Synthesis of indirubin-N′-glycosides and their anti-proliferative activity against human cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2008.04.003

The first indirubin-N′-glycosides were prepared based on reactions of isatin-N′-glycosides with indoxyls. The products show a significant anti-proliferative activity against various human cancer cell lines. Good results were observed for an indirubin-N′-mannoside which was shown to have medium to high anti-proliferative activity against all investigated cell lines. The highest activities and selectivities against the MCF-7 breast cancer cell line were observed for indirubin-N′-rhamnosides.

Full text of DOI:10.1016/j.bmc.2008.04.003

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5570–5583
Synthesis of indirubin-N0-glycosides and their
anti-proliferative activity against human cancer cell lines
Stefanie Libnow,a Karen Methling,b Martin Hein,a Dirk Michalik,c Manuela Harms,b
Kristian Wende,b Anke Flemming,a Martin Ko¨ckerling,a Helmut Reinke,a
Patrick J. Bednarski,b Michael Lalkb and Peter Langera,c,
*
aInstitut fu¨r Chemie der Universita¨t Rostock, Albert-Einstein-Straße 3a, D-18059 Rostock, Germany
bInstitut fu¨r Pharmazie, Ernst-Moritz-Arndt-Universita¨t Greifswald, Friedrich-Ludwig-Jahn-Straße 17, D-17487 Greifswald, Germany
cLeibniz Institut fu¨r Katalyse e.V. an der Universita¨t Rostock, Albert-Einstein-Straße 29a, D-18059 Rostock, Germany
Received 2 January 2008; revised 27 March 2008; accepted 1 April 2008
Available online 6 April 2008
Abstract—The first indirubin-N0-glycosides were prepared based on reactions of isatin-N0-glycosides with indoxyls. The products
show a significant anti-proliferative activity against various human cancer cell lines. Good results were observed for an indiru-
bin-N0-mannoside which was shown to have medium to high anti-proliferative activity against all investigated cell lines. The highest
activities and selectivities against the MCF-7 breast cancer cell line were observed for indirubin-N0-rhamnosides.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
can selectively stop centrosome duplication in tumor cells
without affecting normal cells.4 The anti-proliferative
activity of substituted indirubin derivatives against vari-
ous cancer cell lines and CDK2 inhibitory activityhave re-
cently been studied.11 Some years ago, Laatsch et al.
reported the isolation of N-glycosides of indigo.12 In con-
trast to pharmacologically inactive indigo, the indigo-N-
glycosides (akashines) show a significant antiproliferative
activity against various human cancer cell lines. Owing to
the strong activity of the akashines, we set up a program
directed towards the synthesis of N-glycosides of indigo
(blue sugars)13 and of indirubin (red sugars).14 Herein,
we report full details of the synthesis of indirubin-N0-gly-
cosides. With respect to our preliminary communication
in this field,14 we studied the scope of our synthetic strat-
egy and disclose, for the first time, the anti-proliferative
activity of various indirubin-N0-glycosides on human
cancer cells in vitro, which is considerably higher than
the activity of the corresponding non-glycosylated
indirubins.
In recent years, there has been a dramatic renewal of the
interest in indirubin—the red isomer of the well-known
dye indigo—due to the discovery of its great pharmaco-
logical potential.1 The pharmacological activity of indiru-
binhas been known for a long time. In fact, it is the active
ingredient of the traditional Chinese medicinal recipe
Danggui Longhui Wan for the treatment of myelocytic leu-
kemia.2 Recently, Meijer and coworkers showed that
indirubin derivatives selectively inhibit cyclin-dependent
kinases (CDKs), which represent important components
of cell cycles taking place in many human tumors, and
thus have a great potential for the treatment of cancer.3,4
The anti-proliferative effect of indirubin on human cancer
cells is based on the inhibition of genes or proteins which
regulate cell cycle progression.5 The inhibition ofGSK-3b
and CDK5 by indirubin derivatives, important for treat-
ment of Alzheimer’s disease, has also been studied.6,7
Indirubin-30-monoxime was shown to influence p27Kip1
transcription and to inhibit C-Jun NH2-terminal kinase,
which plays an important role in neuronal apoptosis.8–10
In addition, it was reported that indirubin-30-monoxime
2. Results and discussion
2.1. Chemistry
Keywords: Anti-proliferative activity; Carbohydrates; N-heterocycles;
Indirubin.
*
Indirubins are available by reaction of a methanol solu-
tion of indoxyl acetate with isatines under oxygen-free
Corresponding author. Tel.: +49 381 498 6410; fax: +49 381 498
6411; e-mail: peter.langer@uni-rostock.de
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.003

S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
5571
atmosphere. This transformation could be successfully
employed for the synthesis of indirubin-N-glycosides.
The starting materials, N-glycosyl isatines 4a–e, were
prepared from rhamnose, glucose, galactose and man-
nose in three steps (Scheme 1). The reaction of the free
sugars with different anilines and subsequent acetylation
afforded the N-glycosyl anilines 3a–e.15 The AlCl3-med-
iated cyclization16 of 3a–e with oxalyl chloride afforded
the N-glycosyl isatines 4a–e. The isatin-N-rhamnosides
were formed as separable mixtures of anomers a-4a
and b-4a. The major isomer, b-4a, could be readily
isolated in anomerically pure form in good yield. The
a-anomer could be isolated by chromatographic separa-
tion of the combined anomerically enriched fractions of
several reaction batches. Isatine-N-rhamnoside 4b was
prepared in good yield as a mixture of anomers (b/a
> 3.5:1). Recrystallization gave the pure b-anomer
b-4b. The reaction of the latter with 5a afforded the
indirubin-N-rhamnoside b-6b. The cyclization of oxalyl
chloride with N-glycosyl anilines 3c–e, employed as
isomerically enriched b-anomers (b/a > 5:1), gave isa-
tine-N-glucoside b-4c, isatine-N-galactoside b-4d, and
isatine-N-mannoside b-4e. All products were isolated
as the pure b-anomers. Only traces of a-anomers were
formed in these reactions. The configurations of all isa-
tine-N-glycosides were established by spectroscopic
methods. The molecular structures of rhamnosyl deriva-
tives b-4a and b-4b were independently confirmed by X-
ray structure analysis (Figs. 3 and 4).
The Na2CO3-mediated reaction of indoxyl acetate (5a)
with isatine-N-glycoside b-4a (MeOH, 20 ꢁC, 4 h) affor-
ded the deprotected N0-(b-L-rhamnopyranosyl)indirubin
b-6a in 77% yield (Scheme 1, Table 1). During the opti-
mization of this reaction, the use of an excess of sodium
carbonate proved to be important to achieve a complete
cleavage of the acetyl protective groups of the sugar
moiety. For the sake of a convenient structural elucida-
tion, b-6a was transformed, by treatment with acetic
anhydride/pyridine, into its tetra-O-acetate b-7a. The
reaction of 5a with b-4b gave the deprotected N0-(b-L-
rhamnopyranosyl)indirubin b-6b. The condensation of
5a with b-4c, b-4d, and b-4e gave rise to the formation
of N0-(b-D-gluco-pyranosyl)indirubin (b-6c), N0-(b-D-
galactopyranosyl)-indirubin (b-6d), and N0-(b-D-manno-
pyranosyl)indirubin (b-6e) which were transformed into
their acetylated derivatives b-7c, b-7d, and b-7e,
respectively.
The Na2CO3-mediated reaction of isatine-N-rhamnoside
b-4a with chlorinated indoxyl acetate 5b17 and subse-
quent acetylation of the crude product afforded the tet-
ra-O-acetylated indirubin-N0-rhamnoside b-7f in 63%
yield (Scheme 2). The acetylation was necessary, as a
mixture of partially deprotected products was formed
during the condensation. Stirring of a methanol solution
of b-7f in the presence of a catalytic amount of KOtBu
(0.06 equiv) gave the deprotected rhamnoside b-6f in
72% yield.
NH2
1a
NH
OAc
OH
OH
OH
i, ii
+
O
O
H3C
H3C
AcO
OAc
HO
3a
2a
Cl
O
O
iii
OAc
Cl
The reaction of isatine-N-rhamnoside a-4a with indoxyl
acetates 5a and 5b and subsequent acetylation gave the
tetra-O-acetylated indirubin-N0-rhamnosides a-7a and
a-7f, respectively (Scheme 3). The KOtBu-catalyzed
deacetylation of the latter afforded the deprotected
rhamnosides a-6a and a-6f. The rhamnosylated indiru-
bin-3-oxime b-9a was prepared by reaction of O-acety-
lated indirubin-N0-glycoside b-7a with hydroxylamine
hydrochloride (to give b-8a) and subsequent deprotec-
tion (Scheme 4). The glycosylated oxime b-9a was pre-
pared in order to compare its anti-proliferative activity
with that of the aglycon (vide infra). Crystals of the
O-acetylated indirubin-3-oxime derivative b-8a were
suitable for X-ray structural analysis (Fig. 5).
N
H
O
5a
iv
N
O
O
H3C
OAc
O
AcO
OAc
β-4a (23%, β/α > 98:2)
α,β-4a (40%, β/α = 3:1)
N
H
N
O
O
v
5a
H3C
OH
O
HO
OH
The configuration and conformation of the products
was studied in detail by NMR spectroscopy. The assign-
ment of the signals was established by DEPT and
β-6a (77%, from β-4a)
N
H
N
O
two-dimensional H,1H COSY and H,13C correlation
spectra (HETCOR, HSQC). In addition, 1H,1H NOESY
and 1H,13C HMBC spectra were recorded for com-
pounds b-4e, b-6c, b-7a, and b-7c. For example, in the
HMBC spectrum of b-6c the following correlations are
found: NH with C-2, C-3, C-3a, and C-7a; H-40 with
C-30, C-60, and C-7a0; H-60 with C-7a0; H-4 with C-6
and C-7a; H-6 with C-7a. In the NOESY spectrum of
b-6c, NOE cross peaks are observed for protons H-5
1
1
O
H3C
OAc
AcO
OAc
β-7a (70%, from β-4a)
Scheme 1. Synthesis of N-glycosyl indirubines. Reagents and condi-
tions: (i) EtOH, rt, 12 h; (ii) Ac2O, pyridine, 0–4 ꢁC, 12 h; (iii) AlCl3,
55 ꢁC, 1.5h; (iv) Na2CO3, MeOH, 20 ꢁC, 4 h; (v) 1—Na 2CO3, MeOH,
20 ꢁC, 4 h; 2—Ac2O/pyridine = 1:1, 0 ꢁC, 12 h.

5572
S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
Table 1. Synthesis of N0-glycosyl indirubines from N-glycosyl anilines
Series
% (4)a
% (6)a
% (7)a
O
O
O
N
H
N
H
N
O
L-Rhamno
N
N
O
O
O
O
O
H3C
OAc
H3C
OH
H3C
OAc
AcO
OAc
HO
OH
AcO
OAc
b-4a (23%, b/a > 98:2)
a,b-4a (40%, b/a = 3:1)
b-6a (77%, b/a > 98:2)
b-7a (70% based on b-4a, b/a > 98:2)
O
CH3
CH3
H3C
H3C
O
N
H
N
L-Rhamno
N
O
O
O
O
H3C
OAc
H3C
OH
AcO
OAc
HO
OH
a,b-4b (76%, b/a = 3.5:1)
b-6b (62%, b/a > 98:2)
O
O
O
N
H
N
H
N
O
O
D-Gluco
N
N
O
O
O
O
OAc
OH
OAc
AcO
HO
AcO
AcO
OAc
HO
OH
AcO
OAc
b-4c (30%, b/a > 98:2)16
b-6c (70%, b/a > 98:2)
b-7c (75% based on b-6c, b/a > 98:2)
O
O
O
N
H
N
H
N
O
O
D-Galacto
N
N
O
O
O
O
OAc
OH
OAc
AcO
HO
AcO
AcO
OAc
HO
OH
AcO
OAc
b-4d (52%, b/a > 98:2)
b-6d (73%, b/a > 98:2)
b-7d (70% based on b-6d, b/a > 98:2)
O
O
O
N
H
N
H
N
O
O
D-Manno
N
N
O
O
O
O
OAc
OH
OAc
AcO
HO
AcO
AcO
OAc
HO
OH
AcO
OAc
b-4e (21%, b/a > 98:2)
b-6e (59%, b/a > 98:2)
b-7e (60% based on b-6e, b/a > 98:2)
a Yields of isolated products.
with H-4 and H-6; H-50 with H-40 and H-60; H-70 with
H-100 and H-400; H-500 with H-100, H-300 and H-600. These
100 and H-200 indicating a bis-axial position of these pro-
tons. Based on the coupling constants 3J1 ;2 (3J1 ;2 for b-
4d)ꢀ9 Hz the D-gluco and D-galacto pyranosides can be
00 00
0
0
1
findings confirm the assignments given for the H and
13C NMR signals of the indirubin and the sugar moiety.
Furthermore, the stereochemistry of the pyranosyl ring
was proven by the NOE’s found in the NOESY spec-
trum for the protons H-100, H-300, and H-500 (Fig. 1).
considered as b-anomers with a C1 conformation. In
4
case of the D- manno derivatives b-4e, b-6e, and b-7e,
exhibiting smaller coupling constants (3J < 1.5 Hz), the
anomeric configuration could not be assigned based on
the analysis of the coupling constants. However, the b-
configuration could be unambigiously assigned based
on NOESY measurements carried out for b-4e. The cor-
The b-configuration at carbon atom C-100 is also con-
3
00 00
firmed by the coupling constant J1 ;2 ¼ 9:5 Hz for H-

