Water-soluble cationic derivatives of indirubin, the active anticancer component from indigo naturalis

Article abstract of DOI:10.1002/cbdv.201200147

To overcome the problem of poor aqueous solubility and bioavailability of indirubin-3-oximes, the compounds were modified by attaching a quaternary ammonium group at the oxime moiety. Exploring the prodrug concept, an oxime ester with acetyl-l-carnitine was prepared, and the rate of its hydrolysis was investigated to assess its suitability for clinical administration. In addition, the cytotoxic potency of new stable oxime ethers with a choline moiety and their influence on the cell cycle were tested in human cancer cell lines.

Full text of DOI:10.1002/cbdv.201200147

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Water-Soluble Cationic Derivatives of Indirubin, the Active Anticancer
Component from Indigo naturalis
by Werner Ginzingera), Alexander Eggera), Gerhard Mꢀhlgassnera), Vladimir B. Arion*a),
Michael A. Jakupeca)b), Markus Galanskia), Walter Bergerb)c), and Bernhard K. Keppler*a)b)
a) University of Vienna, Institute of Inorganic Chemistry, Wꢀhringer Str. 42, A-1090 Vienna
(phone: þ431427752600; fax: þ431427752680; e-mail: vladimir.arion@univie.ac.at,
bernhard.keppler@univie.ac.at)
b) Research Platform ꢁTranslational Cancer Therapy Researchꢂ, Vienna
c) Medical University of Vienna, Institute of Cancer Research, Borschkeg. 8a, A-1090 Vienna
To overcome the problem of poor aqueous solubility and bioavailability of indirubin-3-oximes, the
compounds were modified by attaching a quaternary ammonium group at the oxime moiety. Exploring
the prodrug concept, an oxime ester with acetyl-l-carnitine was prepared, and the rate of its hydrolysis
was investigated to assess its suitability for clinical administration. In addition, the cytotoxic potency of
new stable oxime ethers with a choline moiety and their influence on the cell cycle were tested in human
cancer cell lines.
Introduction. – Indirubin was found to be the active component of Indigo naturalis,
which is an ingredient of a recipe used for treatment of chronic myelocytic leukemia
(CML) in traditional Chinese medicine. Investigations into the mode of action revealed
that it induces a G2/M cell-cycle arrest and inhibits cyclin-dependent kinases [1][2]. In
the meantime, other targets have been identified for indirubin and several derivatives
[3], e.g., GSK-3b, an enzyme involved not only in cell cycle regulation but also in
inflammations, apoptosis, etc., as well as in neurodegenerative diseases and diabetes [4–
6]; c-Src, which phosphorylates and activates Stat3, a transcriptional factor involved in
cell proliferation, cell survival, angiogenesis, and immune evasion of cancer cells [7];
the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor that
regulates genes involved in xenobiotic metabolism, cell proliferation, and differ-
entiation [8].
However, indirubin and its derivatives display an extremely low solubility in
biological fluids and a poor bioavailability. Attempts to overcome this problem include
the introduction of substituents at the aromatic rings or the modification of the
C(3’)¼O moiety with Grignard reagents or other C nucleophiles in order to increase
the distortion from the planarity of the molecule, which is responsible for the very tight
packing within crystals and, as a consequence, for the poor solubility in biologically
relevant media [9]. The conversion of the C(3’)¼O group to an oxime offers further
opportunities for modifications [10–12]. A dilemma with these modifications is that
bulky hydrophilic moieties would provide increased aqueous solubility, while flat and
lipophilic groups attached will normally mediate better cytotoxic effects in cell cultures
[10]. Indirubin-5-sulfonic acid, for instance, was found to be very potent in in vitro
ꢃ 2012 Verlag Helvetica Chimica Acta AG, Zꢄrich

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kinase assays, while having only limited effects on cell proliferation, most probably for
uptake reasons [1].
All these prompted us to create a soluble oxime ester as a prodrug, capable of
releasing the free oxime upon hydrolysis. A similar concept was recently patented [13].
As solubilizing component, we chose the bulky and charged acetyl-l-carnitine. (3R)-3-
hydroxy-4-(trimethylammonium)butanoate (l-carnitine) is involved in the mitochon-
drial and peroxisomal uptake of activated long-chain fatty acids as well as in the
regulations of these processes, and it is being used as a nutritional supplement [14].
Acetyl-l-carnitine with acetylated 3-OH group showed positive effects on fatigue [15]
as well as depression and cognitive abilities of elderly persons [16]. The compound led
also to improved glucose disposal in type-II diabetes patients [17]. Taking all these into
account, we anticipated that, after cleavage, no side effects will arise from this
component.
