Synthesis and antiproliferative activity of N-glycosyl-3,3-diaryloxindoles

Article abstract of DOI:10.1039/c4ra02627f

N-Glycosylated 3,3-diaryloxindoles were prepared by Lewis acid catalyzed reaction of acetylated N-glycosylisatins with various benzene derivatives and subsequent deprotection. Some of the products showed antiproliferative activity against malignant cutane

Full text of DOI:10.1039/c4ra02627f

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Synthesis and antiproliferative activity of
N-glycosyl-3,3-diaryloxindoles†
Cite this: RSC Adv., 2014, 4, 22828
Dennis Kleeblatt,a Christoph A. Cordes,a Philipp Lebrenz,a Martin Hein,a Holger Feist,a
Abdul Matin,b Rabia Raza,c Jamshed Iqbal,c Omar Munshi,ad Qamar Rahman,d
a
ae
*
Alexander Villinger and Peter Langer
Received 25th March 2014
Accepted 2nd May 2014
N-Glycosylated 3,3-diaryloxindoles were prepared by Lewis acid catalyzed reaction of acetylated N-
glycosylisatins with various benzene derivatives and subsequent deprotection. Some of the products
showed antiproliferative activity against malignant cutaneous melanoma cells HT-144 (ATCC HTB-63)
and lung carcinoma (H157) cell lines (ATCC CRL-5802).
DOI: 10.1039/c4ra02627f
www.rsc.org/advances
Introduction
The 3,3-diaryloxindole core structure is of considerable phar-
macological relevance. For example, oxyphenisatin and its
derivative acetyl-oxyphenisatin were used until the 1970s as
nonprescription laxatives. Later a relationship between oxy-
phenisatin derivatives and chronic liver-diseases was observed
and the molecule was taken from the market.1,2 However, it is
known today that these liver-injuries were not caused by a
general toxicity of oxyphenisatin, but instead by a hypersensi-
tivity of liver cells. Several recent studies showed that oxy-
phenisatin and other 3,3-diaryoxindoles show considerable
antiproliferative activity against various human cancer-cell-
lines.3–6 3,3-Diaryloxindole derivatives can be prepared by
reaction of isatin with arenes in the presence of acid.7,8 3,3-
Diaryloxindoles containing two different aryl groups were
prepared by Grignard reaction of isatin with arylmagnesium
halides followed by acid-catalyzed condensation reaction with
benzene derivatives (Fig. 1).9
Fig. 1 Structure of oxyphenisatine.
considerably higher than that of the non-glycosylated hetero-
cycles. For example, the akashines, indigo-N-glycosides isolated
from Streptomyces sp. GW48/1497, proved to be active against
various human cancer cell lines.10 Indirubin derivatives are
potent selective inhibitors of cyclin-dependent kinases (CDKs),
which play an important role in proliferation, and show
promising anti-cancer activities.11–13 N-Glycosides of thia- and
selenaindirubins show a remarkable activity against melanoma
and lung carcinoma cells, respectively.14 Moreau and Sassatelli
and their coworkers reported the synthesis and anti-cancer
activity of various isoindigo-N-glycosides.15 In this context,
oxindole-N-glycosides were prepared as synthetic building
blocks, (1-(2,3,4-tri-O-acetyl-b-D-xylopyranosyl)isoindigo), so-
called NATURA, represents a pharmacologically active natural
O-glycosylated isoindigo.16 Recently, we reported the synthesis
of a number of N,N-diglycosylated indirubin derivatives.17
Due to the considerable pharmacological relevance of non-
glycosylated 3,3-diaryloxindoles and due to our ongoing interest
in the synthesis and pharmacological evaluation of new N-gly-
cosylated indole and bisindole derivatives, we decided to study
the synthesis of hitherto unknown N-glycosides of these mole-
cules. Herein, we report an efficient synthesis of 3,3-diary-
loxindole-N-glycosides and their anti-proliferative activity
against various cancer cell lines.