S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
Cl
5573
HON
O
AcO
Cl
O
O
N
H
N
H
N
N
N
H
N
N
O
Ac
O
O
O
i
5b
O
O
N
H3C
OAc
O
H3C
OAc
H3C
OAc
O
i, ii
H3C
OAc
AcO
OAc
β-4a
AcO
OAc
AcO
OAc
β-7a
AcO
OAc β-7f (63%)
β-8a (57%)
Cl
HON
O
O
ii
N
H
N
H
N
iii
O
N
O
O
H3C
OH
H3C
OH
β-6f (72%)
HO
OH
β-9a (69%)
HO
OH
Scheme 4. Synthesis of the glycosylated indirubin-3-monoxim b-9a,
Reagents and conditions: (i) H2NOHÆHCl, pyridine, 90 ꢁC, 7 h, (ii):
KOtBu, MeOH, 20 ꢁC, 12 h.
Scheme 2. Synthesis of the indirubin-N0-rhamnoside b-6f. Reagents
and conditions: (i) Na2CO3, MeOH, 20 ꢁC, 2 h; (ii) Ac2O, pyridine,
0 ꢁC, 12 h; (iii) KOtBu (cat.), MeOH, 20 ꢁC, 12 h.
5
7
4
R1
O
3a
AcO
5'
OH
6
4'
3a'
3'
R1
3
6''
O
H
6'
2
5''
O
7a
HO 4''
HO
N
O
N
1''
H1
N
3''
7'
2'
7a'
3''
H
2'' OH
N
R2
N1'
5a,b
H
O
N
H
H
1''
O
O
N
5''
O
O
OH
O
2''
i, ii
6''
4''
H3C
OAc
HO
H3C
OAc
β
-6c
HO
OH
5a (R1 = R2 = H)
4C1
AcO
OAc
β
-D-
gluco
5b (R1 = Cl, R2 = Ac)
AcO
OAc
5
7
α
-4a
4
α
α
-7a (R1 = H, 67%)
-7f (R1 = Cl, 63%)
O
3a
5'
6
4'
R1
3
H
H
6'
2
3a'
7a
2'
1'' N
5''
H
N
6''
O
3'
H1
7'
Me
AcO
7a'
3''
O
H
N
1'
iii
3''
2''
4''
O
AcO
1''
O
N
H
5''
OAc
H3C
2'' OAc
OAc
4''
6''
N
O
β-7a
β
-L-
rhamno
1C4
O
AcO
H3C
OH
α
α
-6a (R1 = H, 90%)
-6f (R1 = Cl, 70%)
Figure 1. Numbering of the atoms for NMR assignment and relevant
NOE correlations for b-6c and b-7a.
HO
OH
Scheme 3. Synthesis of a-6a and a-6f . Reagents and conditions: (i)
Na2CO3, MeOH, 20 ꢁC, 2 h; (ii) Ac2O, pyridine, 0–4 ꢁC, 12 h; (iii)
KOtBu, MeOH, 20 ꢁC, 12 h.
rhamnosides a-6a and a-6f clearly indicate a bis-axial
position of protons H-100 and H-200 confirming a 4C1 con-
formation (Fig. 2). In contrast, the protected derivatives
a-4a, a-7a, and a-7f seem to prefer a twisted boat con-
formation. This can be concluded from the smaller cou-
relations of proton H-10 with H-30 and H-50 prove an ax-
ial position of these protons.
3
3
00 00
0
0
pling constants J1 ;2 ( J1 ;2 for a-4a) = 4.6–5.7 Hz as
well as from the increase of the coupling constants
Those L-rhamno derivatives, which show small vicinal
3
00 00
coupling constants [i.e., J1 ;2 < 1:6 Hz (for b-6a, 6b,
3
0
0
b-6f, b-7a, 7b, b-7f), and J1 ;2 ¼ 1:5 Hz (for b-4a, b-
4b)], have to be assigned to b-anomeric structures with
HO
4''
1
1C4 conformation. The b ꢁ C4 conformation of b-7a
H
H
H
H
5''
O
3''
O
H
N
2''
2''
AcO
was independently confirmed by a NOESY experiment
in which relevant NOE correlations were observed for
protons H-100, H-300, and H-500 proving their axial posi-
tion (Fig. 1). Those L-rhamnosides, which exhibit larger
N
5'' AcO
H
1''
AcO
3''
1''
Me6''
4'' H
OH
H
Me
6''
HO
H
α-L-rhamno
4C1
α-6a,6f
twist-boat
α-4a,7a,7f
vicinal coupling constants [i.e., 3J1 ;2 ¼ 8:5 Hz (a-6a, a-
00 00
3
3
00 00
0
0
6f), J1 ;2 ¼ 4:6–4:7 Hz (a-7a, a-7f), and J1 ;2 ¼ 5:7 Hz
(a- 4a)], have to be assigned to the a-anomers. The cou-
Figure 2. Configurations and conformations of acetylated and deacet-
ylated isatine- and indirubin-N0-b-L-rhamnopyranosides.
3
00 00
pling constants J1 ;2 ¼ 8:5 Hz of the deprotected

5574
3
S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
3
3
3
00 00
00 00
0
0
0
0
J3 ;4 ꢀ J4 ;5 ( J3 ;4 ꢀ J4 ;5 for a-4a) ffi 7 Hz compared to
the corresponding deprotected indirubin-N0-rhamno-
sides. The 1C4 conformation should be excluded, be-
2.2. Biological evaluation
The anti-proliferative activity of indirubin-N0-glycosides
6a– f and 9a against four adherent human cancer cell
lines [bladder (5637), small cell lung (A-427), esophageal
(Kyse-70), and breast (MCF-7)] was studied (Table 2).
The shapes of the dose–response curves were rather
broad over the five concentrations tested, in contrast
to the dose–response curves of many antitumor agents.
The IC50 values obtained for indirubin-N0-glycosides
6a–f and 9a are summarized in Table 1. Rhamnosides
a-6a, b-6a and b-6b were insoluble in the culture medium
at concentrations above 12.5 and 25 lM, respectively;
thus, the growth inhibition could only be examined in
the MCF-7 cell line, where a-6a and b-6b proved to be
very potent. In the other three cell lines, less than 20%
growth inhibition was observed at concentrations of
12.5 lM. The IC50 values of rhamnosides a-6a and b-
6a (tested against MCF-7 cells) are comparable to those
observed for etoposide.
3
3
00 00
00 00
cause coupling constants J3 ;4 and J4 ;5 of at least
9 Hz would have been expected (corresponding to a full
axial arrangement of these protons similar to the
b ꢁ C4 sugars discussed above).
1
The molecular structures of b-4a, b-4b, and b-8a were
independently confirmed by X-ray crystal structure
analyses (Figs. 3–5).18
The rhamnosides a-6f, b-6f, and b-6b, containing a
substituted indirubin moiety, show a lower activity
compared to those rhamnosides containing a non-
substituted indirubin moiety. Noteworthy, rhamnoside
a-6f shows a better selectivity for MCF-7 cancer cells.
Rhamnoside b-6f, containing a chlorinated indirubin
moiety, possesses a higher, but less selective anti-prolif-
erative activity against all cell lines. The galactoside
b-6d exhibits the lowest activity followed by the oxime
b-9a which shows the lowest selectivity of all com-
pounds tested.
Figure 3. ORTEP plot of b-4a (50% probability of the thermal
ellipsoids).
The activities of compounds 6c–e were comparable with
chlorambucil. Comparing the activities of glycosides 6c-
e against all cell lines, mannoside b-6e was found to be
most potent, followed by glucoside b-6c. Galactoside
b-6d required a two to fivefold higher concentration
for a 50% inhibition of cell growth. These experiments
clearly demonstrate that the sugar moiety has an impor-
tant influence on the anti-proliferative activity of indiru-
bin-N0-glycosides. Notably, the anti-proliferative
activity of indirubin-N0-glycosides 6a–f and b-9a is con-
siderably higher than the activity recently reported11 for
non-glycosylated indirubins. In particular, the activity
of rhamnosides a-6a and b-6a against the human breast
cancer cell line MCF-7 is much higher (by the factor 10
to 100) than the activity of all non-glycosylated indiru-
bins tested before.11 Notably, only nitro derivatives of
non-glycosylated indirubins show an activity which is
in the same range.
Figure 4. ORTEP plot of b-4b (30% probability of the thermal
ellipsoids).
The experiments suggest that the deoxy-sugar moiety
present in rhamnosides a-6a and b-6a is the reason for
their higher activity compared to gluco, galacto, and
manno configured glycosides b-6c, b-6d, and b-6e. In
contrast, the configuration of the anomeric carbon atom
of the rhamnoside did not show a major influence. A
substitution in the aromatic ring system considerably
lowers the cytotoxic activity of the tested compounds.
This could be caused by a lower binding affinity to
CDK2 which is described as the main reason for the
cancerostatic properties of the indirubins.11 The reason
Figure 5. ORTEP plot of b-8a (50% probability of the thermal
ellipsoids).

S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
5575
Table 2. Average IC50 values (lM SD, results are from three to six independent determinations)
Compound
Cell lines
5637
A-427
KYSE-70
MCF-7
b-6a
a-6a
b-6f
a-6f
b-6b
b-6c
b-6d
b-6e
>12.5a
>12.5a
> 12.5a
0.76 0.16
0.67 0.08
7.98 1.97
6.96 0.46
6.16 2.33
15.67 2.48
25.75 4.27
4.22 1.07
17.03 2.57
0.50 0.19
1.38 0.29
6.78 5.21
18.4 5.5
>25a
>25a
> 25a
8.23 1.47
12.87 0.7
>12.5a
n.d.b
n.d.
5.94 2.29
15.28 1.28
>12.5a
n.d.
14.36 2.46
24.94 2.26
10.63 1.67
15.53 2.44
0.54 0.30
0.35 0.10
1.73 1.20
6.55 3.46
11.84 2.75
13.16 2.93
5.55 0.67
n.d.
28.92 5.52
41.45 5.38
11.83 2.46
15.40 2.03
0.94 0.23
0.63 0.14
1.59 1.93
38.4 1.7
b-9a
Etoposidec
Cisplatinc
5-Aza-cytidinec
Chlor-ambucilc
0.13 0.10
1.96 0.54
0.63 0.05
9.50 3.47
a An IC50 value could not be determined because a-6a , b-6a and b-6b were insoluble above concentrations of 12.5 and 25 lM, respectively.
b Not determined.
c From Ref. 19.
for the better selectivity of the rhamnoside a-6f com-
pared to compound b-6f remains unclear at present.
Notably, a strong inhibition of the growth of tumor cells
was reported12 for akashine A, B, and C which represent
naturally occurring N-glycosides of indigo.
3. Conclusions
In conclusion, we have reported the synthesis of the
first indirubin-N0-glycosides based on reactions of isa-
tine-N-glycosides with indoxyl acetates. Our strategy
allows the synthesis of unsubstituted indirubin-N0-gly-
cosides as well as derivatives containing different sub-
stitution patterns at both indol moieties of the
indirubin. The products show a significant anti-prolif-
erative activity against various human cancer cell lines.
Good results were observed for an indirubin-N 0-man-
noside, which was shown to have medium to high
anti-proliferative activity against all investigated cell
lines. The highest activity and selectivity against the
MCF-7 breast cancer cell line were observed for the
anomeric indirubin-N0-rhamnosides. The anti-prolifera-
tive activity of the indirubin-N0-glycosides was shown
to be higher than the activity of the corresponding
aglycons in most of the cases.
An interesting result was the highly selective anti-pro-
liferative activity of compounds a-6a and b-6a in the
MCF-7 breast cancer cell line. The poor solubility of
a-6a and b-6b did not allow the determination of
IC50 values for the other three cell lines under investi-
gation. However, less than 20% cell growth inhibition
was observed even at the highest concentrations tested
(i.e., 12.5 and 25 lM, respectively). The growth of
MCF-7 breast cancer cells is sensitive to estrogens
and antiestrogens and this cell line is known to possess
a functional estrogen receptor. Therefore, compounds
a-6a and b-6b may act by interfering with this path-
way. In fact, the planar, conjugated structure of the
indirubin moiety is an integral part of some synthetic
estrogens and antiestrogens.20 However, further studies
are needed to support this hypothesis. Moon et al. sug-
gested11 that the formation of a hydrogen bond be-
tween the backbone oxygen of Leu83 in CDK2 and
the secondary amine function of the indirubine moiety
might play an important role during cell growth inhibi-
tion. This is in agreement with our studies, since the
presence of the sugar residue in indirubin-N0-glycosides
6a-f and 9a should not avoid the formation of such a
hydrogen bond. The lower cytotoxic activity and selec-
tivity of rhamnosyl-oxime b-9a, compared to rhamno-
sides a-6a and b-6a, suggests that the carbonyl group
plays an important role in the biochemical mechanism
of cytotoxic activity.
4. Experimental
4.1. General
1H NMR spectra (250.13, 300.13, and 500.13 MHz) and
13C NMR spectra (62.9, 75.5, and 125.8 MHz) were re-
corded on Bruker spectrometers AV II 250, AV III 300,
and AV 500 in CDCl3, DMSO-d6, and C6D6 as solvents.
The calibration of spectra was carried out on solvent sig-
nals (CDCl3: d (1H) = 7.25, d (13C) = 77.0; DMSO-d6: d
1
13
(
H) = 2.50,
d
(
C) = 39.7; C6D6: d(1H) = 7.16,
d(13 C) = 128.0). Infrared spectra were recorded on a
FTIR spectrometer. Mass spectrometric data (MS) were
obtained by electron ionization (EI, 70 eV), chemical
ionization (CI, isobutane) or electrospray ionization
(ESI). Melting points are uncorrected. Analytical thin
layer chromatography was performed on 0.20 mm
60 A silica gel plates. Column chromatography was per-
formed on 60 A silica gel (60–200 mesh). Crystallo-
graphic data were collected on a Bruker–Nonius
X8Apex-CCD diffractometer with MoKa radiation
Future studies are directed towards the influence of
other substituents present in the indirubin moiety
and of other (more rare) sugar moieties on the anti-
proliferative activity. The effects of such substituents
should give more insight in the pharmacological activ-
ity of glycosylated indirubins and their mode of
action.

