Synthesis and antiproliferative activity of selenoindirubins and selenoindirubin-N-glycosides

Article abstract of DOI:10.1039/c3ob40603b

Selenoindirubins and selenoindirubin-N-glycosides were prepared by the reaction of isatins and isatin-N-glycosides with 3-acetoxy-benzo[b]selenophene, respectively. While selenoindirubin-N-glycosides have not been reported before, three non-glycosylated s

Full text of DOI:10.1039/c3ob40603b

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Synthesis and antiproliferative activity of
selenoindirubins and selenoindirubin-N-glycosides†
Cite this: DOI: 10.1039/c3ob40603b
Friedrich Erben,a Dennis Kleeblatt,a Marcel Sonneck,a Martin Hein,a Holger Feist,a
Thomas Fahrenwaldt,a Christine Fischer,b Abdul Matin,c Jamshed Iqbal,d
Michael Plötz,e Jürgen Eberlee and Peter Langer*a,b
Selenoindirubins and selenoindirubin-N-glycosides were prepared by the reaction of isatins and isatin-N-
glycosides with 3-acetoxy-benzo[b]selenophene, respectively. While selenoindirubin-N-glycosides have
not been reported before, three non-glycosylated selenoindirubins were previously reported, but
without quantities, yields, scales, experimental details and spectroscopic data. In addition, the work
could, in our hands, not be reproduced to prepare pure products. The present paper includes an opti-
mized procedure for the synthesis of selenoindirubins and their complete characterization. Both seleno-
indirubins and selenoindirubin-N-glycosides showed antiproliferative activity in lung cancer cell lines. In
melanoma cells, antiproliferative effects were further accompanied by induced apoptosis in combination
with the death ligand TRAIL.
Received 26th March 2013,
Accepted 29th April 2013
DOI: 10.1039/c3ob40603b
www.rsc.org/obc
In recent years, it has been demonstrated that the pharma-
cological activity of indigo, indirubin and isoindigo derivatives
Introduction
In recent decades, the chemistry of indigo, indirubin and iso- can be considerably increased by the presence of a glycosyl
indigo derivatives has witnessed a dramatic rebirth because it moiety attached to the nitrogen atom.6 Laatsch and coworkers
was discovered that these bis-indoles are not only useful dyes, reported the isolation of the akashines A, B, and C.7 These
but also potent anti-cancer agents (Fig. 1). While indigo is N-glycosylated 5,5′-dichloroindigos show a strong activity against
pharmacologically inactive, indirubin represents a bioactive various human tumor cell lines. Although indigo-N-glycosides
natural product1 which is the active ingredient of Danggui are synthetically available,8 applications are limited by low
Longhui Wan, a recipe used in traditional Chinese medicine for yields and by the unstable character of the molecules. Moreau,
the treatment of myelocytic leukemia.2 Synthetic indirubin Sassatelli and their coworkers reported the synthesis of iso-
derivatives were reported to be selective inhibitors of cyclin- indigo-N-glycosides which exhibit anticancer activity.9 We have
dependent kinases (CDKs) which play an important role in recently reported the synthesis of oxa- and thia-analogous iso-
cancer growth and proliferation.3 Thus, indirubin and its indigo-N-glycosides10 and of isoindigo-N,N′-diglycosides.11
derivatives have great potential in the treatment of cancer.4 Some years ago, we developed the first synthesis of indirubin-
Furthermore, it was shown that centrosome duplication in
tumor cells is selectively stopped by indirubin-3′-monoxime
without affecting normal cells.5
aInstitut für Chemie, Universität Rostock, Albert-Einstein-Str. 3a, 18059 Rostock,
Germany. E-mail: peter.langer@uni-rostock.de; Fax: +49 381 4986412
bLeibniz-Institut für Katalyse e. V. an der Universität Rostock,
Albert-Einstein-Str. 29a, 18059 Rostock, Germany
cInstitute of Biomedical and Genetic Engineering, PO Box: 2891, Sector: G-9/1,
Islamabad, 44000, Pakistan
dDepartment of Pharmaceutical Sciences, COMSATS Institute of Information
Technology, Postal Code 22060, Abbottabad, Pakistan
eDepartment of Dermatology and Allergy, Skin Cancer Center,
University Medical Center Charité, Berlin, Germany
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob40603b
Fig. 1 Indigoid bis-indoles.
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N-glycosides.12,13 These compounds exhibit a considerably (4) which underwent cyclization and decarboxylation to give
higher anti-proliferative activity than the corresponding agly- selenoindoxyl (5). Because of the instability of 5 in air (for-
cons. In 2010, we reported that thia-analogous indirubin-N-gly- mation of selenoindigo by dimerization), it was acetylated to
cosides exhibit an even higher anti-proliferative activity and an form the stable product 6 which can be stored for a long
apoptosis inducing effect on different types of melanoma cell period of time.
lines.14 In contrast, the corresponding aglycons were found to
The synthesis of selenoindirubin-N-glycosides by conden-
be inactive. This result prompted us to study the synthesis and sation of building blocks 1a–h and 6 was next studied
the anti-proliferative and apoptosis-inducing activity of selena- (Scheme 3, Table 1). Due to the sensitivity of selenoindoxyl (5)
analogous indirubin-N-glycosides and of their aglycons. The to oxygen, the reaction was carried out as a one-pot procedure
results of our efforts are reported herein.
under an argon atmosphere. The best yields were obtained by
the following one-pot procedure: first selenoindoxyl (5) was
generated by deacetylation of 6 under Zemplén conditions,
neutralization of the solution by the addition of a small
amount of acetic acid, followed by removal of the solvent
under reduced pressure. Subsequently, the isatin-N-glycoside
1, sodium acetate, acetic acid and acetic anhydride were
added. The reaction of isatin-N-glycosides 1a–h with 6 afforded
the desired acetyl-protected selenoindirubin-N-glycosides 7a–h
in good yields and with high anomeric purity. In those cases
where the isatin-N-glycosides contained a small amount of the
minor α-configured anomer, trace amounts of the α-anomer
were also present in the product. The deprotection of com-
pounds 7a–h was successfully achieved by treatment with cata-
lytic amounts of sodium methoxide in a solution of THF and
methanol (Scheme 3, Table 1). The deacetylated target mole-
cules 8a–h precipitated from the reaction mixture and were iso-
lated with high anomeric purity and in very good yields. In the
case of 8f–h, a small amount of the solvent could not be
removed.
Results and discussion
Selenoindirubin-N-glycosides
Isatin-N-glycoside 1a was prepared by reaction of aniline with
L-rhamnose, acetylation and subsequent cyclization with oxalyl
chloride (Scheme 1). Likewise, isatin-N-glycosides 1b–h were
prepared from the corresponding substituted anilines and
carbohydrates (Table 1). The synthesis, detailed structural
proof and discussion of the anomeric configuration of 1a–h
have been previously reported.11,12b Some of the isatin-N-glyco-
sides could be isolated as pure β-isomers (Table 1, 1a, 1e, 1f,
1h), whereas other derivatives contained a small amount of
the corresponding α-isomer (1b, 1c, 1d, 1g).
3-Acetoxy-benzo[b]selenophene (6) was prepared in
a
multi-step synthesis according to literature procedure
a
(Scheme 2).15,16 However, it is important to note that the pro-
cedure was optimized in terms of yield by small changes of the
conditions. Therefore, the detailed description of the exper-
iments is given in the Experimental section. Selenium was
reduced by sodium hydroxymethanesulfinate to form an
aqueous solution of disodium diselenide (Na2Se2).17 The reac-
tion of Na2Se2 with the diazonium salt generated from anthra-
nilic acid afforded the symmetrical diselanedibenzoic acid 3.
Its reduction with zinc and subsequent condensation with
sodium bromoacetate gave carboxymethylselanylbenzoic acid
The configuration of the C–C double bond was established
1
on the basis of H NMR chemical shifts. Proton H-4′ of 7a–h
appears in the range of δ = 9.15–9.22 ppm. The downfield shift
can be explained by the anisotropic effect of the carbonyl
group located at carbon atom C-3 which is only possible for
Z- but not for E-configured selenoindirubins. Similar results
were previously reported for thiaindirubin-N-glycosides.14
Selenoindirubins
In order to better evaluate the pharmacological relevance of
the carbohydrate moiety of the selenoindirubin-N-glycosides 8,
we decided to prepare a series of non-glycosylated selenoindi-
rubins and to compare their antiproliferative activities with
those of the glycosylated derivatives. As mentioned above, we
have previously observed that non-glycosylated thia-analogous
indirubins are entirely inactive, while thiaindirubin-N-glyco-
sides exhibit a strong anti-proliferative activity against mela-
noma cells.14 In contrast,
a significant anti-proliferative
activity against various cancer cell lines has been reported by
several researchers for non-glycosylated indirubins (containing
the usual two nitrogen atoms).3–5
Selenoindirubins have only scarcely been reported in the lit-
erature to date. Only three derivatives were reported about 100
years ago.18 However, no information related to the quantities
of starting materials and products, yields, and scales were
given. In addition, only elemental analyses and melting
points, but no spectroscopic data were provided, due to the
Scheme 1 Synthesis of 1-(2,3,4-tri-O-acetyl-L-rhamnopyranosyl)-isatin 1a
(according to ref. 11 and 12b). Reagents and conditions: (i) EtOH, 78 °C, 24 h;
(ii) Ac2O, pyridine, 0 °C, 12 h; (iii) (COCl)2 (10 eq.), AlCl3 (1 eq.), 55 °C, 1.5 h.
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Table 1 Synthesis of selenoindirubin-N-glycosides 7a–h and 8a–h
Yield (7)a
Yield (8)a
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Table 1 (Contd.)
Yield (7)a
Yield (8)a
a Yields of isolated products. b A small amount of solvent could not be removed.
Scheme 4 Synthesis of selenoindirubins 10a–h. Reagents and conditions:
(i) Method A: EtOH–THF, cat. KOtBu, 20 °C, 3–4.5 h. Method B: (1) MeOH,
NaOMe (0.1 eq.), 20 °C, 15–20 min, (2) HOAc (0.1 eq.), (3) cat. piperidine,
EtOH, 1.5 h.
Scheme 2 Synthesis of 3-acetoxy-benzo[b]selenophene 6. Reagents and condi-
tions: (i) NaOH, H2O, 20 °C, 5 h; (ii) HCl, NaNO2, H2O, −14 °C; (iii) (1) Na2Se2,
K2CO3, H2O, 0 °C → 100 °C, (2) HCl; (iv) (1) Zn, NaOH, H2O, 100 °C, 0.5 h,
(2) BrCH2COONa, 100 °C, 0.5 h, (3) HCl; (v) Ac2O, Pyridine, 90 °C, 6 h.
lack of methods at that time. The published procedure18 relies
on the reaction of selenoindoxyl (5) with N-unprotected isatins
(EtOH, catalytic amounts of piperidine). As we were unable
to reproduce the published procedure and to prepare pure
products, we decided to reinvestigate the synthesis of
selenoindirubins.
The reaction of 3-acetoxy-benzo[b]selenophene (6) with
N-methylisatin (9a), carried out in EtOH–THF in the presence
of catalytic amounts of KOtBu (method A), afforded N-methyl-
selenoindirubin 10a in 95% yield (Scheme 4, Table 2). The
product was formed by base catalyzed transformation of 6 into
5 and subsequent base catalyzed condensation. It is important
to note that the employment of selenoindoxyl (5) instead of 6
proved to be unsuccessful (formation of a complex mixture).
The reaction of 6 with unprotected isatin (9b), following
method A, afforded parent selenoindirubin (10b), albeit in
only 27% yield. The low yield can be explained by competing
deprotonation of the NH group by the base. The problem
could be solved by the development of method B. 3-Acetoxy-
Scheme 3 Synthesis of N-(L-rhamnopyranosyl)selenoindirubins 7a and 8a.
Reagents and conditions: (i) (1) 0.1 eq. NaOMe, MeOH, 20 °C, 15–20 min:
(2) HOAc, NaOAc, Ac2O, 80 °C, 3–4 h; (ii) NaOMe (cat.), MeOH–THF (2 : 1),
20 °C, 7 h.
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Selenoindirubins are conjugated systems with auxochromic
groups and can absorb light by π→π* and n→π* transitions of
electrons. UV/Vis spectra of 10a–h in acetic acid were recorded.
The spectra of all eight derivatives are very similar to each
other. The values of the wavelengths of the maximum absorp-
tions are shown in Fig. 2 and Table 3. The absorption maxima
at about 510 nm are located in the range of green light
(490–560 nm). Therefore, the compounds show a red to violet
colour.
In a first investigation for monitoring cellular effects of the
new compounds, the anti-proliferative activities of deprotected
selenoindirubin-N-glycosides 8a–e and of the non-glycosylated
selenoindirubin 10h were studied in lung carcinoma cells
(H157) and in human corneal epithelial cells (HCEC). Other
non-glycosylated derivatives were not included because of their
limited solubility in DMSO and water. Assays for the determi-
nation of cell density by the sulforhodamine colorimetric
assay revealed a dose-dependant decrease of cell density in
H157 cells within 24 h by all tested derivatives, whereas the
effects on HCEC cells were only weak (Table 4). At 100 μM, the
percent inhibition in H157 ranged between 17% (indirubin 8a)
and 30% (indirubin 8b). Methotrexate treatment was applied
as a reference.