In recent years, we and others studied the synthesis and
antiproliferative activity of various N-glycosylated indole and
bisindole derivatives. In this context, it was observed that the
pharmacological activity of the N-glycosides is oen
a
¨
¨
Institut fur Chemie, Universitat Rostock, Albert-Einstein-Str. 3a, 18059 Rostock,
Germany. E-mail: peter.langer@uni-rostock.de; Fax: +49 (381)4986412; Tel: +49
(381)4986410
bDepartment of Medical Lab Technology, University of Haripur, Hattar Road, Haripur,
Khyber Pakhtunkhwa, 22620-Pakistan
cDepartment of Pharmaceutical Sciences, COMSATS Institute of Information
Technology, Abbottabad, Postal Code 22060, Pakistan
dAmity University, Lucknow Campus, Viraj Khand-5, Gomti Nagar, Lucknow – 226010,
India
e
¨
¨
Leibniz-Institut fur Katalyse e. V. an der Universitat Rostock, Albert-Einstein-Str. 29a,
Results and discussion
18059 Rostock, Germany
† Electronic supplementary information (ESI) available. CCDC 960490. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ra02627f
Acetyl-protected isatin-N-glycosides 4a–d were prepared as
previously reported.13,18–21 For example, N-(b-D-mannopyranosyl)-
22828 | RSC Adv., 2014, 4, 22828–22839
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isatin 4c was prepared by reaction of D-mannose (1b) with
aniline, followed by acetylation of the sugar moiety and cycli-
zation with oxalyl chloride (Scheme 1). The isatin-N-glycosides of
L-rhamnose 4a, D-glucose 4b and D-galactose 4d were synthesized
under similar conditions.13,18–21
The reaction of isatin-N-glycosides 4 with benzene deriva-
tives was next studied. In the literature, several Lewis and
¨
Bronstedt acids (TiCl4, BF3$OEt2, AlCl3, AlEtCl2, H2SO4, H2SO4–
AcOH, MeSO3H) were used as catalysts for the synthesis of non-
glycosylated 3,3-diaryloxindoles.22–24 To nd the best conditions
for the reaction of N-(2,3,4-tri-O-acetyl-b-L-rhamnopyranosyl)
isatin (4a) with anisole, we systematically varied the catalyst and
the conditions. It proved to be advantageous to use anisole in a
high excess as a solvent. The use of TiCl4 or BF3$OEt2 resulted in
decomposition of the starting material and complex mixtures
(temperature: ꢀ78 ꢁC). Aer much experimentation, only the
employment of AlCl3 as catalyst was successful and gave the
expected 3,3-diaryloxindole-N-glycoside 5c in excellent yield
(Scheme 2).
With optimized reaction conditions in hand, we studied next
the scope of the reaction. The AlCl3 catalyzed reaction of
N-glycosylated isatines 4a–d with different benzene derivatives
afforded the desired 3,3-diaryloxindole-N-glycosides 5a–l
(Table 1). The best yields were obtained for products 5c–e,h,i,l
derived from electron-rich arenes (like anisole and N,N-dime-
thylaniline). In fact, the yields were excellent for all sugar
moieties, due to the high nucleophilicity of the arenes. These
transformations required relatively short reaction times and
reactions had to be performed at low temperatures. The high
yields can be explained by the high nucleolphilic character of
electron-rich arenes. Furthermore, AlCl3 proved to be better
soluble in anisole and N,N-dimethylaniline as compared to
benzene or toluene. The yields of the reaction of benzene with
isatine-N-glycosides 4a–d were dependent on the carbohydrate
moiety. While an excellent yield was obtained for rhamnoside
5a, only moderate yields were obtained for mannoside 5f and
galactoside 5j. The use of electron-poor arenes, such as bromo-
or chlorobenzene, was unsuccessful (no reaction and various
conditions and decomposition of the starting material).
Scheme
2
Synthesis of N-glycosylated 3,3-diaryl-oxindole 5c.
Reagents and conditions: (i) anisole, AlCl3, 20 ꢁC, 10 min; (ii) MeOH,
NaOMe, 12 h, 20 ꢁC.
The deprotection of 5a–l was successfully performed under
´
Zemplen conditions (Scheme 2 and Table 1). Products 5a–l were
dissolved in dry methanol and treated with catalytic amounts of
sodium methoxide at 20 ꢁC. Aer stirring for 12 h, the reactions
were complete. Aer standard work-up procedure, the pure
products were isolated as white solids in 75–88% yields
(Table 1).