In addition, non-hydrolyzable oxime ethers with the less bulky choline moiety,
which also comprise a quaternary trimethylammonium group, were prepared. Some
cephalosporines [18] or serotonin antagonists [19] are examples for this strategy to
increase solubility.
Results and Discussion. – Syntheses. For obvious reasons, the acetyl-l-carnitine
indirubin-3’-monoxime ester (2) was synthesized via the acyl chloride of acetyl-l-
carnitine (Scheme). Since the product turned out to undergo hydrolysis extremely fast,
and to be sensitive to elevated temperature (>408), purification presented a challenge.
Although in SOCl2 a homogenous solution is formed within a short time, facilitating the
generation of the acyl chloride, it was not possible to obtain a sufficiently pure final
product in this way. Therefore, oxalyl chloride with sonication of the heterogenous
reaction mixture was used instead.
Scheme. Synthetic Pathways to New Water-Soluble Indirubin Derivatives
i) (2R)-2-(Acetyloxy)-4-chloro-N,N,N-trimethyl-4-oxobutan-1-ammonium chloride, THF, 308. ii) DMF,
NaH; (2-Bromoethyl)trimethylammonium bromide, 908; H2O, Ag2CO3, r.t.; HCl. iii) DMF, (2-
Chloroethyl)trimethylammonium chloride, K2CO3, 708; HCl.
The choline derivatives 3a and 3b were prepared by nucleophilic substitution
(Scheme). Initially, the oxime was deprotonated with NaH and reacted with (2-
bromoethyl)trimethylammonium bromide. Afterwards, the bromide was removed by

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treatment of the reaction mixture with Ag2CO3. Finally, the chloride salt was obtained
by addition of aqueous HCl. Even more easily, the desired chloride salt can be
synthesized starting from (2-chloroethyl)trimethylammonium chloride and K2CO3 as a
base. This simplified method was used in the case of the 5-bromo derivative. For
purification, the crude products were dissolved in H2O, and small amounts of the
unreacted oximes were removed by filtration or by extraction with AcOEt.
Subsequently, the product was extracted with BuOH. Standard drying agents could
not be used for the wet organic layers, which had been washed with brine. As a
consequence, the H2O-saturated BuOH layer contained some amount of inorganic salts
after separation. Hence, the residues after evaporation were dissolved in mixtures of
BuOH with CH2Cl2 or Et2O, and the salts insoluble therein removed by filtration.
Finally, all products were dissolved in H2O and lyophilized in order to remove traces of
organic solvents.
Characterization. The attachment of a choline moiety to the oximes 1a or 1b led to
only minor shifts of either the 1H-NMR signals (up to 0.17 ppm for NH singlets), or the
13C and 15N resonances (less than 2 ppm). The additional ester groups within 2 shift the
signal of the H-atom at C(4) by anisotropic effects downfield (þ 0.40 ppm).
Furthermore, stronger shifts were observed for the signals of C(3’) (þ 5.6 ppm) and
C(3) (þ 4.8 ppm). The CH2 groups of the acetyl-l-carnitine exhibited the expected
diastereotopic splitting.
While solutions of the free oximes 1a and 1b are both bright red, the substitutions
performed led to a purple color. This was caused by a bathochromic shift of the main
absorption peak with shoulders at both sides in the visible range from 502 (1a) or 503
(1b) to 525 (2), 518 (3a), or 519 nm (3b) in MeOH. Several other peaks showed similar
behaviors. Substitution of H by Br at C(5) shifted the maximum at 212 (1a, 2, and 3a) to
219 nm (1b and 3b). The bromo derivatives are characterized by slightly higher
extinction coefficients.
Positive-ion-mode ESI mass spectra of 2, 3a, and 3b all display the peaks of the
corresponding cations (m/z 463, 363, and 441, resp). For 2, peaks of carnitine (m/z 161),
acetylcarnitine (m/z 204), and the deacetylated cation (m/z 421) were also observed.
Cytotoxicity. The cytotoxic activities of 2, 3a, and 3b were determined in the human
tumor cell lines A549 (non-small cell lung cancer), CH1 (ovarian carcinoma), and
SW480 (colon carcinoma) by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yl-2H-tetrazolium bromide (MTT) assay. For comparison, 1a and 1b were also tested.