Table 2 Synthesis of selenoindirubins 10a–h
9, 10
R1
R2
R3
Method
% (10)a
a
Me
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
A
A
B
B
B
B
B
B
B
95
27
99
84
93
94
86
95
92
b
b
c
d
e
F
Cl
Br
I
fa
g
h
OCF3
a Yields of isolated products.
benzo[b]selenophene (6) was treated with NaOMe–MeOH and
then with AcOH to generate selenoindoxyl (5) in situ (as
described for the synthesis of products 7a–h). Subsequently,
the isatin and catalytic amounts of piperidine were directly
added to the reaction mixture without isolation of 5. This pro-
cedure allowed for the synthesis of selenoindirubins 10b–h in
very good yields and with high purities (Scheme 4, Table 2).
Products 10b and 10f have been previously reported, but, as
mentioned above, without yields and spectroscopic character-
ization.18 Employment of 3-acetoxy-benzo[b]selenophene (6)
rather than selenoindoxyl (5) as the starting material proved to
be mandatory for the success of the reactions (Schemes 5–7).
Thus, in contrast to the sulfur series,14 a significant activity
was observed also for the non-glycosylated selenoindirubin
10h. However, selenoindirubin-N-glycosides showed a much
better solubility than the non-glycosylated derivatives which
precipitated during the biological screening. Although the
activities were not very high, the results were promising in the
sense that a dose-dependant activity was observed which can
be the basis for future studies. In addition, the results may
suggest that the pharmacological activity is based on the
heterocyclic moiety and that the carbohydrate may increase
the solubility and also the bioavailability of the compound.
However, this result is in contrast to the thiaindirubin series
and it has to be taken into account that different cell lines
were tested.
Scheme 5 Numbering of compounds 7 with 7a as an example.
In a previous study, we had identified synergistic proapop-
totic effects of indirubin derivatives and the death ligand
TRAIL in human melanoma cells.12c Thus, we investigated the
effects of indirubin 8e in combination with TRAIL in the mela-
noma cell line A-375. Other indirubin derivatives could not be
tested due to their limited solubility in the melanoma growth
medium. Comparable to HCEC and H157 cells, there were only
moderate effects on melanoma cell density by indirubin 8e
alone (10 μM), as determined in real time. However, the effect
of the death ligand TRAIL was strongly enhanced by the
addition of indirubin 8e (Fig. 3a). This effect on cell density
was clearly correlated with the loss of melanoma cell viability.
Again, indirubin 8e (10 μM) remained without significant
effect, but the effect of TRAIL was enhanced, resulting in a
loss of cell viability of 60% at 48 h in the combination treat-
ment (Fig. 3c).
Scheme 6 Numbering scheme of compounds 8 with 8a as an example.
We furthermore distinguished between reduced cell pro-
liferation, induced cytotoxicity and induced apoptosis. Indiru-
bin 8e revealed a dose-dependent (2.5–10 μM) antiproliferative
Scheme 7 Numbering scheme of compounds 10 with 10a as an example.
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Fig. 2 UV/Vis absorption spectra of compounds 10a–h (measured in acetic acid).
Table 3 Absorptions of selenoindirubins 10a–h
receptor agonists are promising future anti-tumor agents,19
combinations of TRAIL and selenoindirubin derivatives may
be considered.
10
λmax,1 [nm]
λmax,2 [nm]
λmax,3 [nm]
a
b
c
d
e
f
292.5
295.5
296.0
297.5
296.0
296.5
295.5
297.0
351.0
354.0
357.0
349.0
350.0
350.0
352.5
347.0
510.0
512.5
509.5
514.0
514.0
515.5
517.0
514.0
Conclusion
In conclusion, we have reported the synthesis of the first sele-
noindirubin-N-glycosides. We were able to prepare a series of
non-glycosylated selenoindirubins and report, for the first
time, spectroscopic data of this interesting type of indirubin
derivative. The products showed antiproliferative activities in
lung cancer and melanoma cells, as well as enhanced apopto-
sis with the proapoptotic stimulus TRAIL.
g
h
Table 4 Percentage inhibition values of the tested compounds in H-157 and
HCEC cells
100 μM
Compound
HCEC
H157
H157 (blind)a
Experimental section
2,2′-Diselanediyldibenzoic acid (3)
8a
8b
8c
8d
6.9 2.5
6.3 2.2
7.4 3.9
4.4 1.3
6.9 2.5
4.8 2.0
6.5 3.2
16.7 0.7
29.7 0.7
28.4 2.3
18.9 2.5
21.5 1.5
17.4 1. 3
20.1 1. 4
6.3 2.8
6.9 3.4
9.0 4.7
6.5 1.3
8.9 5.9
5.9 0.6
4.3 2.0
A suspension of selenium (2.875 g, 0.036 mol), hydroxymetha-
nesulfinate (2.481 g, 0.021 mol) and NaOH (1.458 g,
0.036 mol) in degased water (28 ml) is stirred at 20 °C for 5 h.
Subsequently, after the addition of K2CO3 (5.039 g, 0.036 mol)
a degased diazonium salt solution* is added dropwise at 0 °C.
Then the reaction mixture is stirred at 100 °C for 15 min. The
raw product is precipitated by the addition of conc. HCl
(20 ml) and separated by filtration. It is dissolved again in a
solution of NaOH (1.604 g, 0.04 mol) in water (25 ml). By
repeated filtration all insoluble impurities are removed. Re-
acidification precipitates the product as a brown solid. After
washing with hot EtOH compound 3 can be obtained as a col-
ourless solid (3.084 g, 42%). Mp 358–360 °C. 1H NMR
(300 MHz, DMSO-d6): δ = 13.67 (s, 2H, 2 × COOH); 8.03 (dd,
8e
10h
Methotrexate
a Inhibition without the presence of the tested compound.
effect on A-375 cells, and this antiproliferative effect was
strongly enhanced in the combination with TRAIL (Fig. 3b).
Whereas there was no effect of indirubin 8b and TRAIL on
induced cytotoxicity, as determined by the release of lactate
dehydrogenase (Fig. 3d), apoptosis was significantly enhanced
by the combination treatment (30% apoptotic cells), as com-
pared to the effect of TRAIL alone (Fig. 3e).
Thus, the new derivatives revealed significant effects on
tumor cell numbers and cell proliferation. In combination
with other proapoptotic stimuli (here TRAIL), apoptosis induc-
tion was strongly enhanced, which resembles the effects
reported previously for other derivatives.12c As TRAIL or TRAIL
4
3
3J = 7.7 Hz, J = 1.4 Hz, 2H, 2 × CHAr); 7.68 (dd, J = 8.0 Hz,
4J = 0.7 Hz, 2H, 2 × CHAr); 7.49 (d“t”, J = 7.3 Hz, J = 8.0 Hz,
3
3
4J = 1.5 Hz, 2H, 2 × CHAr); 7.36 (d“t”, J = 7.5 Hz, J = 7.4 Hz,
4J = 0.9 Hz, 2H, 2 × CHAr). 13C NMR (75 MHz, DMSO-d6):
δ = 168.7 (s, 2 × COOH); 133.8 (2 × Cqu); 133.7, 131.8, 129.7
3
3
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Fig. 3 Effects of indirubin 8e in combination with TRAIL on melanoma cells. (a) Real-time cell analysis (xCELLigence) of A-375 cells treated with 5 μM indirubin 8e,
20 ng ml−1 TRAIL or the combination, as compared to non-treated controls. Seeding density was 1250 cells per microtiter well. The cell index was normalized at
24 h, when treatment had started (arrow). Cell indices indicate cell adhesion. Two independent experiments revealed highly comparable results. (b) Cell proliferation
for A-375 cells expressed as percent of non-treated controls was determined after 48 h by the WST-1 assay. Cells were treated with 2.5–10 μM indirubin and/or
20 ng ml−1 TRAIL. (c) Cell viability for A-375 cells was determined after 24 h by calcein staining. Cells were treated with TRAIL (20 ng ml−1), indirubin (10 μM) or the
combination. (d, e) Values of cytotoxicity (d) and apoptosis (e) are shown after indirubin, TRAIL or a combination treatment. Apoptosis values correspond to percen-
tages of sub-G1 cell populations, as determined after 24 h by flow cytometry after PI staining. LDH-release indicative of cytotoxicity was determined in parallel.
A-375 melanoma cells were treated with indirubin (10 μM) and/or TRAIL (20 ng ml−1), as indicated. (b–d) Mean values SDs of four to six individual values
are shown. Individual values were obtained in at least two independent experiments, which revealed highly comparable results. Statistical significance is indicated
(*, p < 0.05), comparing combined treatment with indirubin alone.
(3s, 6 × CHAr); 129.0 (Cqu); 126.7 (CHAr). MS (EI, 70 eV): m/z (%) = 100 °C. After the addition of zinc (3.302 g, 0.05 mol) stirring is
404 ([M]+, [80Se][82Se], 9), 402 ([M]+, [78Se][82Se], [80Se][80Se], 33), continued for 25 min. Then the excess of zinc is removed by
401 ([M]+, [77Se][82Se], 5), 400 ([M]+, [76Se][82Se], [78Se][80Se], 29), filtration and 2-bromoacetic acid (2.947 g, 0.02 mol) dissolved
399 ([M]+, [77Se][80Se], 10), 398 ([M]+, [76Se][80Se], [78Se][78Se], 16), in water (15 ml) and neutralized with Na2CO3 (1.124 g,
397 ([M]+, [77Se][78Se], 4), 396 ([M]+, [76Se][78Se], [77Se][77Se], 6), 0.01 mol) is added. After stirring at 20 °C for 1.5 h the mixture
395 ([M]+, [76Se][77Se], 2), 394 ([M]+, [76Se][76Se], 1), 322 (52), is heated up to 70 °C for 15 min. By the addition of conc. HCl
201 (100), 184 (89), 156 (45), 77 (20). HRMS (EI): calcd for (15 ml) and continued stirring at 20 °C for 1 h a precipitate is
C14H10O4Se2 ([M]+, [76Se] [78Se]) 395.89388, found 395.895066; formed. This raw product is separated by filtration. Recrystalli-
calcd for C14H10O4Se2 ([M]+, [76Se] [80Se]) 397.89310, found zation in MeOH–water gives compound 4 as a slightly yellow
397.893096; calcd for C14H10O4Se2 ([M]+, [78Se] [78Se]) solid (2.356 g, 45%). Mp 224–226 °C. 1H NMR (300 MHz,
3
397.89198, found 397.893096; calcd for C14H10O4Se2 ([M]+, DMSO-d6): δ = 12.96 (s, 2H, 2 × COOH); 7.98 (dd, J = 7.7 Hz,
3
4
[78Se] [80Se]) 399.89119, found 399.891737; calcd for 4J = 1.5 Hz, 1H, CHAr); 7.62 (dd, J = 8.1 Hz, J = 0.6 Hz, 1H,
C14H10O4Se2 ([M]+, [80Se] [80Se]) 401.89040, found 401.890813. CHAr); 7.52 (d“t”, 3J = 7.2 Hz, 3J = 8.1 Hz, 4J = 1.5 Hz, 1H,
Elemental analysis: calcd for C14H10NO4Se2 (400.15): C, 42.02; CHAr); 7.32–7.26 (m, 1H, CHAr); 3.60 (s, 2H, CH2). 13C NMR
H, 2.52. Found: C, 41.97; H, 2.90.
(63 MHz, DMSO-d6): δ = 172.4, 168.3 (2s, 2 × COOH); 137.3
*Preparation of the diazonium salt solution: Anthranilic (Cqu); 133.1, 131.6, 128.4 (3s, 3 × CHAr); 128.2 (Cqu); 125.2
acid (5 g, 0.036 mol) is suspended in water (30 ml) treated (CHAr); 25.9 (CH2). MS (EI, 70 eV): m/z (%) = 262 ([M]+, [82Se], 4),
with conc. HCl (7.5 ml) and cooled down to −15 °C. Sub- 260 ([M]+, [80Se], 27), 258 ([M]+, [78Se], 13), 257 ([M]+, [77Se], 4),
sequently, a solution of NaNO2 (2.516 g, 0.036 mol) in water 256 ([M]+, [76Se], 5), 201 (100), 156 (9), 145 (9), 77 (9), 51 (7).
(15 ml) is added dropwise.
HRMS (ESI-TOF/MS): calcd for C9H7O4Se ([M − H]−, [78Se])
256.95251, found 256.95287; calcd for C9H7O4Se ([M − H]−,
[80Se]) 258.95154, found 258.95189. Elemental analysis: calcd
for C9H8O4Se (259.12): C, 41.72; H, 3.11. Found: C, 42.30;
H, 3.44.
2-(Carboxylmethylselanyl)benzoic acid (4)
Compound 3 (4.042 g, 0.01 mol) is dissolved in a solution of
NaOH (6.061 g, 0.152 mol) in water (44 ml) and heated up to
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3-Acetoxy-benzo[b]selenophene (6)
C(O)CH3); 168.1 (CvO); 143.2, 142.6, 141.5 (3s, 3 × Cqu); 135.9
(CHAr); 131.6 (Cqu); 131.2 (CHAr); 128.1 (Cqu); 128.0, 127.6,
126.7, 126.4, 123.1 (5s, 5 × CHAr); 121.4 (Cqu); 113.6 (CHAr);
80.8 (C-1′′); 74.1, 70.6, 70.4, 70.1 (4s, C-2′′, C-3′′, C-4′′, C-5′′);
20.8, 20.8, 20.5 (3s, 3 × C(O)CH3); 17.6 (C-6′′). MS (EI, 70 eV):
m/z (%) = 601 ([M]+, [82Se], 4), 599 ([M]+, [80Se], 17), 597 ([M]+,
[78Se], 8), 596 ([M]+, [77Se], 3), 595 ([M]+, [76Se], 3), 327 (23), 273
(21), 153 (90), 111 (71), 43 (100). HRMS (ESI-TOF/MS): calcd for
C28H26NO9Se ([M + H]+, [78Se]) 598.07823, found 598.07804;
calcd for C28H26NO9Se ([M + H]+, [80Se]) 600.07696, found
600.07651.
