.4 (Cipso), 135.2 (Cꢀ7a), 133.9 (Cpara),
3
31.9 (Cꢀ6), 128.9 (2Cmeta), 128.8 (2Cortho), 127.2 (Cꢀ8), 127.1 (Cꢀ4), 122.9 (Cꢀ5), 120.2 (Cꢀ
a), 113.9 (Cꢀ7), 80.4 (Cꢀ1’), 74.0 (Cꢀ5’), 70.6 (Cꢀ3’), 70.4 (Cꢀ2’), 70.1 (Cꢀ4’), 20.8, 20.7,
+
0.5 (3CH CO), 17.6 (Cꢀ6’). MS (EI, 70 eV): m/z (%) = 521 (M , 12), 273 (24), 153 (59).
3
+
HRMS (ESI): calc. for C28
H27NNaO
9
([M+Na] ) 544.15780, found 544.15760.
(
E)ꢀ3ꢀ(2ꢀ(4ꢀMethoxyphenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀ
βꢀLꢀ
rhamnopyranosyl)indolinꢀ2ꢀone (9b)
According to the general procedure purified 8b (210 mg, 0.37 mmol) and 4ꢀ
dimethylamino)pyridine (50 mg, 0.41 mmol) were dissolved in about 2 ml of
dichloromethane. Then triethylamine (57 µl, 0.41 mmol) and methanesulfonyl chloride (32 µl,
.41 mmol) were added carefully to the reaction mixture. After work up 9b could be isolated
after column chromatography (heptane/ ethyl acetate v:v = 6:1 → 3:1) as orange solid (yield:
(
0
1
63 mg, 80 %). mp 87ꢀ90 °C (heptane/ ethyl acetate). R
f
0.45 (heptane/ ethyl acetate, v:v =
1:3).
1
3
3
HꢀNMR (300 MHz, CDCl
3
): δ = 8.14 (d, J4,5 = 7.9 Hz, 1H, Hꢀ4), 8.06 (d, Jo,m = 8.9 Hz, 2H,
3
3
2
Hortho), 7.83 (s, 1H, Hꢀ8), 6.97 (d, Jo,m = 8.9 Hz, 2H, 2Hmeta), 7.50 (d, J7,6 = 7.9 Hz, 1H, Hꢀ
4
3
3
4
3
7
), 7.30 (“dt”, J6,4 = 1.2 Hz, J6,7 ≈ J6,5 ≈ 7.7 Hz, 1H, Hꢀ6), 6.98 (“dt”, J5,7 = 1.2 Hz, J5,4
≈
3
3
3
3
J
5,6 ≈ 7.8 Hz, 1H, Hꢀ5), 5.94 (d, J1’,2’ = 1.4 Hz, 1H, Hꢀ1’), 5.59 (dd, J1’,2’ = 1.5 Hz, J2’,3’
=
3
3
3
3
3
1
7
.0 Hz, 1H, Hꢀ2’), 5.29ꢀ5.17 (m, J2’,3’ = 3.0 Hz, J
= 10.1 Hz, 2H, Hꢀ3’, Hꢀ4’), 3.88 (s,
3’,4’
3
3
H, OCH
3
), 3.80ꢀ3.71 (m, J5’,6’ = 6.1 Hz, J4’,5’ = 9.4 Hz, 1H, Hꢀ5’), 2.09, 1.97, 1.84 (3s, 9H,
3
13
CH CO), 1.35 (d, J
= 6.2 Hz, 3H, 3Hꢀ6’). CꢀNMR (75 MHz, CDCl ): δ = 189.6 (Cꢀ9),
3
5’,6’
3
69.9, 169.7, 169.6 (3CH
3
CO) 167.0 (Cꢀ2), 164.3 (Cpara), 142.9 (Cꢀ3), 134.3 (Cipso), 135.2 (Cꢀ
a), 131.5 (Cꢀ6), 131.3 (2Cmeta), 128.0 (Cꢀ8), 126.9 (Cꢀ4), 122.9 (Cꢀ5), 120.3 (Cꢀ3a), 114.1
(2Cortho), 113.8 (Cꢀ7), 80.4 (Cꢀ1’), 74.0 (Cꢀ5’), 70.6 (Cꢀ3’), 70.4 (Cꢀ2’), 70.1 (Cꢀ4’), 55.6
+
(OCH
3
), 20.8, 20.8, 20.5 (3CH
3
CO), 17.6 (Cꢀ6’). MS (EI, 70 eV): m/z (%) = 551 (M , 30),
+
+
2
73 ([Mꢀaglycone] , 61), 153 (100). HRMS (EI): calc. for C H NO (M ) 551.17860,
2
9
29
10
found 551.17748.

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2
(E)ꢀ3ꢀ(2ꢀ(4ꢀNitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀꢀLꢀ
rhamnopyranosyl)indolinꢀ2ꢀone (9c)
According to the general procedure 7a (200 mg, 0.48 mmol) and 4ꢀnitroacetophenone (159
mg, 0.96 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (97 mg, 0.96
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (97 mg, 0.96 mmol) and
methanesulfonyl chloride (110 mg, 0.96 mmol). After work up 9c could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 212 mg, 78
%). mp 160ꢀ161°C (heptane/ ethyl acetate).