5576
S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
˚
(k = 0.71073 A). The structures were solved by direct
methods using SHELXS-97 and refined against F2 on
all data by full matrix least-squares with SHELXL-
97.21 All non-hydrogen atoms were refined anisotropi-
cally, all hydrogen atoms were refined in the model at
geometrically calculated positions and refined using a
riding model.
mated by a linear least-squares regression of the T/C corr
values versus the logarithm of the substance concentra-
tion and extrapolating to values of 50%. Only test con-
centrations yielding T/C
90% were used in the calculations.
values between 10% and
corr
4.3. General procedure for the synthesis of isatine-N-
glycosides
4.2. Biological screening
To a stirred solution of the acetylated glycosyl aniline in
oxalyl chloride (about 10 equiv) an equivalent of anhy-
drous aluminum chloride was added. The mixture was
stirred for 1.5 h at 55 ꢁC (TLC control). After cooling
to 0 ꢁC, ice water was added to the solution with stir-
ring. The yellow sugar precipitated. The mixture was ex-
tracted three times with EtOAc, the combined organic
layers were washed with a saturated aqueous solution
of sodium bicarbonate and with water and dried
(Na2SO4). The solution was filtered, the filtrate was con-
centrated under reduced pressure, and the residue was
purified by column chromatography (silica gel).
The four adherent human cancer cell lines used for
determination of cytotoxic activity [bladder (5637),
small cell lung (A-427), esophageal(Kyse-70), and breast
(MCF-7)] were obtained from the German Collection of
Microorganisms and Cell Cultures (DSMZ Braun-
schweig). Cells were grown in medium containing 90%
RPMI 1640 medium and 10% FCS, and supplemented
with penicillin G/streptomycin; 1000-fold stock solu-
tions of the compounds for testing were prepared in
DMSO and serially diluted in DMSO to concentrations
500-fold of the desired concentrations (giving series of 5
dilutions). The concentration of substances a-6a, b- 6a,
and 6b ranged in the area of 1.5–100 mM (giving con-
centration ranges of 1.5–100 lM on the test plates).
Twofold dilutions were done for these compounds.
For the testing of compounds a-6a and b-6a with
MCF-7 cells fourfold dilutions were carried out; the sub-
stances were used in a concentration range between
0.024 and 6.25 lmol/l on the microtiter plates. The cells
of all cell lines were plated out 24 h prior to testing in 96-
well microtiter plates at a density of 1000 cells/well in
100 lL of the medium.19 After 24 h, one untreated plate
of each cell line was removed and served later as ‘C,0’
control. The dilution series of the test compounds were
500-fold diluted into the culture medium to give concen-
trations twofold of the final test concentration; 100 lL
of this treated medium was added to each well, giving
a final DMSO concentration of 0.1% in all wells. Two
substances at five concentrations per substance and
one row (i.e., eight wells) per concentration were tested
on each plate. Each plate contained two rows of control
wells with cells only treated with medium containing
0.1% of DMSO. The cells were continuously exposed
to all compounds for a period of 96 h. Growth inhibi-
tion of the cells was measured using the Crystal Violet
assay.19 After 96 h of incubation with the substance,
the medium was discarded from the wells and replaced
for 20 min by a glutaraldehyde buffer solution (1%) to
fix the cells. After discarding the fixing solution the cells
were stored under PBS at 4 ꢁC until staining. Cells were
treated with 100 lL of staining solution (0.02% aqueous
solution of Crystal Violet) for 30 min. The excess of dye
was removed and the cells were washed for 30 min with
water. The cell-bound dye was dissolved in ethanol/
water (70%) and the optical density was subsequently
N-(20,30,40-Tri-O -acetyl-b-L- rhamnopyranosyl)isatine
(b-4a) and N-(20,30,40-tri-O -acetyl-a-L-rhamnopyrano-
syl)isatine (a-4a): Starting with a mixture of the acety-
lated rhamnosyl anilines a/b-3a (1.24 g, 3.4 mmol, b/a
= 2.5:1), b-4a (320 mg, 23%) and a mixed fraction of
a-4a and b-4a (570 mg, 40%, b/a = 3:1) were isolated
after column chromatography (n-heptane/EtOAc = 3:1).
A mixture of enriched a-anomer gave an analytical sam-
ple of pure N-(20,30,40-tri-O-acetyl-a-L-rhamnopyrano-
syl)isatine (a-4a).
4.4. Data of b-4a
23
D
Yellow solid. Mp 94–95 ꢁC; ½aŠ +152.00 (c = 0.82,
1
CHCl3); Rf = 0.54 (n-heptane/EtOAc = 1:3). H NMR
(250 MHz, C63D6): d = 7.41 (ddd, 5J4,7 = 0.6 Hz,
4J5,7 = 1.0 Hz, J6,7 = 8.2 Hz, 1 H, H-7); 7.19 (ddd,
5J4,7 = 0.6 Hz, J4,6 = 1.5 Hz, J4,5 = 7.5 Hz, 1H, H-4);
4
3
4
6.95 (ddd, J4,6 = 1.5 Hz, J5,6 = 7.6 Hz, J6,7 = 8.2 Hz,
3
3
4
3
1H, H-6); 6.48 (d0t0, J5,7 = 1.0 Hz, J4,5 = 7.5 Hz,
3
3
0
0
J5,6 = 7.6 Hz, 1H, H-5); 5.84 (dd, J1 ;2 ¼ 1:5 Hz,
J2 ;3 ¼ 3:0 Hz, 1H, H-20); 5.82 (d, J1 ;2 ¼ 1:5 Hz, 1H,
3
3
0
0
0
0
H-10); 5.46–5.32 (m, 2H, H-30, H-40); 3.31–3.19 (m,
1H, H-50); 1.77, 1.72, 1.44 (3s, 9H, 3xC(O)CH3); 1.17
(d, J5 ;6 ¼ 6:2 Hz, 3H, H-60). 13C NMR (75 MHz,
C6D6): d = 181.8 (C-3); 169.7, 169.5, 169.4
(3xC(O)CH3); 157.2 (C-2); 149.6 (C-7a); 136.9, 124.7,
123.4 (C-4, C-5, C-6); 118.6 (C-3a); 115.6 (C-7); 81.1
(C-10); 74.0, 71.0, 70.9, 70.5 (C-20, C-30, C-40, C-50);
20.3, 20.3, 20.3 (3xC(O)CH3); 17.7 (C-60). MS (EI,
70 eV): m/z (%) = 419 (7) [M+], 273 (42) [M+-isatine],
153 (42) [M+-isatine-2HOAc], 146 (10) [M+-sugar].
HRMS (EI, 70 eV): calcd for C20H21NO9 ([M+]):
419.12108; found: 419.12245. Anal. calcd for
C20H21NO9 (419.38): C, 57.28; H, 5.05; N, 3.34. Found:
C, 57.68; H, 5.39; N, 3.07.
3
0
0
~
measured at m ¼ 570 nm using an Anthos 2010 plate
reader. To construct the dose–response curves the cor-
rected T/C values were calculated as follows: (T/C)corr
(%) = (OD ꢁ OD )/(ODC ꢁ OD ) · 100, with ODT
T
C,0
C,0
as the mean optical density of the treated cells after
staining, ODC as the mean optical density of the con-
trols and ODC,0 as the mean optical density at the time
the substances were added. The IC50 values were esti-
4.5. Data of a-4a
23
Yellow solid. Mp 66–67 ꢁC; ½aŠ ꢁ92.19 (c 0.61,
D
CHCl3); Rf = 0.52 (n -heptane/EtOAc = 1:3). H NMR
1

