Synthesis of thia-analogous indirubin n-glycosides and their influence onmelanoma cell growth and apoptosis

Article abstract of DOI:10.1002/cmdc.200900506

Stopping cancer in its tracks Thia-analogues of indirubin-N-glycosides, prepared by condensation of N-glycosylisatines with thiaindane-3-one and subsequent deprotection, were tested for their activity against malignant melanoma cells. These indirubin-N-gl

Full text of DOI:10.1002/cmdc.200900506

MED
DOI: 10.1002/cmdc.200900506
Synthesis of Thia-Analogous Indirubin N-Glycosides and their Influence on
Melanoma Cell Growth and Apoptosis
Manfred Kunz,[a] Katrin M. Driller,[b] Martin Hein,[b] Stephanie Libnow,[b] Ina Hohensee,[a] Robert Ramer,[c]
Burkhard Hinz,[c] Anja Berger,[d] Jꢀrgen Eberle,[d] and Peter Langer*[b, e]
Glycosylated indoles possess remarkable pharmacological ac-
tivity against different malignant tumor cells. Prominent deriva-
tives with antitumor activity include the natural products staur-
osporine, K-252d, rebeccamycin and tjipanazoles.[1] Indigo, in-
dirubin, and isoindigo contain a bis-indole framework and are
found in a number of natural products. We previously reported
the synthesis of indigo-N-glycosides (blue sugars).[2] This type
of core structure is present in the akashines A–C, which were
isolated by Laatsch et al. from Streptomyces sp. GW48/1497.[3] In
contrast to the inactive parent indigo, the akashines show a re-
markable antitumor activity against various human cancer cell
lines. Indirubin, the red isomer of indigo, is the active ingredi-
ent in the traditional Chinese medicinal recipe Danggui Long-
hui Wan, which has been used for the treatment of myelocytic
leukemia.[4] This substance and its substituted derivatives are
potent inhibitors of several kinases involved in intracellular sig-
naling pathways, such as GSK-3b and cyclin dependent kinases
(CDKs).[5,6] Recently, we reported the synthesis of indirubin-N-
glycosides (red sugars),[7] which showed considerable antiproli-
ferative activity against various human cancer cell lines. Sassa-
telli et al. described the preparation of isoindigo-N-glycosides,
which also possess considerable antiproliferative activity and
kinase inhibitory potency.[8] Notably, both the deprotected and
protected isoindigo-N-glycosides are of pharmacological rele-
vance. For example, the biological activity of ‘Natura‘, acetyl-
protected b-d-xylopyranosyl-N-isoindigo, was reported to be
higher than the activity of its deprotected analogue.[9] Herein,
we report the first synthesis of thia-analogues of indirubin-N-
glycosides and their influence on melanoma cell growth, apop-
tosis and intracellular signaling.
sation products with isatin, where only the reactive b-carbonyl
group of the isatin is involved in the reaction. N-Glycosylated
isatins are also suitable for such condensation reactions.[7,8] As
starting materials in the synthesis of thia-analogous indirubin-
N-glycosides, we used isatin-N-glycosides with different carbo-
hydrate moieties and thiaindane-3-one. In the case of the
sugar l-rhamnose, acetyl-protected N-(b-l-rhamnopyranosyl)-
isatin (4-Rha-b) was prepared from the acetyl-protected ani-
line-N-rhamnoside (3-Rha-a,b), which was obtained by reaction
of l-rhamnose with aniline and subsequent acetylation
(Scheme 1).[7,10] Furthermore, the corresponding isatin-b-N-gly-
cosides of d-mannose, d-glucose and d-galactose were pre-
pared under similar conditions.[7] Thiaindane-3-one was pre-
pared from thiosalicylic acid and ethyl acetoacetate in the
presence of sulfuric acid according to a procedure published
by Friedlꢁnder.[11]
Reaction of the acetyl-protected isatin-N-glycosides (4-b)
with thiaindane-3-one under mildly acidic conditions resulted
in the formation of thia-analogous indirubin-N-glycosides in
good to very good yields (see example given in Scheme 2).[12]
Compounds with a reactive methylene group like indoxyl, 3-
coumaranone or thiaindane-3-one are known to give conden-
[a] Prof. Dr. M. Kunz, I. Hohensee
Scheme 1. Synthesis of the acetyl-protected N-b-l-rhamnopyranosylisatin (4-
Rha-b). Reagents and conditions: a) PhNH2, EtOH, 208C, 12 h, 96%; b) Ac2O,
pyridine, 0!48C, 8–12 h, 85%; c) oxalyl chloride, AlCl3, 558C, 1.5 h, 63%.