1
3
H NMR (300 MHz, CDCl ): δ = 8.39 (d (br, overlapped), 1H, Hꢀ4), 8.36 (d, J = 8.9 Hz,
3
o,m
3
3
2
H, 2Hmeta), 8.23 (d, Jo,m = 8.9 Hz, 2H, 2Hortho), 7.80 (s, 1H, Hꢀ8), 7.53 (d (br), J7,6 = 8.1 Hz,
4
3
3
4
1
H, Hꢀ7), 7.36 (“dt”, J6,4 = 1.2 Hz, J6,7 ≈ J6,5 ≈ 8.0 Hz, 1H, Hꢀ6), 7.03 (“dt”, J5,7 = 1.0 Hz,
3
3
3
3
J5,4 ≈ J ≈ 7.7 Hz, 1H, Hꢀ5), 5.92 (d, J = 1.5 Hz, 1H, Hꢀ1’), 5.59 (dd, J2’,1’= 1.5 Hz,
5,6
1’,2’
3
J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.30ꢀ5.18 (m, 2H, Hꢀ3’, Hꢀ4’), 3.82ꢀ3.71 (m, 1H, Hꢀ5’), 2.09, 1.98,
3
13
1
1
1
.84 (3s, 9H, 3CH
89.1 (Cꢀ9), 170.0, 169.7, 169.7 (3CH
32.9 (CH), 129.7 (2CH), 127.7, 124.6 (2CH), 124.1 (2CH), 123.2 (CH), 120.0 (C), 114.1
CO),
7.7 (Cꢀ6’). MS (EI, 70eV): m/z (%) = 556 (M , 12), 294 (9), 273 (63), 153 (80), 111 (72).
3
CO), 1.36 (d, J5’,6’ = 6.2 Hz, 3H, 3Hꢀ6’). C NMR (63 MHz, CDCl
3
): δ =
3
CO), 166.7 (Cꢀ2), 150.6, 143.9, 142.1, 137.2 (4C),
(
CH), 80.6, 74.1, 70.5, 70.3, 70.1 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’), 20.8, 20.8, 20.5 (3CH
3
+
1
+
+
27 2 26 2
HRMS (ESI): calc. for C28H N O11 ([M+H] ) 567.16094 and for C28H N NaO11 ([M+Na] )
589.14288, found 567.16135 and 589.14317.
(
E)ꢀ3ꢀ(2ꢀ(4ꢀChlorophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀ
ꢀLꢀ
rhamnopyranosyl)indolinꢀ2ꢀone (9d)
According to the general procedure 7a (200 mg, 0.48 mmol) and 4ꢀchloroacetophenone (149
mg, 0.96 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (97 mg, 0.96
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (97 mg, 0.96 mmol) and
methanesulfonyl chloride (110 mg, 0.96 mmol). After work up 9d could be isolated after

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2
3
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 131 mg, 49
%). mp 160ꢀ162°C (heptane/ ethyl acetate).
1
3
3
H NMR (300 MHz, CDCl
3
): δ = 8.24 (d (br), J4,5 = 7.9 Hz, 1H, Hꢀ4), 8.02 (“d”, 2H, Jo,m
=
=
=
=
3
8
8
1
1
2
.6 Hz, 2Hortho), 7.78 (s, 1H, Hꢀ8), 7.51 (d (br, overlapped), 1H, Hꢀ7), 7.49 (“d”, 2H, Jo,m
4
3
3
4
.6 Hz, 2Hmeta), 7.32 (“dt”, J6,4 = 1.3 Hz, J6,7 ≈ J6,5 ≈ 7.9 Hz, 1H, Hꢀ6), 7.00 (“dt”, J5,7
3
3
3
3
.0 Hz, J ≈ J ≈ 7.8 Hz, 1H, Hꢀ5), 5.93 (d, J
= 1.5 Hz, 1H, Hꢀ1’), 5.59 (dd, J
2’,1’
4
,5
5,6
1’,2’
3
.5 Hz, J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.29ꢀ5.17 (m, 2H, Hꢀ3’, Hꢀ4’), 3.81ꢀ3.71 (m, 1H, Hꢀ5’),
3
13
3
.08, 1.96, 1.82 (3s, 9H, 3CH CO), 1.34 (d, J5’,6’ = 6.2 Hz, 3H, 3Hꢀ6’). C NMR (75 MHz,
CDCl ): δ = 189.6 (Cꢀ9), 169.8, 169.6, 169.6 (3CH CO), 166.7 (Cꢀ2) 143.3, 140.3, 135.7,
3
3
1
1
2
1
35.6 (4C), 132.0 (CH), 130.1 (2CH), 129.1 (2CH), 127.1 (CH), 126.1 (CH), 122.9 (CH),
20.0 (C), 113.8 (CH), 80.4, 73.9, 70.5, 70.2, 70.0 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’), 20.6, 20.6,
+
35
+ 37
0.4 (3CH CO), 17.5 (Cꢀ6’). MS (EI, 70 eV): m/z (%) = 555 (M [ Cl], 22), 557 (M [ Cl],
3
35
1), 312 (9), 283 (13), 273 (70), 153 (89), 111 (84). HRMS (ESI): calc. for C28H27 ClNO
9
+
35
+
(
[M+H] ) 556.13689 and for C28
9
H26 ClNaNO ([M+Na] ) 578.11883, found 556.13773 and
578.11996.