The compounds displayed IC50 values in the low mm range or even lower, in the general
order CH1<SW480<A549 (Table). The concentrationꢀeffect curves of the oxime
ester 2 are nearly identical to those of the oxime 1a, suggesting that cleavage of the
ester (extracellular due to hydrolysis and/or via intracellular esterases), takes place
prior to distinct interaction with target molecules. Generally, the IC50 values of the
bromo derivatives are lower than those of the corresponding unsubstituted oxime or
oxime ethers. Those of the stable choline ethers 3a and 3b are comparable to the
corresponding precursors 1a and 1b, although doseꢀeffect curves differ significantly.
While the oxime ethers are typically more potent than the oximes up to concentrations
corresponding to the IC50 values, they become much less potent in higher concen-
trations (Fig. 1). The only exception is the pair 1a/3a in cell line CH1, which exhibited
almost identical effects throughout the applied concentrations. Cytocidal effects

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. Concentrationꢀeffect curves of indirubine derivatives 1a, 1b, 2, 3a, and 3b, obtained by MTT assay
in the human cancer cell lines A549, CH1, and SW480 (continuous exposure for 96 h). Values are meansꢁ
standard deviations from at least three independent experiments.
(corresponding to T/C values<20,<11 and<4% in the case of A549, SW480, and CH1
cells, resp.) were observed for all oximes, but not for the corresponding ethers in all cell
lines with the exception of CH1, where all compounds exhibited cytocidal behavior at
high concentrations. All in all, the results suggest different modes of action of oximes
on the one hand and oxime ethers on the other.

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Table. Cytotoxicity (IC50 [mm]) of Oximes 1a and 1b, Carnitine Oxime Ester 2, and Choline Oxime Ethers
3a and 3b in Three Human Cancer Cell Linesa)
Compound
A549
CH1
SW480
1a
1b
2
3a
3b
7.9ꢁ1.2
1.6 ꢁ0.2
7.6ꢁ3.6
13ꢁ4
1.6 ꢁ0.4
5.2ꢁ1.3
1.1ꢁ0.3
5.6ꢁ1.7
2.5ꢁ0.4
0.52ꢁ0.27
0.52ꢁ0.04
1.8ꢁ0.5
1.3ꢁ0.2
1.0 ꢁ0.3
0.28ꢁ0.17
a) 50% Inhibitory concentrations in A549, CH1, and SW480 cells after exposure for 96 h in the MTT
assay. Values are meansꢁstandard deviations obtained from at least three independent experiments.
Hydrolysis of the Oxime Ester. The hydrolysis of acetyl-l-carnitine indirubin-3’-
monoxime ester 2 was examined by UV/VIS spectroscopy (Fig. 2). For this purpose, a
6 mm solution of 2 in 10 mm phosphate buffer, at pH 7.2, containing 5% glucose, was
prepared. This medium is suitable for infusion. Note that the solubility of 2 in saline
(0.9% NaCl) is much lower (<0.5 mm). The optimal pH was found experimentally.
The solution, which was kept at 218, became rapidly turbid. Hence, aliquots were taken
periodically, filtered, diluted to 0.12 or 0.06 mm with the same medium, and absorption
spectra (250–650 nm) were recorded immediately. An exponential fit on the plot of the
absorbance at 326 or 520 nm vs. time under the assumption of first-order kinetics
revealed for the hydrolysis a rate of 24ꢁ2%/h.
Fig. 2. Hydrolysis of 2, monitored by UV/VIS spectroscopy at l of 326 nm
This behavior is in line with the MTTassays. The conversion of 2 to 1a and acetyl-l-
carnitine occurred rapidly within the incubation time. In addition, the high hydrolysis
rate of 2 precludes its intravenous administration.
Analogous experiments under the same conditions with the oxime ethers 3a and 3b
showed that they remained completely intact for at least 20 h.
Flow-Cytometric Study of Cell-Cycle Phase Distribution. To investigate whether 3a
and 3b are capable of inducing shifts in cell-cycle distribution, exponentially growing
A549 cells were treated with these compounds in varying concentrations for 24 h,
stained with propidium iodide, and analyzed for their DNA content by flow cytometry.
As with the cytotoxicity, 1a and 1b were included in this study. While 1a caused a

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marked G2/M arrest (ca. 49% at 40 mm compared to 10% in untreated control), the
effect of 1b was appreciably smaller (ca. 20% at 40 mm; Fig. 3). This is remarkable, since
1b displayed substantially lower IC50 values than 1a for various CDK/Cyc complexes
and GSK-3b in a cell-free assay, testing inhibitory activity on 85 kinases [20]. The
oxime ethers 3a and 3b did not influence the phase distribution significantly.