Comprehensive Center for Inflammation Medicine
University of Schleswig-Holstein, Campus Lꢀbeck, 23562 Lꢀbeck (Germany)
[b] K. M. Driller, Dr. M. Hein, Dr. S. Libnow, Prof. Dr. P. Langer
Institute of Organic Chemistry, University of Rostock
Albert-Einstein-Str. 3a, 18059 Rostock (Germany)
Fax: (+49)381-4986-412
E-mail: peter.langer@uni-rostock.de
[c] Dr. R. Ramer, Prof. Dr. B. Hinz
Institute of Toxicology and Pharmacology, University of Rostock
18055 Rostock (Germany)
[d] A. Berger, Dr. J. Eberle
Department of Dermatology and Allergy, Skin Cancer Center Charitꢁ
Charitꢁ-Universitꢂtsmedizin Berlin, 10117 Berlin (Germany)
[e] Prof. Dr. P. Langer
Leibniz Institute of Catalysis e.V., 18059 Rostock (Germany)
Scheme 2. Synthesis of 3-[thiaindan-3’-on-2’-(Z)-ylidene]-1-(2’’,3’’,4’’-tri-O-
acetyl-b-l-rhamnopyranosyl)oxindol (5-Rha-b). Reagents and conditions:
a) Ac2O, AcOH, NaOAc, 808C, 1 h.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900506.
534
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 534 – 539

Interestingly, only one p-diastereomer could be isolated (Z-
isomer). The condensation reaction was carried out with the
isatin-N-glycosides of l-rhamnose (4-Rha-b), d-Mannose (4-
Man-b), d-glucose (4-Glc-b) and d-galactose (4-Gal-b). The
yields and reaction times are given in Table 1.
Table 1. Results of the synthesis of the thia-analogous indirubin-N-glyco-
sides of l-rhamnose, d-mannose, d-glucose und d-galactose.
Scheme 3. Deacetylation of thia-analogous indirubin-N-glycosides (synthesis
of the deprotected 3-[thiaindan-3’-one-2’-(Z)-ylidene]-1-(b-l-rhamnopyrano-
syl)oxindol, 6-Rha-b). Reagents and conditions: a) NaOMe, anhyd MeOH, RT;
b) 1% HCl in anhyd MeOH, RT.
Glycoside
Product (Z-Isomer)
Yield
[%]
Trxn
[h]
5-Rha-b
b-l-rhamnose
90
95
72
93
1
1
1
1
Table 2. Results of the deacetylation of the thia-analogous indirubin-N-
glycosides.
Glycoside
Product (Z-Isomer)
Yield
[%]
Trxn
[h]
6-Rha-b
5-Man-b
5-Glc-b
5-Gal-b
b-l-rhamnose
78
84
75
56
3
12
12
12
b-d-mannose
b-d-glucose
b-d-galactose
6-Man-b
6-Glc-b
6-Gal-b
b-d-mannose
b-d-glucose
b-d-galactose
In the case of the sulfur-analogous indirubin derivatives, de-
protection of the acetyl groups could be carried out under
base catalysis (Zemplꢃn conditions),[13,8c] and resulted in the
formation of the desired products 6-b in 56–84% yield (see ex-
ample given in Scheme 3).
