Synthesis and cytotoxicity of novel indirubin-5-carboxamides

Article abstract of DOI:10.1016/j.bmc.2010.04.066

Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility. We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substituents with basic centers. Quaternization or protonation of these alkylamino substituents provided indirubins with significantly improved solubility without loss of bioactivity.

Full text of DOI:10.1016/j.bmc.2010.04.066

Bioorganic & Medicinal Chemistry 18 (2010) 4509–4515
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxicity of novel indirubin-5-carboxamides
*
Xinlai Cheng, Paul Rasqué, Sandra Vatter, Karl-Heinz Merz , Gerhard Eisenbrand
Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Erwin-Schrödinger-Str. 52, PO Box 3049, 67663 Kaiserslautern, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and
of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility.
We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substit-
uents with basic centers. Quaternization or protonation of these alkylamino substituents provided indiru-
bins with significantly improved solubility without loss of bioactivity.
Received 12 November 2009
Revised 19 April 2010
Accepted 21 April 2010
Available online 28 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Indirubin
Chemical synthesis
Antitumor agents
Protein kinase inhibitors
1. Introduction
2. Results and discussion
Dangui Luhui Wan (or named Danggui Longhui Wan) is a recipe
found by Zhengheng Zhu in 14811 that has been used in Traditional
Chinese Medicine (TCM) for treatment of various diseases includ-
ing Chronic Myelogenous Leukemia (CML).2a During the 7th decade
of the last century, indirubin (1, Fig. 1) has been shown to act as an
active ingredient thereof.2b In 1999, indirubins were found to inhi-
bit cyclin dependent kinases (CDKs) binding to the ATP pocket.3
Indirubin became a new lead structure for targeting CDKs,3 GSK-
3b,4 and several tyrosine kinases including c-Src.5 However, due
to strong intermolecular and intramolecular forces in their crystal
lattice,6 indirubins are extremely poorly soluble in nearly all sol-
vents resulting in low bioavailability.7 For improvement of their
aqueous solubility some pharmaceutical formulations8 and chem-
ical modifications9 have been explored.
The general synthesis of indirubin has already been developed
in 1881, based on the basic condensation of isatin and indoxyl.11a
It was modified later in 1969 by replacing indoxyl with the more
stable 3-indoxyl acetate (3).11b
For the synthesis of indirubin-5-carboxamides, we first had to
prepare 5-carboxyisatin (2) serving as a coupling partner for 3-in-
doxyl acetate (Scheme 1). The synthesis of 2 started by converting
p-aminobenzoic acid (10) into 4-carboxyisonitrosoacetanilide (11)
using the Sandmeyer method.12 Cyclization of 11 with concen-
trated sulfuric acid afforded a mixture of 2 and 5-carboxyisatoxime
(12),13 that was difficult to separate. To obtain pure 2 we com-
pletely converted this mixture with hydroxylamin hydrochloride
into 12. Reduction of 5-carboxyisatoxime with Sn/HCl as reducing
Here we focus on chemical modifications of substituents. Struc-
ture–activity studies suggest that substituents in 5- and/or 30-posi-
tion, which face the ribose/triphosphate canal (Fig. 1), are eligible
to enhance solubility and bioactivity. Thus, we succeeded in syn-
thesizing indirubin-30-oxime ethers with potent inhibitory activity
and improved water solubility that carry a sugar moiety linked by a
C2-spacer to the oxime.10a Previously, several 6-substituted indiru-
bin-30-oxime ethers with basic substituents targeting GSK-3b have
been synthesized.10b,c We introduced amide substituents in 5-posi-
tion with basic functional groups for improvement of solubility and
bioactivity.
ribose/triphosphate canal
5
6
O
4
4'
7
3'
hydrophobic pocket
3
5'
6'
open to solvent
2'
N
1'
N
1
2
O
H
7'
H
hinge region
* Corresponding author. Tel.: +49 631 205 2976; fax: +49 631 205 3085.
E-mail address: khmerz@rhrk.uni-kl.de (K.-H. Merz).
Figure 1. Outline of indirubin (1) bound to the ATP-binding pocket (based on the
crystal structure of CDK2 in complex with indirubin-5-sulfonic acid).3
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.066

4510
X. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 4509–4515
OH
N
OH
O
N
O
HOOC
HOOC
HOOC
HOOC
a
b
+
O
N
N
H
NH2
N
H
O
H
11
2
12
10
c
OH
O
Cl
NH3
O
N
d
e
HOOC
HOOC
HOOC
O
O
N
N
N
H
H
H
12
13
2
Scheme 1. Synthesis of 5-carboxyisatin (2). Reactants and conditions: (a) chloral hydrate, H2NOHꢀHCl, Na2SO4; (b) H2SO4 concd.,
D
T; (c) H2NOHꢀHCl; (d) Sn/HCl, H2S; (e)
FeCl3.
N
agent provided 3-amino oxindole hydrochloride (13), which was
quantitatively oxidized by FeCl3 to give 2.14
N
N
F
N
b
a
a
For esterifications and amidations the 5-carboxyl group of 2
was activated by pentafluorophenyl ester formation. Two ways
proved practicable: Activation of 2 before coupling with 3-indoxyl
acetate (Scheme 2, d–f) or activating 5-carboxyindirubin (4,
Scheme 2, a–c). Activating 5-carboxyindirubin yielded the more
stable pentafluorophenyl indirubin-5-carboxylate (6), directly en-
abling amidations with different amino compounds.
Synthesis of 1-(4-aminophenyl)-4-methylpiperazine (15) was
achieved by nucleophilic aromatic substitution of 1-fluoro-4-nitro-
benzol with 1-methylpiperazine and subsequent reduction with
Pd/C (Scheme 3, I).15 1-(2-aminoethyl)-4-methyl-piperazine 19
was prepared by hydrogenation of 2-(4-methylpiperazino) aceto-
nitrile (17) with lithium aluminium hydride. Compound 17 was
obtained by nucleophilic substitution of chloroacetonitrile with
1-methylpiperazine (Scheme 3, II).16
I
NO2
NH2
NO2
14
15
N
N
N
N
N
b
II
N
H
N
NH2
19
17
Scheme 3. I: Synthesis of 15. Reactants and conditions: (a) NaOAc, EtOH, 1-
methylpiperazine; (b) H2, Pd/C, MeOH; II: Synthesis of 19. Reactants and
conditions: (a) chloroacetonitrile, K2CO3, acetonitrile; (b) LiAlH4, dry Et2O.
R1
To further improve solubility, selected indirubin-5-carboxam-
ides were converted into the corresponding 30-oximes using
hydroxylamine hydrochloride in pyridine. Two of the oximes were
alkylated with substituted alkyl halogenides to produce 5-carbox-
amido-indirubin-30-oxime ethers (9, Scheme 4). Tetra-o-acetyl-(2-
R2
O
R1
R1
O
O
HO
O
O
a
b
N
N
NH
N
NH
NH
N
N
H
O
H
H
O
O
bromoethyl)-b-D-glucopyranose, used for preparing 9b, was syn-
8
7
9
thesized according to the Königs–Knorr method.17
Scheme 4. Synthesis of 30-hydroxyimino-indirubin-5-carboxamides (8) and 5-
carboxamido-indirubin-30-oxime ethers (9) (R1 and R2 see Table 2). Reactants and
conditions: (a) H2NOHꢀHCl, pyridine; (b) 1,1,3,3-tetramethylguanidine, R2–Cl or R2–
Br.