A degased solution of compound 4 (3.368 g, 13.00 mmol) in
pyridine (3.2 ml, 39.00 mmol) and acetic anhydride (12.5 ml)
is stirred at 90 °C for 6 h. After the completion of the reaction
the mixture is poured into iced water and extracted two times
with EtOAc. Drying over Na2SO4, filtration, evaporation of the
solvent under reduced pressure and column chromatography
(heptanes–EtOAc 100 : 1) gives compound 6 as an oil (2.593 g,
83%), which is slightly coloured by the side-product 2,2′-disele-
1
3
noindigo. H NMR (300 MHz, CDCl3): δ = 7.87 (d, J = 7.6 Hz,
3
1H, H-4 or H-7); 7.78 (s, 1H, H-2); 7.68 (d (br), J = 7.6 Hz, 1H,
3
4
(Z)-1-(2,3,4-Tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-methyl-
3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]indolin-2-one (7b).
According to the general procedure, 3-acetoxy-benzo[b]seleno-
phene (6) (350 mg, 1.46 mmol), a solution of NaOMe (0.3 ml,
0.5 M) and later acetic acid (18 mg, 0.3 mmol) are brought to
reaction in MeOH (2 ml). The second step is carried out in
acetic acid (4 ml) and acetic anhydride (3 ml) by the addition
of 1-(2′,3′,4′-tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-methylisatin
(1b) (221 mg, 0.51 mmol) and sodium acetate (376 mg,
4.59 mmol). After column chromatography (heptanes–EtOAc
8 : 1 → 4 : 1) compound 7b is isolated as a purple solid
(161 mg, 52%, α,β-anomeric mixture in a ratio of β : α = 6 : 1).
Mp 156–158 °C. 1H NMR (300 MHz, CDCl3): (β-anomer) δ =
H-4 or H-7); 7.42 (dt, J = 7.5 Hz, J = 1.1 Hz, 1H, H-5 or H-6);
3
4
7.34 (dt, J = 7.5 Hz, J = 1.1 Hz, 1H, H-5 or H-6); 2.38 (s, 3H,
C(O)CH3). 13C NMR (63 MHz, CDCl3): δ = 168.3 (COCH3);
142.1, 137.3, 134.2 (3s, C-3, C-3a, C-7a); 125.9, 125.3, 124.5,
122.3 (4s, C-4, C-5, C-6, C-7); 113.5 (C-2); 20.9 (COCH3). MS
(EI, 70 eV): m/z (%) = 242 ([M]+, [82Se], 3), 240 ([M]+, [80Se], 19),
238 ([M]+, [78Se], 9), 237 ([M]+, [77Se], 3), 236 ([M]+, [76Se], 3),
198 (100), 196 (49), 194 (19), 169 (30), 89 (26). HRMS (EI): calcd
for C10H8O2Se ([M]+, [80Se]) 239.96840, found 239.969002.
Elemental analysis: calcd for C10H8O2Se (239.13): C, 50.23;
H, 3.37. Found: C, 50.03; H, 3.49.
General procedure for the synthesis of selenoindirubin-
N-glycosides 7a–h
3
8.98 (s, 1H, H-4′); 7.84 (d, J = 7.4 Hz, 1H, CHAr); 7.58–7.44 (m,
3H, 3 × CHAr); 7.32–7.26 (m, 1H, CHAr); 7.21–7.16 (m, 1H,
3
3
A solution of 6 in degased MeOH is treated with 0.1 eq. of CHAr); 5.98 (d, J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.60 (dd, J1′′,2′′ = 1.5
NaOMe and stirred for 15 min at 20 °C. After neutralization Hz, J2′′,3′′ = 2.9 Hz, 1H, H-2′′), 5.29–5.18 (m, 2H, H-3′′, H-4′′);
3
with acetic acid, all solvents are evaporated. The residue is dis- 3.81–3.70 (m, 1H, H-5′′); 2.40 (s, 3H, CH3); 2.09, 1.97, 1.88 (3s,
3
solved in a degased mixture of acetic acid and acetic anhy- 9H, 3 × C(O)CH3); 1.35 (d, J5′′,6′′ = 6.2 Hz, 3H, H-6′′).
dride. After the addition of glycosylated isatin 1 and of sodium (α-anomer) δ = 9.03 (s, 1H, H-4′); 7.89–7.86 (m, 1H, CHAr);
acetate, the mixture is stirred at 80 °C for 4 h. Then the 7.58–7.44 (m, 3H, 3 × CHAr); 7.41–7.34 (m, 1H, CHAr);
3
remaining sodium acetate is removed by filtration and all sol- 7.14–7.10 (m, 1H, CHAr); 5.94 (d, J1′′,2′′ = 1.5 Hz, 1H, H-1′′);
3
3
vents are evaporated in vacuo. The target compound 7 is puri- 5.60 (dd, J1′′,2′′ = 1.5 Hz, J2′′,3′′ = 2.9 Hz, 1H, H-2′′), 5.29–5.18
fied by column chromatography (flash silica gel). (m, 2H, H-3′′, H-4′′); 3.81–3.70 (m, 1H, H-5′′); 2.37 (s, 3H, CH3);
3
(Z)-1-(2,3,4-Tri-O-acetyl-β-L-rhamnopyranosyl)-3-[3-oxo-benzo- 2.08, 1.96, 1.88 (3s, 9H, 3 × C(O)CH3); 1.33 (d, J5′′,6′′ = 6.2 Hz,
[b]selenophen-2(3H)-ylidene]indolin-2-one (7a). According to 3H, H-6′′). 13C NMR (75 MHz, CDCl3): (β-anomer) δ = 192.8
the general procedure, 3-acetoxy-benzo[b]selenophene (6) (CvO); 170.0, 169.7, 169.6 (3s, 3 × C(O)CH3); 168.2 (CvO);
(359 mg, 1.50 mmol), a solution of NaOMe (0.3 ml, 0.5 M) and 143.4, 142.1, 139.4 (3s, 3 × Cqu); 135.8 (CHAr); 132.5 (Cqu);
later acetic acid (18 mg, 0.3 mmol) are brought to reaction in 131.9 (CHAr); 131.7, 128.4 (2s, 2 × Cqu); 127.9, 127.9, 126.7,
MeOH (2 ml). The second step is carried out in acetic 126.3 (4s, 4 × CHAr); 121.5 (Cqu); 113.3 (CHAr); 80.7 (C-1′′); 74.0,
acid (4 ml) and acetic anhydride (3 ml) by the addition of 70.6, 70.4, 70.1 (4s, C-2′′, C-3′′, C-4′′, C-5′′); 21.1 (CH3); 20.9,
1-(2′,3′,4′-tri-O-acetyl-β-L-rhamnopyranosyl)-isatin (1a) (419 mg, 20.8, 20.5 (3s, 3 × C(O)CH3); 17.6 (C-6′′). MS (EI, 70 eV): m/z (%) =
1.00 mmol) and sodium acetate (738 mg, 9.00 mmol). After 615 ([M]+, [82Se], 9), 613 ([M]+, [80Se], 41), 611 ([M]+, [78Se], 19),
column chromatography (heptanes–EtOAc 6 : 1 → 4 : 1), com- 610 ([M]+, [77Se], 7), 609 ([M]+, [76Se], 6), 341 (65), 273 (24), 153
pound 7a is isolated as a purple solid (446 mg, 75%). Mp (100), 111 (87), 43 (94). HRMS (ESI-TOF/MS): calcd for
1
3
150–152 °C. H NMR (300 MHz, CDCl3): δ = 9.16 (dd, J4′,5′
=
C29H27NNaO9Se ([M
8.0 Hz, J4′,6′ = 0.8 Hz, 1H, H-4′); 7.86–7.82 (m, 1H, CHAr); 634.07580; calcd for C29H27NNaO9Se ([M
7.60–7.57 (m, 1H, CHAr); 7.56–7.51 (m, 2H, 2 × CHAr); 7.38 636.07457, found 636.07490. Elemental analysis: calcd for
+
Na]+, [78Se]) 634.07585, found
Na]+, [80Se])
4
+
3
3
4
(d“t”, J = 7.5 Hz, J = 8.1 Hz, J = 1.3 Hz, 1H, CHAr); 7.33–7.27 C29H27NO9Se (612.49): C, 56.87; H, 4.44; N, 2.29. Found: C,
(m, 1H, CHAr); 7.12 (d“t”, J = 7.5 Hz, J = 7.8 Hz, J = 1.1 Hz, 56.93; H, 4.95; N, 2.17.
1H, CHAr); 6.00 (d, J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.61 (dd, J1′′,2′′
3
3
4
3
3
=
(Z)-1-(2,3,4-Tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-ethyl-
3
1.5 Hz, J2′′,3′′ = 2.9 Hz, 1H, H-2′′), 5.27–5.19 (m, 2H, H-3′′, 3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]indolin-2-one (7c).
H-4′′); 3.81–3.72 (m, 1H, H-5′′); 2.09, 1.97, 1.85 (3s, 9H, 3 × According to the general procedure, 3-acetoxy-benzo[b]seleno-
C(O)CH3); 1.36 (d, J5′′,6′′ = 6.2 Hz, 3H, H-6′′). 13C NMR phene (6) (375 mg, 1.57 mmol), a solution of NaOMe (0.3 ml,
3
(75 MHz, CDCl3): δ = 192.8 (CvO); 170.0, 169.7, 169.6 (3s, 3 × 0.5 M) and later acetic acid (18 mg, 0.3 mmol) are brought to
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3
reaction in MeOH (2 ml). The second step is carried out in 1.4 Hz, J2′′,3′′ = 2.9 Hz, 1H, H-2′′), 5.27–5.18 (m, 2H, H-3′′,
3
acetic acid (4 ml) and acetic anhydride (3 ml) by the addi- H-4′′); 3.81–3.70 (m, 1H, H-5′′); 2.99 (sep, J = 6.9 Hz, 1H, CH-
3
tion of 1-(2′,3′,4′-tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-ethylisa- (CH3)2); 2.09, 1.97, 1.85 (3s, 9H, 3 × C(O)CH3); 1.34 (d, J5′′,6′′
=
3
tin (1c) (468 mg, 1.05 mmol) and sodium acetate (772 mg, 6.2 Hz, 3H, H-6′′); 1.31 (d, J = 6.9 Hz, 3H, CH(CH3)2); 1.30 (d,
4
9.41 mmol). After column chromatography (heptanes–EtOAc 3J = 6.8 Hz, 3H, CH(CH3)2). (α-anomer) δ = 9.15 (d, J4′,6′
=
8 : 1 → 4 : 1) compound 7c is isolated as a purple solid 1.7 Hz, 1H, H-4′); 7.91–7.86 (m, 1H, CHAr); 7.57–7.52 (m, 2H,
3
(486 mg, 74%, α,β-anomeric mixture in a ratio of β : α = 8 : 1). CHAr); 7.49 (d, J = 8.4 Hz, 1H, CHAr); 7.33–7.28 (m, 1H, CHAr);
4
3
3
Mp 138–140 °C. 1H NMR (250 MHz, CDCl3): (β-anomer) δ = 7.26 (dd, J = 1.9 Hz, J = 8.2 Hz, 1H, CHAr); 6.04 (dd, J1′′,2′′
=
4
3
3
3
9.03 (d, J4′,6′ = 1.6 Hz, 1H, H-4′); 7.85 (d (br), J = 7.6 Hz, 1H, 6.5 Hz, J2′′,3′′ = 3.7 Hz, 1H, H-2′′); 5.86 (d, J1′′,2′′ = 6.5 Hz, 1H,
3
3
H-4 or H-7); 7.59–7.50 (m, 2H, H-4 or H-7, H-5 or H-6); 7.48 (d, H-1′′); 5.78 (dd, J2′′,3′′ = 3.7 Hz, J3′′,4′′ = 5.9 Hz, 1H, H-3′′); 5.01
3J6′,7′ = 8.3 Hz, 1H, H-7′); 7.33–7.26 (m, 1H, H-5 or H-6); 7.21 (dd, J3′′,4′′ = 5.9 Hz, J4′′,5′′ = 5.0 Hz, 1H, H-4′′); 4.15–4.05 (m,
3
3
4
3
3
(dd, J4′,6′ = 1.8 Hz, J6′,7′ = 8.3 Hz, 1H, H-6′); 5.98 (d, J1′′,2′′
=
1H, H-5′′); 2.99 (sep, 3J = 6.9 Hz, 1H, CH(CH3)2); 2.16, 2.15,
3
3
3
1.4 Hz, 1H, H-1′′); 5.60 (dd, J1′′,2′′ = 1.4 Hz, J2′′,3′′ = 2.8 Hz, 1H, 1.99 (3s, 9H, 3 × C(O)CH3); 1.43 (d, J5′′,6′′ = 6.8 Hz, 3H, H-6′′);
H-2′′), 5.31–5.17 (m, 2H, H-3′′, H-4′′); 3.83–3.69 (m, 1H, H-5′′); 1.31 (d, 3J = 6.9 Hz, 3H, CH(CH3)2); 1.30 (d, 3J = 6.8 Hz, 3H,
2.70 (q, J = 7.5 Hz, 2H, CH2CH3); 2.09, 1.97, 1.87 (3s, 9H, 3 × CH(CH3)2). 13C NMR (75 MHz, CDCl3): (β-anomer) δ = 192.7
3
C(O)CH3); 1.35 (d, 3J5′′,6′′ = 6.1 Hz, 3H, H-6′′); 1.28 (t, 3J = 7.5 Hz, (CvO); 169.9, 169.6, 169.6 (3s, 3 × C(O)CH3); 168.2 (CvO);
4
3H, CH2CH3). (α-anomer) δ = 9.03 (d, J4′,6′ = 1.6 Hz, 1H, H-4′); 143.8, 143.3, 142.0, 139.5 (4s, 4 × Cqu); 135.8 (CHAr); 131.7
7.85 (d (br), 3J = 7.6 Hz, 1H, H-4 or H-7); 7.59–7.50 (m, 2H, H-4 (Cqu); 129.3 (CHAr); 128.5 (Cqu); 127.9, 126.7, 126.2, 125.7 (4s,
or H-7, H-5 or H-6); 7.48 (d, 3J6′,7′ = 8.3 Hz, 1H, H-7′); 7.33–7.26 4 × CHAr); 121.5 (Cqu); 113.4 (CHAr); 80.7 (C-1′′); 74.0, 70.6,
(m, 1H, H-5 or H-6); 7.21 (dd, 4J4′,6′ = 1.8 Hz, 3J6′,7′ = 8.3 Hz, 1H, 70.4, 70.1 (4s, C-2′′, C-3′′, C-4′′, C-5′′); 33.9 (CH(CH3)2); 24.2,
3
3
H-6′); 5.95 (d, J1′′,2′′ = 1.4 Hz, 1H, H-1′′); 5.60 (dd, J1′′,2′′ = 1.4 24.1 (2s, CH(CH3)2); 20.8, 20.7, 20.5 (3s, 3 × C(O)CH3); 17.6
Hz, J2′′,3′′ = 2.8 Hz, 1H, H-2′′), 5.31–5.17 (m, 2H, H-3′′, H-4′′); (C-6′′). MS (EI, 70 eV): m/z (%) = 643 ([M]+, [82Se], 7), 641 ([M]+,
3
3.83–3.69 (m, 1H, H-5′′); 2.70 (q, 3J = 7.5 Hz, 2H, CH2CH3); [80Se], 25), 639 ([M]+, [78Se], 13), 638 ([M]+, [77Se], 5), 637 ([M]+,
2.08, 1.96, 1.86 (3s, 9H, 3 × C(O)CH3); 1.35 (d, J5′′,6′′ = 6.1 Hz, [76Se], 4), 369 (48), 273 (23), 153 (93), 111 (76), 43 (100). HRMS
3
3H, H-6′′); 1.28 (t, 3J = 7.5 Hz, 3H, CH2CH3). 13C NMR (63 MHz, (ESI-TOF/MS): calcd for C31H32NO9Se ([M
+
H]+, [78Se])
CDCl3): (β-anomer) δ = 192.8 (CvO); 170.0, 169.7, 169.6 (3s, 3 × 640.12526, found 640.1256; calcd for C31H32NO9Se ([M + H]+,
C(O)CH3); 168.2 (CvO); 143.3, 142.1, 139.5, 139.2 (4s, 4 × Cqu); [80Se]) 642.12395, found 642.12521. Elemental analysis: calcd
135.8 (C-4 or C-7); 131.7 (Cqu); 130.8 (C-6′); 128.5 (Cqu); 127.9 for C31H31NO9Se (640.54): C, 58.13; H, 4.88; N, 2.19. Found:
(C-4 or C-7); 127.0 (C-4′); 126.7, 126.3 (2s, C-5, C-6); 121.5 (Cqu); C, 58.13; H, 5.25; N, 2.35.