Deacetylation is quite fast under the chosen basic conditions
and no side reactions (ring opening reactions) at the thiain-
dan-3-one carbonyl group (C-3) were observed. An alternative
method for the deprotection using acidic conditions (1%
methanolic HCl)[14] was also tested. Cleavage of the acetyl
groups was possible but longer reaction times (3–5 days) were
required and the deprotection product was isolated in lower
yields (65% in the case of the deprotection of 5-Rha-b).
Table 2 shows the results of the deprotection of the acetylated
compounds 5-b under Zemplꢃn conditions.
Two additional compounds were synthesized and used as
reference compounds in the cell-based assays. Compound 7-
Rha-b is a glycosylated indirubin[7] and compound 8-Rha-b is a
related derivative, which could be synthesized by aldol addi-
tion of 4-methoxyacetophenone to 4-Rha-b and subsequent
dehydration.[15]
ChemMedChem 2010, 5, 534 – 539
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
535

MED
with 6-Rha-b and 8-Rha-b (10 mm) and assayed for sub-G1 cell
populations after PI staining, indicating DNA fragmentation
due to apoptosis. Both glycosylated indirubin analogues signif-
icantly increased the numbers of sub-G1 cells in the four mela-
noma cell lines after 48 h and 72 h. The percentage of apop-
totic cells after 72 h reached more than 20% and was five- to
tenfold that of the untreated control (Figure 1a). At the same
time, the number of cells in G1 was significantly reduced (Fig-
ure 1b). Apoptosis was also investigated by a second assay
monitoring DNA fragmentation via an ELISA. In line with the
subG1 populations, DNA fragmentation revealed largely paral-
lel results, namely significantly increased apoptosis after 48 h
and 72 h of treatment with the glycosylated indirubin ana-
logues (Figure 1c).
As mentioned above, indirubin glycosides are strong inhibi-
tors of intracellular signaling kinases and may affect the cell
proliferation of a variety of different tumor cells.[16,17] Here, the
antiproliferative activities of thia-analogous indirubin-N-glyco-
sides 6-b were tested in four different metastatic melanoma
lines (SK-Mel-19, SK-Mel-29, SK-Mel-103, SK-Mel-147). For this
purpose, IC50 values of each of the tested thia-analogous indir-
ubin-N-glycosides were determined using a colorimetric assay
with WST-1 as a substrate (Table 3). The IC50 values showed
The effects of glycosylated indirubin analogues on melano-
ma cell proliferation were monitored by real-time cell analysis
(RTCA). Whereas the cell density of untreated SK-Mel-103 and
SK-Mel-147 in culture continuously increased during the obser-
vation period of 70 h after seeding, 6-Rha-b and 8-Rha-b-treat-
ed cells showed dramatically decreased attached cell numbers
and cell density after only 10 h (Figure 2).
Table 3. Inhibition of metastatic melanoma cell lines by different glycosy-
lated indirubins and analogous compounds.
The cell lines SK-Mel-19 and SK-Mel-29 responded similarly,
however, the effect of 8-Rha-b was somewhat less pronounced
(data not shown). Thus, the data show that thia-analogous in-
dirubin-N-glycosides not only inhibit cell growth but also
induce early apoptotic events in melanoma cells. In support of
these findings, induction of apoptosis in tumor cells by indiru-
bin derivatives was shown in a series of earlier reports. Indiru-
bin-3-monooxime induced apoptosis in Hep-2 laryngeal carci-
noma cells.[18] Moreover, indirubin-3’-oximes potently blocked
constitutive Stat3 signaling in human breast and prostate
cancer cells. Consecutive down-regulation of the Stat3 target
genes encoding antiapoptotic proteins Mcl-1 and survivin was
accompanied by induction of apoptosis in these cells.[19]
Melanoma cell growth and survival involves different intra-
cellular signaling pathways, such as Ras/Raf/MEK/ERK and
PI(3)K pathways.[20] It is well accepted that the transcription
factor cJun is a downstream target of Ras/Raf/MEK/ERK and
JNK/SAPK pathways.[21] Moreover, cJun plays an important role
in melanoma biology.[22,23] To address whether thia-analogous
indirubin-N-glycosides may influence cJun and JNK phosphory-
lation/activation as a possible explanation for their growth in-
hibitory effects, melanoma cell lines were treated with 10 mm
6-Rha-b, and phosphorylation of dually phosphorylated cJun
on Ser63 and Ser73, and dually phosphorylated JNK1/2/3 on
Thr183 and Tyr185, was analyzed by immunoblotting
(Figure 3).