(
E)ꢀ3ꢀ(2ꢀ(4ꢀFluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4ꢀtriꢀOꢀacetylꢀ
ꢀLꢀ
rhamnopyranosyl)indolinꢀ2ꢀone (9e)
According to the general procedure 7a (200 mg, 0.48 mmol) and 4ꢀfluoroacetophenone (133
mg, 0.96 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (97 mg, 0.96
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (97 mg, 0.96 mmol) and
methanesulfonyl chloride (110 mg, 0.96 mmol). After work up 9e could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 168 mg, 65
%). mp 200ꢀ202°C (heptane/ ethyl acetate).
1
3
3
H NMR (300 MHz, CDCl
3
): δ = 8.21 (d (br), J4,5 = 7.8 Hz, 1H, Hꢀ4), 8.11 (“dd”, 2H, Jo,m =
4
3
8
.9 Hz, Jo,F = 5.3 Hz, 2Hortho), 7.79 (s, 1H, Hꢀ8), 7.51 (d (br), J6,7 = 8.1 Hz, 1H, Hꢀ7), 7.31
4
3
3
3
3
(
“dt”, J = 1.3 Hz, J ≈ J ≈ 7.9 Hz, 1H, Hꢀ6), 7.19 (“t”, 2H, J ≈ J ≈ 8.6 Hz,
6
,4
6,7
6,5
o,m
m,F
4
3
3
3
2H
meta), 6.99 (“dt”, J5,7 = 1.0 Hz, J5,4 ≈ J5,6 ≈ 7.7 Hz, 1H, Hꢀ5), 5.93 (d, J1’,2’ = 1.5 Hz, 1H,
3
3
Hꢀ1’), 5.60 (dd, J2’,1’ = 1.5 Hz, J2’,3’ = 3.0 Hz, 1H, Hꢀ2’), 5.29ꢀ5.18 (m, 2H, H3’, Hꢀ4’),
3
3
.81ꢀ3.72 (m, 1H, Hꢀ5’), 2.09, 1.98, 1.84 (3s, 9H 3CH CO), 1.36 (d, J5’,6’= 6.0 Hz, 3H, 3Hꢀ
3

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2
4
13
6
’). C NMR (63 MHz, CDCl
3
3
): δ = 189.4 (Cꢀ9), 169.9, 169.7, 169,7 (3CH CO), 166.9 (Cꢀ
1
4
2
), 165.5 (d, JC,F = 256.3 Hz, Cpara), 143.2, 135.4 (2C), 133.9 (d, JC,F = 2.7 Hz, Cipso), 132.0
3
2
(
CH), 131.6 (d, JC,F = 9.6 Hz, 2CHortho), 127.1, 126.6, 123.0 (3CH), 120.1 (C), 116.1 (d, JC,F
=
22.0 Hz, 2CHmeta), 113.9 (CH), 80.4, 74.0, 70.5, 70.3, 70.1 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’),
19
2
0.7, 20.7, 20.5 (3CH
3
3
CO), 17.6 (Cꢀ6’). F NMR (282 MHz, CDCl ): δ = 103.34 (s, CF). MS
+
(
EI, 70eV): m/z (%) = 539 (M , 22), 296 (10), 273 (66), 171 (24), 153 (90), 111 (75). HRMS
+
(EI): calc. for C28
H26FNO
9
(M ) 539.15861, found 539.15921.
(E)ꢀ3ꢀ(2ꢀ(4ꢀNitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀ
mannopyranosyl)indolinꢀ2ꢀone (9f)
According to the general procedure 7b (200 mg, 0.42 mmol) and 4ꢀnitroacetophenone (139
mg, 0.84 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (43 mg, 0.84
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (43 mg, 0.84 mmol) and
methanesulfonyl chloride (96 mg, 0.84 mmol). After work up 9f could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 184 mg, 70
%). mp 187ꢀ189 °C (heptane/ ethyl acetate).