Consequently, it is more likely that the mode of action is based on interference with
other targets, in line with the different shapes of concentrationsꢀeffect curves in the
MTT experiments. Therefore, the cytotoxicities must be caused by a mechanism which
is independent from cell-cycle regulation.
Fig. 3. Concentration-dependent impact of indirubine-3’-monoximes 1a, 1b, 3a, and 3b on the cellꢀcycle
phase distribution of A549 cells after exposure for 24 h. DNA Content of PI-stained cells was analyzed by
flow cytometry.
Preliminary Toxicological Investigations. An orientating preliminary study in mice,
to which L1210 leukamia cells were implanted, did not give any indication of a
therapeutical window, but revealed severe toxicity, with a dosage of 50 mg/kg/day of 3a,
administered on three consecutive days, being lethal. Using 1a, the same regime was
reported to be non-toxic to male SpragueꢀDawley rats, although continuation of the
treatment led to hepatic damage [21]. So, the acute toxicity of 3a must be ascribed to
the choline moiety.

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Conclusions. – New water-soluble indirubin derivatives containing a quaternary
ammonium group have been synthesized. The first, an oxime ester with acetyl-l-
carnitine designed according to the prodrug concept, releases the free oxime 1a, whose
interesting properties are well established in the literature. Unfortunately, the rate of
hydrolysis turned out to exceed clinical requirements for intravenous administration.
Alternatively, stable oxime ethers, 3a and 3b with a permanently attached choline
moiety were prepared. In contrast to some other water-soluble indirubin derivatives,
these compounds still had an acceptable impact on the cell proliferation, albeit no
influence on the cell-cycle distribution was detected, suggesting a mode of action
independent of inhibition of related protein kinases. However, for 3a toxicity was
observed in preliminary animal experiments. Therefore, a prodrug with a more
retarded release of the active indirubine species is required. Besides, attention should
be paid to potential side-effects, including hepatotoxicity during the development of
new derivatives.
The authors are indebted to the FFG (Austrian Research Promotion Agency, project no. 811591
FA526003), the Austrian Council for Research and Technology Development, and COST (European
Cooperation in the Field of Scientific and Technical Research) for financial support. We also thank
Anatoly Dobrov for recording the ESI mass spectra. Irene Herbacek (Institute of Cancer Research,
Department of Medicine I, Medical University of Vienna) is gratefully acknowledged for flow cytometry
analyses, and Peter Rauko (Cancer Research Institute of the Slovak Academy of Sciences in Bratislava)
for preliminary animal experiments.
Experimental Part
General. Solvents and reagents, if available in anal. grade, were obtained from commercial suppliers
and used without further purification. Water from a reverse osmotic demineralization facility was
distilled before use. The precursors indirubin-3’-monoxime and 5-bromoindirubin-3’-monoxime were
prepared according to literature procedures [1][22][23]. Kinetic measurements of 2 were performed in
10 mm phosphate buffer, at pH 7.2, containing 5% glucose. UV/VIS Spectra were recorded on a Perkin-
Elmer Lambda 650 spectrophotometer using samples dissolved in MeOH (800–210 nm); lmax in nm (e in
mꢀ1 cmꢀ1). For the description of shoulders (sh), the wavelength of the appropriate local minimum of the
first-derivative absolute value was applied. IR Spectra were obtained by a Bruker Vertex 70 instrument
equipped with a Specac Golden Gate Single Reflection Diamond ATR unit (4000–550 cmꢀ1); ˜n in cmꢀ1
.
1H, 13C, 1H,1H-COSY, 1H,1H-TOCSY, 1H,1H-ROESY, 1H,13C-HSQC, 1H,13C-HMBC, 1H,13C-HSQC-
TOCSY, 1H,15N-HSQC, and 1H,15N-HMBC NMR spectra were recorded in (D6)DMSO, CD3OD, or D2O
with a Bruker Avance III 500 MHz FT-NMR spectrometer, at 500.10, 125.81, and 50.70 MHz for 1H, 13C,
and 15N, resp; d in ppm, J in Hz. The 2D-NMR spectra were recorded in a gradient-enhanced mode. The
residual 1H and 13C signals present in the solvents were used as internal references. 15N Shifts are quoted
rel. to external NH4Cl, abreviation: Lc, l-carnitine. MS: An Esquire 3000 ion-trap mass spectrometer
(Bruker Daltonics, D-Bremen) with an orthogonal ESI ion source (pos.-ion mode) was used; in m/z. The
solns. in MeOH were introduced via flow injection by a Cole-Parmer 74900 single-syringe infusion pump
(Vernon Hills, IL). Calculated and experimental isotope distributions were compared. Elemental
analyses were carried out on Perkin-Elmer (2400 CHN) and Mettler-Toledo (DL 21) microanalyzers at
the Microanalytical Laboratory of the University of Vienna.