Some indirubin-5-carboxamides with basic centre placed in the
amide substituent were transformed into their quarternary alkyl
ammonium salts or hydrochlorides (18) (Scheme 5).
Inhibition of tumor cell proliferation by indirubin compounds
was evaluated in human large cell lung cancer cells (LXFL529L
cells) using the SRB assay.18 Water solubility was determined spec-
trophotometrically as reported previously.19 Chemical structures,
IC50 values of tumor cell growth inhibition and solubility data are
listed in Tables 1–3.
More than 50% of the newly synthesized indirubin-5-carboxam-
ides 7a–7k effected potent tumor cell growth inhibition (IC50
<5
lM, Table 1). From these, 50% show adequate to excellent
(>500 mg/L) water solubility. Within that group, compound 7a,
O
OH
HOOC
O PFP
NH
R1
O
O
O
O
N
O
OAc
O
O
H
NH
NH
b
c
a
N
N
N
N
H
H
H
H
O
O
6
O
7
3
4
f
O
O
O
O
O
HOOC
R1
d
e
PFP
O
O
O
O
O
N
N
N
H
H
H
2
16
5
Scheme 2. Synthesis of indirubin-5-carboxamides (7). Reactants and conditions: (a) Na2CO3, MeOH; (b) pentafluorophenyl trifluoroacetate (PFP-trifluoroacetate), DMAP,
pyridine, DMF; (c) R1–H (R1 see Table 1), DMAP, dioxane; (d) PFP-trifluoroacetate, DMAP, pyridine, DMF; (e) R1–H (R1 see Table 1), DMAP, DMF; (f) Na2CO3, MeOH.

X. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 4509–4515
4511
Table 2
R3
O
R1
O
Structure, water solubility and inhibitory activity on tumor cell proliferation of 30-
hydroxyimino-indirubin-5-carboxamides (8) and 30-(subst)-alkoxyimino-indirubin-
5-carboxamides (9)
a/b/c
O
O
NH
NH
R2
R1
N
N
O
H
H
O
O
O
18
7
N
Scheme 5. Conversion of indirubin-5-carboxylic acid alkylaminoalkyl-amides (7)
into corresponding ammonium salts 18. Reactants and conditions: (a) HCl/THF; (b)
MeI/MeOH; (c) dimethyl sulfate, xylene, nitrobenzene. R3 see Table 3.
NH
N
H
O
Compds R1
R2
H
Solubility mg/L IC50, lM
HN
N
N
8a
8b
8c
8d
1600
523
4.2
5.9
4.5
2.2
Table 1
Structure, water solubility and inhibitory activity on tumor cell proliferation of
indirubin-5-carboxamides (7)
OH
H
H
H
2
N
R1
O
H
179
N
OH
N
O
N
6400
NH
N
H
O
OH
OH
O
9a
21
43
OH
Glc
R1
2
N
H
N
Compds
Solubility mg/L
41
IC50
7
, lM
4
OH
9b
>380
>10
N
HN
N
N
7a
520
0.54
Glc: b-D-glucopyranoside.
OH OH
H
N
7b
21.6
> 30
OH
OH OH
N
Table 3
7c
55.5
> 10
HN
Water solubility and inhibitory activity on tumor cell proliferation of alkylammonium
indirubin-5-carboxamide salts (18)
HN
HN
N
7d
7e
580
700
1.2
7.8
R3
O
N
N
O
OH
7f
6.8
9.5
13
4.7
9.2
2.3
5.4
NH
N
2
N
H
O
H
7g
7h
7i
N
Compds
R3
Solubility mg/L
>2000
IC50
6
, lM
OH
N
H
N
HN
N
N
Cl
H
18a
N
35.4
H
N
N
18b
18c
53,000
1600
8.6
5.7
N
CH3SO4
I
7j
1000
4.2
3.9
3.2
N
N
N
N
7k
N
NH
18d
18e
2450
2.5
3.5
N
N
N
N
I
60,000
CH3SO4
carrying a methylpiperazino-phenylamino substituent attached to
5-carboxyindirubin, with a water solubility of 520 mg/L, was the
most potent with an IC50 value of 0.54 lM. Replacing the methyl-
piperazine moiety by dimethylamine (7d) resulted in a slight
reduction of inhibitory activity as well as a slight increase of solu-
bility. Replacement of the phenyl moiety of 7a by an C2-spacer (7e)
resulted in moderately enhanced solubility but markedly attenu-
ated antitumor activity. The attachment of 30-aminopyridine (7c)
or aminosorbitol (7b) to 5-carboxyindirubin resulted in abrogation
of in vitro cell growth. Indirubins with hydroxylated alkylamines
(7f and 7g) linked to the 5-carboxy group display decreased inhib-
itory activity and water solubility as compared to alkylaminoalkyl-
amines (7h and 7i). The methylpiperazino substituent (7j) led to a
greater increase in water solubility as compared to the piperazino
substituent (7k).
inhibitory activity (9a and 9b), presumably due to the additional
steric crowding, counteracting binding affinity into ATP-binding
sites of kinases driving tumor cell proliferation. Analogous effects
have been noticed for indirubin-5-sulfonamides.20
Quaternization of amino substituents (Scheme 5) caused a dras-
tic enhancement of water solubility, while inhibitory activity on
cell proliferation, as expressed by IC50 values, mostly was pre-
served (Table 3).
To gain insight into the mechanism of action of indirubin-5-
carboxamides, inhibitory activity of 7a was tested with 30 differ-
ent protein kinases at two concentrations (ProQinase, Freiburg,
Germany). The table of inhibition data is given in supporting
Transformation of the indirubin-5-carboxamides into the corre-
sponding 30-oximes generally brought about elevation of water
solubility, and maintaining antiproliferative activity (Table 2).
However, reaction of the oximes to oxime ethers abrogates growth
information. At 10 lM, the residual activity of 16 kinases was
<50%, including ABL, CDKs (CDK1/CyclinB1, CDK2/CyclinA/E,
CDK4/CyclinD1 and CDK5/p35), GSK-3b as well as several recep-
tor tyrosine kinases, for example, FGR, EGFR and FLT3 (listed in

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X. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 4509–4515
supporting information). At 1 lM, only the residual activity of
GSK3-b was found to be <50%. Compared to indirubins with
5. Experimental
5.1. Reagents
potent antiproliferative activity and moderate water solubility re-
ported earlier,10a such as 5-methoxyindirubin-30-(2-b-
D
-glucopyr-
anosylethyl)-oxime ether (E729) with a water solubility of 42 mg/
L and an IC50 value of 0.5 M for growth inhibition of LXFL529L
Solvents and reagents obtained from commercial suppliers
were at least of reagent grade and were distilled or dried according
to prevailing methods prior to use, if necessary. The syntheses
were done under argon atmosphere, when required. Argon 4.8
was purchased from Air Liquide and was dried over phosphorus
pentoxide. For monitoring the reactions, Alugram SIL G/UV254
sheets for TLC (Macherey & Nagel) were used. Column chromatog-
raphy was accomplished using Silica Gel 60 (Macherey & Nagel,
0.063–0.200 mm), for flash chromatography Silica Gel 60 (Mache-
rey & Nagel, 0.040–0.063 mm) was used.