1
3
3
H NMR (250 MHz, CDCl ): δ = 8.41ꢀ8.34 (m, 3H, 2Hmeta, Hꢀ4), 8.23 (“d”, Jo,m = 8.9 Hz,
3
4
2
H, 2Hortho), 7.81 (s, 1H, Hꢀ8), 7.50 (d (br), J6,7 = 8.1 Hz, 1H, Hꢀ7), 7.35 (“dt”, J4,6 = 1.2 Hz,
3
3
4
3
3
J
6,7 ≈ J5,6 ≈ 7.9 Hz, 1H, Hꢀ6), 7.04 (“dt”, J5,7 = 1.0 Hz, J4,5 ≈ J5,6 ≈ 7.7 Hz, 1H, Hꢀ5), 5.97
3
3
3
(
(
(
(
d, J1’,2’ = 1.5 Hz, 1H, Hꢀ1’), 5.61 (dd, J2’,1’ = 1.5 Hz, J2’,3’ = 3.3Hz, 1H, Hꢀ2’), 5.46ꢀ5.28
m, 2H, Hꢀ3’, Hꢀ4’), 4.35ꢀ4.19 (m, 2H, 2Hꢀ6’), 3.93ꢀ3.86 (m, 1H, Hꢀ5’), 2.11, 2.09, 1.99, 1.84
13
4s, 12H, 4CH
4CH CO), 166.7 (Cꢀ2), 150.5, 143.6, 142.0, 137.0 (4C), 132.8 (CH), 129.7 (2CH), 127.7,
24.7 (2CH), 124.1 (2CH), 123.2 (CH), 120.0 (C), 114.0 (CH), 80.6, 75.5, 70.5, 69.9, 65.2
3 3
CO). C NMR (63 MHz, CDCl ): δ = 189.0 (Cꢀ9), 170.4, 169.7, 169.6, 169.6
3
1
3
(Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’), 62.2 (Cꢀ6’), 20.7, 20.7, 20.6, 20.4 (4CH CO). MS (EI, 70eV):
+
m/z (%) = 624 (M , 20), 331 (66), 294 (13), 169 (94), 127 (19), 109 (73). HRMS (EI): calc.
+
for C H N O (M ) 624.15859, found 624.16003.
30
28
2
13
(
E)ꢀ3ꢀ(2ꢀ(4ꢀChlorophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
ꢀDꢀ
mannopyranosyl)indolinꢀ2ꢀone (9g)

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5
According to the general procedure 7b (200 mg, 0.42 mmol) and 4ꢀchloroacetophenone (130
mg, 0.84 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (43 mg, 0.84
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (43 mg, 0.84 mmol) and
methanesulfonyl chloride (96 mg, 0.84 mmol). After work up 9g could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 134 mg, 52
%). mp 80 ꢀ 82 °C (heptane/ ethyl acetate).
1
3
3
H NMR (300 MHz, CDCl
3
): δ = 8.25 (d (br), J4,5 = 7.9 Hz, 1H, Hꢀ4), 8.02 (“d”, Jo,m = 8.6
4
Hz, 2H, 2Hortho), 7.79 (s, 1H, Hꢀ8), 7.47ꢀ7.52 (m, 3H, 2Hmeta, Hꢀ7), 7.31 (“dt”, J = 1.3 Hz,
4,6
3
3
4
3
3
J
6,7 ≈ J5,6 ≈ 7.9 Hz, 1H, Hꢀ6), 7.01 (“dt”, J5,7 = 1.0 Hz, J4,5 ≈ J5,6 ≈ 7.8 Hz, 1H, Hꢀ5), 5.97
3
3
3
(
(
(
(
(
d, J1’,2’ = 1.5 Hz, 1H, Hꢀ1’), 5.61 (dd, J2’,1’ = 1.5 Hz, J2’,3’ = 3.3 Hz, 1H, Hꢀ2’), 5.46ꢀ5.29
m, 2H, Hꢀ3’, Hꢀ4’), 4.34ꢀ4.20 (m, 2H, 2Hꢀ6’), 3.92ꢀ3.86 (m, 1H, Hꢀ5’), 2.11, 2.09, 1.99, 1.84
13
4s, 12H, 4CH
4CH CO), 166.8 (Cꢀ2), 143.2, 140.4, 135.7, 135.5 (4C), 132.0 (CH), 130.1 (2CH), 129.2
2CH), 127.2, 126.3, 123.0 (3CH), 120.1 (C), 113.9 (CH), 80.5, 75.4, 70.5, 70.0, 65.3 (Cꢀ1’,
CO). MS (EI, 70eV): m/z
%) = 613 (M [ Cl], 20), 615 (M [ Cl], 11), 441 (12), 331 (35), 169 (100), 146 (13), 127
3 3
CO). C NMR (63 MHz, CDCl ): δ = 189.6 (Cꢀ9), 170.4, 169.6, 169.5, 169.5
3
Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’), 62.2 (Cꢀ6’), 20.7, 20.6, 20.6, 20.4 (4CH
3
+
35
+ 37
(
35
+
(12), 109 (36). HRMS (ESI): calc. for C30
H
29 ClNO11 ([M+H] ) 614.14236 and for
35
+
C
30
H28 ClNaNO11 ([M+Na] ) 636.12431, found 614.14306 and 636.12571.
(
E)ꢀ3ꢀ(2ꢀ(4ꢀFluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
βꢀDꢀ
mannopyranosyl)indolinꢀ2ꢀone (9h)
According to the general procedure 7b (200 mg, 0.42 mmol) and 4ꢀfluoroacetophenone (116
mg, 0.84 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (43 mg, 0.84
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (43 mg, 0.84 mmol) and
methanesulfonyl chloride (96 mg, 0.84 mmol). After work up 9h could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 158 mg, 63
%). mp 158 ꢀ 160 °C (heptane/ ethyl acetate).