(2R)-2-(Acetyloxy)-4-({[(2Z,3E)-2-(1,2-dihydro-2-oxo-3H-indol-3-ylidene)-1,2-dihydro-3H-indol-3-
ylidene]amino}oxy)-N,N,N-trimethyl-4-oxobutan-1-aminium Chloride (2). Acetyl-l-carnitine hydro-
chloride (0.48 g, 2.0 mmol) was suspended in cooled (08), freshly destilled oxalyl chloride (7.0 ml,
81 mmol) under Ar. After 10 min, the mixture was heated to 308 and stirred vigorously at this temp. for
2 h. The stirring was interrupted every 20 min for a short sonication (2 min). The formed emulsion (which
separated into two phases on standing) was evaporated to dryness in vacuo. The solid residue was

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
suspended in abs. CH2Cl2 (10 ml) by using an ultrasonic bath. Then, the solvent was removed again in
vacuo. This operation was repeated with abs. THF (10 ml) in order to remove traces of unreacted oxalyl
chloride. Subsequently, the acyl chloride was suspended in abs. THF (10 ml), and a soln. of indirubin-3’-
monoxime (1a; 0.67 g, 2.4 mmol) in abs. THF (20 ml) was added. After 3 h stirring at 308, the flask was
closed under Ar and allowed to stand in the refrigerator (þ28) for 48 h. Then, abs. EtOH (10 ml) was
added under vigorous stirring, which was continued for further 40 min. The precipitate formed was
filtered off, washed quickly with abs. EtOH (5 ml) and Et2O (5 ml), and dried in vacuo. The crude
product was dissolved in cold H2O (150 ml, 28) and filtered by suction immediately, the filtrate was frozen
with liquid N2 and lyophilized. The substance was stored under Ar at ꢀ208. Yield: 0.46 g (44%). UV/
VIS: 549 (sh, 9.79ꢂ103), 525 (1.16ꢂ104), 494 (sh, 8.91ꢂ103), 372 (sh, 1.42ꢂ103), 356 (sh, 2.38ꢂ103), 334
(sh, 7.84ꢂ103), 324 (8.79ꢂ103), 294 (sh, 2.34ꢂ104), 288 (2.63ꢂ104), 249 (2.02ꢂ104), 211 (3.32ꢂ104).
IR (selected bands): 3212, 1739, 1660, 1613, 1569, 1483, 1464, 1327, 1308, 1222, 1145, 1100, 1039, 966, 917,
876, 813, 787, 744, 700. 1H-NMR ((D6)DMSO): 11.61 (br. s, H–N(1’)); 10.92 (br. s, H–N(1)); 9.06 (d, J¼
7.9, HꢀC(4)); 8.25 (d, J¼7.8, HꢀC(4’)); 7.55 (ddd, J¼7.0, 7.3, 1.2, HꢀC(6’)); 7.49 (d, J¼8.0, HꢀC(7’)); 7.22
(ddd, J¼7.6, 7.6, 1.1, HꢀC(6)); 7.09 (ddd, J¼7.7, 7.9, 1.0, HꢀC(5’)); 6.99 (ddd, J¼7.8, 7.6, 1.1, HꢀC(5));
6.93 (d, J¼7.4, HꢀC(7)); 5.69 (m, HꢀC(3)(Lc)); 3.98 (dd, J¼8.5, 14.4, Ha –C(4)(Lc)); 3.86 (dd, J¼1.3,
14.3, Hb –C(4)(Lc)); 3.38 (dd, J¼6.2, 16.9, Ha –C(2)(Lc)); 3.33 (dd, J¼5.3, 16.8, Hb –C(2)(Lc)); 3.18 (s, 3
Me(Lc)); 2.09 (s, Ac). 13C-NMR ((D6)DMSO): 171.1 (C(2)); 170.2 (COMe); 166.8 (C(1)(Lc)); 157.5
(C(3’)); 147.8 (C(7a’)); 142.3 (C(2’)); 140.2 (C(7a)); 135.3 (C(6’)); 130.2 (C(4’)); 128.3 (C(6)); 125.4
(C(4)); 122.1 (C(3a)); 122.0 (C(5’)); 121.5 (C(5)); 115.5 (C(3a’)); 113.0 (C(7’)); 109.6 (C(7)); 104.2
(C(3)); 67.2 (C(4)(Lc)); 65.2 (C(3)(Lc)); 53.6 (3 Me(Lc)); 36.0 (C(2)(Lc)); 21.5 (COMe). 1H,15N-
HMBC ((D6)DMSO): 94.0 (N(1’)); 114.6 (N(1)). ESI-MS: 161 ([carnitine]þ ), 204 ([acetylcarnitine]þ ),
421 ([MꢀAcþH]þ ), 463 ([MꢀCl]þ ). Anal. calc. for C25H27ClN4O5 ·1.5 H2O: C 57.09, H 5.75, N 10.65,
Cl 6.74; found: C 56.75, H 5.84, N 10.46, Cl 6.43.