113.4 (C-7′); 80.7 (C-1′′); 74.0 (C-5′′); 70.6 (C-3′′); 70.4 (C-2′′);
(Z)-1-(2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl)-3-[3-oxo-
70.1 (C-4′′); 28.7 (CH2CH3); 20.8, 20.8, 20.5 (3s, 3 × C(O)CH3); benzo[b]selenophen-2(3H)-ylidene]indolin-2-one (7e). Accord-
17.6 (C-6′′) 16.1 (CH2CH3). MS (EI, 70 eV): m/z (%) = 629 ([M]+, ing to the general procedure, 3-acetoxy-benzo[b]selenophene
[82Se], 11), 627 ([M]+, [80Se], 51), 625 ([M]+, [78Se], 24), 624 (6) (359 mg, 1.50 mmol), a solution of NaOMe (0.3 ml, 0.5 M)
([M]+, [77Se], 9), 623 ([M]+, [76Se], 8), 355 (84), 171 (27), 153 and later acetic acid (18 mg, 0.3 mmol) are brought to
(100), 111 (90), 43 (95). HRMS (ESI-TOF/MS): calcd for reaction in MeOH (2 ml). The second step is carried out in
C30H30NO9Se ([M + H]+, [78Se]) 626.10958, found 626.11046; acetic acid (4 ml) and acetic anhydride (3 ml) by the addition
calcd for C30H30NO9Se ([M + H]+, [80Se]) 628.10829, found of 1-(2′,3′,4′,6′-tetra-O-acetyl-β-D-mannopyranosyl)-isatin (1e)
628.10946. Elemental analysis: calcd for C30H29NO9Se (626.51): (478 mg, 1.00 mmol) and sodium acetate (738 mg,
C, 57.51; H, 4.67; N, 2.24. Found: C, 57.61; H, 5.15; N, 2.17.
9.00 mmol). After column chromatography (heptanes–EtOAc
(Z)-1-(2,3,4-Tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-isopropyl- 6 : 1 → 3 : 1), compound 7e is isolated as a purple solid
3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]indolin-2-one (7d). (579 mg, 88%). Mp 135–137 °C. 1H NMR (300 MHz, CDCl3): δ =
3
4
According to the general procedure, 3-acetoxy-benzo[b]seleno- 9.15 (dd, J4′,5′ = 7.9 Hz, J4′,6′ = 0.8 Hz, 1H, H-4′); 7.82 (d (br),
phene (6) (359 mg, 1.50 mmol), a solution of NaOMe (0.3 ml, 3J = 7.5 Hz, 1H, CHAr); 7.58–7.48 (m, 3H, 3 × CHAr); 7.37 (d“t”,
3
4
0.5 M) and later acetic acid (18 mg, 0.3 mmol) are brought to 3J = 7.6 Hz, J = 8.0 Hz, J = 1.2 Hz, 1H, CHAr); 7.32–7.26 (m,
3
4
reaction in MeOH (2 ml). The second step is carried out in 1H, CHAr); 7.12 (d“t”, J = 7.8 Hz, J = 0.9 Hz, 1H, CHAr); 6.05
3
3
3
acetic acid (4 ml) and acetic anhydride (3 ml) by the addition (d, J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.64 (dd, J1′′,2′′ = 1.4 Hz, J2′′,3′′
=
of 1-(2′,3′,4′-tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-isopropylisa- 3.3 Hz, 1H, H-2′′), 5.43 (“t”, 3J3′′,4′′ = 10.1 Hz, 3J4′′,5′′ = 9.9 Hz,1H,
tin (1d) (462 mg, 1.00 mmol) and sodium acetate (738 mg, H-4′′); 5.33 (dd, 3J2′′,3′′ = 3.3 Hz, 3J3′′,4′′ = 10.2 Hz, 1H, H-3′′); 4.31
2
3
9.00 mmol). After column chromatography (heptanes–EtOAc (dd, J6a′′,6b′′ = 12.4 Hz, J5′′,6a′′ = 5.2 Hz, 1H, H-6a′′); 4.23 (dd,
8 : 1 → 5 : 1), compound 7d is isolated as a purple solid 2J6a′′,6b′′ = 12.4 Hz, J5′′,6a′′ = 2.5 Hz, 1H, H-6b′′); 3.96–3.89 (m,
3
(503 mg, 79%, α,β-anomeric mixture in a ratio of β : α = 8 : 1). 1H, H-5′′); 2.10, 2.09, 1.98, 1.85 (4s, 12H, 4 × C(O)CH3).
Mp 144–146 °C. 1H NMR (300 MHz, CDCl3): (β-anomer) δ = 13C NMR (75 MHz, CDCl3): δ = 192.7 (CvO); 170.5, 169.7,
4
9.10 (d, J4′,6′ = 1.8 Hz, 1H, H-4′); 7.90–7.85 (m, 1H, CHAr); 169.6, 169.5, 168.1 (5s, 4 × C(O)CH3, CvO); 143.1, 142.8, 141.4
7.57–7.52 (m, 2H, CHAr); 7.49 (d, 3J = 8.4 Hz, 1H, CHAr); (3s, 3 × Cqu); 135.9 (CHAr); 131.6 (Cqu); 131.1 (CHAr); 128.0
4
3
7.33–7.28 (m, 1H, CHAr); 7.26 (dd, J = 1.9 Hz, J = 8.2 Hz, 1H, (Cqu); 128.0, 127.6, 126.7, 126.4, 123.2 (5s, 5 × CHAr); 121.5
3
3
CHAr); 5.98 (d, J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.60 (dd, J1′′,2′′
=
(Cqu); 113.6 (CHAr); 80.8 (C-1′′); 75.6, 70.6, 70.1, 65.3 (4s, C-2′′,
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C-3′′, C-4′′, C-5′′); 62.3 (C-6′′); 20.7, 20.7, 20.7, 20.5 (4s, 4 × C(O)- a purple solid (450 mg, 74%, α,β-anomeric mixture in a ratio
CH3). MS (EI, 70 eV): m/z (%) = 659 ([M]+, [82Se], 6), 657 ([M]+, of β : α = 7 : 1). Mp 133–135 °C. 1H NMR (300 MHz, CDCl3):
4
[80Se], 24), 655 ([M]+, [78Se], 11), 654 ([M]+, [77Se], 4), 653 ([M]+, (β-anomer) δ = 9.09 (d, J4′,6′ = 1.7 Hz, 1H, H-4′); 7.86 (d (br),
[76Se], 4), 327 (23), 169 (100), 127 (17), 109 (58), 43 (93). HRMS 3J = 7.5 Hz, 1H, CHAr); 7.58–7.50 (m, 2H, 2 × CHAr); 7.46 (d, 3J =
(ESI-TOF/MS): calcd for C30H28NO11Se ([M + H]+, [78Se]) 8.3 Hz, 1H, CHAr); 7.33–7.21 (m, 2H, 2 × CHAr); 6.03 (d, 3J1′′,2′′
=
3
3
656.08377, found 656.08379; calcd for C30H28NO11Se ([M + H]+, 1.5 Hz, 1H, H-1′′); 5.62 (dd, J1′′,2′′ = 1.4 Hz, J2′′,3′′ = 3.3 Hz, 1H,
[80Se]) 658.08248, found 658.08306. Elemental analysis: calcd H-2′′), 5.42 (“t”, 3J3′′,4′′ = 10.1 Hz, 3J4′′,5′′ = 9.9 Hz,1H, H-4′′); 5.32
3
3
for C30H27NO11Se (656.50): C, 54.89; H, 4.15; N, 2.13. Found: (dd, J2′′,3′′ = 3.3 Hz, J3′′,4′′ = 10.2 Hz, 1H, H-3′′); 4.30 (dd,
3
C, 55.12; H, 4.53; N, 2.27.