For the conrmation of the structure of compounds 5 and 6,
several NMR experiments, like H and 13C NMR, H,1H COSY,
1
1
1H,1H NOESY and HMBC, were performed. The H,1H NOESY-
1
spectra show correlations between H-1, H-3 and H-5 for each
series of the N-glycosides. These correlations indicate a 1C4-
chair conformation with b-conguration for rhamno-con-
4
gurated products. In contrast, a C1-chair conformation with
b-conguration was observed for gluco-, manno- and galacto-
congurated derivatives. As a typical example, the diagnostic
1
1H, H NOESY correlations of 5e and 5h are shown in Fig. 2.
The structure of 5a was independently conrmed by X-ray
crystal structure analysis (Fig. 3). Compound 5a crystallized in a
orthorhombic crystal lattice with the space-group P212121. The
1C4-chair conformation and the b-conguration of the sugar-
moiety are clearly visible. Bond-lengths and angles do not show
any signicant divergences to theoretically expected values.
Antiproliferative activity
3,3-Diaryloxindoles have been reported for their anti-
proliferative activity. Natarajan et al. found that this group of
compounds depletes intra-cellular Ca2+ stores leading to inhi-
bition of translation initiation.25 Uddin et al.26 evaluated oxy-
phenisatin as potent antiproliferative agent, particularly against
the human breast carcinoma cell line. Keeping in view this
property, we evaluated our synthesized N-glycosylated 3,3-dia-
ryloxindoles for their antiproliferative activities against two
Scheme 1 Synthesis of acetyl protected N-(b-D-mannopyranosyl)
isatin 4b. Reagents and conditions: (i) EtOH (abs.), PhNH2, 78 ꢁC, 12 h;
(ii) pyridine, Ac2O, 0 ꢁC, 24 h; (iii) oxalyl chloride, AlCl3, 55 ꢁC, 1.5 h.
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Table 1 Syntheses of N-glycosylated 3,3-diaryloxindoles 5a–l and 6a–l
4
Time [min]
Temp. [ꢁC]
% (5)a
% (6)a
120
80
120
80
20
20
20
10
10
10
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Table 1 (Contd.)
4
Time [min]
Temp. [ꢁC]
% (5)a
% (6)a
120
80
120
80
10
20
10
20
120
80
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Table 1 (Contd.)
4
Time [min]
Temp. [ꢁC]
% (5)a
% (6)a
120
80
10
20
a
Yields are given for isolated products.
Fig. 2 Diagnostic 1H,1H NOESY correlations of compound 5e and 5h.
cancer cell lines, i.e. H157 cancer cell line derived from human
oral squamous cell carcinoma and HT-144 cell line derived from
malignant cutaneous melanoma and a normal cell line HCEC
derived from human corneal epithelial cells. All the experi-
ments were performed by exposing cells to 1 mM, 25 mM, 50 mM,
and 100 mM end concentration of compounds. The results
obtained revealed some of the compounds as antiproliferative
agents while rest of the compounds were almost inactive
against both cancer cell lines. The active compounds inhibited
the growth of cancer cell lines at 100 mM concentration while Fig. 3 ORTEP plot of compound 5a.
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below this concentration no considerable inhibition was
observed. The results given in Table 2 show the percentage
antiproliferative activity of the compounds against H157 cells,
HT-144 cells and HCEC cells along with standard deviation
(Fig. 4).
The results obtained aer exposing the cancer to a range of
concentration of test compounds revealed that some of the
compounds were active in inhibiting the growth of cancer cells
at 100 mM end concentration, with 60% inhibition as the best
value (compound 6i) while the standard dug methotrexate
showed only up to 21% growth inhibition while vincristine
exhibited 66% growth inhibition at the same concentration. A
very positive aspect of the results was that the compounds did
not show any inhibition activity towards normal cell line HCEC.