S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
5577
4
(250 MHz, C6D6): d = 7.21 (dd, 4J4,6 = 1.5 Hz,
3J6,7 = 8.0 Hz, 1H, H-6); 6.52 (d0t0, J5,7 = 0.8 Hz,
3J4,5 = 7.5 Hz, 1H, H-4); 7.03 (br d, J6,7 = 8.0 Hz, 1H,
3J4,5 = 7.6 Hz, J5,6 = 7.8 Hz, 1H, H-5); 5.91 (dd,
3
3
H-7); 6.91 (d0t0, 4J4,6 = 1.5 Hz, 3J5,6 = 7.3 Hz,
J1 ;2 ¼ 9:3 Hz, J2 ;3 ¼ 10:0 Hz, 1H, H-20); 5.67 (d,
3
3
3
3
2
3
0
0
0
0
3
3J6,7 = 8.0 Hz, 1H, H-6); 6.48 (d0t0, J5,7 = 1.0 Hz,
J1 ;2 ¼ 9:3 Hz, 1H, H-10); 5.49 (dd, J4 ;5 ¼ 1:0 Hz,
3
0
0
0
0
3
3J5,6 = 7.3 Hz, J4,5 = 7.5 Hz, 1H, H-5); 6.28 (dd,
J3 ;4 ¼ 3:2 Hz, 1H, H-40); 5.23 (dd, J3 ;4 ¼ 3:2 Hz,
3
0
0
0
0
J2 ;3 ¼ 3:7 Hz, J1 ;2 ¼ 5:7 Hz, 1H, H-20); 6.01 (dd,
J2 ;3 ¼ 10:0 Hz, 1H, H-30); 4.05 (dd, J5 ;6a0 ¼ 6:2 Hz,
3
3
3
3
3
3
3
0
0
0
0
0
0
0
J2 ;3 ¼ 3:7 Hz, J3 ;4 ¼ 6:7 Hz, 1H, H-30); 5.60 (d,
J6 a0;6b ¼ 11:4 Hz, 1H, H-6a0); 3.95 (dd, J5 ;6b
¼
3
3
0
0
0
0
0
0
0
J1 ;2 ¼ 5:7 Hz, 1H, H-10); 5.16 (dd, J4 ;5 ¼ 5:7 Hz,
6:8 Hz, J6a0;6b ¼ 11:4 Hz, 1H, H-6b0); 3.28 (d0t0,
3
2
0
0
0
0
0
J3 ;4 ¼ 6:7 Hz, 1H, H-40); 3.78 (dq, J4 ;5 ¼ 5:7 Hz,
J4 ;5 ¼ 1:0 Hz, J5 ;6a0 ¼ 6:2 Hz, J5 ;6b ¼ 6:8 Hz, 1H,
3
3
3
3
0
0
0
0
0
0
0
0
0
J5 ;6 ¼ 6:7 Hz, 1H, H-50); 1.67, 1.65, 1.61 (3s, 9H,
H-50); 1.69, 1.65, 1.59, 1.46 (4s, 12H, 4xC(O)CH3). 13C
NMR (75 MHz, C6D6): d = 181.8 (C-3); 169.7, 169.7,
169.5, 169.5 (4xC(O)CH3); 157.7 (C-2); 148.7 (C-7a);
137.6 (C-6); 125.3 (C-4); 124.0 (C-5); 118.5 (C-3a);
113.5 (C-7); 80.5 (C-10); 73.2 (C-50); 71.4 (C-30); 67.5
(C-40); 65.8 (C-20); 61.3 (C-60); 20.1, 20.1, 20.0, 19.7
(4xC(O)CH3). MS (EI, 70 eV): m/z (%) = 477 (8) [M+],
331 (100) [M+ -isatine], 169 (100) [M+-isatine-HOAc-
Ac2O]. HRMS (EI, 70 eV): calcd for C22H23NO11
([M+]): 477.12656; found: 477.12687. Anal. calcd for
C22H23NO11 (477.42): C, 55.35; H, 4.86; N, 2.93. Found:
C, 55.28; H, 5.04; N, 2.71.
0
0
3xC(O)CH3); 1.14 (d, J5 ;6 ¼ 6:7 Hz, 3H, H-60). 13C
NMR (75 MHz, C6D6): d = 181.7 (C-3); 169.4, 169.3,
168.9 (3xC(O)CH3); 157.7 (C-2); 149.7 (C-7a); 137.6,
125.0, 124.0 (C-4, C-5, C-6); 118.6 (C-3a); 113.1 (C-7);
78.0 (C-10); 71.8, 71.3, 69.4, 66.9 (C-20, C-30, C-40, C-
50); 20.3, 20.2, 20.1 (3xC(O)CH3); 16.5 (C-60). MS (CI,
isobutane): m/z (%)=420 (13) [M+H]+, 273 (100) [M+-
isatine]. HRMS (CI, isobutane): calcd for C20H21NO9
([M+]): 419.12108; found: 419.12130. Anal. calcd for
C20H21NO9 (419.38): C, 57.28; H, 5.05; N, 3.34. Found:
C, 57.68; H, 5.39; N, 3.07.
3
0
0
4.6. 4,6-Dimethyl-N-(20,30,40-tri-O-acetyl-b-L-rhamno-
pyranosyl)isatine (b-4b)
4.8. N-(20,30,40,60-Tetra-O-acetyl-b-D-mannopyrano-
syl)isatine (b-4e)
Starting with 3,5-dimethyl-N-(20,30,40-tri-O-acetyl-b/a-L-
rhamnopyranosyl)aniline (a/b-3b) (1.00 g, 2.5 mmol, b/
a
N-(20,30,40,60-tetra-O-acetyl-a,b-D-mannopyranosyl)ani-
line (a/b-3e) (1.00 g, 2.4 mmol) was transformed into the
yellow colored N-(20,30,40,60-tetra-O-acetyl-b-D-manno-
= 2:1), 4,6-dimethyl-N-(20,30,40-tri-O-acetyl-b/a-L-
rhamnopyranosyl)isatine (a/b-4b) was isolated as a yel-
low solid (860 mg, 76%, b/a = 3.5:1). Recrystallization
pyranosyl)isatine (b-4e) (0.24 g, 21%). Mp 82–83 ꢁC;
22
½aŠ +126.36 (c 0.3, CHCl3); Rf = 0.43 (n-heptane/
D
(n -heptane/ EtOAc) gave the pure anomer b-4b. Mp
EtOAc = 1:3). 1H NMR (500 MHz, CDCl3): d = 7.58
(m, 1H, H-4); 7.55–7.51 (m, 2H, H-6, H-7); 7.18 (m,
22
191–193 ꢁC; ½aŠ +201.87 (c 0.78, CHCl3); Rf = 0.58
D
(n-heptane/EtOAc = 1:3). H NMR (250 MHz, CDCl3):
1
1H, H-5); 5.88 (d, J1 ;2 ¼ 1:5 Hz, 1H, H-10); 5.58 (dd,
3
0
0
3
3
3
3
d = 7.14, 6.68 (2br s, 2H, H-5, H-7); 5.82 (d, J1 ;2
¼
J1 ;2 ¼ 1:5 Hz, J2 ;3 ¼ 3:5 Hz, 1H, H-20); 5.39 (‘t’,
0
0
0
0
0
0
3
3
3
1:5 Hz, 1H, H-10); 5.54 (dd,
J1 ;2 ¼ 1:5 Hz,
J3 ;4 ¼ J4 ;5 ¼ 10:0 Hz, 1H, H-40); 5.28 (dd, J2 ;3
¼
0
0
0
0
0
0
0
0
3
3
3
J2 ;3 ¼ 2:8 Hz, 1H, H-20); 5.26–5.15 (m, J2 ;3
¼
3:5 Hz,
0
J3 ;4 ¼ 10:0 Hz, 1H, H-30); 4.31 (dd,
0
0
0
0
0
0
2:8 Hz, J4 ;5 ¼ 9:4 Hz, 2H, H-30, H-40); 3.80–3.66 (m,
J5 ;6a0 ¼ 5:4 Hz, 2J
= 12.5 Hz, 1H, H-6a0); 4.20
3
3
6a0,6b0
2
0
0
J4 ;5 ¼ 9:4 Hz, J5 ;6 ¼ 6:2 Hz, 1H, H-50); 2.49, 2.37
(dd, J5 ;6b ¼ 2:5 Hz, J6a0;6b ¼ 12:5 Hz, 1H, H-6b0);
3
3
3
0
0
0
0
0
0
0
3
3
3
0
0
0
0
0
(2s, 6H, 2xCH3); 2.09, 1.97, 1.89 (3s, 9H, 3xC(O)CH3);
3.89 (ddd, J5 ;6b ¼ 2:5 Hz, J5 ;6a0 ¼ 5:3 Hz, J4 ;5 ¼
1.34 (d, J5 ;6 ¼ 6:2 Hz, 3H, H-60). 13C NMR
(63 MHz, CDCl3): d = 181.4 (C-3); 170.1, 169.6, 169.4
(3xC(O)CH3); 157.7 (C-2); 149.4, 149.0, 141.0 (C-4, C-
6, C-7a); 126.8, 113.6 (C-5, C-7); 111.3 (C-3a); 80.2
(C-10); 74.2, 70.5, 70.4, 70.0 (C-20, C-30, C-40, C-50);
22.9, 18.2 (2xCH3); 20.8, 20.8, 20.5 (3xC(O)CH3); 17.6
(C-60). MS (EI, 70 eV): m/z (%) = 447 (11) [M+], 273
(54) [M+-dimethylisatine], 174 (39) [dimethylisatine],
153 (100) [M+-dimethylisatine-2HOAc]. Anal. calcd for
C22H25NO9 (447.44): C, 59.06; H, 5.63; N, 3.13. Found:
C, 59.18; H, 5.88; N, 3.01.
10:0 Hz, 1H, H-50); 2.10, 2.08, 1.97, 1.87 (4s, 12H,
4xC(O)CH3). 13C NMR (126 MHz, CDCl3): d = 181.6
(C-3); 170.4, 169.7, 169.5, 169.5 (4xC(O)CH3); 157.0
(C-2); 149.0 (C-7a); 137.7 (C-6); 125.2 (C-4); 124.2
(C-5); 118.0 (C-3a); 115.6 (C-7); 80.5 (C-10); 75.6 (C-
50); 70.4 (C-30); 70.0 (C-20); 65.1 (C-40); 62.1 (C-60);
20.7, 20.7, 20.6, 20.4 (4xC(O)CH3). MS (EI, 70 eV):
m/z (%) = 477 (12) [M+], 331 (72) [M+-isatine], 169
(100) [M+-isatine-HOAc-Ac2O], 146 (13) [M+-
sugar].HRMS (EI, 70 eV): calcd for C22H23NO11
([M+]): 477.12656; found: 477.12599. Anal.: calcd for
C22H23NO11 (477.42): C, 55.35; H, 4.86; N, 2.93. Found:
C, 55.52; H, 5.17; N, 2.73.
3
0
0
4.7. N-(20,30,40,60 0-Tetra-O-acetyl-b-D-galactopyrano-
syl)isatine (b-4d)
4.9. General procedure 1 for the synthesis of the
O-acetylated indirubin-N0-glycosides
Starting with N-(20,30,40,60-tetra-O-acetyl- a,b-D-galac-
topyranosyl)aniline (a/b-3d) (0.80 g, 1.9 mmol), N-
(20,30,40,60-tetra-O-acetyl-b-D-galactopyranosyl)isatine
To a stirred degassed MeOH solution of the acetylated
glycosyl isatine 4, the corresponding indoxyl acetate 5
(1.0 equiv) and sodium bicarbonate (2.0–3.0 equiv) were
added (under Argon atmosphere). The mixture was stir-
red for 2 h at 20 ꢁC during which time the yellow to or-
ange color of the solution changed to red to violet. The
mixture was neutralized with IR 120 (H+), filtered and
b-D-galactopyranosyl)isatine (b-4d) was isolated as a
22
yellow solid (0.47 g, 52%). Mp 78–80 ꢁC; ½aŠ -76.76
D
(c 1.02, CHCl3); Rf = 0.45 (n-heptane/EtOAc = 1:3); H
1
3
NMR (250 MHz, C6D6): d = 7.44 (0d0, J6,7 = 8.0 Hz,
3
1H, H-7); 7.23 (dd,4J4,6 = 1.5 Hz, J4,5 = 7.6 Hz, 1H,
H-4); 7.05 (d0t0, 4J4,6 = 1.5 Hz, 3J5,6 = 7.8 Hz,