l
tumor cells), the overall kinase inhibitory activity of 7a was found
inferior, especially with respect to inhibition of CDKs and receptor
(VEGFRs) and nonreceptor (Src) tyrosine kinases, whereas GSK-3b
inhibitory activity (IC50: 0.1 0.01 lM, unpublished result) was
retained. Still the antiproliferative activity was quite similar, pos-
sibly a reflection of retained GSK-3b inhibition or of other as yet
undiscovered mechanisms accounting for antiproliferative activity
of 7a. Although the role of GSK-3b inhibition for antiproliferative
activity is not yet clear, potent and selective GSK-3b inhibitors
have been found to have moderately antiproliferative activity.10c
Of note, the parent compound, indirubin (E211), was markedly
inferior not only with respect to its extremely poor water solubil-
ity but also showing only moderate antiproliferative effectiveness
5.2. Analytical methods
1H and 13C NMR spectra were recorded on a Bruker AMX-400
(1H NMR: 400 MHz, 13C NMR: 100 MHz) or on a Bruker AMX-600
(1H NMR: 600 MHz, 13C NMR: 150 MHz). Chemical shifts are re-
ported in ppm from tetramethylsilane with solvent as the internal
standard (1H CDCl3: d 7.26; 13C CDCl3: d 77.0; 1H DMSO-d6: d 2.49;
13C DMSO-d6: d 39.5).
(IC50
(>2
:
l
10
lM, LXFL529L) and low CDK inhibitory potential
M).10a
3. Conclusions
Mass spectra (MS) were recorded on a Bruker ApexQe hybrid
9.4 T FT-ICR (ESI) or JEOL JMS-700 sector field (EI, FAB). MALDI-
TOF mass spectra were recorded on a Bruker BIFLEX III.
Elemental analyses were performed on an Element Analyzer
Perkin–Elmer EA 240 or 2400 CHN at the University of Kaiserslau-
tern, Department of Chemistry.
The synthesis of novel indirubin derivatives aimed at improv-
ing water solubility and antitumor activity is described. Indirubin-
5-carboxamides are potent inhibitors of tumor cell proliferation
with IC50-values within or beneath the low lM range. Quaterniza-
tion or protonation of amides bearing an alkyl amino group in the
amide substituent provides better water soluble indirubin com-
pounds with potent antiproliferative activity on human tumor
cells.
5.3. Intermediates
As yet unpublished NMR data of the intermediates 2, 11–15, 17
and 19, synthesized following literature methods, are given in Sup-
plementary data.
4. Materials and methods
4.1. Cell culture
5.4. Pentafluorophenyl isatin-5-carboxylate (5)
LXFL529L cells were grown at 37 °C in RPMI 1640 (Invitrogen,
Karlsruhe, Germany), supplemented with 10% heat-inactivated fe-
tal bovine serum, penicillin (100 units/mL), streptomycin (100 mg/
mL), in a humidified atmosphere of 5% CO2. The cells were tested
routinely for absence of mycoplasm contamination.16
Under argon atmosphere, pentafluorophenyl trifluoroacetate
(1.08 g, 3.86 mmol) and pyridine (324 mg, 4.10 mmol) were added
to a solution of 2 (350 mg, 1.83 mmol) in dry DMF. The mixture
was stirred for 5.5 h at room temperature, diluted with ethyl ace-
tate and washed with brine. After removal of the solvent the resi-
due was dried in vacuo to yield 5 (635 mg, 97%). 1H NMR
(400 MHz; DMSO-d6): 7.13 (d, 1H, 3J = 8.2 Hz), 8.14 (d, 1H,
4J = 1.6 Hz), 8.35 (dd, 1H, 3J = 8.2 Hz, 4J = 1.6 Hz), 11.61 (s, 1H);
13C-{1H} NMR (150 MHz; DMSO-d6): 112.8, 119.6, 126.0, 136.2,
138.7, 139.5, 140.0, 141.94, 155.6, 159.6, 161.2, 182.6. Anal. Calcd
for C15H4F5NO4: C, 50.44; H, 1.13; N, 3.92. Found: C, 50.00; H, 1.30;
N, 3.74.
4.2. Sulforhodamine B (SRB) assay
Effects on cell growth were determined according to the meth-
od of Skehan et al. with slight modifications. Briefly, cells were
seeded into 24-well plates and allowed to grow for 24 h before
treatment. Thereafter, cells were incubated with the respective
drug for 3 days in serum containing medium. Incubation was
stopped by addition of trichloroacetic acid (50% solution). After
1 h at 4 °C, plates were washed four times with water. The dried
plates were stained with a 0.4% solution of sulforhodamine B.
The dye was eluted with Tris-buffer (10 mM, pH 10.5) and quanti-
fied photometrically at 570 nm. Cytotoxicity was determined as
percent survival, determined by the number of treated over control
cells ꢁ100 (% T/C).18,19
5.5. Isatin-5-carboxylic acid diethanolamide (16)
Under argon atmosphere a mixture of 5 (300 mg, 0.84 mmol),
diethanolamine (170 mg, 1.24 mmol) pyridine (10 mL) and aceto-
nitrile (20 mL) was stirred at room temperature for 2.5 h. After re-
moval of the solvents the residue was purified by column
chromatography on silica gel using ethyl acetate/methanol (4:1)
as eluent to provide 16 as a orange solid (66.2 mg, 0.25 mmol,
30%). 1H NMR (400 MHz; DMSO-d6): 3.32–3.58 (m, 8H), 4.81 (s,
2H), 6.93 (d, 1H, 3J = 7.8 Hz), 7.57 (s, 1H), 7.62 (d, 1H, 3J = 7.8 Hz),
11.17 (s, 1H); 13C-{1H} NMR (150 MHz; DMSO-d6): 47.4, 51.7,
111.9, 117.3, 123.4, 131.6, 137.2, 150.9, 159.4, 169.7, 184.0. Anal.
Calcd for C13H14N2O5: C, 56.11; H, 5.07; N, 10.07. Found: C,
55.98; H, 4.86; N, 10.00.
4.3. Solubility in water
Standard solutions of the indirubin compounds were prepared
in ethanol and maxima of absorbance were defined. Solubility
was determined spectrophotometrically at maximum of absor-
bance by calibration method.19

X. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 4509–4515
4513
5.6. Indirubin-5-carboxylic acid (4)
11.12 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 42.8, 63.3,
69.4, 71.5, 71.9, 72.3, 105.7, 108.8, 113.6, 119.6, 121.3, 121.6
(C50), 124.4, 124.5, 128.1, 128.2, 137.2, 138.9, 142.9, 152.5, 167.0,
171.2, 188.6. Anal. Calcd for C23H23N3O8ꢀH2O: C, 56.67; H, 5.17;
N, 8.62. Found: C, 56.69; H, 5.08; N, 8.60.