It was also observed that there is a relation between growth
inhibition potency of compounds with a little difference in
activity against both cell lines. Among all the tested N-glycosyl-
3,3-diaryloxindoles, 6i was most active with 60.21 ꢂ 5.22 growth
inhibition against H157 cells and 53.1 ꢂ 4.81% growth inhibi-
tion against HT-144 cells while the second highest growth
inhibition was exhibited by compound 5g with 55.6 ꢂ 5.23%
growth inhibition against H157 cells and 51.55 ꢂ 6.34% activity
Fig. 4 Numbering of compound 5a and related structures (5, 6).
against HT-144 cells. By observing the chemical structures of
active compounds, it can be inferred that simultaneous pres-
ence of hydroxyl and dimethyl amino groups imparts good
growth inhibition characteristics to the compound as can be
seen in compound 6i. Similar substitution of functional groups
was present in compounds 6d but the replacement of one
hydroxyl group with methyl group in 6d resulted in a big change
in activity of compound thus making it much lesser active than
6i. In case of series 5, the compound 5g exhibited maximum
growth inhibition which can be result of simultaneous substi-
tution of O-acetyl and methyl groups on tested compound.
Moreover, it was also observed that stereochemistry of the
compounds also play a vital role in biological activity of the
compounds. This can be observed in the activity difference of 5g
and 5k which contain the same heterocyclic moiety, but a
different stereochemistry. The same type of difference can be
observed for the activities of 6e and 6h. In conclusion, some of
the compounds tested in this study provide a direction towards
the discovery of potent and antiproliferative compounds.
Further work on such compounds with varying substitution
patterns may lead to the discovery of some novel and value able
antiproliferative compounds. Additionally, inactivity of these
compounds towards normal cell lines renders them an inter-
esting class of compounds for cancer studies.
Table 2 Antiproliferative activity of compounds against H157, HT-144
and HCEC cells
H157
HT-144
HCEC
GI ꢂ STDa [%]
GI ꢂ STDb [%]
GI ꢂ STDc [%]
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
15.93 ꢂ 4.53
38.97 ꢂ 4.35
23.54 ꢂ 2.87
18.52 ꢂ 1.09
28.94 ꢂ 6.12
17.21 ꢂ 7.91
55.6 ꢂ 5.23
14.52 ꢂ 1.11
20.91 ꢂ 5.44
16.52 ꢂ 1.14
18.98 ꢂ 5.78
13.39 ꢂ 8.45
28.53 ꢂ 4.81
20.14 ꢂ 7.33
32.94 ꢂ 2.81
14.65 ꢂ 9.41
52.79 ꢂ 4.65
17.91 ꢂ 4.88
18.88 ꢂ 6.94
12.59 ꢂ 7.09
60.21 ꢂ 5.22
16.28 ꢂ 3.45
21.07 ꢂ 9.83
9.33 ꢂ 5.22
21.03 ꢂ 1.1
66.07 ꢂ 9.1
18.76 ꢂ 5.84
36.72 ꢂ 7.33
21.38 ꢂ 4.57
19.27 ꢂ 6.05
29.76 ꢂ 3.12
16.43 ꢂ 7.17
51.55 ꢂ 6.34
10.28 ꢂ 4.88
16.18 ꢂ 3.78
20.15 ꢂ 5.13
21.77 ꢂ 6.55
16.59 ꢂ 4.98
27.7 ꢂ 8.77
29.87 ꢂ 5.76
30.81 ꢂ 6.31
17.6 ꢂ 3.16
44.58 ꢂ 6.95
19.35 ꢂ 5.37
21.31 ꢂ 7.13
9.17 ꢂ 3.62
53.1 ꢂ 4.81
27.34 ꢂ 7.56
16.35 ꢂ 5.33
14.89 ꢂ 4.54
20.71 ꢂ 0.9
68.01 ꢂ 3.2
4.43 ꢂ 1.27
6.94 ꢂ 2.44
6.25 ꢂ 2.19
6.82 ꢂ 3.93
4.92 ꢂ 1.84
4.4 ꢂ 1.27
8.12 ꢂ 4.82
7.4 ꢂ 3.81
7.25 ꢂ 4.65
5.4 ꢂ 0.14
7.56 ꢂ 4.83
4.8 ꢂ 1.91
6.94 ꢂ 2.44
6.25 ꢂ 2.19
4.92 ꢂ 1.84
6.4 ꢂ 2.17
3.4 ꢂ 1.44
4.4 ꢂ 1.27
7.25 ꢂ 4.65
5.4 ꢂ 0.14
6.9 ꢂ 3.32
7.56 ꢂ 4.83
5.4 ꢂ 0.14
5.4 ꢂ 0.14
3.1 ꢂ 0.13
6.2 ꢂ 0.76
Conclusion
In conclusion, N-glycosylated 3,3-diaryloxindoles were prepared
by Lewis acid catalyzed reaction of acetylated N-glycosylisatins
with various benzene derivatives and subsequent deprotection.