5578
S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
3
9H, 3xC(O)CH3); 1.34 (d, J5 ;6 ¼ 6:7 Hz, 3H, H-600).
13C NMR (75 MHz, CDCl3): d = 188.1 (C-3); 170.7,
169.9, 169.7 (3xC(O)CH3); 169.7 (C-20); 151.4 (C-7a);
139.8, 139.7 (C-2, C-7a0); 137.0 (C-6); 129.2 (C-60);
125.3 (C-4); 125.3 (C-40); 123.4, 121.9 (C-5, C-50);
121.5, 119.9 (C-3a, C-3a0); 112.0, 110.5 (C-7, C-70);
105.9 (C-30); 78.3 (C-100); 71.7 (C-400); 70.3 (C-500); 69.6
(C-300); 67.3 (C-200); 20.9, 20.8, 20.7 (3xC(O)CH3); 16.7
(C-600). MS (EI, 70 eV): m/z (%) = 534 (74) [M+], 273
(13) [M+-indirubin], 262 (100) [indirubin], 234 (12)
[indirubin-(C@O)]; HRMS (EI, 70 eV): calcd for
C28H26N2O9 ([M+]): 534.16328; found: 534.16329.
00 00
the filtrate was concentrated under reduced pressure.
The crude product was acetylated with acetic anhy-
dride/pyridine (v:v = 1:1) and stirred overnight at 0 ꢁC.
To the solution was added ice water with stirring. The
mixture was extracted three times with EtOAc. The
combined organic layers were washed with a saturated
aqueous solution of sodium bicarbonate and with water
and dried (Na2SO4). The solution was filtered and the
filtrate was concentrated under reduced pressure. The
residue was purified by column chromatography (n-hep-
tane/EtOAc = 2:1).
4.10. N0-(200,300,400-Tri-O-acetyl-b-L-rhamnopyranosyl)-
indirubin (b-7a)
4.12. 5-Chloro-10-(200,300,400-tri-O-acetyl-b-L-rhamnopyr-
anosyl)indirubin (b-7f)
Starting with b-4a (200 mg, 0.48 mmol), b-7a was isolated
as a red solid (178 mg, 70%).Mp 160–161 ꢁC; Rf = 0.56 (n
Starting with N-(20,30,40-tri-O-acetyl-b-L-rhamnopyrano-
syl)isatine (b-4a) (150 mg, 0.36 mmol), b-7f was isolated
(130 mg, 63%) as a red solid. Mp 159–161 ꢁC; Rf = 0.64
-heptane/EtOAc = 1:3). 1H NMR (500 MHz, CDCl3):
4
0
0
d = 10.42 (s, 1H, NH); 8.81 (dd, J4 ;6 ¼ 1:0 Hz,
3
J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.65 (d, J4,5 = 7.6 Hz, 1H,
(n-heptane/EtOAc = 1:3). 1H NMR (250 MHz, CDCl3):
3
0
0
H-4); 7.55 (d, J6 ;7 ¼ 7:9 Hz, 1H, H-70); 7.45 (d0t0,
d = 10.46 (s, 1H, NH); 8.82 (dd, J4 ;6 ¼ 1:3 Hz,
3
4
0
0
0
0
4J4,6 = 1.0 Hz, J5,6 = 7.6 Hz, J6,7 = 7.9 Hz, 1H, H-6);
J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.65 (d, J4,6 = 2.2 Hz, 1H,
3
3
4
3
0
0
7.22 (d0t0, J4 ;6 ¼ 1:0 Hz, J5 ;6 ¼ 7:5 Hz, J6 ;7
¼
H-4); 7.55 (d, J6 ;7 ¼ 8:0 Hz, 1H, H-70); 7.44 (dd,
4
3
3
3
0
0
0
0
0
0
0
0
3
7:9 Hz, 1H, H-60); 7.06 (d0t0,
J5 ;7 ¼ 1:0 Hz,
4J4,6 = 2.2 Hz, J6,7 = 8.5 Hz, 1H, H-6); 7.26 (d0t0,
4
0
0
J5 ;6 ¼ 7:5 Hz, J4 ;5 ¼ 7:9, 1H, H-50); 6.98 (‘t’,
J4 ;6 ¼ 1:3 Hz, J5 ;6 ¼ 7:6 Hz, J6 ;7 ¼ 8:0 Hz, 1H, H-
3
3
4
3
3
0
0
0
0
0
0
0
0
0
0
3J4,5 = 3J5,6 = 7.6 Hz, 1H, H-5); 6.91 (d, J6,7 = 7.9 Hz,
60); 7.09 (d0t0, J5 ;7 ¼ 1:1 Hz, J5 ;6 ¼ 7:6 Hz, J4 ;5
¼
3
4
3
3
0
0
0
0
0
0
1H, H-7); 5.98 (d, J1 ;2 ¼ 1:6 Hz, 1H, H-100); 5.65 (dd,
7:9 Hz, 1H, H-50); 6.93 (d, 3J6,7 = 8.5 Hz, 1H, H-7); 5.96
3
00 00
J1 ;2 ¼ 1:6 Hz, J2 ;3 ¼ 3:5 Hz, 1H, H-200); 5.33 (dd,
(d, 3J1 ;2 ¼ 1:5 Hz, 1H, H-100); 5.61 (dd, 3J1 ;2 ¼1:5 Hz,
3
3
3
3
3
00 00
00 00
00 00
00 00
J2 ;3 ¼ 3:5 Hz, J3 ;4 ¼ 10:0 Hz, 1H, H-300); 5.26 (‘t’,
J2 ;3 ¼ 3:2 Hz, 1H, H-200); 5.30 (dd, J2 ;3 ¼ 3:2 Hz,
3
3
3
3
3
3
00 00
00 00
00 00
00 00
J4 ;5 ¼ 9:5 Hz, J3 ;4 ¼ 10:0 Hz, 1H, H-400); 3.82 (dq,
J3 ;4 ¼ 10:2 Hz, 1H, H-300); 5.24 (dd, J4 ;5 ¼ 9:2 Hz,
3
3
00 00
00 00
00 00
00 00
J5 ;6 ¼ 6:3 Hz, J4 ;5 ¼ 9:5 Hz, 1H, H-500); 2.10, 1.98,
J3 ;4 ¼ 10:2 Hz, 1H, H-400); 3.79 (dq, J5 ;6 ¼ 6:2 Hz,
3
3
00 00
00 00
00 00
00 00
3
1.81 (3s, 9H, 3xC(O)CH3); 1.38 (d, J5 ;6 ¼ 6:3 Hz, 3H,
H-600). 13C NMR (126 MHz, CDCl3): d = 187.9 (C-3);
170.0, 169.8, 169.8 (3xC(O)CH3); 169.5 (C-20); 151.4 (C-
7a); 139.7, 139.2 (C-2, C-7a0); 136.9 (C-6); 128.5 (C-60);
125.1 (C-4); 125.0 (C-40); 122.7 (C-50); 121.7 (C-5); 121.4
(C-3a0); 119.9 (C-3a); 113.2 (C-70); 112.0 (C-7); 105.5
(C-30); 80.4 (C-100); 74.0 (C-500); 70.7, 70.4, 70.3 (C-200, C-
300, C-400); 20.8, 20.8, 20.6 (3xC(O)CH3); 17.7 (C-600). MS
(EI, 70 eV): m/z(%) = 534 (96) [M+], 273 (21) [M+-indiru-
bin], 262 (100) [indirubin]. HRMS (EI, 70 eV): calcd for
C28H26N2O9 ([M+]): 534.16328; found: 534.16316.
J4 ;5 ¼ 9:2 Hz, 1H, H-500); 2.10, 1.98, 1.80 (3s, 9H,
00 00
00 00
3xC(O)CH3); 1.38 (d, J5 ;6 ¼ 6:2 Hz, 3H, H-600). 13C
NMR (63 MHz, CDCl3): d = 186.8 (C-3); 170.0, 170.0,
169.9 (3xC(O)CH3); 169.5 (C-20); 149.6 (C-7a); 139.5,
139.3 (C-2, C-7a0); 136.5 (C-6); 129.1 (C-60); 127.3 (C-5);
125.2, 124.9 (C-40, C-4); 122.9 (C-50); 121.1, 120.9 (C-3a,
C-3a0); 113.4, 113.2 (C-7, C-70); 106.5 (C-30); 80.4 (C-
100); 74.1 (C-500); 70.6, 70.3, 70.2 (C-200, C-300, C-400); 20.8,
20.8, 20.6 (3xC(O)CH3); 17.7 (C-600). MS (EI, 70 eV): m/
z (%) = 570 (19) [M+; 37Cl]; 568 (51) [M+; 35Cl]; 298 (23)
[5-chloroindirubin; 37Cl]; 296 (65) [5-chloroindirubin;
35Cl]; 273 (19) [M+- 5-chloroindirubin]; 153 (100) [M+-
aglycon-2HOAc]. HRMS (EI, 70 eV): calcd for
C28H25ClN2O9 ([M+]): 568.12431; found: 568.12338.
3
00 00
4.11. N0-(200,300,400-Tri-O-acetyl-a-L-rhamnopyranosyl)-
indirubin (a-7a)
Starting with a-4a (120 mg, 0.286 mmol), a-7a was iso-
lated by column chromatography (n-heptane/ EtOAc=
2:1) -heptane/EtOAc = 2:1) as a red solid (102 mg,
4.13. 5-Chloro-10-(200,300,400-tri-O-acetyl-a-L-rhamnopyr-
anosyl)indirubin (a-7f)
67%).
Mp 105–106 ꢁC; Rf = 0.54 (n-heptane/
Starting with N-(20,30,40-tri-O-acetyl-a-L-rhamnopyrano-
syl)isatine (a-4a) (300 mg, 0.72 mmol), a-7f was isolated
(256 mg, 63%) as a red solid. Mp 93–95 ꢁC; Rf = 0.6 (n-
EtOAc=1:3). -heptane/EtOAc = 1:3). 1H NMR (250
MHz, CDCl3): d = 10.56 (s, 1H, NH); 8.94 (‘d‘,
3
J4 ;5 ¼ 7:7 Hz, 1H, H-40); 7.73 (‘d‘, J4,5 = 7.6 Hz, 1H,
heptane/EtOAc=1:3). 1H NMR (300 MHz, CDCl3):
3
0
0
4
3
3
H-4); 7.50 (ddd, J4,6 = 1.3 Hz, J = 7.5 Hz, J =8.0 Hz,
4
0
0
d = 10.57 (s, 1H, NH); 8.91 (dd, J4 ;6 ¼ 1:2 Hz,
1H, H-6); 7.31 (ddd, J4 6 ¼ 1:3 Hz, 3J =7.3 Hz,
J4 ;5 ¼ 7:8 Hz, 1H, H-40); 7.69 (‘d’, J4,6 = 2.1 Hz, 1H,
4
4
3
0
0
0
0
3J = 8.0 Hz, 1H, H-60); 7.24–7.14 (m, 4J = 1.3 Hz,
4
H-4); 7.46 (dd, J4,6 = 2.1 Hz, J6,7 = 8.5 Hz, 1H, H-6);
3
3
J = 7.3 Hz, J4 ;5 ¼ 7:7 Hz, 2H, H-50, H-70); 7.06–6.95
7.36–7.30 (m, 1H, H-60); 7.21 (‘d’, J6 ;7 ¼ 7:8 Hz, 1H,
3
3
0
0
0
0
(m, 4J = 0.8 Hz, J4,5 = 7.6 Hz, 2H, H-5, H-7); 6.19
H-70); 7.19 (d0t0, J5 ;7 ¼ 1:2 Hz,
J5 ;6 ¼ 7:6 Hz,
3
4
3
0
0
0
0
3
(dd, J2 ;3 ¼4:0 Hz, J1 ;2 ¼ 4:7 Hz, 1H, H-200); 5.95
J6 ;7 ¼ 7:8 Hz, 1H, H-50); 6.95 (d, J6,7 = 8.5 Hz, 1H,
3
3
3
00 00
00 00
0
0
(dd, J2 ;3 ¼ 4:0 Hz, J3 ;4 ¼ 7:3 Hz, 1H, H-300); 5.71
H-7); 6.18 (‘t‘, J1 ;2 ¼ 4:6 Hz, J2 ;3 ¼ 4:0 Hz, 1H, H-
3
3
3
3
00 00
00 00
00 00
00 00
3
3
3
H-100);
5.06
(‘t’,
200); 5.93 (dd, J2 ;3 ¼ 4:0 Hz, J3 ;4 ¼ 7:3 Hz, 1H, H-
00 00
00 00
00 00
(d,
J1 ;2 ¼ 4:8 Hz,
1H,
J3 ;4 ¼ J4 ;5 ¼ 6:9 Hz, 1H, H-400); 3.99 (‘quintet‘,
300); 5.69 (d, J1 ;2 ¼ 4:6 Hz, 1H, H-100); 5.06 (‘t’,
3
3
3
00 00
00 00
00 00
J4 ;5 ¼ J5 ;6 ¼ 6:7 Hz, 1H, H-500); 2.13, 2.11, 2.07 (3s,
J4 ;5 ¼ 7:0 Hz, 3J3 ;4 ¼ 7:3 Hz, 1H, H-400); 3.97 (‘quin-
3
3
3
00 00
00 00
00 00
00 00