Compd
IC50 values [mm]
SK-Mel-19
SK-Mel-29
SK-Mel-103
SK-Mel-147
6-Rha-b
6-Man-b
6-Gal-b
6-Glc-b
7-Rha-b
8-Rha-b
12.08ꢀ1.21
5.81ꢀ1.17
6.29ꢀ1.29
3.96ꢀ1.25
5.57ꢀ1.33
8.39ꢀ1.26
86.60ꢀ1.53
11.92ꢀ1.14
10.38ꢀ1.16
6.69ꢀ1.17
6.94ꢀ1.39
6.06ꢀ1.19
383.30ꢀ2.29
46.41ꢀ1.20
7.52ꢀ1.14
4.28ꢀ1.13
6.27ꢀ1.24
4.98ꢀ1.17
n.d.
24.36ꢀ1.52
17.10ꢀ1.35
65.39ꢀ1.17
13.93ꢀ1.10
27.17ꢀ1.23
slight variations between the different cell lines; 6-Gal-b and
6-Glc-b were less active in SK-Mel-19 cells compared with the
other cell lines tested. 7-Rha-b and 8-Rha-b were used as con-
trol substances. The IC50 values of 7-Rha-b, which had no
effect on melanoma cell proliferation in preliminary experi-
ments (data not shown), were far greater than those of other
substances and can be considered as inactive. 8-Rha-b was
less active in the SK-Mel-103 and SK-Mel-147 cell lines, but had
comparable activity to compounds 6-b in SK-Mel-19 and SK-
Mel29 cells. Altogether, compounds 6-b appear to be active in
melanoma cells.
Similar results were recently obtained for related glycosylat-
ed indirubin derivatives tested in other human cancer cell
lines, such as 5637 (bladder), A-427 (small-cell lung), Kyse-70
(esophageal) and MCF-7 (breast), with IC50 values ranging from
0.67 mm to 15.28 mm.[16] Interestingly, the IC50 values of these
compounds were in the range of well-known cytostatic agents,
such cisplatin and chlorambucil.[16] These findings suggest in-
dirubin-N-glycosides as active agents against malignant mela-
noma and possibly also other malignant tumor types.
Indeed, melanoma cell lines SK-Mel-19 and SK-Mel-29 re-
vealed dramatic reductions in JNK and cJun phosphorylation
after 48 h and 72 h, respectively. In contrast, the negative con-
trol indirubin 7-Rha-b only effected JNK phosphorylation, and
had no effect on cJun phosphorylation. 8-Rha-b had a less pro-
nounced effect on JNK phosphorylation compared with 6-Rha-
b, but again significantly reduced cJun phosphorylation. These
findings support the role of cJun, but not of JNK, in melanoma
cell biology. Obviously, cJun and JNK phosphorylation/activa-
tion at the tested amino acid sites occur independently. The
Impaired apoptosis regulation is a critical feature of metasta-
sizing tumors such as malignant melanoma. In order to test
the hypothesis that thia-analogous indirubin-N-glycosides
induce apoptosis, the four melanoma cell lines were treated
536
www.chemmedchem.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 534 – 539

particular role of the cJun phos-
phorylation at Ser63 and Ser73
for cJun activity, has been de-
scribed.[24]
Taken together, the presented
data support the important role
for cJun phosphorylation in cell
proliferation. However, addition-
al mechanisms involved in the
antiproliferative activity of indir-
ubin-N-glycosides and thia-anal-
ogous compounds cannot be
ruled out.