Some of the products showed good antiproliferative activity
against malignant cutaneous melanoma cells HT-144 (ATCC
HTB-63) and Lung carcinoma (H157) cell lines (ATCC CRL-5802)
and no effect towards normal body cells.
Methotrexate
Vincristine
Experimental
a
All solvents were anhydrous and commercially available and all
reactions were performed under argon atmosphere. Yields refer
to isolated products. 1H NMR spectra (250.13 MHz, 300.13 MHz
and 500.13 MHz) and 13C NMR spectra (62.9 MHz, 75.5 MHz
and 125.8 MHz) were recorded on Bruker spectrometers AV 250,
AV 300 and AV 500 in CDCl3 and acetone-d6 as solvents. The
Growth Inhibition (GI) (%) of compounds along with standard
deviation (STD) when tested against H157 cells at 100 mM end
b
concentration. Growth inhibition (GI) (%) of compounds along with
standard deviation (STD) when tested against HT-144 cells at 100 mM
c
end concentration. Growth inhibition (GI) (%) of compounds along
with standard deviation (STD) when tested against HCEC cells at 100
mM end concentration.
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calibration of spectra was carried out on solvent signals (CDCl3: 7.31 (m, 1H, Ar), 7.20–7.06 (m, 8H, Ar), 7.01–6.98 (m, 2H, Ar),
3
3
d (1H) ¼ 7.25, d (13C) ¼ 77.0; acetone-d6: d (1H) ¼ 2.05, d (13C) ¼ 6.02 (d, 3J1 ,2 ¼ 1.5 Hz, 1H, H-1 ), 5.63 (dd, J2 ,1 ¼ 1.5 Hz, J2 ,3
¼
0
0
0
0
0
0
0
30.8). Mass spectrometric data (MS) were obtained by electron 3.4 Hz, 1H, H-2 ), 5.38 (dd, J3 ,2 ¼ 3.4 Hz, 3J3 ,4 ¼ 10.2 Hz, 1H,
3
0
0
0
0
0
ionization (EI, 70 eV) or electrospray ionization (ESI). The mass H-30), 5.24 (“t”, J ¼ 9.6 Hz, 1H, H-40), 4.10–4.00 (m, 1H, H-50),
spectra and high-resolution mass spectra (HRMS) were recor- 2.29, 2.27 (2 s, 2 ꢃ Me), 2.09, 1.94, 1.78 (3 s, 3 ꢃ OCOCH3), 1.34
ded on the following MS instruments: Time-of-Flight LC/MS (d, 3J6 ,5 ¼ 6.2 Hz, 3H, H-6 ). 13C NMR (63 MHz, acetone-d6): d ¼
6210 (Agilent Technologies) for ESI and Finnigan MAT 95 XP 178.3 (C-2), 171.4, 171.2, 171.1 (3 ꢃ OCOCH3), 142.4, 141.6,
(Thermo Electron Corporation) for EI. Elemental analyses were 140.0, 138.9, 138.4, 134.7 (4 ꢃ CQu), 130.8 (2 ꢃ C–H), 130.7 (2 ꢃ
performed on a C,H,N,S analyser (Thermo Quest Flash EA 1112). C–H), 130.3 (2 ꢃ C–H), 129.9 (2 ꢃ C–H), 129.5 (C–H), 127.4
The melting points were measured with a polarizing micro- (C–H), 124.4 (C–H), 116.2 (C–H), 82.4 (C-10), 75.3 (C-50), 72.4
scope (Leitz Laborlux 12 Pol-S) equipped with a hot stage (C-40), 71.9 (C-30), 71.8 (C-20), 63.1 (C-3), 21.9 (2 ꢃ Me), 21.7, 21.7,
(Mettler FP 90). Thin layer chromatography was performed on 21.4 (3 ꢃ OCOCH3), 18.8 (C-60). MS (EI, 70 eV): m/z (%) ¼ 585
silica-gel foil (Merck DC-foil, silica gel 60, F254) and silica gel (M+, 30), 312 (43), 284 (14), 273 (34), 222 (13), 213 (16), 171 (19),
0
0
0
plates (Merck HPTLC precoated plates, silica gel 60, F254
)
153 (100), 111 (53). HRMS (ESI): calcd for C34H36NO8 [M + H]+
pursues. Detection was carried out via UV absorbance at 254 nm 586.24354 and for C34H35NaNO8 [M + Na]+ 608.22549. Found
or 366 nm and/or development with 10% methanolic sulfuric 586.24393 and 608.2261.