Under argon atmosphere a suspension of 3-indoxyl acetate
(1.34 g, 7.67 mmol), 5-carboxyisatin (1.53 g, 8.03 mmol) and so-
dium carbonate (1.73 g, 16.32 mmol) in degassed methanol
(80 mL) was stirred at room temperature for 20 h, diluted with
water and filtered to get a reddish solid (2.26 g, 7.40 mmol,
92.0%). 1H NMR (400 MHz; DMSO-d6): 6.97 (d, 1H, 3J = 8.2 Hz),
7.04 (t, 1H, 3J = 7.4 Hz), 7.42 (d, 1H, 3J = 8.1 Hz), 7.58 (t, 1H,
3J = 7.7 Hz), 7.66 (d, 1H, 3J = 7.5 Hz), 7.87 (dd, 1H, 3J = 8.2 Hz,
4J = 1.6 Hz), 9.43 (d, 1H, 4J = 1.2 Hz), 11.08 (s, 1H), 11.22 (s, 1H),
12.63 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 105.3, 109.1,
113.5, 119.0, 121.3, 121.5, 123.8, 124.4, 126.1, 130.8, 137.1, 139.0,
144.2, 152.4, 167.5, 171.2, 188.6. Anal. Calcd for C17H10N2O4: C,
66.67; H, 3.29; N, 9.15. Found: C, 66.68; H, 3.20; N, 9.05.
5.8.3. 5-[N-(Pyridin-3-yl)-aminocarbonyl]-indirubin (7c)
1H NMR (400 MHz; DMSO-d6): 7.03–7.07 (m, 2H), 7.42 (dd, 1H,
1J = 8.4 Hz), 7.45 (d, 1H, 3J = 8.4 Hz), 7.60 (t, 1H, 3J = 8.4 Hz), 7.70 (d,
3J = 7.8 Hz, 1H), 7.92 (dd, 1H, 3J = 8.4 Hz, 4J = 1.8 Hz), 8.23 (m, 1H),
8.32 (dd, 1H, 3J = 4.8 Hz, 4J = 1.2 Hz), 9.00 (d, 1H, 4J = 2.4 Hz), 9.40
(d, 1H, 4J = 1.8 Hz), 10.44 (s, 1H), 11.13 (s, 1H), 11.26 (s, 1H); 13C–
{1H} NMR (150 MHz; DMSO-d6): 105.7, 108.8, 113.6, 119.6,
121.4, 121.7, 123.5, 124.6, 124.9, 127.1, 127.9, 128.8, 136.2,
137.2, 139.1, 141.7, 143.2, 144.2, 152.6, 166.4, 171.2, 188.6. Anal.
Calcd for C22H14N4O3ꢀH2O: C, 66.00; H, 4.03; N, 13.99. Found: C,
65.80; H, 3.92; N, 13.95.
5.7. Pentafluorophenyl indirubin-5-carboxylate (6)
Under argon atmosphere a suspension of 4 (1.53 g, 5.0 mmol),
pentafluorophenyl trifluoroacetate (2.42 g, 8.7 mmol), pyridine
(643 mg, 8.14 mmol) and catalytic amount of dimethylaminopyri-
dine (DMAP) in dry DMF (40 mL) was stirred at room temperature
for 4 h, diluted with ethyl acetate and washed with 0.1 N HCl
(200 mL). After removal of the solvent, the residue was dried in va-
cuo to yield a reddish solid (2.22 g, 4.7 mmol, 94.0%). 1H NMR
(400 MHz; DMSO-d6): 7.04 (t, 3J = 7.5 Hz), 7.12 (d, 1H, 3J = 8.3 Hz),
7.43 (d, 1H, 3J = 8.1 Hz), 7.59 (t, 1H, 3J = 7.7 Hz), 7.67 (d, 1H,
3J = 7.5 Hz), 8.08 (dd, 1H, 3J = 8.3, 4J = 1.8 Hz), 9.63 (d,1H,
4J = 1.7 Hz), 11.18 (s, 1H), 11.54 (s, 1H); 13C–{1H} NMR (150 MHz;
DMSO-d6): 104.1, 110.1, 113.7, 118.2, 119.0, 121.9, 122.0, 124.7,
124.9, 126.6, 132.0, 136.8, 137.1, 137.4, 139.8, 140.7, 146.2,
152.6, 162.3, 171.1, 188.9. Anal. Calcd for C23H9F5N2O4: C, 58.49;
H, 1.92; N, 5.93. Found: C, 58.38; H, 1.76; N, 5.84.
5.8.4. 5-[N-(4-Dimethylaminophenyl)-aminocarbonyl]-indirubin
(7d)
1H NMR (400 MHz; DMSO-d6): 3.10 (s, 6H), 6.78 (s, 2H), 6.97 (d,
1H, 3J = 5.6 Hz), 7.04 (t, 1H, 3J = 5.2 Hz), 7.43 (d, 1H, 3J = 5.2 Hz),
7.57–7.61 (m, 3H), 7.67 (d, 1H, 3J = 5.2 Hz), 7.84 (1H, dd, 3J = 5.6 Hz,
4J = 1.2 Hz), 9.36 (d, 1H, 4J = 1.2 Hz), 10.42 (s, 1H), 11.10 (s, 1H),
11.26 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 105.7, 108.8,
113.6, 119.0, 121.3, 121.6, 124.5, 124.7, 128.5, 128.9, 137.3, 138.9,
143.0, 152.5, 165.4, 171.2, 188.6. Anal. Calcd for C25H20N4O3: C,
70.74; H, 4.75; N, 13.20. Found: C, 70.70; H, 5.13; N, 12.80.
5.8.5. 5-{N-[2-(4-Methylpiperazino)-ethyl]-aminocarbonyl}-
indirubin (7e)
1H NMR (400 MHz; DMSO-d6): 2.12–2.60 (m, 15H), 6.93 (d, 1H,
3J = 5.5 Hz), 7.04 (dd, 1H, 3J = 9.4 Hz, 4J = 4.5 Hz), 7.42 (d, 1H,
3J = 5.5 Hz), 7.58 (m, 1H), 7.61 (d, 1H, 3J = 4.9 Hz), 7.70 (1H, dd,
3J = 5.4 Hz, 4J = 1.2 Hz), 8.16 (s, 1H), 9.23 (s, 1H), 11.07 (s, 1H),
11.14 (s, 1H). Anal. Calcd for C24H25N5O3: C, 66.81; H, 5.84; N,
16.23. Found: C, 66.62; H, 5.63; N, 16.13.
5.8. General procedure for the synthesis of indirubin-5-
carboxamides (7)
Under argon atmosphere a suspension of 5 (1.0 mmol), amino
compound (1.8 mmol) and DMAP (1.2 mmol) in dry dioxane
(40 mL) was refluxed for several hours till no educt could be de-
tected by TCL. After cooling the mixture was diluted with 0.1 N
HCl (150 mL). The precipitate was filtered and dried in vacuo to af-
ford indirubin-5-carboxamides (7a, 92.0%; 7b, 87.0%; 7c, 99.9%; 7d,
14.7%; 7e, 84.0%; 7f, 60.2%; 7g, 69.0%; 7h, 88.0%; 7i, 64.0%, 7j,
73.0%; 7k, 47.0%).
5.8.6. 5-[N,N-Bis(2-hydroxyethyl)-aminocarbonyl]-indirubin
(7f)
1H NMR (400 MHz; DMSO-d6): 3.35–3.70 (m, 8H), 4.65–4.90 (s,
2H), 6.91 (d, 1H, 3J = 8.0 Hz), 7.02 (t, 1H, 3J = 7.4 Hz), 7.30 (d, 1H,
3J = 8.0 Hz), 7.42 (d, 1H, 3J = 8.0 Hz), 7.58 (t, 1H, 3J = 7,7 Hz), 7.65
(d, 1H, 3J = 7.5 Hz), 8.79 (d, 1H, 4J = 1.3 Hz), 11.03 (s, 1H), 11.07
(s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 51.9, 58.9, 105.8),
109.0, 113.5, 119.0, 121.0, 121.5, 123.3, 124.5, 128.1, 130.2,
137.3, 138.9, 141.2, 152.5, 170.9, 171.3, 188.7. Anal. Calcd for
C21H19N3O7ꢀ2H2O: C, 58.74; H, 5.40; N, 9.79. Found: C,58.60; H,
5.67; N, 9.40.