Based on these findings, it
might be reasonable to combine
indirubin derivatives with other
chemical agents, for example,
those targeting the mammalian
target of rapamycin (mTOR), a
downstream
component
of
PI(3)K signaling. When used
alone, rapamycin had little effect
on
malignant
melanoma.[25]
More recent developments, such
as the small-molecule MEK inhib-
itor AZD6244, are currently
being evaluated in clinical trials
in patients with advanced mela-
noma.[26] However, data from
these trials have not been pub-
lished so far. These compounds
might also be good candidates
for combination therapy with in-
dirubins since they may act syn-
ergistically by addressing differ-
ent signaling pathways.
In conclusion, the first synthe-
sis of thia-analogues of indiru-
bin-N-glycosides has been re-
ported. Our current studies sug-
gest that the strategy outlined
herein is quite general and can
be successfully applied to the
synthesis of a variety of deriva-
tives containing different carbo-
hydrate moieties. Notably, it is
also possible to prepare oxa-
analogous indirubin-N-glycosides
by condensation of N-glycosylat-
ed isatines with 3-coumaranone,
but the deprotection of the
acetyl groups seems to be more
difficult due to side reactions.
Figure 1. Induction of apoptosis in melanoma cells by glycosylated indirubin analogues. a) Four melanoma cell
lines (SK-Mel-19, SK-Mel-29, SK-Mel-103 and SK-Mel-147) were treated with 10 mm of 6-Rha-b (&) or 8-Rha-b (&),
or vehicle control (&). After incubation for 24, 48 and 72 h, respectively, cell cycle analyses were performed using
PI staining and flow cytometry. The sub-G1 cell populations indicating apoptotic cells (in percent) are shown.
Data shown reflect the means ꢀSD of one representative experiment with triple values. The whole experiment
has been performed three times showing highly comparable results. b) Representative cell cycle analyses are
shown for the cell lines SK-Mel-29 and SK-Mel-147 after 48 h (treated versus untreated). c) The amount of DNA
fragmentation reflecting apoptosis was also determined by an alternative assay (cell death detection ELISA, as de-
scribed in the Supporting Information). As before, cells were treated with 10 mm of 6-Rha-b (&) or 8-Rha-b (&),
or vehicle control (&). Relative values are given as compared with untreated controls. Data shown reflect the
means ꢀSD of one representative experiment with triple values. The whole experiment has been repeated once
showing highly comparable results.
These derivatives are active
against malignant melanoma
cells in vitro and are consequent-
ly good candidates for preclinical
ChemMedChem 2010, 5, 534 – 539
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
537

MED
and perhaps subsequent clinical studies. They not only stop
melanoma cell proliferation at concentrations similar to cur-
rently used chemotherapeutic agents, but also induce a signifi-
cant rate of apoptosis. Finally, the thia-analogues of indirubin-
N-glycosides interfere with a well-defined intracellular signaling
pathway active in melanoma cells.
Experimental Section
The Supporting Information contains:
1) Detailed experimental procedures for the synthesis of com-
pounds 5-b and 6-b.
2) Analytical data (1H NMR, 13C NMR, MS, HRMS and melting
points, where possible) of compounds 5-b and 6-b.
3) Growth conditions of melanoma cell lines.
4) Determination of IC50 values of indirubins for different melano-
ma cell lines, immunoblotting procedures and apoptosis
assays.
Acknowledgements
Financial support by the State of Mecklenburg-Western Pommer-
ania (scholarship for Mrs. Stefanie Libnow) and by the Deutsche
Krebshilfe (Melanomverbund; grant number 108008) are grateful-
ly acknowledged.
Figure 2. Glycosylated indirubin analogues interfere with cell confluence of
melanoma cells. Melanoma cell lines a) SK-Mel-103 and b) SK-Mel-147 were
seeded at a density of 5000 cells per microtiter well. Cells were treated with
indirubin analogues 6-Rha-b and 8-Rha-b (mm) 26 h after seeding and moni-
tored for 70 h. The normalized cell index gives a relative measurement of
cell numbers (set to 1 at 26 h). The experiment was performed twice, each
in triplicate. Data correspond to the means of one representative experi-
ment.