1
3
3
H NMR (300 MHz, CDCl ): δ = 8.21 (d (br), J = 7.8 Hz, 1H, Hꢀ4), 8.11 (“dd”, 2H, J =
o,m
3
4,5
4
3
8
.9 Hz, Jo,F = 5.4 Hz, 2Hortho), 7.79 (s, 1H, Hꢀ8), 7.48 (d (br), J6,7 = 8.1 Hz, 1H, Hꢀ7), 7.30

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2
6
4
3
3
3
3
(
“dt”, J4,6 = 1.3 Hz, J6,7 ≈ J5,6 ≈ 7.9 Hz, 1H, Hꢀ6), 7.19 (“t”, 2H, Jo,m ≈ Jm,F ≈ 8.6 Hz,
4
3
3
3
2
Hmeta), 7.00 (“dt”, J5,7 = 1.0 Hz, J4,5 ≈ J5,6 ≈ 7.7 Hz, 1H, Hꢀ5), 5.97 (d, J1’,2’ = 1.5 Hz, 1H,
3
3
Hꢀ1’), 5.61 (dd, J2’,1’ = 1.5 Hz, J2’,3’ = 3.3 Hz, 1H, Hꢀ2’), 5.29ꢀ5.45 (m, 2H, Hꢀ3’, Hꢀ4’),
.34ꢀ4.20 (m, 2H, 2Hꢀ6’), 3.93ꢀ3.86 (m, 1H, Hꢀ5’), 2.11, 2.09, 1.98, 1.84 (4s, 12H, 4CH CO).
CO), 166.9
4
3
1
3
C NMR (75 MHz, CDCl
3
): δ = 189.4 (Cꢀ9), 170.5, 169.7, 169.6, 169.6 (4CH
3
1
4
(
Cꢀ2), 166.3 (d, J = 256.8 Hz, Cpara), 143.1, 135.3 (2C), 133.8 (d, J = 2.8 Hz, Cipso),
C,F
C,F
3
1
32.0 (CH), 131.6 (d, JC,F = 9.5 Hz, 2CHortho), 127.2, 126.8, 123.1 (3CH), 120.1 (C), 116.1
2
(
d, JC,F = 22.1 Hz, 2CHmeta), 113.9 (CH), 80.5, 75.5, 70.6, 70.0, 65.3 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’,
+
Cꢀ5’), 62.2 (Cꢀ6’), 20.8, 20.7, 20.7, 20.5 (4CH CO). MS (EI, 70eV): m/z (%) = 597 (M , 48),
3
331 (74), 296 (12), 267 (22), 239 (14), 211 (16), 169 (100), 123 (63), 109 (81). HRMS (EI):
+
calc. for C30
H
28FNO11 [M] 597.16409, found 597.16456.
(
E)ꢀ3ꢀ(2ꢀOxoꢀ2ꢀ(4ꢀtrifluoromethylphenyl)ethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
ꢀDꢀ
mannopyranosyl)indolinꢀ2ꢀone (9i)
According to the general procedure 7b (200 mg, 0.42 mmol) and 4ꢀ
trifluoromethylacetophenone (158 mg, 0.84 mmol) were dissolved in about 4 ml of ethanol.
Then triethylamine (43 mg, 0.84 mmol) was added to start the reaction. After work up, the
crude product was dissolved in about 4 ml of abs. dichloromethane and reacted with
triethylamine (43 mg, 0.84 mmol) and methanesulfonyl chloride (96 mg, 0.84 mmol). After
work up 9i could be isolated after column chromatography (heptane/ ethyl acetate v:v = 3:1)
as orange solid (yield: 188 mg, 69 %). mp 98 ꢀ 100 °C (heptane/ ethyl acetate).
1
3
3
H NMR (300 MHz, CDCl
3
): δ = 8.34 (d (br), J4,5 = 7.8 Hz, 1H, Hꢀ4), 8.18 (“d” (br), 2H,
3
J
o,m = 8.2 Hz, 2Hortho), 7.82 (s, 1H, Hꢀ8), 7.79 (“d” (br), 2H, Jo,m = 8.2 Hz, 2Hmeta), 7.49 (d
3
4
3
3
(
br), J6,7 = 8.1 Hz, 1H, Hꢀ7), 7.33 (“dt”, J4,6 = 1.3 Hz, J6,7 ≈ J5,6 ≈ 7.9 Hz, 1H, Hꢀ6), 7.03
4
3
3
3
(
“dt”, J = 1.0 Hz, J ≈ J ≈ 7.8 Hz, 1H, Hꢀ5), 5.97 (d, J
= 1.5 Hz, 1H, Hꢀ1’), 5.62
1’,2’
5
,7
4,5
5,6
3
3
(
dd, J2’,1’ = 1.5 Hz, J2’,3’ = 3.3 Hz, 1H, Hꢀ2’), 5.29ꢀ5.46 (m, 2H, Hꢀ3’, Hꢀ4’), 4.20ꢀ4.35 (m,
1
3
2
3
H, 2Hꢀ6’), 3.87ꢀ3.93 (m, 1H, Hꢀ5’), 2.11, 2.09, 1.99, 1.84 (4s, 12H, 4CH CO). C NMR (75
MHz, CDCl ): δ = 189.7 (Cꢀ9), 170.4, 169.6, 169.6, 169.5 (4CH CO), 166.7 (Cꢀ2), 143.4 (C),
3
3
5
2
1
40.1 (q, JC,F = 1.0 Hz, Cipso), 136.3 (C), 134.8 (q, JC,F = 32.7 Hz, Cpara), 132.4 (CH), 129.0
3
1
(
2CHortho), 127.4 (CH), 125.8 (q, JC,F = 3.6 Hz, 2CHmeta), 125.5 (CH), 123.5 (q, JC,F = 273.0
Hz, CF ), 123.1 (CH), 120.0 (C), 113.9 (CH), 80.5, 75.4, 70.5, 70.0, 65.2 (Cꢀ1’, Cꢀ2’, Cꢀ3’,
3
19
3 3
Cꢀ4’, Cꢀ5’), 62.1 (Cꢀ6’), 20.7, 20.6, 20.6, 20.4 (4CH CO). F NMR (282 MHz, CDCl ): δ =

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2
7
+
113.86 (s, CF
3
). MS (EI, 70eV): m/z (%) = 647 (M , 42), 331 (80), 317 (28), 289 (18), 173
+
(57), 169 (100), 145 (50), 127 (39), 109 (82). HRMS (EI): calc. for C31
H
28
F
3
NO11 (M )
647.16090, found 647.16114.