5.8.1. 5-{N-[4-(4-Methylpiperazino)-phenyl]-aminocarbonyl}-
indirubin (7a)
1H NMR (400 MHz; DMSO-d6): 2.21 (s, 3H), 2.44–2.45 (m, 4H),
3.08–3.10 (m, 4H), 6.92 (d, 2H, 3J = 6.0 Hz), 6.98 (d, 1H, 3J = 5.6 Hz),
7.03 (t, 1H, 3J = 5.2 Hz), 7.43 (d, 1H, 3J = 5.6 Hz), 7.58 (t, 1H,
3J = 5.2 Hz), 7.62 (d, 2H, 3J = 6.0 Hz), 7.67 (d, 1H, 3J = 5.2 Hz), 7.83
(1H, dd, 3J = 5.6 Hz, 4J = 0.8 Hz), 9.32 (d, 1H, 4J = 0.8 Hz), 9.99 (s,
1H), 11.10 (s, 1H), 11.18 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-
d6): 45.8, 48.6, 54.7, 105.7, 108.8, 113.6, 115.5, 119.0, 121.3, 121.6,
121.3, 124.5, 124.7, 128.6, 128.9, 131.5, 137.3, 138.9, 143.0, 147.3,
152.5, 165.5, 171.2, 188.6. Anal. Calcd for C28H25N5O3ꢀ0.5 H2O: C,
68.84; H, 5.36; N, 14.34. Found: C, 69.16; H, 5.18; N, 14.12.
5.8.7. 5-[N-(2-Hydroxyethyl)-aminocarbonyl]-indirubin (7g)
1H NMR (400 MHz; DMSO-d6): 3.35 (q, 2H, 3J = 5.9 Hz), 3.52 (q,
2H, 3J = 5.5 Hz), 4.73 (t, 1H, 3J = 5.0 Hz), 6.92 (d, 1H, 3J = 8.1 Hz),
7.03 (t, 1H, 3J = 7.4 Hz), 7.42 (d, 1H, 3J = 8.0 Hz), 7.58 (t, 1H,
3J = 7.7 Hz), 7.67 (d, 1H, 3J = 7.5 Hz), 7.73 (dd, 1H, 3J = 8.2 Hz,
4J = 1.5 Hz), 8.21 (t, 1H, 3J = 5.4 Hz), 9.25 (d, 1H, 4J = 1.3 Hz), 11.07
(s, 1H), 11.11 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 42.2,
59.9, 105.8, 108.7, 113.6, 119.0, 121.2, 121.5, 124.4, 124.5, 128.2,
128.6, 128.3, 138.8, 142.9, 152.5, 166.8, 171.2, 188.5. Anal. Calcd
for C19H15N3O4ꢀ0.25H2O: C, 64.49; H, 4.42; N, 11.88. Found: C,
64.77; H, 4.50; N, 11.87.
5.8.2. 1-(Indirubin-5-carbonyl)-amino-1-deoxy-glucitol (7b)
1H NMR (400 MHz; DMSO-d6): 3.25–3.81 (m, 8H), 4.32 (d, 1H,
3J = 5.6 Hz), 4.37 (d, 1H, 3J = 6.4 Hz), 4.44 (d, 1H, 3J = 5.2 Hz), 4.47
(d, 1H, 3J = 4.8 Hz), 4.92 (d, 1H, 3J = 4.4 Hz), 6.93 (d, 1H,
3J = 8.0 Hz), 7.04 (t, 1H, 3J = 7.2 Hz), 7.42 (d, 1H, 3J = 8.0 Hz), 7.58
(t, 1H, 3J = 8.0 Hz), 7.67 (d, 1H, 3J = 7.2 Hz), 7.75 (d, 1H,
3J = 8.4 Hz), 8.11 (t, 1H, 3J = 5.6 Hz), 9.26 (s, 1H), 11.07 (s, 1H),
5.8.8. 5-[N-(2-Dimethylaminoethyl)-aminocarbonyl]-indirubin
(7h)
1H NMR (400 MHz; DMSO-d6): 2.43 (s, 6H), 2.72 (t, 2H,
3J = 6.5 Hz), 3.46 (q, 2H, 3J = 6.2 Hz), 6.92 (d, 1H, 3J = 8.1 Hz), 7.03

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X. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 4509–4515
(t, 1H, 3J = 7.4 Hz), 7.42 (d, 1H, 3J = 8.1 Hz), 7.58 (t, 1H, 3J = 7.7), 7.66
(d, 1H, 3J = 7.5 Hz), 7.71 (dd, 1H, 3J = 8.6 Hz, 4J = 1.2 Hz), 8.32 (t, 1H,
3J = 5.5 Hz), 9.25 (d, 1H, 4J = 1.3 Hz), 11.08 (s, 1H), 11.16 (s, 1H);
13C–{1H} NMR (150 MHz; DMSO-d6): 36.2, 44.1, 57.4, 105.7,
108.7, 113.6, 119.0, 121.2, 121.5, 124.4, 124.4, 127.9, 128.1,
137.2, 138.8, 143.0, 152.5, 166.9, 171.2, 188.5. MS (EI) m/z: 376.1
[M]+; calcd for C21H20N4O3: 376.154.
170.9, 171.8. Anal. Calcd for C21H20N4O5ꢀH2O: C, 59.15; H, 5.20;
N, 13.14. Found: C, 59.37; H, 4.80; N, 12.85.
5.9.3. 30-Hydroxyimino-5-[N-(2-hydroxyethyl)-aminocarbonyl]-
indirubin (8c)
1H NMR (400 MHz; DMSO-d6): 3.37 (q, 2H, 3J = 5.9 Hz), 3.52 (q,
2H, 3J = 6.1 Hz), 6.91 (d, 1H, 3J = 8.3 Hz), 7.02 (m, 1H), 7.38–7.42 (m,
2H), 7.64 (dd, 1H, 3J = 7.8 Hz, 4J = 1.5 Hz), 7.97 (s, 1H), 8.25 (t, 1H,
3J = 7.8 Hz), 9.01 (d, 1H, 3J = 1.5 Hz), 10.94 (s, 1H), 11.76 (s, 1H);
13C–{1H} NMR (150 MHz; DMSO-d6): 43.2, 61.0, 99.2, 108.9,
112.5, 117.5, 122.6, 122.9, 123.3, 126.3, 128.3, 128.9, 132.9,
141.3, 145.6, 146.9, 152.3, 168.2, 172.1. Anal. Calcd for
C19H16N4O4ꢀ0.5H2O: C, 61.12; H, 4.59; N, 15.01. Found: C, 61.05;
H, 4.45; N, 14.88.