acid and subsequent heat treatment. Column chromatography
1-(2,3,4-Tri-O-acetyl-b-L-rhamnopyranosyl)-3,3-bis(4-methoxy-
was performed on Macherey-Nagel silica gel 60 (particle size 63– phenyl)indolin-2-one (5c). Starting with 4a (300 mg, 0.72 mmol),
200 nm, 70–230 mesh).
anisole (20 mL) and AlCl3 (200 mg, 1.50 mmol) 5c was isolated as
a colorless solid (433 mg, 98%), mp ¼ 114–116 ꢁC.1H NMR (300
MHz, acetone-d6): d ¼ 7.62–7.60 (m, 1H, Ar), 7.21–7.15 (m, 1H,
Ar), 7.05–6.99 (m, 3H, Ar), 6.97–6.94 (m, 1H, Ar), 6.92–6.86 (m,
General procedure A: preparation of the N-glycosylated 3,3-
diarylindolin-2-ones 5a–l
2H, Ar), 6.75–6.67 (m, 4H, Ar), 5.87 (d, 3J1 ,2 ¼ 1.5 Hz, 1H, H-10),
0
0
The isatin-N-glycoside 4 was dissolved in the corresponding 5.49 (dd, 3J2 ,1 ¼ 1.5 Hz, 3J2 ,3 ¼ 3.6 Hz, 1H, H-20), 5.24 (dd, J3 ,2
3
0
0
0
0
0
0
aromatic compound and AlCl3 was added. Aer heating for 3 ¼ 3.5 Hz, 3J3 ,4 ¼ 10.3 Hz, 1H, H-30), 5.10 (“t”, J ¼ 9.8 Hz, 1H, H-
0
0
h at 80 ꢁC the reaction was complete (TLC-control). Water was 40), 3.95–3.85 (m, 1H, H-50), 3.63, 3.62 (2 s, 2 ꢃ OMe), 1.95, 1.80,
given to the reaction mixture and the organic layer was 1.65 (3 s, 3 ꢃ OCOCH3), 1.19 (d, J6 ,5 ¼ 6.2 Hz, 3H, H-60). 13C
separated. The aqueous layer was extracted three times with NMR (63 MHz, acetone-d6): d ¼ 178.6 (C-2), 171.4, 171.2, 171.1
ethyl acetate. The combined organic layer was washed three (3 ꢃ OCOCH3), 161.0, 160.7, 142.3, 136.6, 135.1, 134.9 (6 ꢃ CQu),
times with 1 M NaHCO3-solution, one time with water and 131.5 (2 ꢃ C–H), 131.1 (2 ꢃ C–H), 129.4 (C–H), 127.4 (C–H),
dried over sodium sulfate. Aer evaporation the residue was 124.4 (C–H), 116.2 (C–H),115.4 (2 ꢃ C–H), 82.4 (C-10), 75.3 (C-50),
puried by column chromatography (heptane–ethyl acetate 72.4 (C-40), 71.9 (C-30), 71.8 (C-20), 62.4 (C-3), 56.5, 56.4 (2 ꢃ
3
0
0
¼ 3 : 1).
OMe), 21.7, 21.7, 21.4 (3 ꢃ OCOCH3), 18.9 (C-60). MS (EI, 70 eV):
1-(2,3,4-Tri-O-acetyl-b-L-rhamnopyranosyl)-3,3-diphenylindolin- m/z (%) ¼ 617 (M+, 20), 344 (97), 316 (16), 273 (21), 238 (12), 207
2-one (5a). Starting with 4a (200 mg, 0.48 mmol), benzene (20 mL) (28), 171 (12), 153 (54), 111 (35). HRMS (EI): calcd for C34H35NO10
and AlCl3 (200 mg, 1.50 mmol) 5a was isolated as a colorless solid [M]+ 617.22555. Found 617.22491. Anal. calc. for C36H37NO12
(250 mg, 94%), mp ¼ 265–267 ꢁC. 1H NMR (300 MHz, CDCl3): d ¼ (617.64): C, 66.12; H, 5.71; N, 2.27. Found: C, 66.33; H, 5.53;
7.78–7.76 (m, 1H, Ar), 7.37–7.19 (m, 10H, Ar), 7.13–7.06 (m, 3H, Ar), N, 2.36.