(
E)ꢀ3ꢀ(2ꢀ(4ꢀNitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
ꢀDꢀ
glucopyranosyl)indolinꢀ2ꢀone (9j)
According to the general procedure 7c (200 mg, 0.42 mmol) and 4ꢀnitroacetophenone (139
mg, 0.84 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (43 mg, 0.84
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (43 mg, 0.84 mmol) and
methanesulfonyl chloride (96 mg, 0.84 mmol). After work up 9j could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 186 mg, 71
%). mp 205 ꢀ 207 °C (heptane/ ethyl acetate).
1
3
H NMR (300 MHz, acetoneꢀd6): δ = 8.37ꢀ8.47 (m, 4H, 2Hmeta, 2Hortho), 8.36 (d (br), J4,5
=
=
3
4
7
.8 Hz, 1H, Hꢀ4), 7.88 (s, 1H, Hꢀ8), 7.60 (d (br), J7,6 = 8.0 Hz, 1H, Hꢀ7), 7.51 (“dt”, J4,6
3
3
4
3
3
1
.2 Hz, J6,7 ≈ J5,6 ≈ 7.8 Hz, 1H, Hꢀ6), 7.12 (“dt”, J5,7 = 1.1 Hz, J4,5 ≈ J5,6 ≈ 7.7 Hz, 1H, Hꢀ
3
3
3
5
), 5.97 (d, J1’,2’ = 9.4 Hz, 1H, Hꢀ1’), 5.72 (“t”, J2’,1’ ≈ J2’,3’ ≈ 9.4 Hz, 1H, Hꢀ2’), 5.55 (“t”,
3
3
3
3
J
4’,3’ ≈ J4’,5’ ≈ 9.5 Hz, 1H, Hꢀ4’), 5.36 (“t”, J3’,2’ ≈ J3’,4’ ≈ 9.5 Hz, 1H, Hꢀ3’), 4.21ꢀ4.35 (m,
13
3
H, H5’, 2Hꢀ6’), 2.05, 2.04, 1.96, 1.83 (4s, 12H, 4CH
δ = 190.6 (Cꢀ9), 170.7, 170.2, 170.1, 169.6 (4CH CO), 167.7 (Cꢀ2), 151.6, 144.0, 143.0,
37.2 (4C), 134.3 (CH), 130.9 (2CH), 128.2, 126.4 (2CH), 124.9 (2CH), 123.9 (CH), 120.8
3
CO). C NMR (63 MHz, acetoneꢀd6):
3
1
(C), 113.5 (CH), 80.4, 75.2, 73.9, 68.9, 68.6 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’), 62.7 (Cꢀ6’), 20.6,
+
20.6, 20.5, 20.2 (4CH
3
CO). MS (EI, 70eV): m/z (%) = 624 (M , 10), 331 (59), 294 (9), 169
+
(100), 150 (20), 127 (19), 109 (73). HRMS (EI): calc. for C30
H
28
N
2
O
13 (M ) 624.15859,
found 624.15896.
(
E)ꢀ3ꢀ(2ꢀOxoꢀ2ꢀ(4ꢀtrifluoromethylphenyl)ethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
ꢀDꢀ
glucopyranosyl)indolinꢀ2ꢀone (9k)
According to the general procedure 7c (200 mg, 0.42 mmol) and 4ꢀ
trifluoromethylacetophenone (158 mg, 0.84 mmol) were dissolved in about 4 ml of ethanol.

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8
Then triethylamine (43 mg, 0.84 mmol) was added to start the reaction. After work up, the
crude product was dissolved in about 4 ml of abs. dichloromethane and reacted with
triethylamine (43 mg, 0.84 mmol) and methanesulfonyl chloride (96 mg, 0.84 mmol). After
work up 9k could be isolated after column chromatography (heptane/ ethyl acetate v:v = 3:1)
as orange solid (yield: 182 mg, 67 %). mp 160 ꢀ 162 °C (heptane/ ethyl acetate).