2J6a′′,6b′′ = 12.4 Hz, J5′′,6a′′ = 5.2 Hz, 1H, H-6a′′); 4.22 (dd,
(Z)-1-(2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl)-5-methyl- 2J6a′′,6b′′ = 12.4 Hz, J5′′,6a′′ = 2.4 Hz, 1H, H-6b′′); 3.94–3.87 (m,
3
3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]indolin-2-one (7f). 1H, H-5′′); 3.00 (sep, 3J = 6.9 Hz, 1H, CH(CH3)2); 2.10, 2.09,
3
According to the general procedure, 3-acetoxy-benzo[b]seleno- 1.97, 1.85 (4s, 12H, 4 × C(O)CH3) 1.31 (d, J = 6.9 Hz, 3H, CH-
phene (6) (350 mg, 1.46 mmol), a solution of NaOMe (0.3 ml, (CH3)2); 1.31 (d, 3J = 6.8 Hz, 3H, CH(CH3)2). (α-anomer) δ = 9.15
0.5 M) and later acetic acid (18 mg, 0.3 mmol) are brought (d, 4J4′,6′ = 1.7 Hz, 1H, H-4′); 7.86 (d (br), 3J = 7.5 Hz, 1H, CHAr);
to reaction in MeOH (2 ml). The second step is carried out 7.58–7.50 (m, 2H, 2 × CHAr); 7.46 (d, 3J = 8.3 Hz, 1H, CHAr);
in acetic acid (3.9 ml) and acetic anhydride (2.9 ml) by 7.21–7.16 (m, 2H, 2 × CHAr); 6.00 (d, 3J1′′,2′′ = 1.5 Hz, 1H, H-1′′);
3
3
the addition of 1-(2′,3′,4′,6′-tetra-O-acetyl-β-D-mannopyranosyl)- 5.62 (dd, J1′′,2′′ = 1.4 Hz, J2′′,3′′ = 3.3 Hz, 1H, H-2′′), 5.42
3
3
5-methylisatin (1f) (479 mg, 0.97 mmol) and sodium acetate (“t”, J3′′,4′′ = 10.1 Hz, J4′′,5′′ = 9.9 Hz,1H, H-4′′); 5.32 (dd,
3
2
(720 mg, 8.77 mmol). After column chromatography (hep- 3J2′′,3′′ = 3.3 Hz, J3′′,4′′ = 10.2 Hz, 1H, H-3′′); 4.30 (dd, J6a′′,6b′′
=
3
2
tanes–EtOAc 8 : 1 → 3 : 1) compound 7f is isolated as a purple 12.4 Hz, J5′′,6a′′ = 5.2 Hz, 1H, H-6a′′); 4.22 (dd, J6a′′,6b′′ = 12.4
solid (382 mg, 58%). Mp 135–137 °C. 1H NMR (300 MHz, Hz, J5′′,6a′′ = 2.4 Hz, 1H, H-6b′′); 3.94–3.87 (m, 1H, H-5′′); 3.00
3
CDCl3): δ = 8.97 (s, 1H, H-4′); 7.83 (d, J = 7.4 Hz, 1H, CHAr); (sep, 3J = 6.9 Hz, 1H, CH(CH3)2); 2.10, 2.07, 1.96, 1.86 (4s, 12H,
3
7.58–7.49 (m, 2H, 2 × CHAr); 7.43 (d, 3J = 8.3 Hz, 1H, CHAr); 4 × C(O)CH3) 1.31 (d, 3J = 6.9 Hz, 3H, CH(CH3)2); 1.31 (d,
7.32–7.26 (m, 1H, CHAr); 7.17 (dd, J = 8.3 Hz, J = 1.1 Hz, 1H, 3J = 6.8 Hz, 3H, CH(CH3)2). 13C NMR (75 MHz, CDCl3):
3
4
3
3
CHAr); 6.02 (d, J1′′,2′′ = 1.4 Hz, 1H, H-1′′); 5.62 (dd, J1′′,2′′ = 1.4 (β-anomer) δ = 192.7 (CvO); 170.5, 169.7, 169.6, 169.5, 168.2
Hz, J2′′,3′′ = 3.2 Hz, 1H, H-2′′), 5.42 (“t”, J3′′,4′′ = 10.1 Hz, J4′′,5′′ (5s, 4 × C(O)CH3, CvO); 143.9, 143.2, 142.3, 139.4 (4s,
= 9.9 Hz, 1H, H-4′′); 5.32 (dd, J2′′,3′′ = 3.3 Hz, J3′′,4′′ = 10.2 Hz, 4 × Cqu); 135.8 (CHAr); 131.7 (Cqu); 129.3 (CHAr); 128.4 (Cqu);
1H, H-3′′); 4.30 (dd, J6a′′,6b′′ = 12.5 Hz, J5′′,6a′′ = 5.2 Hz, 1H, 127.9, 126.7, 126.3, 125.7 (4s, 4 × CHAr); 121.5 (Cqu); 113.4
3
3
3
3
3
2
3
2
3
H-6a′′); 4.22 (dd, J6a′′,6b′′ = 12.4 Hz, J5′′,6a′′ = 2.5 Hz, 1H, (CHAr); 80.7 (C-1′′); 75.6, 70.6, 70.1, 65.3 (4s, C-2′′, C-3′′, C-4′′,
H-6b′′); 3.95–3.86 (m, 1H, H-5′′); 2.40 (s, 3H, CH3); 2.10, 2.09, C-5′′); 62.3 (C-6′′); 34.0 (CH(CH3)2); 24.2, 24.2 (2s, CH(CH3)2);
1.98, 1.87 (4s, 12H, 4 × C(O)CH3). 13C NMR (63 MHz, CDCl3): 20.8, 20.7, 20.7, 20.5 (4s, 4 × C(O)CH3). MS (EI, 70 eV): m/z (%)
δ = 192.8 (CvO); 170.5, 169.7, 169.6, 169.5, 168.1 (5s, 4 × C(O)- = 701 ([M]+, [82Se], 16), 699 ([M]+, [80Se], 58), 697 ([M]+, [78Se],
CH3, CvO); 143.3, 142.3, 139.2 (3s, 3 × Cqu); 135.9 (CHAr); 27), 696 ([M]+, [77Se], 10), 695 ([M]+, [76Se], 8), 369 (48), 354
132.6 (Cqu); 131.8 (CHAr); 131.6, 128.3 (2s, 2 × Cqu); 128.0, (21), 169 (100), 109 (66), 43 (96). HRMS (ESI-TOF/MS): calcd for
127.9, 126.7, 126.4 (4s, 4 × CHAr); 121.5 (Cqu); 113.3 (CHAr); C33H34NO11Se ([M + H]+, [80Se]) 700.12947, found 700.12832;
80.7 (C-1′′); 75.5, 70.7, 70.1, 65.3 (4s, C-2′′, C-3′′, C-4′′, C-5′′); calcd for C33H33NNaO11Se ([M + Na]+, [80Se]) 722.11142, found
62.3 (C-6′′); 21.2 (CH3); 20.8, 20.7, 20.7, 20.5 (4s, 4 × C(O)CH3). 722.11093.
MS (EI, 70 eV): m/z (%) = 673 ([M]+, [82Se], 6), 671 ([M]+, [80Se],
(Z)-1-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-3-[3-oxo-
25), 669 ([M]+, [78Se], 13), 668 ([M]+, [77Se], 5), 667 ([M]+, [76Se], benzo[b]selenophen-2(3H)-ylidene]indolin-2-one (7h). Accord-
4), 341 (40), 169 (100), 127 (18), 109 (56), 43 (85). HRMS ing to the general procedure, 3-acetoxy-benzo[b]selenophene
(ESI-TOF/MS): calcd for C31H29NNaO11Se ([M + Na]+, [78Se]) (6) (280 mg, 1.17 mmol), a solution of NaOMe (0.3 ml, 0.5 M)
692.08139, found 692.08213; calcd for C31H29NNaO11Se and later acetic acid (18 mg, 0.3 mmol) are brought to reaction
([M + Na]+, [80Se]) 694.08009, found 694.08143. Elemental ana- in MeOH (2 ml). The second step is carried out in acetic acid
lysis: calcd for C31H29NO11Se (670.52): C, 55.53; H, 4.36; N, (3 ml) and acetic anhydride (2.3 ml) by the addition of
2.09. Found: C, 55.18; H, 4.66; N, 2.05.
1-(2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranosyl)-isatin (1h) (308 mg,
(Z)-1-(2,3,4,6-Tetra-O-acetyl-α,β-D-mannopyranosyl)-5-isopropyl- 0.65 mmol) and sodium acetate (575 mg, 7.01 mmol). After
3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-indolin-2-one (7g). column chromatography (heptanes–EtOAc 6 : 1 → 3 : 1) com-
According to the general procedure, 3-acetoxy-benzo[b]seleno- pound 7h is isolated as a purple solid (355 mg, 69%). Mp
1
3
phene (6) (311 mg, 1.30 mmol), a solution of NaOMe (0.26 ml, 235–237 °C. H NMR (300 MHz, CDCl3): δ = 9.22 (dd, J4′,5′
=
4
3
0.5 M) and later acetic acid (18 mg, 0.3 mmol) are brought to 7.9 Hz, J4′,6′ = 0.7 Hz, 1H, H-4′); 7.87 (d (br), J = 7.5 Hz, 1H,
3
3
reaction in MeOH (2 ml). The second step is carried out in CHAr); 7.61–7.51 (m, 2H, 2 × CHAr); 7.46 (d“t”, J = 7.9 Hz, J =
acetic acid (3.5 ml) and acetic anhydride (2.6 ml) by the 7.8 Hz, 4J = 1.2 Hz, 1H, CHAr); 7.36–7.29 (m, 1H, CHAr);
addition of 1-(2′,3′,4′,6′-tetra-O-acetyl-α,β-D-mannopyranosyl)- 7.28–7.26 (m, 1H, CHAr); 7.20 (d“t”, 3J = 7.9 Hz, 3J = 7.7 Hz, 4J =
3
5-isopropylisatin (1g) (450 mg, 0.87 mmol) and sodium 1.0 Hz, 1H, CHAr); 5.84 (d, J1′′,2′′ = 9.4 Hz, 1H, H-1′′); 5.68 (“t”,
acetate (640 mg, 7.80 mmol). After column chromatography 3J1′′,2′′ = 9.4 Hz, J2′′,3′′ = 9.3 Hz,1H, H-2′′), 5.42 (“t”, J2′′,3′′ = 9.3
3
3
3
3
3
(heptanes–EtOAc 8 : 1 → 3 : 1) compound 7g is isolated as Hz, J3′′,4′′ = 9.5 Hz,1H, H-3′′); 5.28 (“t”, J4′′,5′′ = 9.9 Hz, J3′′,4′′
=
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2
3
9.7 Hz,1H, H-4′′); 4.26 (dd, J6a′′,6b′′ = 12.5 Hz, J5′′,6a′′ = 4.6 Hz, general procedure, compound 7b (164 mg, 0.27 mmol) is
2
3
1H, H-6a′′); 4.20 (dd, J6a′′,6b′′ = 12.5 Hz, J5′′,6a′′ = 2.5 Hz, 1H, brought to reaction with a methanolic solution of NaOMe
H-6b′′); 3.98–3.92 (m, 1H, H-5′′); 2.09, 2.08, 2.00, 1.82 (4s, 12H, (0.03 ml, 0.5 M) in abs. MeOH (4 ml) and abs. THF (2 ml).
4 × C(O)CH3). 13C NMR (63 MHz, CDCl3): δ = 192.6 (CvO); Compound 8b is isolated as a wine red solid (115 mg, 88%).
170.4, 169.9, 169.5, 169.0, 168.8 (5s, 4 × C(O)CH3, CvO); Mp 326–328 °C. 1H NMR (300 MHz, DMSO-d6): δ = 8.90 (s, 1H,
143.0, 142.8, 140.3 (3s, 3 × Cqu); 136.0, 131.9 (2s, 2 × CHAr); H-4′); 7.86 (“t”, 3J = 7.8 Hz, 3J = 8.1 Hz, 2H, 2 × CHAr); 7.72
3
4
3
131.6 (Cqu); 128.1, 128.0 (2s, 2 × CHAr); 128.0 (Cqu); 126.7, (d“t”, J = 7.5 Hz, J = 1.3 Hz, 1H, CHAr); 7.59 (d, J = 8.3 Hz,
3
4
126.5, 123.7 (3s, 3 × CHAr); 121.3 (Cqu); 111.4 (CHAr); 80.2 1H, CHAr); 7.43 (d“t”, J = 7.5 Hz, J = 0.9 Hz, 1H, CHAr); 7.24
(C-1′′); 74.8, 73.3, 67.9, 67.8 (4s, C-2′′, C-3′′, C-4′′, C-5′′); 61.8 (dd (br), 3J = 8.4 Hz, 4J = 1.3 Hz, 1H, CHAr); 5.63 (d, J1′′,2′′
=
3
3
3
(C-6′′); 20.7, 20.5, 20.5, 20.2 (4s, 4 × C(O)CH3). MS (EI, 70 eV): 1.0 Hz, 1H, H-1′′); 5.12 (d, J = 4.8 Hz, 1H, OH); 4.99 (d, J =
m/z (%) = 659 ([M]+, [82Se], 4), 657 ([M]+, [80Se], 19), 655 ([M]+, 4.7 Hz, 1H, OH); 4.88 (d, J = 5.4 Hz, 1H, OH); 3.88–3.82 (m,
3
[78Se], 9), 654 ([M]+, [77Se], 4), 653 ([M]+, [76Se], 3), 327 (22), 169 1H, CHglyk); 3.56–3.35 (m, 3H, 3 × CHglyk); 2.36 (s, 3H, CH3);
(100), 127 (18), 109 (71), 43 (75). HRMS (ESI-TOF/MS): calcd for 1.26 (d, J5′′,6′′ = 5.4 Hz, 3H, H-6′′). 13C NMR: could not be
3
C30H28NO11Se ([M + H]+, [78Se]) 656.08377, found 656.08473; measured, due to solubility problems. MS (EI, 70 eV): m/z (%)
calcd for C30H28NO11Se ([M + H]+, [80Se]) 658.08248, found = 489 ([M]+, [82Se], 17), 487 ([M]+, [80Se], 56), 485 ([M]+, [78Se],
658.08313. Elemental analysis: calcd for C30H27NO11Se 25), 484 ([M]+, [77Se], 8), 483 ([M]+, [76Se], 8), 341 (100), 326
(656.50): C, 54.89; H, 4.15; N, 2.13. Found: C, 54.66; H, 4.35; (31), 233 (25), 146 (62), 43 (33). HRMS (ESI-TOF/MS): calcd for
N, 2.21.
C23H22NO6Se ([M + H]+, [78Se]) 486.06204, found 486.06190;
calcd for C23H22NO6Se ([M + H]+, [80Se]) 488.06084, found
488.06013. Elemental analysis: calcd for C23H21NO6Se (486.38):
C, 56.80; H, 4.35; N, 2.88. Found: C, 56.95; H, 4.25; N, 2.85.
(Z)-5-Ethyl-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-
General procedure for the synthesis of deprotected
selenoindirubin-N-glycosides 8a–h
The corresponding acetylated compound is dissolved in a solu-
tion of abs. MeOH and abs. THF (v : v = 2 : 1) and treated with 1-(β-L-rhamnopyranosyl)indolin-2-one (8c). According to the
a methanolic solution of NaOMe (0.5 M) (0.02 eq. per acetyl general procedure, compound 7c (411 mg, 0.66 mmol) is
group). After stirring for 7 h at 20 °C the precipitated products brought to reaction with a methanolic solution of NaOMe
are obtained by filtration, washing with EtOH and drying (0.08 ml, 0.5 M) in abs. MeOH (5 ml) and abs. THF (2.5 ml).
in vacuo.
Compound 8c is isolated as a wine red solid (277 mg, 84%).