2-({[(2Z,3E)-2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,2-dihydro-3H-indol-3-ylidene]amino}oxy)-
N,N,N-trimethylethanaminium Chloride (3a). Compound 1a (0.28 g, 1.0 mmol) was dissolved in abs.
DMF (5.0 ml) under Ar. The red soln. was cooled in an ice bath, before NaH (60% dispersion in mineral
oil, 0.10 g, 2.5 mmol) was added. The mixture was stirred vigorously at 08 for 45 min, interrupted by a
short sonication (1–2 min) after 15 min. Then, (2-bromoethyl)trimethylammonium bromide (0.59 g,
2.3 mmol) was added. Ten min later, after a second short sonication the mixture was slowly heated to 908
and stirred at this temp. for 22 h. After cooling of the mixture to r.t., DMF was removed in vacuo. The
residue was dissolved in H2O (150 ml), Ag2CO3 (0.73 g, 2.6 mmol) was added, and the mixture was
stirred at r.t. under light protection for 1.5 h. The suspension was filtered through a suction filter, the
filtrate was treated with conc. HCl (37%, 2.4 ml, 29 mmol), followed by another portion of Ag2CO3
(0.73 g, 2.6 mmol). After 30 min of vigorous stirring, the precipitate was filtered off. The filtrate was
transferred into a separatory funnel and shaken with BuOH (40 ml). Then, NaCl (50 g, 856 mmol) was
added, and shaking was continued. After separation, the aq. layer was extracted with BuOH (20 ml) once
more. The combined org. layers were repeatedly washed with brine (5ꢂ70 ml). Finally, the accurately
separated org. layer was evaporated to dryness in vacuo. The crude product was dissolved in BuOH
(25 ml), mixed with CH2Cl2 (75 ml), and filtered to remove traces of NaCl. The filtrate was evaporated to
dryness in vacuo again and, the remaining solid was dissolved in H2O (30 ml) and filtered. The clear
purple soln. was frozen with liquid N2 and lyophilized. The lyophilization was repeated with H2O (20 ml)
slightly acidified with HCl (37%, 30 ml, 0.36 mmol). Yield: 0.17 g (40%). UV/VIS: 549 (sh, 8.56ꢂ103),
518 (1.19ꢂ104), 491 (sh, 9.61ꢂ103), 455 (sh, 5.02ꢂ103), 365 (sh, 1.50ꢂ103), 339 (9.43ꢂ103), 327 (9.70ꢂ
103), 286 (2.78ꢂ104), 252 (1.96ꢂ104), 212 (3.21ꢂ104). IR (selected bands): 3366, 3238, 1655, 1611, 1563,
1482, 1464, 1393, 1327, 1309, 1260, 1225, 1190, 1147, 1097, 1024, 978, 961, 877, 863, 809, 786, 770, 745, 699,
682, 634. 1H-NMR ((D6)DMSO): 11.70 (br. s, H–N(1’)); 10.87 (br. s, H–N(1)); 8.58 (d, J¼7.8, HꢀC(4));
8.24 (d, J¼7.7, HꢀC(4’)); 7.47 (dd, J¼8.0, 7.1, HꢀC(6’)); 7.43 (d, J¼7.4, HꢀC(7’)); 7.18 (dd, J¼7.5, 7.6,
HꢀC(6)); 7.04 (dd, J¼6.9, 7.9, HꢀC(5’)); 7.02 (dd, J¼7.6, 7.8, HꢀC(5)); 6.94 (d, J¼7.6, HꢀC(7)); 5.10 (m,
CH2O); 4.01 (m, CH2N); 3.25 (s, 3 Me). 13C-NMR ((D6)DMSO): 171.3 (C(2)); 152.8 (C(3’)); 146.4
(C(7a’)); 143.9 (C(2’)); 139.4 (C(7a)); 133.7 (C(6’)); 129.1 (C(4’)); 127.1 (C(6)); 124.1 (C(4)); 122.6
(C(3a)); 122.0 (C(5’)); 121.3 (C(5)); 116.4 (C(3a’)); 112.4 (C(7’)); 109.6 (C(7)); 101.4 (C(3)); 70.5
(OCH2); 64.4 (CH2N); 53.4 (3 CH3). 1H,15N-HMBC ((D6)DMSO): 96.0 (N(1’)); 115.0 (N(1)). ESI-MS:

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2183
363 ([MꢀCl]þ ). Anal. calc. for C21H23ClN4O2 ·1.5 H2O: C 59.22, H 6.15, N 13.15, Cl 8.32; found: C 59.59,
H 5.93, N 12.98, Cl 8.19.