S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
5579
3
3
tet‘, J5 ;6 ¼ 6:6 Hz, J4 ;5 ¼ 7:0 Hz, 1H, H-500); 2.13,
4.16. N0-(200,300,400,600-Tetra-O-acetyl-b-D-galactopyrano-
syl)indirubin (b-7d)
00 00
00 00
2.11, 2.08 (3s, 9H, 3xC(O)CH3); 1.34 (d,
00 00
J5 ;6 ¼ 6:6 Hz, 3H, H-600). 13C NMR (126 MHz,
3
CDCl3): d = 186.9 (C-3); 170.6, 169.9, 169.7, 169.7
(3xC(O)CH3, C-20); 149.7, 140.0, 139.4 (C-7a, C-7a0,
C-2); 136.6 (C-6); 129.7 (C-60); 127.4 (C-5); 125.5,
125.0, 123.6 (C-4, C-40, C-50); 121.3, 120.9 (C-3a, C-
3a0); 113.1, 110.6 (C-7, C-70); 106.8 (C-30); 78.5 (C-100);
71.7 (C-400); 70.2 (C-500); 69.6 (C-300); 67.3 (C-200); 20.9,
20.8, 20.7 (3xC(O)CH3); 16.8 (C-600). MS (EI, 70 eV):
m/z (%) = 570 (12) [M+; 37Cl]; 568 (31) [M+; 35Cl]; 298
(18) [5-chloroindirubin; 37Cl]; 296 (51) [5-chloroindiru-
bin; 35Cl]; 273 (23) [M+-5-chloroindirubin]; 153 (63)
[M+-aglycon-2HOAc]. HRMS (EI, 70 eV): calcd for
C28H25ClN2O9 ([M+]): 568.12431; found: 568.12427.
Starting with b-6d (50 mg, 0.118 mmol), b-7d was isolated
(49 mg, 70%) as a red solid. Mp 249 ꢁC (polymorphism),
1
268–269 ꢁC; Rf = 0.48 (n -heptane/EtOAc = 1:3). H
NMR (250 MHz, CDCl3): d = 10.46 (s, 1H, NH); 8.93
3
(br d, J4 ;5 ¼ 7:8 Hz, 1H, H-40); 7.74 (br d,
0
0
4
3J4,5 = 7.6 Hz, 1H, H-4); 7.51 (d0t0, J4,6 = 1.1 Hz,
3
3J5,6 = 7.6 Hz, J6,7 = 8.0 Hz, 1H, H-6); 7.42–7.28 (m,
4
3
3
0
0
0
0
0
0
J4 ;6 ¼ 1:0 Hz, J5 ;6 ¼ 7:8 Hz, J6 ;7 ¼ 8:0 Hz, 2H, H-
60, H-70); 7.18 (‘t’, 3J4 ;5 ¼ 7:8 Hz , 1H, H-50); 7.07–6.97
0
0
3
3
(m, J5,6 = 7.6 Hz, J6,7 = 8.0 Hz, 2H, H-5, H-7); 5.95–
5.75 (br, 2H, H-100, H-200); 5.57 (d, J3 ;4 ¼ 3:2 Hz, 1H,
3
00 00
H-400); 5.26 (dd, J3 ;4 ¼ 3:2 Hz, J2 ;3 ¼ 10:0 Hz, 1H,
3
3
00 00
00 00
H-300);
4.28–4.08 (m, 3J = 4.5 Hz, 3J = 9.8 Hz,
2
J6a00;6b ¼ 12:6 Hz, 3H, H-500, H-6a00, H-6b00); 2.30, 2.04,
2.00, 1.86 (4s, 12H, 4xC(O)CH3); 13C NMR (75 MHz,
CDCl3): d = 187.9 (C-3); 170.4, 170.3, 170.0, 169.9 (4s,
4xC(O)CH3); 169.1 (C-20); 151.3 (C-7a); 139.8, 138.3 (C-
7a0, C-2); 137.0, 129.2, 125.6, 125.4, 123.3, 122.0 (C-4,
C-40, C-5, C-50, C-6, C-60); 121.2, 120.0 (C-3a, C-3a0);
111.9, 111.2 (C-7, C-70), 105.6 (C-30); 79.6 (C-100); 73.2,
71.6, 67.3, 65.6 (C-200, C-300, C-400, C-500); 61.5 (C-600);
20.8, 20.7, 20.6, 20.4 (4s, 4xC(O)CH3). MS (EI, 70 eV):
m/z (%) = 592 (86) [M+], 331 (17) [M+-indirubin], 262
(100) [indirubin]. HRMS (EI, 70 eV): calcd for
C30H28N2O11 ([M+]): 592.16876; found: 592.16879.
00
4.14. General procedure 2 for the synthesis of the O-
acetylated indirubin-N0-glycosides
To a stirred solution of a deprotected indirubin-N-gly-
coside 6 in pyridine was added acetic anhydride at
0 ꢁC. The mixture was stirred overnight at 0 ꢁC. To
the solution was added ice water with stirring. The mix-
ture was extracted three times with EtOAc. The com-
bined organic layers were washed with a saturated
aqueous solution of sodium bicarbonate and with water.
The solution was dried (Na2SO4), filtered and the filtrate
was concentrated under reduced pressure. The residue
was purified by column chromatography (n-heptane/
EtOAc = 2:1).
4.17. N0-(200,300,400,600-Tetra-O-acetyl-b-D-mannopyrano-
syl)indirubin (b-7e)
4.15. N0-(200,300,400,600-Tetra-O-acetyl-b-D-glucopyrano-
syl)indirubin (b-7c)
Starting with 10-b-D-mannopyranosylindirubin (b-6e) (60
mg, 0.141 mmol), b-7e was yielded as a red solid (49 mg,
Starting with b-6c (100 mg, 0.236 mmol), b-7c was
isolated as a red solid (105 mg, 75%). Mp 176 ꢁC
(polymorphism), 294-295 ꢁC; Rf = 0.46 (n-heptane/
60%).
Mp
132–134 ꢁC;
Rf = 0.46
(n-heptane/
1
EtOAc = 1:3). H NMR (250 MHz, CDCl3): d = 10.42
4
3
0
0
0
0
(br, 1H, NH); 8.83 (dd, J4 ;6 ¼ 1:0 Hz, J4 ;5 ¼ 7:9 Hz,
1
3
EtOAc = 1:3). H NMR (500 MHz, CDCl3): d = 10.46
1H, H-40); 7.68 (d, J4,5 = 7.6 Hz, 1H, H-4); 7.54–7.44
4
3
3
(br, 1H, NH); 8.92 (dd, J4 ;6 ¼ 1:2 Hz, J4 ;5 ¼ 7:9 Hz,
(m, J6;7¼3J6 ;7 ¼ 7:9 Hz, 2H, H-6, H-70); 7.20 (d0t0,
0
0
0
0
0
0
3
1H, H-40); 7.73 (br d, J4,5 = 7.6 Hz, 1H, H-4); 7.51
J4 ;6 ¼ 1:2 Hz, J6 ;7 ¼ 7:9 Hz, 1H, H-60); 7.06 (d0t0,
4
4
3
0
0
0
0
(d0t0, J4,6 = 1.0 Hz, J5,6 = 3J6,7 = 7.9 Hz, 1H, H-6);
J5 ;7 ¼ 1:1 Hz, J4 ;5 ¼ J5 ;6 ¼ 7:9 Hz, 1H, H-50); 7.00
4
3
3
3
0
0
0
0
0
0
7.32 (d0t0, J4 ;6 ¼ 1:2 Hz, J5 ;6 ¼ J6 ;7 ¼ 7:9 Hz, 1H,
(‘t’, 3J4,5 = 3J5,6 = 7.6 Hz, 1H, H-5); 6.93 (d,
4
3
3
0
0
0
0
0
0
3
H-60); 7.22 (br d, J6 ;7 ¼ 7:9 Hz, 1H, H-70); 7.17 (d0t0,
J6,7 = 7.9 Hz, 1H, H-7); 6.03 (d, J1 ;2 ¼ 1:4 Hz, 1H,
3
3
0
0
3
00 00
J5 ;7 ¼ 1:0 Hz, J4 ;5 ¼ J5 ;6 ¼ 7:9 Hz, 1H, H-50); 7.02
H-100); 5.65 (dd, J1 ;2 ¼ 1:4 Hz, J2 ;3 ¼ 3:1 Hz, 1H,
4
3
3
3
0
0
0
0
0
0
00 00
00 00
(‘t’, 3J4,5 = 3J5,6 = 7.6 Hz, 1H, H-5); 6.99 (d,
3J6,7 = 7.9 Hz, 1H, H-7); 5.78 (br, 2H, H-100, H-200);
H-200); 5.46 (‘t’, J4 ;5 ¼ 9:5 Hz, J3 ;4 ¼ 10:0 Hz, 1H,
3
3
00 00
00 00
H-400); 5.38 (dd, J2 ;3 ¼ 3:1 Hz, J3 ;4 ¼ 10:0 Hz, 1H,
3
3
00 00
00 00
2
5.43 (‘t’, J2 ;3 ¼ J3 ;4 ¼ 9:5 Hz, 1H, H-300); 5.30 (‘t’,
H-300); 4.27 (dd, J5 ;6a00 ¼ 2:8 Hz, J6a00;6b ¼ 12:5 Hz,
3
3
3
00 00
00 00
00
00
J3 ;4 ¼ 9:5 Hz, J4 ;5 ¼ 10:0, 1H, H-400); 4.26 (dd,
1H, H-6a00); 4.33 (dd, J5 ;6b ¼ 4:7 Hz, J6a00;6b
¼
3
3
3
3
2
00 00
00 00
00
00
00
J5 ;6a00 ¼ 4:5 Hz, J6a00;6b ¼ 12:5 Hz, 1H, H-6a00); 4.21
12:5 Hz, 1H, H-6b00); 3.98 (ddd, J5 ;6a00 ¼ 2:8 Hz,
2
3
00
00
00
3
(dd, J5 ,6b = 2.5 Hz, J6a00;6b ¼ 12:5 Hz, 1H, H-6b00);
J5 ;6b ¼ 4:7 Hz, J4 ;5 ¼ 9:5 Hz, 1H, H-500); 2.11, 2.10,
2
3
3
00
00
00
00
00 00
00
3.97 (ddd, J5 ;6a00 ¼ 4:5 Hz, J4 ;5 ¼ 10:0, J5 ;6b
¼
1.99, 1.81 (4s, 12H, 4xC(O)CH3); 13C NMR (63 MHz,
CDCl3): d = 188.0 (C-3); 170.6, 169.8, 169.8, 169.7,
169.5 (5s, 4xC(O)CH3, C-20); 151.4 (C-7a); 140.0,
139.0 (C-2, C-7a0); 137.0 (C-6); 128.5 (C-60); 125.3 (C-4);
125.0 (C-40); 122.8 (C-50); 121.9 (C-5); 121.4, 119.9 (C-
3a0, C-3a); 113.2, 112.0 (C-7, C-70); 105.4 (C-30); 80.4
(C-100); 75.5 (C-500); 70.7 (C-300); 70.1 (C-200); 65.4 (C-400);
62.3 (C-600); 20.7, 20.7, 20.7, 20.5 (4s, 4xC(O)CH3). MS
(EI, 70 eV): m/z (%) = 592 (57) [M+], 331 (7) [M+-indiru-
bin], 262 (100) [indirubin], 169 (91) [M+-indirubin-
HOAc-Ac2O], 133 (12) [indoxyl]. HRMS (EI, 70 eV):
calcd for C30H28N2O11 ([M+]): 592.16876; found:
592.16850.
3
3
3
00
00 00
00
00
2:5 Hz, 1H, H-500); 2.08, 2.08, 2.01, 1.83 (s, 12H,
4xC(O)CH3). 13C NMR (126 MHz, CDCl3): d = 188.0
(C-3); 170.5, 170.4, 170.1, 169.5, 168.9 (4xC(O)CH3, C-
20); 151.3 (C-7a); 139.9 (C-2); 138.1 (C-7a0); 137.0 (C-
6); 129.2 (C-60); 125.6 (C-40); 125.4 (C-4); 123.4 (C-50);
122.0 (C-5); 121.3 (C-3a0); 120.0 (C-3a); 112.0 (C-7);
110.9 (br, C-70); 105.4 (C-30); 79.5 (C-100); 74.7 (C-500);
73.5 (C-300); 68.0 (C-400); 67.9 (C-200); 61.9 (C-600); 20.7,
20.6, 20.6, 20.3 (4xC(O)CH3). MS (EI, 70 eV): m/z
(%) = 592 (94) [M+], 331 (9) [M+-indirubin], 262 (100)
[indirubin]. HRMS (EI, 70 eV): calcd for C30H28N2O11
([M+]): 592.16876; found: 592.16834.