5.8.9. 5-[N-(2-Dimethylaminoethyl)-N-methyl-amino-carbonyl]-
indirubin (7i)
1H NMR (400 MHz; DMSO-d6): 2.15 (s, 6 H), 2.52 (s, 2H), 3.02 (s,
3H), 3.48 (t, 1H, 3J = 6.4 Hz), 6.95 (d, 1H, 3J = 7.9 Hz), 7.04 (t, 1H,
3J = 7.3 Hz), 7.29 (d, 3J = 7.8 Hz), 7.38 (d, 3J = 8.1 Hz), 7.56 (t,
3J = 7.5 Hz), 7.65 (d, 3J = 7.5 Hz), 8.83 (s, 1H), 10.72 (s, 1H), 10.86
(s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 37.9, 45.2, 48.7,
56.5, 100.7, 109.1, 113.5, 118.9, 120.8, 121.5, 123.1, 124.3, 128.3,
129.5, 137.2, 138.8, 141.4, 152.5, 170.1, 171.0, 188.7. Anal. Calcd
for C22H22N4O3: C, 67.68; H, 5.68; N, 14.35. Found: C, 67.29; H,
5.87; N, 13.95.
5.9.4. 30-Hydroxyimino-5-[N-(2-dimethylaminoethyl)-N-methyl-
aminocarbonyl]-indirubin (8d)
1H NMR (400 MHz; DMSO-d6): 2.77 (s, 6 H), 3.07 (s, 3 H), 3,31 (s,
2H), 3.77 (t, 2H, 3J = 6.3 Hz), 6.96 (d, 1H, 3J = 7.9 Hz), 7,04 (t, 1H,
3J = 7.4 Hz), 7.23 (dd, 1H, 3J = 6.5 Hz, 4J = 1.3 Hz), 7.33 (d, 1H,
3J = 8.0 Hz), 7.40 (t, 1H, 3J = 7.6 Hz), 8.27 (d, 1H, 3J = 7.5 Hz), 8,70
(s, 1H) 10,56 (s, 1H), 11.71 (s, 1H), 13.60 (s, 1H); 13C–{1H} NMR
(150 MHz; DMSO-d6): 37.8, 40.0, 42.5, 53.4, 98.2, 108.4, 111.7,
116.5, 121.8, 122.0, 126.0, 127.9, 128.0, 132.1, 139.2, 144.7, 146.0,
151.4, 171.0, 171.7, 188.9. Anal. Calcd for C22H23N5O3ꢀ1.5 HClꢀ2.5
H2O: C, 52.31; H, 5.89; N, 13.86. Found: C, 52.59; H, 5.68; N, 13.51.
5.8.10. 5-(4-Methylpiperazinocarbonyl)-indirubin (7j)
1H NMR (400 MHz; DMSO-d6,): 2.25 (s, 3H), 2.39 (t, 4H,
3J = 4.9 Hz), 3.55 (t, 4H, 3J = 4.9 Hz), 6.96 (d, 1H, 3J = 7.9 Hz), 7.04
(t, 1H, 3J = 7.4 Hz), 7.30 (dd, 1H, 3J = 9.3 Hz, 4J = 1.4 Hz), 7.38 (d,
1H, 3J = 7,9 Hz), 7.57 (t, 1H, 3J = 7.6 Hz), 7.66 (d, 1H, 3J = 7.5 Hz),
8.8 (s, 1H), 10.72 (s, 1H), 10.83 (s, 1H); 13C-{1H} NMR (150 MHz;
DMSO-d6): 45,4, 54,4, 105.6, 109.0, 113.4, 118.9, 120.9, 121.4,
123.5, 124.3, 128.4, 128.6, 137.1, 138.8, 141.6, 152.4, 169.2,
170.0, 188.5. Anal. Calcd for C22H20N4O3ꢀHClꢀ0.33 H2O: C, 61.32;
H, 5.07; N, 13.00. Found: C, 61.79; H, 5.47; N, 12.36.
5.10. General procedure for the synthesis of indirubin-30-oxime
ethers (9)
1,1,3,3-Tetramethylguanidine (582 lL) and alkyl-halogenide
5.8.11. 5-(Piperazinocarbonyl)-indirubin (7k)
(5.4 mmol) were added to indirubin-30-oxime (0.5 mmol) in etha-
nol (7 mL) with stirring. The mixture was refluxed for 2 h, cooled
to 0 °C and diluted with water (20 mL) and 1 N HCl (20 mL). The
precipitate was filtered, washed with water and dried to afford 9
(9a, 63.9%, 9b, 17.0%).
1H NMR (400 MHz; DMSO-d6): 2.07 (s, 1H), 2.72 (s, 4H), 3.46 (s,
4H), 6.94 (d, 1H, 3J = 7.9 Hz), 7.02 (t, 1H, 3J = 7.3 Hz), 7.29 (dd, 1H,
3J = 9.1 Hz, 3J = 1.2 Hz), 7.41 (d, 1H, 3J = 8.0 Hz), 7.57 (t, 1H,
3J = 7.6 Hz), 7.66 (d, 1H, 3J = 7.8 Hz), 8.82 (s, 1H), 11.08 (m, 2H);
13C–{1H} NMR (150 MHz; DMSO-d6): 30.7, 45.8, 105.7, 109.3,
113.6, 119.0, 121.0, 121.5, 123.6, 124.5, 128.6, 128.8, 137.2,
138.9, 141.8, 152.6, 169.3, 171.8, 188.8. Anal. Calcd for
C21H18N4O3ꢀ0.5 HClꢀ1.5 H2O: C, 60.10; H, 5.16; N, 13.35. Found:
C, 60.48; H, 5.02; N, 13.05.
5.10.1. 30-(2,3-Dihydroxypropyloxyimino)-5-[N,N-bis(2-hydroxy-
ethyl)-aminocarbonyl]-indirubin (9a)
1H NMR (400 MHz; DMSO-d6): 3.35–3.70 (m, 8H), 3.51 (d, 2H,
3J = 5.4 Hz), 3.97 (m, 1H), 4.50 (dd, 1H, 3J = 10.7 Hz, 3J = 6.3 Hz),
4.59 (dd, 1H, 3J = 10.8 Hz, 3J = 3.8 Hz), 4.75 (m, 3H), 5.09 (s, H),
6.91 (d, 1H, 3J = 7.9 Hz), 7.05 (m, 1H), 7.21 (dd, 1H, 3J = 7,9 Hz,
4J = 1.0 Hz), 7.41–7.45 (m, 2H), 8.17 (d, 1H, 3J = 7.6 Hz), 8.71 (s,
1H), 10.91 (s, 1H), 11.69 (s, 1H); 13C–{1H} NMR (150 MHz;
DMSO-d6): 36.2, 62.8), 69.9, 78.6, 98.4, 108.3, 111.8, 116.2, 121.6,
121.8, 122.0, 125.2, 128.8, 129.4, 132.8, 138.9, 144.5, 145.3,
151.1, 170.9, 171.0. Anal. Calcd for C24H26N4O7ꢀ0.5H2O: C, 58.65;
H, 5.54; N, 11.40. Found: C, 58.81; H, 5.65; N, 11.58.
5.9. General procedure for the synthesis of indirubin-30-oximes
(8)
A mixture of indirubins (1.1 mmol) and hydroxylamine hydro-
chloride (24.4 mmol) in pyridine (7 mL) was refluxed for 4 h. The
mixture was diluted with 2 N HCl (50 mL) to yield a reddish solid
8 (8a, 67.9%; .8b, 49.0%; 8c, 90.0%; 8d, 82.0%).