(Z)-3-[3-Oxo-benzo[b]selenophen-2(3H)-ylidene]-1-(β-L-rham- Mp 320–322 °C. 1H NMR (300 MHz, DMSO-d6): δ = 8.94 (d,
nopyranosyl)indolin-2-one (8a). According to the general pro- 4J4′,6′ = 1.1 Hz, 1H, H-4′); 7.90–7.83 (m, 2H, 2 × CHAr); 7.71 (“t”,
cedure, compound 7a (365 mg, 0.61 mmol) is brought to 3J = 7.8 Hz, 3J = 7.4 Hz, 1H, CHAr); 7.61 (d, 3J = 8.2 Hz, 1H,
3
3
3
reaction with a methanolic solution of NaOMe (0.07 ml, 0.5 M) CHAr); 7.42 (“t”, J = 7.4 Hz, J = 7.6 Hz, 1H, CHAr); 7.27 (dd, J
in abs. MeOH (5 ml) and abs. THF (2.5 ml). Compound 8a is = 8.3 Hz, 4J = 1.6 Hz, 1H, CHAr); 5.63 (s (br), 1H, H-1′′); 5.13 (d,
isolated as a wine red solid (255 mg, 89%). Mp 333–335 °C. 1H 3J = 5.0 Hz, 1H, OH); 4.98 (d, 3J = 4.8 Hz, 1H, OH); 4.87 (d, 3J =
3
4
NMR (300 MHz, DMSO-d6): δ = 9.02 (dd, J4′,5′ = 7.9 Hz, J4′,6′
=
5.7 Hz, 1H, OH); 3.89–3.83 (m, 1H, CHglyk); 3.56–3.34 (m, 3H, 3
0.6 Hz, 1H, H-4′); 7.81 (“t”, 3J = 7.7 Hz, 3J = 8.1 Hz, 2H, 2 × × CHglyk); 2.65 (q, 3J = 7.5 Hz, 2H, CH2CH3); 1.26 (d, J5′′,6′′
=
3
CHAr); 7.69 (“t”, J = 7.9 Hz, J = 7.0 Hz, 1H, CHAr); 7.67 (d“t”, 5.2 Hz, 3H, H-6′′); 1.23 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR
3
3
3
3J = 7.7 Hz, 4J = 1.2 Hz, 1H, CHAr); 7.44–7.35 (m, 2H, 2 × CHAr); (75 MHz, DMSO-d6): δ = 192.7, 167.7 (2s, 2 × CvO); 143.3,
3
3
4
7.09 (d“t”, J = 7.5 Hz, J = 7.9 Hz, J = 0.9 Hz, 1H, CHAr); 5.65 141.6, 140.6, 137.6 (4s, 4 × Cqu); 136.6, 131.4 (2s, 2 × CHAr);
(s, 1H, H-1′′); 5.15 (d, J = 4.9 Hz, 1H, OH); 5.00 (d, 3J = 4.9 Hz, 131.4, 129.0 (2s, 2 × Cqu); 127.7, 127.5, 126.9, 125.6 (4s, 4 ×
3
1H, OH); 4.89 (d, 3J = 5.8 Hz, 1H, OH); 3.91–3.85 (m, 1H, CHAr); 120.6 (Cqu); 115.6 (CHAr); 82.9 (C-1′′); 75.5, 73.1, 72.0,
3
CHglyk); 3.57–3.37 (m, 3H, 3 × CHglyk); 1.28 (d, J5′′,6′′ = 5.4 Hz, 71.5 (4s, C-2′′, C-3′′, C-z, C-5′′); 28.3 (CH2CH3); 18.2 (CH2CH3);
3H, H-6′′). 13C NMR (75 MHz, DMSO-d6, 60 °C): δ = 192.4, 16.3 (C-6′′). MS (EI, 70 eV): m/z (%) = 503 ([M]+, [82Se], 8), 501
167.5 (2s, 2 × CvO); 143.3, 142.9. 140.6 (3s, 3 × Cqu); 136.2, ([M]+, [80Se], 23), 499 ([M]+, [78Se], 11), 498 ([M]+, [77Se], 4), 497
131.5 (2s, 2 × CHAr); 131.2, 128.6 (2s, 2 × Cqu); 127.4, 127.2, ([M]+, [76Se], 4), 355 (100), 340 (15), 326 (14), 312 (9), 160 (20).
126.6, 126.1, 122.0 (5s, 5 × CHAr); 120.4 (Cqu); 115.4 (CHAr); HRMS (EI): calcd for C24H23NO6Se ([M]+, [78Se]) 499.06930,
82.9 (C-1′′); 75.3, 73.0, 71.7, 71.4 (4s, C-2′′, C-3′′, C-4′′, C-5′′); found 499.070223; calcd for C24H23NO6Se ([M]+, [80Se])
17.9 (C-6′′). MS (EI, 70 eV): m/z (%) = 475 ([M]+, [82Se], 8), 473 501.06851, found 501.069507. Elemental analysis: calcd for
([M]+, [80Se], 27), 471 ([M]+, [78Se], 13), 470 ([M]+, [77Se], 4), 469 C24H23NO6Se (500.40): C, 57.60; H, 4.63; N, 2.80. Found: C,
([M]+, [76Se], 4), 327 (100), 299 (9), 285 (8), 219 (15), 132 (12). 57.27; H, 4.63; N, 2.94.
HRMS (EI): calcd for C22H19NO6Se ([M]+, [78Se]) 471.03800,
(Z)-5-Isopropyl-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-
found 471.038892; calcd for C22H19NO6Se ([M]+, [80Se]) 1-(α,β-L-rhamnopyranosyl)indolin-2-one (8d). According to the
473.03721, found 473.037626. Elemental analysis: calcd for general procedure, compound 7d (429 mg, 0.67 mmol) is
C22H19NO6Se (472.35): C, 55.94; H, 4.05; N, 2.97. Found: C, brought to reaction with a methanolic solution of NaOMe
55.79; H, 4.05; N, 3.05.
(0.08 ml, 0.5 M) in abs. MeOH (6 ml) and abs. THF (3 ml).
(Z)-5-Methyl-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-1- Compound 8d is isolated as a wine red solid (252 mg, 73%,
(β-L-rhamnopyranosyl)indolin-2-one (8b). According to the α,β-anomeric mixture in
a ratio of β : α = 17 : 1). Mp
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307–309 °C. H NMR (250 MHz, DMSO-d6): (β-anomer) δ = 9.00
Organic & Biomolecular Chemistry
1
(Z)-1-(β-D-Mannopyranosyl)-5-methyl-3-[3-oxo-benzo[b]sele-
4
(d, J4′,6′ = 1.7 Hz, 1H, H-4′); 7.88–7.80 (m, 2H, 2 × CHAr); 7.69 nophen-2(3H)-ylidene]indolin-2-one (8f). According to the
3
4
3
(d“t”, J = 7.5 Hz, J = 1.2 Hz, 1H, CHAr); 7.62 (d, J = 8.4 Hz, general procedure, compound 7f (218 mg, 0.33 mmol) is
1H, H-7′); 7.40 (“t” (br), 3J = 7.5 Hz, 3J = 8.3 Hz, 1H, CHAr); 7.31 brought to reaction with a methanolic solution of NaOMe
3
4
(dd, J6′,7′ = 8.4 Hz, J4′,6′ = 1.8 Hz, 1H, H-6′); 5.63 (s (br), 1H, (0.03 ml, 0.5 M) in abs. MeOH (5 ml) and abs. THF (2.5 ml).
3
3
H-1′′); 5.14 (d, J = 4.9 Hz, 1H, OH); 4.99 (d, J = 4.9 Hz, 1H, Compound 8f is isolated as a wine red solid (130 mg, 80%)
OH); 4.87 (d, J = 5.7 Hz, 1H, OH); 3.90–3.82 (m, 1H, CHglyk); containing some solvent impurities. 1H NMR (250 MHz,
3
3.58–3.34 (m, 3H, 3 × CHglyk); 2.93 (sep, J = 6.9 Hz, 1H, CH- DMSO-d6): δ = 8.87 (br s, 1H, H-4′); 7.83 (d, 3J = 7.5 Hz, 1H,
3
(CH3)2); 1.32–1.21 (m, 9H, H-6′′, CH(CH3)2). (α-anomer) δ = 9.08 CHAr); 7.81 (d, 3J = 7.4 Hz, 1H, CHAr); 7.68 (“t”, 3J = 7.5 Hz, 1H,
(s (br), 1H, H-4′); 7.88–7.80 (m, 2H, 2 × CHAr); 7.73–7.65 (m, CHAr); 7.63 (d, J7,6 = 8.3 Hz, 1H, H-7); 7.40 (“t”, 3J = 7.5 Hz,
3
2H, 2 × CHAr); 7.44–7.36 (m, 2H, 2 × CHAr); 5.79 (d, 3J1′′,2′′ = 8.7 1H, CHAr); 7.22 (br d, J6,7 = 8.3 Hz 1H, H-6); 5.63 (br s, 1H,
3
3
3
3
3
Hz, 1H, H-1′′); 5.17 (d, J = 3.8 Hz, 1H, OH); 5.08 (d, J = 3.9 H-1′′); 5.11 (d, J = 4.8 Hz, 1H, OH); 4.94, 4.90 (2d, J = 4.4 Hz,
Hz, 1H, OH); 5.02 (d, J = 5.9 Hz, 1H, OH); 4.49–4.40 (m, 1H, 3J = 4.7 Hz, 2H, 2 × OH); 4.58 (“t”, 3J = 5.5 Hz, 1H, OH(6′′));
3
CHglyk); 4.01–3.95 (m, 1H, CHglyk); 3.66–3.60 (m, 1H, CHglyk); 3.89–3.29 (m, 6H, 6 × CHglyk); 2.35 (s, 3H, CH3).
3.58–3.34 (m, 1H, CHglyk); 2.93 (sep, 3J = 6.9 Hz, 1H, CH-
(Z)-5-Isopropyl-1-(β-D-mannopyranosyl)-3-[3-oxo-benzo[b]-
3
(CH3)2); 1.39 (d, J5′′,6′′ = 7.2 Hz, 3H, H-6′′); 1.32–1.21 (m, 6H, selenophen-2(3H)-ylidene]indolin-2-one (8g). According to the
CH(CH3)2). 13C NMR (63 MHz, DMSO-d6): (β-anomer) δ = 192.7, general procedure, compound 7g (377 mg, 0.54 mmol) is
167.7 (2s, 2 × CvO); 143.3, 142.3, 141.6, 140.6 (4s, 4 × Cqu); brought to reaction with a methanolic solution of NaOMe
136.5 (CHAr); 131.4 (Cqu); 130.1 (CHAr); 129.1 (Cqu); 127.8, (0.09 ml, 0.5 M) in abs. MeOH (5 ml) and abs. THF (2.5 ml).
127.5, 126.8, 124.2 (4s, 4 × CHAr); 120.6 (Cqu); 115.6 (CHAr); Compound 8g is isolated as a wine red solid (248 mg, 87%)
82.9 (C-1′′); 75.5, 73.1, 72.0, 71.5 (4s, C-2′′, C-3′′, C-4′′, C-5′′); containing some solvent impurities. 1H NMR (250 MHz,
4
33.5 (CH(CH3)2); 24.3, 24.3 (2s, CH(CH3)2); 18.2 (C-6′′). MS (EI, DMSO-d6): δ = 9.00 (d, J4′,6′ = 1.5 Hz, 1H, H-4′); 7.88–7.83 (m,
3
3
70 eV): m/z (%) = 517 ([M]+, [82Se], 8), 515 ([M]+, [80Se], 21), 513 2H, 2 × CHAr); 7.70 (“t”, J = 7.7 Hz, 1H, CHAr); 7.67 (d, J7,6
=
([M]+, [78Se], 10), 512 ([M]+, [77Se], 4), 511 ([M]+, [76Se], 3), 369 8.4 Hz, 1H, H-7); 7.42 (“t”, 3J = 7.6 Hz, 1H, CHAr); 7.31 (dd, 3J6,7
4
3
(100), 354 (43), 328 (24), 57 (21), 44 (26). HRMS (EI): calcd for = 8.4 Hz, J6,4 = 1.5 Hz, 1H, H-6); 5.64 (d, J1′′,2′′ = 1.0 Hz, 1H,
C25H25NO6Se ([M]+, [78Se]) 513.08495, found 513.085364; calcd H-1′′); 5.13 (d, J = 4.1 Hz, 1H, OH); 4.94, 4.89 (2d, J = 3.5 Hz,
3
3
for C25H25NO6Se ([M]+, [80Se]) 515.08416, found 515.084997. 3J = 3.7 Hz, 2H, 2 × OH); 4.57 (“t”, 3J = 5.5 Hz, 1H, OH(6′′));
3
Elemental analysis: calcd for C25H25NO6Se (514.43): C, 58.37; 3.88–3.29 (m, 6H, 6 × CHglyk); 2.94 (sept, J = 6.9 Hz, 1H, CH-
H, 4.90; N, 2.72. Found: C, 57.93; H, 4.95; N, 2.90.
(CH3)2); 1.27 (d, 3J = 6.9 Hz, 6H, CH(CH3)2).
(Z)-1-(β-D-Mannopyranosyl)-3-[3-oxo-benzo[b]selenophen-2-
(Z)-1-(β-D-Glucopyranosyl)-3-[3-oxo-benzo[b]selenophen-2-
(3H)-ylidene]indolin-2-one (8e). According to the general pro- (3H)-ylidene]indolin-2-one (8h). Similar to the general pro-
cedure, compound 7e (489 mg, 0.74 mmol) is brought to reac- cedure, 7h (300 mg, 0.46 mmol) is suspended in a mixture of
tion with a methanolic solution of NaOMe (0.06 ml, 0.5 M) in 3 ml of abs. methanol and 6 ml of abs. pyridine (instead of
abs. MeOH (10 ml) and abs. THF (5 ml). Compound 8e is iso- THF). After the addition of a methanolic solution of NaOMe
lated as a wine red solid (339 mg, 93%). Mp 282–284 °C. 1H (0.07 ml, 0.5 M), the mixture is stirred for 6 hours at 20 °C.