5580
S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
4.18. General procedure 1 for the synthesis of indirubin-
N0-glycosides
30); 82.3 (C-100); 75.6, 71.7 (C-400, C-500); 73.5 (C-300);
72.0 (C-200); 22.9, 22.0 (2xCH3); 18.3 (C-600). MS (EI,
70 eV): m/z (%) = 436 (12) [M+], 290 (79) [aglyconH],
273 (59) [M+-aglycon], 161 (57) [4,6-dimethyloxindol].
HRMS (EI, 70 eV): calcd for C24H24N2O6 ([M+]):
436.16289; found: 436.16259.
To a stirred, degassed methanol solution of glycosyl isa-
tine 4, indoxyl acetate 5 (1.0 equiv) and sodium carbonate
(4.0 equiv) were added under Argon atmosphere. The
mixture was stirred for1.5–4 h at 20 ꢁC during which time
the yellow to orange color of the solution changed to red
to violet. The mixture was neutralized with IR 120 (H+),
filtered and the filtrate was concentrated under reduced
pressure. Column chromatography of the residue gave
the desired indirubin glycosides as red to violet solids.
4.21. N0-b-D-Glucopyranosylindirubin (b-6c)
Starting with b-4c (160 mg, 0.335 mmol) (reaction time:
4 h), b-6c was isolated (100 mg, 70%) by column chro-
matography (CHCl3/MeOH=20:1) as
a red solid.
Mp=221–222 ꢁC; Rf = 0.5 (CHCl3/MeOH = 5:1). 1H
NMR (500 MHz, DMSO): d = 11.11 (s, 1H, NH); 8.88
4.19. N 0-b-L-Rhamnopyranosylindirubin (b-6a)
4
3
(dd, J4 ;6 ¼ 1:2 Hz, J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.67
0
0
0
0
3
4
Starting with b-4a (300 mg, 0.72 mmol) (reaction time:
4 h), b-6a (224 mg, 77%) was isolated by column chroma-
tography (CHCl3/MeOH = 20:1) as a red solid. Mp 287–
288 ꢁC; Rf = 0.76 (CHCl3/MeOH = 5:1). 1H NMR
(500 MHz, DMSO): d = 10.08 (s, 1H, NH); 8.80 (dd,
(0d0, J4,5 = 7.5 Hz, 1H, H-4); 7.60 (ddd, J4,6 = 1.0 Hz,
3J5,6 = 7.5 Hz, J6,7 = 8.0 Hz, 1H, H-6); 7.43 (d, J6,7
=
¼
3
3
8.0 Hz, 1H, H-7); 7.32 (d0t0, J4 ;6 ¼ 1:2 Hz, J5 ;6
4
3
0
0
0
0
J6 ;7 ¼ 7:9 Hz, 1H, H-60); 7.26 (0d0, J6 ;7 ¼ 7:9 Hz,
3
3
0
0
0
0
1H, H-70); 7.11 (d0t0, J5 ;7 ¼ 1:0 Hz, J4 ;5 ¼ J5 ;6
¼
4
3
3
0
0
0
0
0
0
J4 ;6 ¼ 1:0 Hz, J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.67 (d,
7:9 Hz, 1H, H-50); 7.04 (‘t’, J4,5 = 3J5,6 = 7.5 Hz, 1H,
3
4
3
0
0
0
0
3
3
3
J4,5 = 7.6 Hz, 1H, H-4); 7.64 (d, J6 ;7 ¼ 7:9 Hz, 1H,
H-5); 5.33 (d, J1 ;2 ¼ 9:5 Hz, 1H, H-100); 4.57-4.03 (br
0
0
00 00
4
3
3
H-70); 7.60 (d0t0, J4,6 = 1.0 Hz, J5,6 = 7.6 Hz, J6,7
7.9 Hz, 1H, H-6); 7.42 (d, J6,7 = 7.9 Hz, 1H, H-7); 7.23
=
m, 4H, 4xOH); 3.94 (br, 1H, H-200); 3.75 (dd,
3
J5 ;6a00 ¼ 1:8 Hz, J6a00;6b ¼ 12:0 Hz, 1H, H-6a00); 3.49
3
2
00
00
(d0t0, J4 ;6 ¼ 1:0Hz, J5 ;6 ¼ 7:5 Hz, J6 ;7 ¼ 7:9 Hz,
(dd, J5 ;6b ¼ 6:2 Hz, J6a00;6b ¼ 12:0 Hz, 1H, H-6b00);
4
3
3
3
2
0
0
0
0
0
0
00
00
00
1H, H-60); 7.06-7.02 (m, 2H, H-5, H-50); 5.62 (s, 1H,
3.37 (ddd, J5 ;6a00 ¼ 1:8 Hz, J5 ;6b ¼ 6:2 Hz, J4 ;5
¼
3
3
3
00
00
00 00
00
3
H-100); 5.12 (d, J2 ;OH ¼ 5:0 Hz, 1H, OH ); 4.96 (d,
9:3 Hz, 1H, H-500); 3.35 (‘t’, J2 ;3 ¼ J3 ;4 ¼ 8:8 Hz,
3
3
00
00
00 00
00 00
ð2 Þ
3
3
3
3
J4 ;OH ¼ 5:0 Hz, 1H, OH ); 4.85 (d, J3 ;OH ¼ 6:0 Hz,
1H, H-300); 3.28 (‘t’, J3 ;4 ¼ 8:8 Hz, J4 ;5 ¼ 9:3 Hz,
1H, H-400). 13C NMR (126 MHz, DMSO): d = 188.7
(C-3); 169.1 (C-20); 152.6 (C-7a); 139.7 (C-7a0);
138.9 (C-2); 137.4 (C-6); 129.1 (C-60); 124.7 (C-4);
124.6 (C-40); 122.0 (C-50); 121.7 (C-5); 121.2 (C-3a0);
119.2 (C-3a); 113.7 (C-7); 111.6 (C-70); 105.7 (C-30);
82.0 (C-100); 80.2 (C-500); 77.6 (C-300); 70.1 (C-400);
68.9 (C-200); 61.3 (C-600). MS (EI, 70 eV): m/z (%) =
424 (30) [M+], 262 (100) [indirubin]. HRMS (EI,
70 eV): calcd for C22H20N2O7([M+]): 424.12650; found:
424.12741.
00
00
00
00 00
00 00
ð4 Þ
1H, OH ); 3.86 (m, 1H, H-200); 3.50 (m, 1H, H-300);
00
ð3 Þ
3.41-3.35 (m, 2H, H-400, H-500); 1.27 (d, J5 ;6 ¼ 5:8 Hz,
3
00 00
3H, H-600). 13C NMR (126 MHz, DMSO): d = 188.6
(C-3); 168.5 (C-20); 152.4 (C-7a); 140.9 (C-7a0); 138.8
(C-2); 137.3 (C-6); 128.5 (C-60); 124.6 (C-4); 123.7 (C-
40); 121.6, 121.5 (C-5,50); 120.7 (C-3a0); 119.2 (C-3a);
114.9 (C-70); 113.6 (C-7); 105.5 (C-30); 82.3 (C-100); 75.5
(C-400); 73.3 (C-300); 72.0 (C-200); 71.5 (C-500), 18.2 (C-600).
MS (EI, 70 eV): m/z (%) = 408 (13) [M+], 262 (100)
[indirubin], 234 (35) [indirubin-(C@O)], 147 (22) [M+-
indirubin]. HRMS (ESI): calcd for C22H20N2NaO6
([M+Na]+) 431.12136; found 431.12162.
4.22. N0-b-D-Galactopyranosylindirubin (b-6d)
4.20. 40,60-Dimethyl-10-b-L-rhamnopyranosylindirubin (b-
6b)
Starting with b-4d (200 mg, 0.419 mmol) (reaction time:
3 h), b-6d was isolated (130 mg, 73%) by column chro-
matography (CHCl3/MeOH = 20:1) as a red solid. Mp
275–276 ꢁC; Rf 0.46 (CHCl3/MeOH = 5:1). 1H NMR
(250 MHz, DMSO): d = 11.09 (s, 1H, NH); 8.87 (dd,
Starting with 4,6-dimethyl-1-b-L-rhamnopyranosylisa-
tine (b-4b) (250 mg, 0.56 mmol), b-6b was isolated
(150 mg, 62%) by column chromatography (CHCl3/
EtOH = 30:1 ! 20:1) as a red solid. Mp 210–213 ꢁC;
Rf = 0.79 (CHCl3/EtOH 5:1). 1H NMR (300 MHz,
4
3
J4 ;6 ¼ 1:0 Hz, J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.67 (0d0,
0
0
0
0
4
3J4,5 = 7.5 Hz, 1H, H-4); 7.60 (ddd, J4,6 = 1.1 Hz,
3
3J5,6 = 7.5 Hz, J6,7 = 8.0 Hz, 1H, H-6); 7.48 (0d0,
3
3
3
J6 ;7 ¼ 8:0Hz, 1H, H-70); 7.43 (0d0, J6,7 = 8.0 Hz, 1H,
0
0
DMSO): d = 11.07 (s, 1H, NH); 7.65 (d, J4,5 = 7.5 Hz,
4
3
1H, H-4); 7.58 (d0t0, J4,6 = 1.2 Hz, J5,6 = 7.5 Hz,3J6,7
= 8.0 Hz, 1H, H-6); 7.37 (d, J6,7 = 8.0 Hz, 1H, H-7);
H-7); 7.30 (d0t0,
J4 ;6 ¼ 1:2 Hz,
J5 ;6 ¼ 7:8 Hz,
4
3
0
0
0
0
3
J6 ;7 ¼ 8:0 Hz, 1H, H-60); 7.14–7.00 (m, 2H, H-5, H-
3
0
0
3
7.31 (s, 1H, H-70); 7.03 (‘t’, J5,6 = 7.5 Hz, 1H, H-5);
6.72 (s, 1H, H-50); 5.58 (s, 1H, H-100); 5.16 (d,
50); 5.35 (d, J1 ;2 ¼ 9:1 Hz, 1H, H-100); 5.19–4.69 (br
3
00 00
3
3
00 00
00 00
m, 4H, 4xOH); 4.18 (‘t’, J1 ;2 ¼ J2 ;3 ¼ 9:1 Hz, 1H,
3
3
3
J2 ;OH ¼ 4:8 Hz, 1H, OH ); 4.96 (d, J4 ;OH ¼ 4:9 Hz,
H-200); 3.81 (d, J3 ;4 ¼ 2:8 Hz, 1H, H-400); 3.63-3.50
00
00
00
00 00
ð2 Þ
1H, OH ); 4.80 (d, J3 ;OH ¼ 6:0 Hz, 1H, OH );
(m, 4H, H-300, H-500, H-6a00, H-6b00). 13C NMR
(63 MHz, DMSO): d = 188.7 (C-3); 168.9 (C-20); 152.6
(C-7a); 139.7, 138.9 (C-7a0, C-2); 137.4 (C-6); 129.0
(C-60); 124.7 (C-4); 124.5 (C-40); 122.0 (C-50); 121.7 (C-
5); 121.2, 119.3 (C-3a, C-3a0); 113.8 (C-7); 112.2 (C-
70); 106.0 (C-30); 82.1 (C-100); 78.0, 74.2 (C-300, C-500);
68.5 (C-400); 66.3 (C-200); 61.0 (C-600). MS (EI, 70 eV):
m/z (%) = 424 (8) [M+], 262 (100) [indirubin], 234 (35)
3
00
00
00
ð4 Þ
ð3 Þ
3.84 (m, 1H, H-200); 3.51–3.44 (m, 1H, H-300); 3.35–3.25
(m, 2H, H-400, H-500); 2.29, 2.08 (2s, 6H, 2xCH3); 1.26
(d, J5 ;6 ¼ 5:3 Hz, 3H, H-600). 13C NMR (75 MHz,
3
00 00
DMSO): d = 186.7 (C-3); 169.5 (C-20); 151.4, 141.9,
137.6 (C-2, C-7a0, C-7a); 136.7 (C-6); 136.5, 135.5 (C-
40, C-60); 124.9 (C-50); 124.3 (C-4); 121.5 (C-5); 119.4,
118.3 (C-3a, C-3a0); 113.3 (C-7); 112.7 (C-70); 106.0 (C-