5.9.1. 30-Hydroxyimino-5-{N-[4-(4-methylpiperazino)-phenyl]-
aminocarbonyl}-indirubin (8a)
5.10.2. 30-[(2-b-
D-Glucopyranosylethyl)-oxyimino]-5-[N-(2-di-
methylaminoethyl)-N-methyl-aminocarbonyl]-indirubin (9b)
1H NMR (400 MHz; DMSO-d6): 2.17 (s, 6H), 2.39 (m, 2H), 3.11
(m, 2H), 3.00–5.02 (m, 6.91 (s, 1H), 7.03 s, 1H), 7.41 (m, 1H), 7.62
(s, 1H), 8.01 (s, 1H), 8.21 (s, 1H), 9.17 (s, 1H), 11.65 (m, 4H); 13C-
{1H} NMR (150 MHz; DMSO-d6,): 30.5, 37.3, 45.1–76.8, 99.6,
103.0, 108.0 (C7), 111.7, 116.2, 121.5, 122.2, 122.9, 125.2, 125.8,
127.2, 128.5, 132.7, 145.6, 151.2, 166.9, 171.3. Anal. Calcd for
1H NMR (400 MHz; DMSO-d6): 2.21 (s, 3H), 2.44–2.45 (m, 4H),
3.08–3.10 (m, 4H), 6.89–7.01 (m, 4H), 7.32–7.37 (m, 2H), 7.62–
7.63 (m, 3H), 8.28 (d, 1H, 3J = 6.6 Hz), 9.09 (s, 1H), 9.79 (s, 1H),
10.86 (s, 1H), 11.98 (s, 1H). Anal. Calcd for C28H26N6O3ꢀ0.5 H2O:
C, 66.79; H, 5.40; N, 16.69. Found: C, 66.79; H, 5.18; N, 16.79.
.
5.9.2. 30-Hydroxyimino-5-[N,N-bis(2-hydroxyethyl)-aminocarbo-
nyl]-indirubin (8b)
C29H35N5O9 H2O: C, 56.58; H, 6.06; N, 11.38. Found: C, 56.38; H,
6.01; N, 11.18.
1H NMR (400 MHz; DMSO-d6): 3.36–3.63 (m, 10 H), 6.90 (d, 1H,
3J = 7.9 Hz), 7.03 (m, 1H), 7.15 (dd, 1H, 3J = 7.9 Hz, 4J = 1.4 Hz),
7.40–7.41 (m, 2H), 8.23 (d, 1H, 3J = 7.6 Hz), 8.59 (s, 1H), 10.86 (s,
1H), 11.80 (s, 1H), 13.54 (s, 1H); 13C–{1H} NMR (150 MHz;
DMSO-d6): 51.9 58.8, 98.3, 108.1, 111.5, 116.5, 121.0, 121.6,
122.2, 124.7, 128.0, 129.4, 132.0, 138.5, 144.6, 145.8, 151.4,
5.11. 5-{N-[2-(4-Methylpiperazino)-ethyl]-aminocarbonyl}-
indirubin hydrochloride 18a
To a solution of 7f (100 mg, 0.23 mmol) in dried THF was added
HCl saturated THF. The suspension was stirred for 2 h at room tem-

X. Cheng et al. / Bioorg. Med. Chem. 18 (2010) 4509–4515
4515
perature. The precipitate was filtered, washed with THF and dried
in vacuo to yield 18a (86 mg, 18.4 mmol, 80.0%). 1H NMR
(400 MHz; D2O/DMSO-d6): 2.86–3.68 (m, 15H), 6.96 (d, 1H,
3J = 7.9 Hz), 7.04 (t, 1H, 3J = 7.5 Hz), 7.41 (d, 1H, 3J = 7.9 Hz), 7.57
(t, 1H, 3J = 7.5 Hz), 7.67 (d, 1H, 3J = 7.4 Hz), 7.81 (1H, dd,
3J = 8.3 Hz, 4J = 1.8 Hz), 9.29 (d, 1H, 4J = 1.3 Hz). Anal. Calcd for
C24H25N5O3. HCl: C, 61.60; H, 5.60; N, 14.97. Found: C, 61.43; H,
5.77; N, 14.58.
7.38 (d, 1H, 3J = 8.0 Hz), 7.43 (d, 1H, 3J = 8.0 Hz), 7.57–7.64 (m,
2H), 8.91 (s, 1H), 11.11 (s, 1H), 11.14 (s, 1H); 13C–{1H} NMR
(150 MHz; DMSO-d6): 40.1, 50.5, 60.1, 105.4, 109.3, 113.7, 118.9,
121.3, 121.6, 124.1, 124.5, 127.0, 128.8, 137.4, 139.1, 142.3,
152.6, 169.5, 170.9, 188.8. Anal. Calcd for C24H26N4O7Sꢀ1.5 H2O:
C, 53.23; H, 5.40; N, 10.35. Found: C, 53.00; H, 4.90; N, 10.60.
Acknowledgements
5.12. Procedures for the Quaternization of indirubin-5-carbox-
amides (18b–e)
We are grateful to Mrs. C. Müller for recording the NMR spectra,
and Mrs. E. Biel and Mrs. B. Dusch for performing CHN analyses.
Methode 1: To
a suspension of indirubin-5-carboxamide
Supplementary data
(0.1 mmol) in methanol (10 mL) was added methyl iodide
(5 mmol). The mixture was stirred for 24 h at room temperature.
The solvent and excess MeI were eliminated. The residue was dis-
solved in methanol. After dilution with ether the precipitate was
filtered and dried to afford 18c (62.0%) or 18d (82.0%).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.066.
References and notes
Methode 2: To
a suspension of indirubin-5-carboxamide
1. Zhu, Z. H. In Dan Xi Xin Fa; Wang, Y., Zhu, J. P., Jiang, L. Z., Eds.; People’s medical
publishing, 2005, ISBN 7-117-06711-X/Rꢀ6712.
(0.1 mmol) in xylene (1 mL) and nitrobenzene (2 mL) was added
dimethylsulfate (1 mmol). The mixture was heated for 45 min
and cooled. After addition of ether the precipitate was filtered
and dried in vacuo to yield 18b (82.0%) or 18e (74.0%).
2. (a) Tang, W.; Eisenbrand, G. Chinese drugs of plant origin: Chemistry,
Pharmacology, and Use in Traditional and Modern Medicine; Springer: Berlin,
Heidelberg, New York, 1990; (b) Institute of Hematology; Chinese Academy of
Medical Sciences; Chengdu traditional Chinese Medical College; Sichuang
Institute of Traditional Medicine. Transf. Hematol. 1978, 2, 17–20.
3. Hössel, R.; Leclerc, S.; Endicott, J. A.; Nobel, M.; Lawrie, A.; Tunnah, P.; Leost, M.;
Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.; Tang, W. C.; Eisenbrand, G.;
Meijer, L. Nat. Cell Biol. 1999, 1, 60.
5.12.1. 2-(Indirubin-5-carbonylamino)-ethyl-trimethyl-ammo-
nium methosulfate (18b)
1H NMR (400 MHz; DMSO-d6): 3.16 (s, 9H), 3.36 (s, 3H), 3.52 (t,
2H, 3J = 6.4 Hz), 3.70 (q, 2H, 3J = 5.7 Hz), 6.97 (d, 1H, 3J = 8.2 Hz),
7.04 (t, 1H, 3J = 7.4 Hz), 7.43 (d, 1H, 3J = 8.0 Hz), 7.59 (t, 1H,
3J = 7.4 Hz), 7.66 (d, 1H, 3J = 7.5 Hz), 7.73 (dd, 1H, 3J = 8.2 Hz,
4J = 1.4 Hz), 8.63 (t, 1H, 3J = 5.4 Hz), 9.28 (s, 1H), 11.10 (s, 1H),
11.17 (s, 1H). Anal. Calcd for C23H26N4O7Sꢀ1.5 H2O: C, 52.16; H,
5.52; N, 10.58. Found: C, 52.06; H, 5.32; N, 10.88. MS (MALDI-
TOF) m/z: 391.0 [M]+; calcd for C22H23N4O3+: 391,177.