2-({[(2Z,3E)-2-(5-Bromo-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-1,2-dihydro-3H-indol-3-ylidene]-
amino}oxy)-N,N,N-trimethylethanaminium Chloride (3b). To a mixture of 5-bromoindirubin-3’-oxime
(1b; 0.71 g, 2.0 mmol), K2CO3 (0.42 g, 3.0 mmol), and (2-chloroethyl)trimethylammonium chloride
(0.65 g, 4.0 mmol) was added abs. DMF (14 ml). After a short sonication (1–2 min), the mixture was
stirred vigorously at 708 for 18 h. The stirring was interrupted by short sonications (1–2 min) after 1 and
17 h. Subsequently, DMF was removed in vacuo. The residue was transferred to H2O (100 ml), acidified
with aq. HCl (37%, 0.6 ml, 7 mmol), and carefully sonicated for 15 min. After vigorous stirring for
further 60 min, H2O (150 ml) was added, and the mixture was extracted with AcOEt (5ꢂ160 ml),
followed by BuOH (6ꢂ70 ml). The combined BuOH layers were washed with H2O (3ꢂ25 ml) acidified
with HCl (37%, 3ꢂ0.1 ml). After addition of Et2O (60 ml) and the removal of the small amounts of H2O
which separated thereupon, the BuOH was removed in vacuo. The residue was suspended in CH2Cl2
(120 ml) under sonication and allowed to stand in refrigerator (28) for 48 h. Then, the crude product was
filtered off, washed with CH2Cl2 (3ꢂ8 ml), and dried in vacuo. Finally, the product was dissolved in H2O
(60 ml), the soln. was filtered, and the filtrate was acidified with HCl (37%, 40 ml), frozen with liquid N2,
and lyophilized. Yield: 0.61 g (60%). UV/VIS: 550 (sh, 9.79ꢂ103), 519 (1.36ꢂ104), 491 (sh, 1.09ꢂ104),
455 (sh, 5.57ꢂ103), 364 (sh, 9.54ꢂ102), 338 (9.62ꢂ103), 326 (1.01ꢂ104), 295 (3.94ꢂ104), 289 (sh, 3.60ꢂ
104), 255 (2.26ꢂ104), 219 (3.43ꢂ104). IR (selected bands): 3377, 3136, 1655, 1612, 1562, 1482, 1463, 1440,
1313, 1270, 1223, 1189, 1147, 1101, 1028, 979, 961, 875, 813, 783, 745, 726, 691, 660. 1H-NMR
((D6)DMSO): 11.73 (br. s, H–N(1’)); 11.02 (br. s, H–N(1)); 8.79 (d, J¼1.9, HꢀC(4)); 8.25 (d, J¼7.7,
HꢀC(4’)); 7.51–7.45 (m, HꢀC(6’), HꢀC(7’)); 7.33 (dd, J¼2.0, 8.2, HꢀC(6)); 7.07 (dd, J¼7.8, 7.5,
HꢀC(5’)); 6.90 (d, J¼8.2, HꢀC(7)); 5.08 (m, CH2O); 4.07 (m, CH2N); 3.26 (s, 3 Me). 13C-NMR
((D6)DMSO): 170.9 (C(2)); 152.9 (C(3’)); 146.3 (C(7a’)); 145.0 (C(2’)); 138.3 (C(7a)); 133.9 (C(6’));
129.0 (C(4’) or C(6)); 128.9 (C(4’) or C(6)); 126.2 (C(4)); 124.8 (C(3a)); 122.4 (C(5’)); 116.3 (C(3a’));
112.9 (C(5)); 112.8 (C(7’)); 111.2 (C(7)); 100.0 (C(3)); 70.6 (CH2O); 64.2 (CH2N); 53.4 (3 Me). 1H,15N-
HMBC ((D6)DMSO): 98.4 (N(1’)); 114.6 (N(1)). ESI-MS: 441 ([MꢀCl]þ ). Anal. calc. for
C21H22BrClN4O2 ·1.8 H2O: C 49.44, H 5.06, N 10.98, Cl 6.94; found: C 49.50, H 4.74, N 10.97, Cl 7.05.