3
NMR (300 MHz, DMSO-d6): δ = 9.04 (d, J4′,5′ = 7.8 Hz, 1H, Then, ethanol (40 ml) is added and the mixture is cooled. The
3
3
H-4′); 7.87 (d, J = 7.7 Hz, 1H, CHAr); 7.83 (d, J = 7.7 Hz, 1H, formed precipitate is worked-up as given in the general pro-
CHAr); 7.76 (d, 3J = 8.1 Hz, 1H, CHAr); 7.71 (“t”, 3J = 7.4 Hz, 3J = cedure. Compound 8h is isolated as a red solid (168 mg; 75%)
3
7.6 Hz, 1H, CHAr); 7.46–7.37 (m, 2H, 2 × CHAr); 7.11 (“t”, J = containing some solvent impurities. 1H NMR (250 MHz,
3
3
7.5 Hz, 3J = 7.8 Hz, 1H, CHAr); 5.67 (s (br), 1H, H-1′′); 5.13 (d, 3J DMSO-d6): δ = 9.13 (d, J = 7.9 Hz, 1H, CHAr); 7.88 (d, J = 7.7
= 4.1 Hz, 1H), 4.95 (d, 3J = 3.3 Hz, 1H), 4.90 (d, 3J = 2.8 Hz, 1H) Hz, 1H, CHAr); 7.84 (d, 3J = 7.7 Hz, 1H, CHAr), 7.71 (“t”, 3J = 7.6
3
3
3
(OH(2′′), OH(3′′), OH(4′′)); 4.58 (t, J = 5.3 Hz, 1H, OH(6′′)); 3.87 (s Hz, 1H, CHAr); 7.52 (“t”, J = 7.8 Hz, 1H, CHAr); 7.42 (“t”, J =
(br), 1H, CHglyk); 3.83–3.74 (m, 1H, H-6a′′); 3.62–3.50 (m, 3H, 3 7.5 Hz, 1H, CHAr); 7.34 (d, 3J = 7.9 Hz, 1H, CHAr); 7.20 (“t”, 3J =
× CHglyk); 3.38–3.33 (m, 1H, CHglyk). 13C NMR (75 MHz, DMSO- 7.7 Hz, 1H, CHAr); 5.41–5.29 (m, 2H, H-1′′, OH); 5.17, 5.13 (2d,
d6): δ = 192.7, 167.7 (2s, 2 × CvO); 143.5, 143.2, 141.0 (3s, 3 × 3J = 4.5 Hz, J = 4.6 Hz, 2H, 2 × OH); 4.62 (“t”, J6a′′/b′′,OH = 5.5
3
3
3
C
qu); 136.6, 131.9 (2s, 2 × CHAr); 131.3, 128.7 (2s, 2 × Cqu); Hz, 1H, OH(6′′)); 3.92 (br, 1H, H-2′′); 3.75 (dd, J6a′′,OH = 5.8 Hz,
127.7, 127.6, 126.9, 126.3, 122.2 (5s, 5 × Cqu); 120.5 (Cqu); 116.3 3J6a′′,6b′′ = 11.3 Hz, 1H, H-6a′′); 3.56–3.26 (m, 4H, 4 × CHglyk).
(CHAr); 83.0 (C-1′′); 81.0, 73.4, 71.9, 66.4 (4s, C-2′′, C-3′′, C-4′′, (Z)-1-Methyl-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-
C-5′′); 61.4 (C-6′′). MS (EI, 70 eV): m/z (%) = 491 ([M]+, [82Se], indolin-2-one (10a) (Method A). 3-Acetoxy-benzo[b]seleno-
10), 489 ([M]+, [80Se], 24), 487 ([M]+, [78Se], 11), 486 ([M]+, phene (6) (224 mg, 0.94 mmol), N-methyl-isatin (9a) (150 mg,
[77Se], 4), 485 ([M]+, [76Se], 4), 327 (100), 219 (15), 132 (18), 105 0.93 mmol) and KOtBu (11 mg, 0.09 mmol) were dissolved in a
(10), 43 (13). HRMS (ESI-TOF/MS): calcd for C22H19NNaO7Se mixture of abs. EtOH (2 ml) and abs. THF (1 ml) and stirred at
([M + Na]+, [78Se]) 510.02323, found 510.02338; calcd for 20 °C for 3 h. The precipitated product is separated by fil-
C22H19NNaO7Se ([M
+
Na]+, [80Se]) 512.02204, found tration. After washing with EtOH and drying in vacuo, com-
512.02282. Elemental analysis: calcd for C22H19NO7Se (488.35): pound 10a was obtained as a red solid (302 mg, 95%). Due to
C, 54.11; H, 3.92. Found: C, 53.89; H, 3.90.
the low solubility, 13C NMR spectra could not be obtained.
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Mp 292–294 °C. 1H NMR (250 MHz, CDCl3): δ = 9.17 (d, 3J4′,5′
=
0.5 M) are brought to reaction in MeOH (3 ml). Subsequent
7.9 Hz, 1H, H-4′); 7.88 (d, 3J = 7.7 Hz, 1H, CH Ar); 7.59–7.53 (m, addition of 7-fluoro-isatin (9c) (115 mg, 0.70 mmol) gave 10c
2H, CHAr); 7.44 (“t”, 3J = 7.4 Hz, 3J = 7.9 Hz, 1H, CHAr); as an orange solid (201 mg, 84%). Mp 324–326 °C. 1H NMR
3
3
7.36–7.28 (m, 1H, CHAr); 7.16 (“t”, J = 7.6 Hz, J = 8.0 Hz, 1H, (300 MHz, DMSO-d6): δ = 11.79 (s, 1H, NH); 8.85 (d, 3J4′,5′ = 8.1
3
3
3
CHAr); 6.89 (d, J = 7.7 Hz, 1H, CH Ar); 3.35 (s, 3H, N-CH3). IR Hz, 1H, H-4′); 7.85 (“t” (br), J = 8.0 Hz, J = 8.4 Hz, 2H, CHAr);
(ATR, cm−1): ˜ν = 3333 (w), 3216 (w), 3113 (w), 3051 (m), 3018 7.72 (d“t”, 3J = 7.3 Hz, 3J = 7.6 Hz, 4J = 0.9 Hz, 1H, CHAr);
(w), 2929 (w), 2881 (w), 2101 (w), 1957 (w), 1927 (w), 1907 (w), 7.47–7.34 (m, 2H, 2 × CHAr); 7.16–7.05 (m, 1H, CHAr). 19F NMR
1812 (w), 1681 (w), 1662 (s), 1605 (s), 1586 (s), 1556 (w), 1487 (282 MHz, DMSO-d6): δ = −132.82 (s, C-F). IR (ATR, cm−1): ˜ν =
(m), 1469 (m), 1446 (m), 1423 (m), 1377 (s), 1341 (m), 1319 (w), 3139 (m), 3041 (m), 2817 (m), 1698 (s), 1672 (s), 1642 (s), 1586
1310 (w), 1284 (s), 1264 (m), 1230 (m), 1218 (m), 1194 (w), 1164 (m), 1551 (w), 1492 (m), 1443 (m), 1415 (w), 1367 (w), 1333 (s),
(w), 1131 (m), 1110 (m), 1079 (m), 1045 (s), 1024 (w), 1017 (m). 1304 (w), 1283 (s), 1261 (m), 1240 (w), 1215 (s), 1154 (w), 1114
MS (EI, 70 eV): m/z (%) = 343 ([M]+, [82Se], 42), 341 ([M]+, [80Se], (w), 1078 (s), 1056 (s), 1001 (s). MS (EI, 70 eV): m/z (%) = 347
100), 339 ([M]+, [78Se], 61), 338 ([M]+, [77Se], 35), 337 ([M]+, ([M]+, [82Se], 23), 345 ([M]+, [80Se], 100), 343 ([M]+, [78Se], 43),
[76Se], 27), 312 (24), 233 (42), 204 (32), 146 (74), 44 (39). HRMS 342 ([M]+, [77Se], 19), 341 ([M]+, [76Se], 16), 237 (50), 214 (40),
(ESI-TOF/MS): calcd for C17H11NNaO2Se ([M + Na]+, [80Se]) 94 (72), 57 (64), 39 (30). HRMS (ESI/TOF-MS): calcd for
363.98479, found 363.9854. Elemental analysis: calcd for C16H8FNNaO2Se ([M
+
Na]+, [80Se]) 367.95971, found
C17H11NO2Se (340.23): C, 60.01; H, 3.26; N, 4.12. Found: C, 367.95915; calcd for C16H8FNNaO2Se ([M + Na]+, [78Se])
59.69; H, 3.63; N, 4.22.
365.96081, found 365.95993; calcd for C16H8FNNaO2Se ([M +
Na]+, [76Se]) 363.96236, found 363.96232. Elemental analysis:
calcd for C16H8FNO2Se (344.20): C, 55.83; H, 2.34; N, 4.07.
Found: C, 55.82; H, 2.25; N, 3.83.
General procedure for the synthesis of non-glycosylated
selenoindirubins 10b–h (Method B)
A solution of 6 in degased MeOH is treated with 0.1 eq. of
(Z)-5-Fluoro-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-
NaOMe and stirred for 15 min at 20 °C to give 5. After neutrali- indolin-2-one (10d). According to the general procedure
zation with acetic acid the corresponding isatin 9 is added and (method B), 3-acetoxy-benzo[b]selenophene (6) (200 mg,
dissolved completely. Then, catalytic amounts of piperidine 0.84 mmol) and a methanolic solution of NaOMe (0.17 ml,
are added. After stirring at 20 °C for 1.5 h the precipitated 0.5 M) are brought to reaction in MeOH (3 ml). Subsequent
product is separated by filtration, washed with EtOH and dried addition of 5-fluoro-isatin (9d) (115 mg, 0.70 mmol) gave 10d
1
in vacuo. Due to the low solubility, 13C NMR spectra could not as a red brown solid (224 mg, 93%). Mp 360–362 °C. H NMR
be obtained.
(300 MHz, DMSO-d6): δ = 11.27 (s, 1H, NH); 8.87–8.75 (m, 1H,
(Z)-3-[3-Oxo-benzo[b]selenophen-2(3H)-ylidene]indolin-2-one CHAr); 7.90–7.79 (m, 2H, 2 × CHAr); 7.78–7.68 (m, 1H, CHAr);
(10b). According to the general procedure (method B), 7.49–7.39 (m, 1H, CHAr); 7.37–7.25 (m, 1H, CHAr); 7.04–6.91
3-acetoxy-benzo[b]selenophene (6) (200 mg, 0.84 mmol) and a (m, 1H, CHAr). 19F NMR (282 MHz, DMSO-d6): δ = −121.32 (s,
methanolic solution of NaOMe (0.17 ml, 0.5 M) are brought to C–F). IR (ATR, cm−1): ˜ν = 3139 (m), 3113 (m), 3083 (m), 3014
reaction in MeOH (3 ml). Subsequent addition of isatin (9b) (m), 2855 (m), 2792 (m), 2755 (m), 2689 (m), 2645 (m), 1942
(103 mg, 0.70 mmol) gave 10b as a dark red solid (225 mg, (w), 1915 (w), 1865 (w), 1834 (w), 1789 (w), 1687 (m), 1663 (m),
99%). Mp 300–302 °C. 1H NMR (250 MHz, DMSO-d6): δ = 11.25 1651 (m), 1644 (m), 1586 (m), 1574 (m), 1552 (w), 1538 (w),
(s, 1H, NH); 9.01 (d, 3J4′,5′ = 7.7 Hz, 1H, H-4′); 7.88–7.80 (m, 1H, 1488 (w), 1465 (s), 1445 (s), 1395 (w), 1370 (w), 1311 (s), 1278
CH Ar); 7.71 (“t”(br), 3J = 7.4 Hz, 3J = 7.6 Hz, 1H, CHAr); (s), 1261 (m), 1247 (m), 1219 (w), 1189 (m), 1160 (w), 1147 (m),
3
3
7.49–7.38 (m, 2H, CHAr); 7.10 (“t”, J = 7.6 Hz, J = 7.9 Hz, 1H, 1107 (w), 1060 (w), 1041 (m), 1002 (s). MS (EI, 70 eV): m/z (%) =
CHAr); 6.98 (d, 3J = 7.7 Hz, 1H, CH Ar). IR (ATR, cm−1): ˜ν = 3152 347 ([M]+, [82Se], 35), 345 ([M]+, [80Se], 100), 343 ([M]+, [78Se],
(m), 3060 (m), 3010 (m), 2951 (m), 2877 (m), 2796 (m), 2667 46), 342 ([M]+, [77Se], 17), 341 ([M]+, [76Se], 16), 289 (17), 265
(w), 1941 (w), 1918 (w), 1889 (w), 1835 (w), 1811 (w), 1688 (m), (21), 237 (93), 208 (39), 150 (19). HRMS (ESI/TOF-MS): calcd for
1662 (s), 1651 (s), 1644 (s), 1615 (m), 1587 (m), 1575 (m), 1458 C16H8FNNaO2Se ([M
+
Na]+, [82Se]) 367.95971, found
(m), 1445 (m), 1400 (w), 1337 (s), 1301 (m), 1277 (s), 1230 (m), 367.95949; calcd for C16H8FNNaO2Se ([M + Na]+, [80Se])
1186 (w), 1159 (w), 1147 (w), 1118 (w), 1099 (m), 1081 (w), 1061 365.96081, found 365.96066. Elemental analysis: calcd for
(w), 1045 (s), 1022 (w), 1014 (m), 1000 (s). MS (EI, 70 eV): C16H8FNO2Se (344.20): C, 55.83; H, 2.34; N, 4.07. Found: C,
m/z (%) = 329 ([M]+, [82Se], 4), 327 ([M]+, [80Se], 23), 325 ([M]+, 55.80; H, 2.26; N, 4.13.