S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
5581
[M+-indirubin-(C@O)]. HRMS (EI): calcd for
J2 ;3 ¼ 3:5 Hz, J3 ;4 ¼ 3:8 Hz, J3 ;OH ¼ 4:0Hz, 1H,
3
3
3
00 00
00 00
00
C22H20N2O7 ([M+]): 424.12650; found: 424.12566.
H-300); 3.96 (dq, J4 ;5 ¼ 2:8Hz, J5 ;6 ¼ 7:0 Hz, 1H, H-
3
3
00 00
00 00
3
3
3
500); 3.61 (d0t0, J4 ;5 ¼ 2:8 Hz, J4 ;OH¼ J3 ;4 ¼ 3:8 Hz,
00 00
00
00 00
4.23. N0-b-D-Mannopyranosylindirubin (b-6e)
1H, H-400); 1.37 (d, 3J5 ;6 ¼ 7:0 Hz, 3H, H-600). 13C NMR
00 00
(75 MHz, DMSO): d = 188.7 (C-3); 169.4 (C-20); 152.6
(C-7a); 140.4 (C-7a0); 138.9 (C-2); 137.4 (C-6); 129.1 (C-
60); 124.7, 124.6 (C-4, C-40); 122.0, 121.7 (C-5, C-50);
121.3, 119.3 (C-3a, C-3a0); 113.7 (C-7); 111.6 (C-70); 106.0
(C-30); 76.1 (C-100); 74.6 (C-500); 72.4 (C-300); 72.4 (C-400);
63.7 (C-200); 16.9 (C-600). MS (EI, 70 eV): m/z (%) = 408
(26) [M+], 262 (100) [indirubin], 234 (24) [indirubin-
(C@O)]. HRMS (EI, 70 eV): calcd for C22H20N2O6
([M+]): 408.13159; found: 408.13061.
Starting with b-4e (190 mg, 0.398 mmol) (reaction time:
3 h), b-6e was isolated (100 mg, 59%) by column chro-
matography (CHCl3/MeOH = 20:1) as a red solid. Mp
1
137–138 ꢁC; Rf = 0.55 (CHCl3/MeOH = 5:1). H NMR
(250 MHz, DMSO): d = 11.09 (s, 1H, NH); 8.80 (dd,
4
3
3
J4 ;6 ¼ 1:0 Hz, J4 ;5 ¼ 7:8 Hz, 1H, H-40); 7.68 (0d0,
0
0
0
0
3
J6 ;7 ¼ 7:9 Hz, 1H, H-70); 7.67 (0d0, J4,5 = 7.5 Hz, 1H,
0
0
H-4); 7.59 (ddd, 4J4,6 = 1.2 Hz, 3J5,6 = 7.5 Hz,
3J6,7 = 8.0 Hz, 1H, H-6); 7.42 (0d0, J6,7 = 8.0 Hz, 1H,
3
H-7); 7.22 (d0t0,
J4 ;6 ¼ 1:2 Hz,
J5 ;6 ¼ 7:8 Hz,
4.26. 5-Chloro-10-b-L-rhamnopyranosylindirubin (b-6f)
4
3
0
0
0
0
3
J6 ;7 ¼ 8:0 Hz, 1H, H-60); 7.05–7.00 (m, 2H, H-5, H-
0
0
50); 5.63 (d, J1 ;2 ¼ 1:0 Hz, 1H, H-100); 5.13 (d,
Starting with b-7f (162 mg, 0.28 mmol), b-6f was iso-
lated (91 mg, 72%) by column chromatography
(CHCl3/EtOH = 20:1 ! 10:1) as a red solid. Mp 285–
286 ꢁC; Rf = 0.52 (CHCl3 /EtOH = 5:1). 1H NMR
3
00 00
3J = 4.8 Hz, 1H, OH ); 4.94 (d, 3J = 4.5 Hz, 1H),
00
ð2 Þ
4.89 (d, 3J = 4.5 Hz, 1H) (OH , OH ); 4.59 (t,
00
00
ð3 Þ
ð4 Þ
3J = 5.5 Hz, 1H, OH ); 3.88–3.85 (m, 1H, H-200);
00
ð6 Þ
3.82–3.78 (m, 1H, H-6a00); 3.59–3.50 (m, 3H, H-300, H-
400, H-6b00); 3.40-3.28 (m, 1H, H-500). 13C NMR
(63 MHz, DMSO): d = 188.6 (C-3); 168.6 (C-20); 152.5
(C-7a); 141.1 (C-7a0); 138.9 (C-2); 137.4, 128.7 (C-6,
C-60); 124.7, 123.7 (C-4, C-40); 121.7, 121.6 (C-5, C-500);
120.8, 119.3 (C-3a, C-3a0); 115.4, 113.7 (C-7, C-7 );
105.7 (C-30); 82.4 (C-100); 81.1 (C-500); 73.7, 66.6 (C-300,
C-400); 72.0 (C-200); 61.5 (C-600). MS (EI, 70 eV): m/z
(%) = 424 (24) [M+], 262 (100) [indirubin], 234 (28)
[M+-indirubin-(C@O)]. HRMS (EI, 70 eV): calcd for
C22H20N2O7 ([M+]): 424.12650; found: 424.12557.
(250 MHz, DMSO): d = 11.17 (s, 1H, NH); 8.78 (0d0,
3
J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.69–7.60 (m, 3H, H-4, H-
0
0
3
6, H-70); 7.45 (0d0, J6,7 = 8.5 Hz, 1H, H-7); 7.24 (d0t0,
4
3
3
0
0
0
0
0
0
J4 ;6 ¼ 1:2 Hz, J5 ;6 ¼ 7:5 Hz, J6 ;7 ¼ 7:9 Hz 1H, H-
J6 ;7 ¼ 7:9 Hz, 1H, H-50); 5.62 (br s, 1H, H-100); 5.14
4
3
60); 7.03 (d0t0,
J5 ;7 ¼ 1:0 Hz,
J5 ;6 ¼ 7:5 Hz,
0
0
0
0
3
0
0
(d, 3J = 4.8 Hz, 1H, OH); 4.99 (d, 3J = 4.8 Hz, 1H,
OH); 4.89 (d, 3J = 6.0 Hz, 1H, OH); 3.88–3.83 (m,
3
1H, H-200); 3.52-3.35 (m, J5 ;6 ¼ 5:3 Hz, 3H, H-300,
00 00
H-400, H-500); 1.27 (d, J5 ;6 ¼ 5:3 Hz, 3H, H-600). 13C
3
00 00
NMR (63 MHz, DMSO): d = 187.5 (C-3); 168.4 (C-
20); 151.1 (C-7a); 141.3 (C-7a0); 138.6 (C-2); 136.7 (C-
6); 129.0 (C-60); 125.7 (C-5); 123.9 (C-4); 123.9 (C-40);
121.6 (C-50); 120.7, 120.5 (C-3a0, C-3a); 115.4, 115.1
(C-7, C-70); 106.5 (C-30); 82.4 (C-100); 75.6 (C-400);
73.4, 72.1 (C-200, C-300); 71.6 (C-500); 18.3 (C-600). MS
(EI, 70 eV): m/z (%) = 444 (6) [M+, 37Cl]; 442 (17)
[M+, 35Cl]; 298 (35) [5- chloroindirubin, 37Cl]; 296
(100) [5- chloroindirubin, 35Cl]; 209 (9) [5-chloroindiru-
bin-C 6H4N+2H]; 207 (36) [5-chloroindirubin-C 6H4
N + 2H]; 129 (27) [4-chloroaniline, 37Cl]; 127 (7) [4-
chloroaniline, 35Cl]; 93 (13) [aniline]. HRMS (EI,
70 eV): calcd for C22H19ClN2O6 ([M+]): 442.09079;
found: 442.09120.
4.24. General procedure 2 for the synthesis of indirubin-
N0-glycosides
To a methanol solution of acetylated indirubin-N-glyco-
side 7 was added KOtBu (0.02 equiv per acetyl group).
The mixture was stirred for 10–12 h at 20 ꢁC. The mix-
ture was neutralized by addition of IR 120 (H+) and was
subsequently filtered. The filtrate was concentrated un-
der reduced pressure. The residue was purified by col-
umn chromatography to give the desired indirubine
glycoside as a red to violet solid.
4.25. N 0-a-L-Rhamnopyranosylindirubin (a-6a)
4.27. 5-Chloro-10-a-L-rhamnopyranosylindirubin (a-6f)
Starting with a-7a (70 mg, 0.13 mmol), a-6a was isolated
(48 mg, 90%) as a red solid by column chromatography
(CHCl3 /MeOH = 30:1 ! 20:1). Mp 287–288 ꢁC;
Starting with a-7f (110 mg, 0.19 mmol), a-6f was isolated
(60 mg, 70%) by column chromatography (CHCl3/
EtOH = 20:1 ! 10:1) as a red solid. Mp=247-250 ꢁC;
Rf = 0.49 (CHCl3 /EtOH = 5:1). 1H NMR (300 MHz,
DMSO): d = 11.20 (s, 1H, NH); 8.84 (0d0,
1
Rf = 0.74 (CHCl3/MeOH = 5:1). H NMR (250 MHz,
DMSO): d = 11.10 (s, 1H, NH); 8.87 (0d0,
3
3
J4 ;5 ¼ 7:9 Hz, 1H, H-40); 7.67 (d, J4,5 = 7.5 Hz, 1 H,
0
0
4
3
3
4
H-4); 7.59 (d0t0, J4,6 = 1.2 Hz, J5,6 = 7.5 Hz, J6,7
=
J4 ;5 ¼ 7:8 Hz, 1H, H-40); 7.68 (d, J4,6 = 2.0 Hz, 1H,
3
0
0
3
4
H-4); 7.63 (dd, J4,6 = 2.0 Hz, J6,7 = 8.5 Hz, 1H, H-6);
3
7.9 Hz, 1H, H-6); 7.41 (0d0, J6,7 = 7.9 Hz, 1H, H-7);
7.38 (dd, 4J5 ;7 ¼ 1:2 Hz, 3J6 ;7 ¼ 8:0 Hz, 1H, H-70); 7.31
7.45 (d, 3J6,7 = 8.5 Hz, 1H, H-7); 7.40-7.29 (m, 2H, H-60,
0
0
0
0
4
3
3
(ddd, J4 ;6 ¼ 1:2 Hz, J5 ;6 ¼ 7:6 Hz, J6 ;7 ¼ 7:9 Hz,
H-70); 7.11 (d0t0,3J5 ;6 ¼ 7:2 Hz, 3J4 ;5 ¼ 7:8 Hz , 1H, H-
0
0
0
0
0
0
0
0
0
0
1H, H-60); 7.10 (d0t0,4J5 ;7 ¼ 1:2 Hz , J5 ;6 ¼ 7:6 Hz,
50); 5.82 (d, J1 ;2 ¼ 8:5 Hz, 1H, H-100); 5.15 (d,
3
3
0
0
0
0
00 00
4
J4 ;5 ¼ 7:9 Hz, 1H, H-50); 7.03 (d0t0, J5,7 = 0.8 Hz,
J4 ;OH ¼ 3:8 Hz, 1H, OH ); 5.03 (d, 3J2 ;OH ¼ 4:0 Hz,
3
3
0
0
00
00
00
ð4 Þ
J4,5 = 3J5,6 = 7.5 Hz, 1H, H-5); 5.83 (d, J1 ;2 ¼ 8:5 Hz,
1H, OH ); 4.93 (d, J2 ;OH ¼ 6:3 Hz, 1H, OH ); 4.51
3
3
3
00 00
00
00
00
ð3 Þ
ð2 Þ
1H, H-100); 5.16 (d, J4 ;OH ¼ 3:8 Hz, 1H, OH ); 5.03
(ddd, J2 ;3 ¼ 3:4 Hz, J2 ;OH ¼ 6:3Hz, J1 ;2 ¼ 8:5 Hz,
3
3
3
3
00
00
00 00
00
00 00
ð4 Þ
4.90
00 00
3
(d,
J3 ;OH ¼ 4:0 Hz,
1H,
00
OH );
ð3 Þ
(d,
1H, H-200); 4.02-3.92 (m, 2H, H, H-300, H-500); 3.60 (m,
00
00
J2 ;OH ¼ 6:3 Hz, 1H, OH ); 4.52 (ddd, 3J2 ;3 ¼3:5 Hz,
1H, H-400); 1.37 (d, J5 ;6 ¼ 7:0 Hz, 3H, H-600). 13C
3
3
00
00 00
ð2 Þ
J2 ;OH ¼ 6:3Hz, J1 ;2 ¼ 8:5 Hz, 1H, H-200); 4.00 (0q0,
NMR (75 MHz, DMSO): d = 187.6 (C-3); 169.2 (C-20);
3
3
00
00 00

5582
S. Libnow et al. / Bioorg. Med. Chem. 16 (2008) 5570–5583
151.2 (C-7a); 140.6, 138.6 (C-2, C-7a0); 136.7 (C-6); 129.5
(C-60); 124.7, 123.9 (C-4, C-40); 122.1 (C-50); 125.7 (C-5);
121.1, 120.5 (C-3a0, C-3a); 115.5, 111.7 (C-7, C-70); 107.0
(C-30); 76.1 (C-100); 74.6 (C-500); 72.4 (C-300); 72.4 (C-400);
63.7 (C-200); 16.9 (C-600). MS (CI, isobutane): m/z
(%)=445 (41) [M++H; 37Cl]; 443 (77) [M++H; 35Cl].
HRMS (CI, isobutane): calcd for C22H19ClN2O6 ([M+]):
443.10044; found: 443.10063.
500); 18.3 (C-600). MS (CI, Isobutane): m/z (%) = 424
(100) [M++H]. HRMS (EI, 70 eV): calcd. for
C22H21N3O6 ([M+]): 423.14249; found: 423.14240.
Acknowledgments
Financial support by the Deutsche Forschungsgemeins-
chaft (LA 1301/9-1) and by the State of Mecklenburg-
Vorpommern (scholarship for S.L. and Exzellenzfo¨rder-
programm UR 07 068) is gratefully acknowledged.
4.28. 10-(200,300,400-Tri-O-acetyl-b-L-rhamnopyrano-
syl)indirubin-3-monoxim (b-8a)
To a pyridine solution of b-7a hydroxylamine hydrochlo-
ride (2.0 equiv) was added. The mixture was stirred for 7 h
at 90 ꢁC. The solvent was removed under reduced
pressure and the residue was purified by column chro-
matography (heptane/EtOAc = 5:1 ! 1:1). Starting
with b-7a (200 mg, 0.37 mmol), b-8a was isolated as a
red solid(118 mg, 57%). Mp 94–96 ꢁC; Rf = 0.52 (n-hep-
tane/EtOAc = 1:3). 1H NMR (300 MHz, CDCl3):
d = 11.43 (s, 1H, NH); 9.53 (br s, 1H, OH); 8.43 (dd,
References and notes
1. Indirubin, the red shade of indigo in editions Life in
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4
3
J4 ;6 ¼ 1:1 Hz, J4 ;5 ¼ 7:9 Hz, 1H, H-40); 8.21 (0d0,
0
0
0
0
3
3
J4,5 = 7.7 Hz, 1H, H-4); 7.57 (0d0 J6 ;7 ¼ 7:9 Hz, 1H,
0
0
H-70); 7.29–7.07 (2 dt, 2H, H-50, H-60); 6.98–6.82 (m,
3
2H, H-5, H-6); 6.83 (0d0, J6,7 = 7.9 Hz, 1H, H-7); 6.06
(d, J1 ;2 ¼ 1:5 Hz, 1H, H-100); 5.67 (dd, J1 ;2
¼
3
3
00 00
3
00 00
`
4. (a) Beauchard, A.; Ferandin, Y.; Frere, S.; Lozach, O.;
Blairvacq, M.; Meijer, L.; Thiery, V.; Besson, T. Bioorg.
1:5 Hz, J2 ;3 ¼ 3:2 Hz, 1H, H-200); 5.39–5.22 (m, 2H,
00 00
´
H-300, H-400); 3.80 (dq, J5 ;6 ¼ 6:2 Hz, J4 ;5 ¼ 9:0 Hz,
3
3
00 00
00 00
Med. Chem. 2006, 14, 6434; (b) Ferandin, Y.; Bettayeb, K.;
Kritsanida, M.; Lozach, O.; Polychronopoulos, P.; Magia-
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49, 4638; (c) Ribas, J.; Bettayeb, K.; Ferandin, Y.;
1H, H-500); 2.11, 2.00, 1.85 (3s, 9H, 3xC(O)CH3); 1.38
(d, J5 ;6 ¼ 6:2 Hz, 3H, H-600). 13C NMR (75 MHz,
3
00 00
CDCl3): d = 170.6, 170.1, 169.8 (3xC(O)CH3); 169.2 (C-
20); 152.6, 146.6, 144.4, 136.8 (C-2, C-3, C-7a, C-7a0);
132.2, 129.2, 125.3, 123.2 (C-4, C-40, C-6, C-60); 122.5,
116.9 (C-3a, C-3a0); 121.9, 121.7 (C-5, C-50); 112.5, 110.3
(C-7, C-70); 98.1 (C-30); 80.5 (C-100); 74.0, 70.8, 70.7, 70.5
(C-200, C-300, C-400, C-500); 20.9, 20.8, 20.6 (3xC(O) CH3);
17.7 (C-600). MS (EI, 70 eV): m/z (%)=549 (7) [M+]; 277
(7) [indirubin-3-monoxim]; 153 (11) [M+-aglycon-
2HOAc]; 135 (30). HRMS (EI, 70 eV): calcd for
C28H27N3O9 ([M+]): 549.17418; found: 549.17541.
´
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4.29.10-b-L-Rhamnopyranosylindirubin-3-monoxim (b-9a)
The synthesis of b-9a was carried out following the pro-
cedure as given for the preparation of a-6a. Starting
with b-8a (100 mg, 0.18 mmol), b-9a was isolated
(53 mg, 69%) by column chromatography (CHCl3/
MeOH = 20:1 ! 10:1) as a red solid. Mp 295–297
ꢁC; Rf = 0.7 (CHCl3 /MeOH = 5:1). 1H NMR
(250 MHz, DMSO)22: d = 11.77 (s, 1H, NH); 8.71 (dd,
4
3
J4 ;6 ¼ 1:2 Hz, J4 ;5 ¼ 7:9 Hz, 1H, H-40); 8.25 (d,
0
0
0
0
3
4
0
0
J4,5 = 7.6 Hz, 1H, H-4); 7.62 (dd, J5 ;7 ¼ 1:0 Hz,
3
J6 ;7 ¼ 8:0 Hz, 1H, H-70); 7.42-7.39 (m, 2H), 7.14-6.92
0
0
(m, 3H) (H-5, H-50, H-6, H-60, H-7); 5.66 (s, 1H, H-
100); 5.20 (d, 3J = 5.2 Hz, 1H, OH); 4.97 (br s, 1H,
OH); 4.85 (br s, 1H, OH); 3.89 (br s, 1H, H-200); 3.60-
3.40 (m, 3H, H-300, H-400, H-500); 1.27 (d,
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3
00 00
DMSO): d = 168.5 (C-20); 151.5, 146.1, 144.7, 138.4
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