Cell Lines and Culture Conditions [24]. CH1 Cells originate from an ascites sample of a patient with
a papillary cystadenocarcinoma of the ovary and were a generous gift from Lloyd R. Kelland, CRC
Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK. SW480 (Adenocarcinoma of
the colon) and A549 (non-small cell lung cancer) cells were kindly provided by Brigitte Marian (Institute
of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria). All cell culture
reagents were obtained from Sigma–Aldrich Austria. Cells were grown in 75 cm2 culture flasks (Iwaki)
as adherent monolayer cultures in Minimal Essential Medium (MEM) supplemented with 10% heat-
inactivated fetal calf serum, 1 mm sodium pyruvate, 4 mm l-glutamine, and 1% non-essential amino acids
(100ꢂ). Cultures were maintained at 378 in a humidified atmosphere containing 5% CO2.
MTT Assay Conditions [24][25]. Cytotoxicity was determined by the colorimetric MTT (¼ 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, purchased from Sigma-Aldrich) micro-
culture assay. For this purpose, cells were harvested from culture flasks by trypsinization and seeded into
96-well microculture plates (Iwaki). Cell densities of 1.5ꢂ103 (CH1), 2.5ꢂ103 (SW480), and 4ꢂ103
cells/well (A549) were chosen in order to ensure exponential growth of the untreated controls
throughout drug exposure. Cells were allowed to settle and resume adherent growth in drug-free
complete culture medium for 24 h. Stock solns. of the test compounds in DMSO were diluted in complete
culture medium such that the highest effective DMSO content did not exceed 0.5% (especially in case of
1a and 1b, this procedure yielded opaque but colloidal solns. from which no precipitates could be
separated by centrifugation). These dilutions were added to the microcultures, and cells were exposed to
the test compounds for 96 h. At the end of exposure, all media were replaced by 100 ml/well RPMI1640
culture medium (supplemented with 10% heat-inactivated fetal bovine serum and 4 mm l-glutamine)
plus 20 ml/well MTT solution in phosphate-buffered saline (5 mg/ml). After incubation for 4 h, the
supernatants were removed, and the formazan crystals formed by viable cells were dissolved in 150 ml
DMSO per well. Optical densities at 550 nm were measured with a microplate reader (Tecan Spectra
Classic), using a reference wavelength of 690 nm. The quantity of viable cells was expressed in terms of

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
T/C values by comparison to untreated control microcultures, and 50% inhibitory concentrations (IC50)
were calculated from concentration–effect curves by interpolation. Evaluation is based on means from at
least three independent experiments, each comprising six replicates per concentration level.
Cell-Cycle Analyses. A549 Cells (1ꢂ106 cells) were seeded into Petri dishes and allowed to recover
for 24 h. Cells were then exposed for 24 h to the test compounds by addition of stock soln. as described for
the MTTassays. Control and treated cells were collected, washed with PBS, fixed in 70% ice-cold EtOH,
and stored at ꢀ208. To determine cell-cycle distributions, cells were transferred in physiological NaCl
soln. into PBS, incubated with 10 mg/ml RNAse A for 30 min at 378, followed by treatment with 5 mg/ml
propidium iodide for 30 min, and their fluorescence was measured by flow cytometry, using a FACS
Calibur instrument (Becton Dickinson, Palo Alto, CA). The resulting DNA histograms were quantified
by Cell Quest Pro software (Becton Dickinson and Company, New York, USA).
Preliminary Toxicological Investigations. Animal experiments were performed in accordance with
the European Community Guidelines for the use of experimental animals in the animal facility at the
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic. L1210 Murine
leukemia cells (1ꢂ105) were injected in a volume of 0.5 ml into DBA/2J mice (weighing 18–22 g)
intraperitoneally on day 0. The test compound 3a (25 and 50 mg/kg body weight) was administered
intraperitoneally in a volume of 0.5 ml aqua ad injectionem per mouse per day in a split-dose regimen
(days 1, 2, and 3 after implantation of cells) to groups of two animals per dose.
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Received April 26, 2012