Keywords: antitumor agents · apoptosis · carbohydrates ·
indirubin analogues · kinases
[1] a) Review: G. W. Gribble, S. J. Berthel in Studies in Natural Products
Chemistry, Vol. 12, Elsevier Science, New York, 1993, pp. 365–409; isola-
tion of staurosporin: b) S. Omura, Y. Iwai, A. Hirano, A. Nakagawa, J.
Awaya, H. Tsuchiya, Y. Takahashi, R. Masuma, J. Antibiot. 1977, 30, 275–
282; synthesis: c) J. T. Link, S. Raghavan, M. Gallant, S. J. Danishefsky,
T. C. Chou, L. M. Ballas, J. Am. Chem. Soc. 1996, 118, 2825–2842; phar-
macological activity: d) Y. Yamashita, N. Fujii, C. Murakata, T. Ashizawa,
M. Okabe, H. Nakano, Biochemistry 1992, 31, 12069–12075.
[2] a) M. Hein, D. Michalik, P. Langer, Synthesis 2005, 3531–3534; b) M.
Hein, T. B. P. Nguyen, D. Michalik, H. Gçrls, M. Lalk, P. Langer, Tetrahedron
Lett. 2006, 47, 5741–5745.
[3] R. P. Maskey, I. Grꢀn-Wollny, H. H. Fiebig, H. Laatsch, Angew. Chem.
2002, 114, 623–625; Angew. Chem. Int. Ed. 2002, 41, 597–599.
[4] Z. Xiao, Y. Hao, B. Liu, L. Qian, Leuk. Lymphoma 2002, 43, 1763–1768.
[5] A. Beauchard, Y. Ferandin, S. Frere, O. Lozach, M. Blairvacq, L. Meijer, V.
Thiery, T. Besson, Bioorg. Med. Chem. 2006, 14, 6434–6443.
[6] a) M. J. Moon, S. K. Lee, J.-W. Lee, W. K. Song, S. W. Kim, J. I. Kim, C. Cho,
S. J. Choi, Y.-C. Kim, Bioorg. Med. Chem. 2006, 14, 237–246; b) M. A.
Tucker, A. M. Goldstein, Oncogene 2003, 22, 3042–3052.
[7] S. Libnow, M. Hein, D. Michalik, P. Langer, Tetrahedron Lett. 2006, 47,
6907–6909.
[8] a) M. Sassatelli, E. Saab, F. Anizon, M. Prudhomme, P. Moreau, Tetrahe-
dron Lett. 2004, 45, 4827–4830; b) M. Sassatelli, F. Bouchikhi, S. Mes-
saoudi, F. Anizon, E. Debiton, C. Barthomeuf, M. Prudhomme, P. Moreau,
Eur. J. Med. Chem. 2006, 41, 88–100; c) M. Sassatelli, F. Bouchikhi, B.
Aboab, F. Anizon, F. Doriano, M. Prudhomme, P. Moreau, Anti-Cancer
Drugs 2007, 18, 1069–1074.
[9] a) L. Wang, X. Liu, R. Chen, (Natrogen Therapeutics, Inc., Whitefish Bay,
WI, USA), US6566341, 2003, [Chem. Abstr. 2003, 138, 379213]; b) L.
Wang, X. Liu, R. Chen, (Natrogen Therapeutics, Inc., Whitefish Bay, WI,
USA), WO/2003/051900, 2003, [Chem. Abstr. 2003, 139, 47135].
Figure 3. Glycosylated indirubin analogues interfere with phosphorylation of
intracellular signaling kinase JNK and transcription factor cJun. a) SK-Mel-19
and b) SK-Mel-29 cells were treated with 10 mm 6-Rha-b, 7-Rha-b and 8-Rha-
b. Expression of dually phosphorylated JNK1/2/3 and cJun was analyzed by
immunoblotting using appropriate antibodies. Staining of b-tubulin was
used as loading control.
538
www.chemmedchem.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 534 – 539

[10] a) C. Chavis, C. De Gourcy, F. Dumont, J.-L. Imbach, Carbohydr. Res.