6.04 (d, 3J1 , 2 ¼ 1.5 Hz, 1H, H-10), 5.65 (dd, J2 ,1 ¼ 1.5 Hz, 3J2 ,3
¼
1-(2,3,4-Tri-O-acetyl-b-L-rhamnopyranosyl)-3,3-bis[4-(dimethyl-
3
0
0
0
0
0
0
3
3
3.4 Hz, 1H, H-20), 5.38 (dd, J3 ,2 ¼ 3.4 Hz, J3 ,4 ¼ 10.4 Hz, 1H, H- ), amino)phenyl]indolin-2-one (5d). Starting with 4a (1.0 g, 2.38
0
0
0
0
0
5.24 (“t”, J ¼ 9.8 Hz, 1H, H-4), 4.09–4.00 (m, 1H, H-50), 2.08, 1.94, mmol), N,N-dimethylaniline (20 mL) and AlCl3 (600 mg, 4.50
1.78 (3 s, 3 ꢃ OCOCH3), 1.33 (d, 3J6 ,5 ¼ 6.0 Hz, 3H, H-6 ). 13C NMR mmol) 5d was isolated as a colorless solid (1.380 g, 90%), mp ¼
0
0
0
1
ꢁ
(63 MHz, CDCl3): d ¼ 178.0 (C-2), 171.4, 171.2, 171.1 (3 ꢃ 130–132 C. H NMR (300 MHz, CDCl3): d ¼ 7.58–7.55 (m, 1H,
COOCH3), 144.5, 142.9, 134.3, 134.3 (4 ꢃ CQu), 130.4 (C–H), 130.2 Ar), 7.22–7.10 (m, 4H, Ar), 7.03–6.96 (m, 3H, Ar), 6.65–6.62 (m,
(C–H), 130.1 (C–H), 130.0 (C–H), 129.7 (C–H), 129.3 (C–H), 128.9 4H, Ar), 5.87 (d, 3J1 ,2 ¼ 1.5 Hz, 1H, H-1 ), 5.66 (dd, J2 ,1 ¼ 1.5 Hz,
3
0
0
0
0
0
3
(C–H), 127.5 (C–H), 124.5 (C–H), 116.3 (C–H), 82.5 (C-10), 75.3
J2 ,3 ¼ 2.8 Hz, 1H, H-2 ), 5.28–5.18 (m, 2H, H-3 , H-4 ), 3.73–3.64
0
0
0
0
0
(C-50), 72.4 (C-40), 71.9 (C-30), 71.8 (C-20), 63.8 (C-3), 21.7, 21.7, 21.4 (m, 1H, H-50), 2.91, 2.90 (2 s, 2 ꢃ NMe2), 2.08, 1.97, 1.81 (3 s, 3 ꢃ
(3 ꢃ OCOCH3), 18.8 (C-60). MS (EI, 70 eV): m/z (%) ¼ 557 (M , OCOCH3), 1.34 (d, 3J6 ,5 ¼ 6.2 Hz, 3H, H-60). 13C NMR (63 MHz,
57.47), 314 (10), 285 (40), 269 (25), 273 (61), 256 (22), 213 (19), 171 CDCl3): d ¼ 177.7 (C-2), 170.0, 169.5, 169.5 (3 ꢃ OCOCH3), 149.5,
(25), 153 (100), 111 (73). HRMS (EI): calcd. for C32H31N2O8 [M]+ 149.2, 140.1, 133.9 (4 ꢃ CQu), 129.4 (C–H), 128.6 (C–H), 126.9 (C–
557.20442. Found 557.204724. Anal. calc. for C32H31NO8 (557.59): H), 125.5 (C–H), 122.6 (C–H), 113.7 (C–H), 112.4 (C–H), 112.1 (C–
+
0
0
C, 68.93; H, 5.60; N, 2.51. Found: C, 68.94; H, 5.77; N, 2.46.