4. Meijer, L.; Skaltsounis, A. L.; Magiatis, P.; Polychronopoulos, P.; Knockaert, M.;
Leost, M.; Ryan, X. P.; Vonica, C. A.; Brivanlou, A.; Dajani, R.; Crovace, C.;
Tarricone, C.; Musacchio, A.; Roe, S. M.; Pearl, L.; Greengard, P. Chem. Biol. 2003,
10, 1255.
5. Nam, S.; Buettner, R.; Turkon, J.; Kim, D.; Cheng, J. Q.; Muehlbeyer, S.; Hippe, F.;
Vatter, S.; Merz, K.-H.; Eisenbrand, G.; Jove, R. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 5998.
6. Pandraud, P. H. Acta Crystallogr. 1961, 14, 901.
7. Eisenbrand, G.; Hippe, F.; Jakobs, S.; Muehlbeyer, S. J. Cancer Res. Clin. Oncol.
2004, 130, 627.
8. Fiebig, H.-H.; Schüler, J. B. In vivo Antitumor Activity of 5-Methylindirubin. In
Indirubin, the Red Shade of Indigo; Meijer, L., Guyard, N., Skaltsounis, L.,
Eisenbrand, G., Eds.; Life in Progress Editions; Roscoff: France, 2006; pp 209–
213.
5.12.2. 2-[N-(5-Indirubincarbonyl)-N-methyl]-aminoethyl-
trimethyl-ammonium iodide (18c)
1H NMR (400 MHz; DMSO-d6): 3.82 (s, 3H), 3.16 (s, 6H), 3.65 (s,
2H), 3.89 (s, 2H), 6.96 (d, 1H, 3J = 8.0 Hz), 7.04 (t, 1H, 3J = 7.5 Hz),
7.37 (dd, 1H, 3J = 9.5 Hz, 4J = 1.5 Hz), 7.43 (d, 1H, 3J = 8.1 Hz), 7.59
(t, 1H, 3J = 7.2 Hz), 7.64 (d, 1H, 3J = 7.5 Hz), 8.88 (s, 1H), 11.09 (s,
1H), 11.12 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 40.1,
48.6, 52.5, 61.1, 105.5, 109.2, 113.6, 118.9, 121.1, 121.6, 123.7,
124.5, 128.2, 128.5, 137.4, 139.1, 142.0, 152.6, 170.9, 171.1,
188.9. Anal. Calcd for C23H25IN4O3ꢀH2O: C, 50.19; H, 4.94; N,
10.18. Found: C, 50.03; H, 4.84; N, 10.40.
9. Jautelat, R.; Brumby, T.; Schäfer, M.; Briem, H.; Eisenbrand, G.; Schwahn, S.;
Krüger, M.; Lücking, U.; Prien, O.; Siemeister, G. ChemBioChem 2005, 6, 531.
10. (a) Merz, K.-H.; Eisenbrand, G. Chemistry and Structure-Activity of Indirubins.
In Indirubin, the red shade of indigo; Meijer, L., Guyard, N., Skaltsounis, L.,
Eisenbrand, G., Eds; Life in Progress Editions; Roscoff: France, 2006; pp 135–
145; (b) Polychronopoulos, P.; Magiatis, P.; Skaltsounis, A. L.; Myrianthopoulos,
V.; Mikros, E.; Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.; Leost, M.;
Greengard, P.; Meijer, L. J. Med. Chem. 2004, 47, 935; (c) Vougogiannopoulou,
K.; Ferandin, Y.; Bettayeb, K.; Myrianthopoulos, V.; Lozach, O.; Fan, Y.; Johnson,
C. H.; Magiatis, P.; Skaltsounis, A. L.; Mikros, E.; Meijer, L. J. Med. Chem. 2008,
51, 6421.
11. (a) Baeyer, A. Chem. Ber. 1881, 14, 1741; (b) Russell, G. A.; Kaupp, G. J. Am. Chem.
Soc. 1969, 91, 3851.
12. Sandmeyer, T. Helv. Chim. Acta. 1919, 2, 234.
13. Waldmann, H. J. Prakt. Chem. 1937, 147, 338.
5.12.3. 4-(5-Indirubincarbonyl)-1,1-dimethylpiperazinium iodide
(18d)
14. Giovaninni, E.; Portmann, P. Helv. Chim. Acta 1948, 31, 1392.
15. Romanelli, M. N.; Manetti, D.; Scapecchi, S.; Borea, P. A.; Dei, S.; Bartolini, A.;
Ghelardini, C.; Gualtieri, F.; Guandalini, L.; Varani, K. J. Med. Chem. 2001, 44,
3946.
16. Tapia, I.; Alono-Cires, L.; Lopez-Tudconca, P. L.; Mosquera, R.; Labeaga, L.;
Innerarity, A.; Orjales, A. J. Med. Chem. 1999, 42, 2870.
17. Königs, W.; Knorr, E. Chem. Ber. 1901, 34, 957.
18. Marko, D.; Schätzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer, L.;
Eisenbrand, G. Br. J. Cancer 2001, 84, 283.
1H NMR (400 MHz; DMSO-d6): 3.19 (s, 9H), 3.47 (s, 4H), 3,94 (s,
4H), 6.98 (d, 1H, 3J = 8.0 Hz), 7.04 (t, 1H, 3J = 7.4 Hz), 7.30 (d, 1H,
3J = 8.0 Hz), 7.43 (d, 1H, 3J = 8.0 Hz), 7.6 (m, 2H), 8.9 (s, 1H), 11.11
(s, 1H), 11.15 (s, 1H); 13C–{1H} NMR (150 MHz; DMSO-d6): 48.4,
50.6, 60.1, 105.4, 109.2, 113.4, 118.9, 121.1, 121.5, 123.8, 124.3,
126.9, 128.6, 137.2, 138.9, 142.2, 152.4, 169.4, 170.8, 188.6. Anal.
Calcd for C23H23IN4O3ꢀ1.5 H2O: C, 49.56; H, 4.70; N, 10.05. Found:
C, 49.41; H, 4.45; N, 9.62.
19. Eisenbrand, G.; Cheng, X. L.; Vatter, S.; Merz, K.-H.; AACR 100th Annual
Meeting, 2009; Denver, CO, AACR Abstr. #1808.
20. Merz, K.-H.; Schwahn, S.; Hippe, F.; Muehlbeyer, S.; Jakobs, S.; Eisenbrand, G.
Int. J. Clin. Pharmacol.Ther. 2004, 42, 656.
5.12.4. 4-(5-Indirubincarbonyl)-1,1-dimethylpiperazinium
methosulfate (18e)
1H NMR (400 MHz; DMSO-d6): 3.19 (s, 9H), 3.36 (s, 3H), 3.47 (s,
4H), 3,94 (s, 4H), 6.98 (d, 1H, 3J = 8.0 Hz), 7.04 (t, 1H, 3J = 7.4 Hz),