[78Se], 11), 324 ([M]+, [77Se], 6), 323 ([M]+, [76Se], 5), 219 (21),
190 (12), 78 (72), 63 (100), 44 (49). HRMS (ESI/TOF-MS): calcd indolin-2-one (10e). According to the general procedure
for C16H9NNaO2Se ([M
Na]+, [80Se]) 349.96914, found (method B), 3-acetoxy-benzo[b]selenophene (6) (150 mg,
349.96884. Elemental analysis: calcd for C16H9NO2Se (326.98): 0.63 mmol) and a methanolic solution of NaOMe (0.13 ml,
C, 58.91; H, 2.78; N, 4.29. Found: C, 58.71; H, 3.21; N, 4.39. 0.5 M) are brought to reaction in MeOH (2.5 ml). Subsequent
(Z)-5-Chloro-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]-
+
(Z)-7-Fluoro-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]- addition of 5-chloro-isatin (9e) (95 mg, 0.52 mmol) gave 10e as
indolin-2-one (10c). According to the general procedure a dark red solid (178 mg, 94%). Mp 341–343 °C. 1H NMR
(method B), 3-acetoxy-benzo[b]selenophene (6) (200 mg, (250 MHz, DMSO-d6): δ = 11.38 (s, 1H, NH); 9.04 (s, 1H, H-4′);
0.84 mmol) and a methanolic solution of NaOMe (0.17 ml, 7.90–7.81 (m, 2H, 2 × CHAr); 7.77–7.67 (m, 1H, CHAr);
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7.54–7.39 (m, 2H, 2 × CHAr); 7.07–6.94 (m, 1H, CHAr). IR (ATR, CHAr). IR (ATR, cm−1): ˜ν = 3151 (m), 3111 (m), 3079 (m), 3027
cm−1): ˜ν = 3164 (m), 3117 (m), 3058 (m), 3002 (w), 1880 (w), (m), 2996 (m), 2871 (m), 2824 (m), 2783 (m), 2707 (m), 2667
1689 (m), 1668 (s), 1651 (w), 1642 (m), 1618 (m), 1587 (m), (m), 1981 (w), 1948 (w), 1915 (w), 1880 (w), 1692 (s), 1665 (s),
1574 (w), 1552 (w), 1488 (w), 1455 (m), 1441 (s), 1388 (w), 1370 1651 (s), 1608 (m), 1586 (m), 1575 (m), 1556 (w), 1537 (w), 1520
(w), 1315 (s), 1306 (s), 1279 (s), 1255 (w), 1228 (m), 1182 (m), (w), 1504 (w), 1477 (w), 1444 (s), 1435 (s), 1367 (w), 1349 (w),
1158 (w), 1116 (w), 1078 (w), 1061 (w), 1044 (s), 1020 (w), 1000 1317 (s), 1276 (s), 1227 (s), 1179 (m), 1153 (w), 1120 (w), 1053
(s). MS (EI, 70 eV): m/z (%) = 365 ([M]+, [82Se], [37Cl], 9), 363 (w), 1040 (s), 1021 (m), 1002 (s). MS (EI, 70 eV): m/z (%) = 455
([M]+, [82Se], [35Cl] bzw. [80Se], [37Cl], 49), 361 ([M]+, [80Se], ([M]+, [82Se], 25), 453 ([M]+, [80Se], 100), 451 ([M]+, [78Se], 42),
[35Cl] bzw. [78Se], [37Cl], 100), 360 ([M]+, [77Se], [37Cl], 14), 359 450 ([M]+, [77Se], 14), 449 ([M]+, [76Se], 14), 326 (38), 190 (24),
([M]+, [78Se], [35Cl] bzw. [76Se], [37Cl], 41), 358 ([M]+, [77Se], 163 (18), 128 (18), 44 (25). HRMS (EI): calcd for C16H8INO2Se
[35Cl], 13), 357 ([M]+, [76Se], [35Cl], 13), 326 (25), 281 (14), 255 ([M + Na]+, [80Se]) 452.87594, found 452.877097; calcd for
(15), 253 (48), 190 (23). HRMS (EI): calcd for C16H8ClNO2Se C16H8INO2Se ([M + Na]+, [78Se]) 450.87673, found 450.878138.
([M]+, [82Se], [35Cl]) 362.94051, found 362.939468; calcd for Elemental analysis: calcd for C16H8INO2Se (452.10): C, 42.51;
C16H8ClNO2Se ([M]+, [80Se], [35Cl]) 360.94033, found H, 1.78; N, 3.10. Found: C, 42.63; H, 2.28; N, 3.32.
360.940627; calcd for C16H8ClNO2Se ([M]+, [78Se], [35Cl])
(Z)-5-Trifluoromethoxy-3-[3-oxo-benzo[b]selenophen-2(3H)-
358.94112, found 358.941763; calcd for C16H8ClNO2Se ([M]+, ylidene]indolin-2-one (10h). According to the general pro-
[76Se], [35Cl]) 356.94302, found 356.943702. Elemental analysis: cedure (method B), 3-acetoxy-benzo[b]selenophene (6)
calcd for C16H8ClNO2Se (360.65): C, 53.28; H, 2.24; N, 3.88. (200 mg, 0.84 mmol) and a methanolic solution of NaOMe
Found: C, 53.26; H, 2.23; N, 3.54.
(0.17 ml, 0.5 M) are brought to reaction in MeOH (3 ml). Sub-
(Z)-5-Bromo-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]- sequent addition of 5-trifluoromethoxy-isatin (9h) (161 mg,
indolin-2-one (10f). According to the general procedure 0.70 mmol) gave 10h as a red solid (262 mg, 92%). Mp
(method B), 3-acetoxy-benzo[b]selenophene (6) (200 mg, 335–337 °C. 1H NMR (300 MHz, DMSO-d6): δ = 11.47 (s, 1H,
0.84 mmol) and a methanolic solution of NaOMe (0.17 ml, NH); 9.02 (s, 1H, H-4′); 7.92–7.81 (m, 2H, 2 × CHAr); 7.79–7.67
3
0.5 M) are brought to reaction in MeOH (3 ml). Subsequent (m, 1H, CHAr); 7.51–7.39 (m, 2H, 2 ×CHAr); 7.07 (d, J = 8.7 Hz,
addition of 5-bromo-isatin (9f) (158 mg, 0.70 mmol) gave 10f 1H, CHAr). 19F NMR (282 MHz, DMSO-d6): δ = −57.14 (s,
1
as a dark red solid (243 mg, 86%). Mp 354–355 °C. H NMR OCF3). IR (ATR, cm−1): ˜ν = 3172 (m), 3149 (m), 3120 (m), 3064
(300 MHz, DMSO-d6): δ = 11.36 (s, 1H, NH); 9.19 (s, 1H, H-4′); (m), 3020 (m), 2925 (m), 2861 (m), 2802 (w), 2757 (w), 2698 (w),
7.90–7.82 (m, 2H, 2 × CHAr); 7.77–7.68 (m, 1H, CHAr); 1874 (m), 1823 (w), 1697 (s), 1668 (s), 1634 (m), 1622 (m), 1588
7.66–7.58 (m, 1H, CHAr); 7.48–7.39 (m, 1H, CHAr); 6.99–6.91 (m), 1575 (m), 1556 (w), 1538 (w), 1531 (w), 1519 (w), 1514 (w),
(m, 1H, CHAr). IR (ATR, cm−1): ˜ν = 3333 (m), 3208 (m), 3116 1505 (w), 1464 (s), 1446 (s), 1372 (w), 1315 (m), 1273 (s), 1255
(m), 1693 (s), 1666 (s), 1653 (s), 1634 (m), 1614 (m), 1585 (m), (s), 1205 (s), 1193 (s), 1161 (s), 1149 (s), 1113 (m), 1077 (w),
1575 (m), 1557 (w), 1539 (w), 1520 (w), 1505 (w), 1478 (w), 1453 1059 (w), 1046 (s), 1022 (w), 1004 (s). MS (EI, 70 eV): m/z (%) =
(m), 1443 (m), 1434 (m), 1384 (w), 1366 (w), 1316 (s), 1277 (s), 413 ([M]+, [82Se], 23), 411 ([M]+, [80Se], 100), 409 ([M]+, [78Se],
1254 (m), 1228 (s), 1181 (m), 1153 (w), 1117 (w), 1078 (w), 1058 48), 408 ([M]+, [77Se], 18), 407 ([M]+, [76Se], 19), 369 (24), 331
(w), 1043 (s), 1001 (s). MS (EI, 70 eV): m/z (%) = 409 ([M]+, (20), 303 (72), 206 (21), 76 (21). HRMS (EI): calcd for
[82Se], [81Br], 22), 407 ([M]+, [82Se], [79Br] bzw. [80Se], [81Br], 89), C17H8F3NO3Se ([M]+, [78Se]) 408.96239, found 408.963415;
405 ([M]+, [80Se], [79Br] bzw. [78Se], [81Br], 100), 404 ([M]+, [77Se], calcd for C17H8F3NO3Se ([M]+, [80Se]) 410.96160, found
[81Br], 17), 403 ([M]+, [78Se], [79Br] bzw. [76Se], [81Br], 38), 402 410.962096. Elemental analysis: calcd for C17H8F3NO3Se
([M]+, [77Se], [79Br], 10), 401 ([M]+, [76Se], [79Br], 10), 326 (41), (410.21): C, 49.78; H, 1.97; N, 3.41. Found: C, 49.80; H, 1.86; N,
299 (21), 190 (24), 163 (18), 44 (35). HRMS (EI): calcd for 3.26.
C16H8BrNO2Se ([M]+, [80Se], [81Br]) 406.88777, found
406.889101; calcd for C16H8BrNO2Se ([M]+, [80Se], [79Br])
Cell lines and cell cultures
404.88981, found 404.890256; calcd for C16H8BrNO2Se ([M]+,
[78Se], [79Br]) 402.89060, found 402.891302. Elemental analysis: Lung carcinoma (H157) cell lines (ATCC, CRL-5802) were main-
calcd for C16H8BrNO2Se (405.10): C, 47.44; H, 1.99; N, 3.46. tained in RPMI-1640 medium containing 10% heat-inactivated
Found: C, 47.52; H, 1.91; N, 3.52.
fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, penicil-
(Z)-5-Iodo-3-[3-oxo-benzo[b]selenophen-2(3H)-ylidene]- lin (100 U ml−1) and streptomycin (100 μg ml−1). Immortalized
indolin-2-one (10g). According to the general procedure human corneal epithelial cells (HCEC) were obtained from
(method B), 3-acetoxy-benzo[b]selenophene (6) (200 mg, RIKEN Bio Resource Center (Tsukuba Institute, Ibaraki, Japan)
0.84 mmol) and a methanolic solution of NaOMe (0.17 ml, and were grown in DMEM medium containing 5% heat-inacti-
0.5 M) are brought to reaction in MeOH (3 ml). Subsequent vated fetal bovine serum, insulin (5 μg ml−1), human epider-
addition of 5-iodo-isatin (9g) (190 mg, 0.70 mmol) gave 10g as mal growth factor (10 ng ml−1) and DMSO (0.5%), as described
a dark red solid (300 mg, 95%). Mp 362–364 °C. 1H NMR previously.20 Under these conditions, HCEC exhibited corneal
(300 MHz, DMSO-d6): δ = 11.37 (s, 1H, NH); 9.36 (d, 4J4′,6′ = 1.3 epithelial cell-specific properties. The human melanoma cell
Hz, 1H, H-4′); 7.90–7.82 (m, 2H, 2 × CHAr); 7.79–7.69 (m, 2H, 2 line A-375 (ATCC, CRL-1619) was cultured in DMEM (Gibco,
× CHAr); 7.48–7.39 (m, 1H, CHAr); 6.84 (d, 3J = 7.6 Hz, 1H, Karlsruhe, Germany) supplemented with 10% fetal calf serum
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and antibiotics (Biochrom, Berlin, Germany). All cells were Statistical analyses
continuously kept at 37 °C in a 5% CO2 incubator.
Assays consisted of duplicate or triplicate values, and at least
two independent experiments were performed. Mean values
and SDs were either calculated by including all individual
values of the independent experiments (at least 4 values) or a
representative experiment is shown (triplicate values). Statisti-
cal significance was proven by the Student’s t-test (normal dis-
tribution), and p-values <0.05 were considered as statistically
significant.
Analysis of cell density by the sulforhodamine B assay
H157 and HCEC cells were grown in 96-well plates by inoculat-
ing 104 and 5 × 104 cells per well (100 μl medium), respectively.
At this cell density, confluent monolayers were formed within
24 h, which were subsequently used for treatment. Relative cell
numbers and densities were determined according to the
method of Skehan et al.21 Therefore, the cell monolayers were
washed with Hank’s balanced salt solution (HBSS) to remove
non-adherent cells and were further incubated for 24 h with Acknowledgements
increasing concentrations of indirubin derivatives (1, 25, 50
Financial support from the State of Mecklenburg-Western
and 100 μM). Then, cells were fixed with 50% ice-cold TCA
buffer and were kept for 1 h at 4 °C. Plates were washed 5
times with HBSS and air dried. Fixed cells were stained for
20–30 min with a sulforhodamine B solution (0.4%). Cells
were rinsed with 1% acetic acid and the plates were dried
again. Proteins were resuspended in 10 mM Tris buffer, and
absorption was measured at 565 nm.
Pommerania, the Deutsche Forschungsgemeinschaft and the
Deutsche Krebshilfe (Melanomverbund, grant numbers
108008 TP5 and TP7) is gratefully acknowledged.
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