1
3
H NMR (300 MHz, Acetonꢀd6): δ = 8.28ꢀ8.32 (m, 3H, 2Hortho, Hꢀ4), 7.93 (“d” (br), 2H, Jo,m
3
4
=
8.2 Hz, 2Hmeta), 7.84 (s, 1H, Hꢀ8), 7.56 (d (br), J7,6 = 8.0 Hz, 1H, Hꢀ7), 7.46 (“dt”, J4,6 =
3
3
4
3
3
1
.2 Hz, J6,7 ≈ J5,6 ≈ 7.8 Hz, 1H, Hꢀ6), 7.08 (“dt”, J5,7 = 1.1 Hz, J4,5 ≈ J5,6 ≈ 7.7 Hz, 1H, Hꢀ
3
3
3
5
), 5.95 (d, J
= 9.4 Hz, 1H, Hꢀ1’), 5.70 (“t”, J
≈ J
≈ 9.4 Hz, 1H, Hꢀ2’), 5.53 (“t”,
2’,3’
1
’,2’
2’,1’
3
3
3
3
J
4’,3’ ≈ J4’,5’ ≈ 9.5 Hz, 1H, Hꢀ4’), 5.33 (“t”, J3’,2’ ≈ J3’,4’ ≈ 9.5 Hz, 1H, Hꢀ3’), 4.18ꢀ4.33 (m,
13
3H, H5’, 2Hꢀ6’), 2.02, 2.01, 1.93, 1.80 (4s, 12H, 4CH
3
CO). C NMR (63 MHz, Acetonꢀd6):
5
δ = 190.9 (Cꢀ9), 170.7, 170.2, 170.1, 169.6 (4CH CO), 167.8 (Cꢀ2), 143.9 (C), 141.5 (q, J
3
C,F
2
=
1.1 Hz, Cipso), 136.9 (C), 134.9 (q, JC,F = 32.2 Hz, Cpara), 134.1 (CH), 130.3 (2CHortho),
3
1
1
1
6
28.1 (CH), 126.9 (q, JC,F = 3.7 Hz, 2CHmeta), 126.7 (CH), 124.9 (q, JC,F = 272.5 Hz, CF
3
),
23.8 (CH), 120.8 (C), 113.5 (CH), 80.4, 75.1, 73.9, 68.9, 68.6 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’),
19
2.7 (Cꢀ6’), 20.6, 20.6, 20.5, 20.2 (4CH
3
CO). F NMR (282 MHz, acetoneꢀd6): δ = 113.86
+
(
s, CF
3
). MS (EI, 70eV): m/z (%) = 647 (M , 14), 331 (55), 317 (13), 289 (8), 169 (100), 145
+
(
23), 127 (21), 109 (81). HRMS (EI): calc. for C31
47.16031.
28 3
H F NO11 (M ) 647.16090, found
6
(
E)ꢀ3ꢀ(2ꢀ(4ꢀFluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
ꢀDꢀ
galactopyranosyl)indolinꢀ2ꢀone (9l)
According to the general procedure 7d (200 mg, 0.42 mmol) and 4ꢀfluoroacetophenone (116
mg, 0.84 mmol) were dissolved in about 4 ml of ethanol. Then triethylamine (43 mg, 0.84
mmol) was added to start the reaction. After work up, the crude product was dissolved in
about 4 ml of abs. dichloromethane and reacted with triethylamine (43 mg, 0.84 mmol) and
methanesulfonyl chloride (96 mg, 0.84 mmol). After work up 9l could be isolated after
column chromatography (heptane/ ethyl acetate v:v = 3:1) as orange solid (yield: 161 mg, 64
%).