1983, 113, 1–20; b) J. G. Douglas, J. Honeyman, J. Chem. Soc. 1955,
3674–3678; c) M. N. Preobrazhenskaya, I. V. Yartseva, L. V. Ektova, Nucle-
ic Acid Chem. 1978, 2, 725–727; d) I. V. Yartseva, L. V. Ektova, M. N. Preo-
brazhenskaya, Bioorg. Khim. 1975, 1, 189–194.
[18] T. R. Kameswaran, R. Ramanibai, Biomed. Pharmacother. 2009, 63, 146–
154.
[19] S. Nam, R. Buettner, J. Turkson, D. Kim, J. Q. Cheng, S. Muehlbeyer, F.
Hippe, S. Vatter, K. H. Merz, G. Eisenbrand, R. Jove, Proc. Natl. Acad. Sci.
USA 2005, 102, 5998–6003.
[11] P. Friedlꢁnder, Liebigs Ann. Chem. 1907, 351, 390–420.
[12] Similar conditions were previously employed for the aldol condensation
of thiazolidinones with isatines: a) H. Altuntas, ꢄ. Ates, B. S. Uydes-
Dogan, F. I. Alp, D. Kaleli, O. ꢄzdemir, S. Birteksçz, G. ꢄtꢀk, D. Satana, M.
Uzun, Arzneim.-Forsch. 2006, 56, 239–248; b) K. Fries, A. Hasselbach, L.
Schrçder, Liebigs Ann. Chem. 1914, 405, 346–372.
[13] G. Zemplꢃn, A. Gerecs, I. Hadꢅcsy, Chem. Ber. 1936, 69B, 1827–1829.
[14] A. Pews-Davtyan, A. Piroan, I. Shaljyan, A. A. Awetissjan, H. Reinke, C.
Vogel, J. Carbohydr. Chem. 2003, 22, 939–962.
[20] A. J. Miller, M. C. Mihm Jr. , N. Engl. J. Med. 2006, 355, 51–65.
[21] M. J. Robinson, M. H. Cobb, Curr. Opin. Cell Biol. 1997, 9, 180–186.
[22] V. N. Ivanov, A. Bhoumik, M. Krasilnikov, R. Raz, L. B. Owen-Schaub, D.
Levy, C. M. Horvath, Z. Ronai, Mol. Cell 2001, 7, 517–528.
[23] I. Poser, A. K. Bosserhoff, Histol. Histopathol. 2004, 19, 173–188.
[24] C. C. Franklin, V. Sanchez, F. Wagner, J. R. Woodgett, A. S. Kraft, Proc.
Natl. Acad. Sci. USA 1992, 89, 7247–7251.
[25] V. Gray-Schopfer, C. Wellbrock, R. Marais, Nature 2007, 445, 851–857.
[26] P. Hersey, L. Bastholt, V. Chiarion-Sileni, G. Cinat, R. Dummer, A. M. Eg-
germont, E. Espinosa, A. Hauschild, I. Quirt, C. Robert, D. Schadendorf,
Ann. Oncol. 2009, 20, Suppl 6, vi35–vi40.
[15] M. Becker, P. Langer, unpublished results.
[16] S. Libnow, K. Methling, M. Hein, D. Michalik, M. Harms, K. Wende, A.
Flemming, M. Koeckerling, H. Reinke, P. J. Bednarski, M. Lalk, P. Langer,
Bioorg. Med. Chem. 2008, 16, 5570–5583.
[17] L. Meijer, A. L. Skaltsounis, P. Magiatis, P. Polychronopoulos, M. Knock-
aert, M. Leost, X. P. Ryan, C. A. Vonica, A. Brivanlou, R. Dajani, C. Crovace,
C. Tarricone, A. Musacchio, S. M. Roe, L. Pearl, P. Greengard, Chem. Biol.
2003, 10, 1255–1266.
Received: December 5, 2009
Revised: January 13, 2010
Published online on February 4, 2010
ChemMedChem 2010, 5, 534 – 539
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
539