1
H NMR (300 MHz, acetoneꢀd6): δ = 8.19ꢀ8.25 (m, 3H, 2Hortho, Hꢀ4), 7.83 (s, 1H, Hꢀ8), 7.47ꢀ
3
3
4
7
.58 (m, 2H, Hꢀ6, Hꢀ7), 7.35 (“t”, 2H, Jo,m ≈ J ≈ 8.8 Hz, 2Hmeta), 7.10 (“dt”, J = 1.4
m,F 5,7

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9
3
3
3
3
Hz, J4,5 ≈ J5,6 ≈ 7.6 Hz, 1H, Hꢀ5), 5.97 (d, J1’,2’ = 9.3 Hz, 1H, Hꢀ1’), 5.82 (“t” (br), J2’,1’
≈
3
3
3
3
J
2’,3’ ≈ 9.6 Hz, 1H, Hꢀ2’), 5.60 (dd, J4’,3’ = 3.2 Hz, J4’,5’ = 0.8 Hz, 1H, Hꢀ4’), 5.46 (dd, J3’,2’
3
3
3
3
=
10.0 Hz, J3’,4’ = 3.2 Hz, 1H, Hꢀ3’), 4.58 (ddd, J5’,4’ = 0.8 Hz, J5’,6a’ = 5.6 Hz, J5’,6b’ = 6.8
3
2
3
Hz, 1H, Hꢀ5’), 4.29 (dd, J6a’,5’ = 5.6 Hz, J6a’,6b’ = 11.5 Hz, 1H, Hꢀ6a’), 4.14 (dd, J6b’,5’ = 6.8
2
13
Hz, J6b’,6a’ = 11.5 Hz, 1H, Hꢀ6b’), 2.31, 1.98, 1.95, 1.87 (4s, 12H, 4CH
3
CO). C NMR (75
MHz, acetoneꢀd6): δ = 190.3 (Cꢀ9), 170.6, 170.5, 170.2, 169.8 (4CH CO), 167.7 (Cꢀ2), 167.0
3
1
4
(
d, JC,F = 254.2 Hz, Cpara), 143.7, 136.1 (2C), 135.1 (d, JC,F = 2.8 Hz, Cipso), 133.6, (CH),
3
2
1
32.6 (d, JC,F = 9.9 Hz, 2CHortho), 127.9, 127.6, 123.7 (3CH), 120.9 (C), 116.9 (d, JC,F = 22.2
Hz, 2CHmeta), 113.1 (CH), 80.5, 73.8, 72.0, 68.5, 66.4 (Cꢀ1’, Cꢀ2’, Cꢀ3’, Cꢀ4’, Cꢀ5’), 62.6 (Cꢀ
1
9
6’), 20.7, 20.6, 20.5, 20.3 (4CH
3
CO). F NMR (282 MHz, acetoneꢀd6): δ = 71.74 (s, CF).
+
MS (EI, 70eV): m/z (%) = 597 (M , 30), 331 (94), 317 (13), 289 (12), 211 (11), 169 (98), 146
+
(
17), 127 (26), 123 (60), 109 (68). HRMS (ESI): calc. for C H FNO ([M+H] ) 598.17192,
30
29
11
found 598.17140.
(
E)ꢀ3ꢀ(2ꢀOxoꢀ2ꢀ(4ꢀtrifluoromethylphenyl)ethylidene)ꢀ1ꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀ
ꢀDꢀ
galactopyranosyl)indolinꢀ2ꢀone (9m)
According to the general procedure 7d (200 mg, 0.42 mmol) and 4ꢀ
trifluoromethylacetophenone (158 mg, 0.84 mmol) were dissolved in about 4 ml of ethanol.
Then triethylamine (43 mg, 0.84 mmol) was added to start the reaction. After work up, the
crude product was dissolved in about 4 ml of abs. dichloromethane and reacted with
triethylamine (43 mg, 0.84 mmol) and methanesulfonyl chloride (96 mg, 0.84 mmol). After
work up 9m could be isolated after column chromatography (heptane/ ethyl acetate v:v = 3:1)
as orange solid (yield: 180 mg, 66 %). mp 91 ꢀ 93 °C (heptane/ ethyl acetate).
1
3
H NMR (300 MHz, acetoneꢀd6): δ = 8.33ꢀ8.37 (m, 3H, 2Hortho, Hꢀ4), 7.97 (d (br), Jo,m = 8.2
Hz, 2H, 2Hmeta), 7.88 (s, 1H, Hꢀ8), 7.51ꢀ7.60 (m, 2H, Hꢀ6, Hꢀ7), 7.11ꢀ7.17 (m, 1H, Hꢀ5), 5.97
3
3
3
3
(
d, J1’,2’ = 9.2 Hz, 1H, Hꢀ1’), 5.82 (“t” (br), J2’,1’ ≈ J2’,3’ ≈ 9.6 Hz, 1H, Hꢀ2’), 5.60 (dd, J4’,3’
3
3
3
=
3.3 Hz, J4’,5’ = 0.9 Hz, 1H, Hꢀ4’), 5.46 (dd, J3’,2’ = 10.0 Hz, J3’,4’ = 3.3 Hz, 1H, Hꢀ3’),
3
3
3
3
4
.59 (ddd, J
= 0.9 Hz, J
= 5.7 Hz, J
= 6.8 Hz, 1H, Hꢀ5’), 4.29 (dd, J
= 5.7
6a’,5’
5
’,4’
5’,6a’
5’,6b’
2
3
2
Hz, J6a’,6b’ = 11.5 Hz, 1H, Hꢀ6a’), 4.15 (dd, J6b’,5’ = 6.8 Hz, J6b’,6a’ = 11.5 Hz, 1H, Hꢀ6b’),
1
3
2
1
3
.32, 1.99, 1.96, 1.87 (4s, 12H, 4CH
3
CO). C NMR (63 MHz, acetoneꢀd6): δ = 190.9 (Cꢀ9),
2
70.6, 170.5, 170.2, 169.8 (4CH CO), 167.7 (Cꢀ2), 144.0, 141.5, 137.0 (3C), 134.9 (q, J
=
C,F
3
3
2.3 Hz, Cpara), 134.1 (CH), 130.3 (2CHortho), 128.2 (CH), 126.9 (q, JC,F = 3.8 Hz, 2CHmeta),