H), 80.5 (C-10), 76.4 (C-50), 73.8 (C-40), 70.6 (C-30), 70.4 (C-20), 60.6
1-(2,3,4-Tri-O-acetyl-b-L-rhamnopyranosyl)-3,3-bis(4-methyl- (C-3), 20.7, 20.7, 20.4 (3 ꢃ OCOCH3), 17.6 (C-60). MS (EI, 70 eV):
phenyl)indolin-2-one (5b). Starting with 4a (200 mg, 0.48 m/z (%) ¼ 643 (M+, 24), 370 (18), 157 (33), 135 (9), 115 (25). HRMS
mmol), toluene (20 mL) and AlCl3 (200 mg, 1.50 mmol) 5b was (EI): calcd. for C36H41N3O8 [M]+ 643.28882. Found 643.28907.
isolated as a colorless solid (156 mg, 56%), mp ¼ 114–116 ꢁC. 1H Anal. calc. for C36H41N3O8 (643.73): C, 67.17; H, 6.42; N, 6.53.
NMR (300 MHz, acetone-d6): d ¼ 7.78–7.75 (m, 1H, Ar), 7.36– Found: C, 67.13; H, 6.50; N, 5.97.
22834 | RSC Adv., 2014, 4, 22828–22839
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1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-3,3-bis(4-methoxy-
RSC Advances
1-(2,3,4,6-Tetra-O-acetyl-b-D-mannopyranosyl)-3,3-bis(4-
phenyl)indolin-2-one (5e). Starting with 4b (200 mg, 0.42 mmol), methoxyphenyl)indolin-2-one (5h). Starting with 4c (300 mg,
anisole (10 mL) and AlCl3 (220 mg, 1.65 mmol) 5e was isolated as a 0.63 mmol), anisole (10 mL) and AlCl3 (300 mg, 2.25 mmol) 5h
1
ꢁ
colorless solid (241 mg, 85%), mp ¼ 108–110 C. H NMR (250 was isolated as a colorless solid (403 mg, 95%), mp ¼ 119–
1
ꢁ
MHz, acetone-d6): d ¼ 7.63–7.60 (m, 1H, Ar), 7.38–7.32 (m, 1H, Ar), 121 C. H NMR (300 MHz, acetone-d6): d ¼ 7.78–7.75, (m, 1H,
7.25–7.22 (m, 1H, Ar), 7.16–7.04 (m, 5H, Ar), 6.90–6.84 (m, 4H, Ar), Ar), 7.35–7.29 (m, 1H, Ar), 7.18–6.98 (m, 6H, Ar), 6.88–6.80
3
3
3
5.94 (d, 3J2 ,1 ¼ 9.5 Hz, 1H, H-10), 5.73 (“t”, J ¼ 9.4 Hz, 1H, H-20), (m, 4H, Ar), 6.10 (d, J1 ,2 ¼ 1.7 Hz, 1H,H-10), 5.60 (dd, J2 ,1
¼
0
0
0
0
0
0
3
3
3
5.49 (“t”, J ¼ 9.4 Hz, 1H, H-30), 5.37 (“t”, J ¼ 9.4 Hz, 1H, H-40), 1.7 Hz, J2 ,3 ¼ 3.0 Hz, 1H, H-20), 5.55–5.44 (m, 2H, H-30, H-40),
4.32–4.20 (m, 3H, H-50, H-60), 3.76, 3.75 (2 s, 6H, 2 ꢃ OMe2), 2.04, 4.35–4.20 (m, 2H, H-50, H-60), 3.76, 3.75 (2 s, 2 ꢃ OMe), 2.07,
2.03, 1.92, 1.41 (4 s, 4 ꢃ OCOCH3). 13C NMR (63 MHz, acetone-d6): 2.07, 1.94, 1.77 (4 s, 4 ꢃ OCOCH3). 13C NMR (63 MHz, acetone-
d ¼ 179.2 (C-2), 171.6, 171.1, 171.0, 170.2 (4 ꢃ OCOCH3) 160.9, d6): d ¼ 178.6 (C-2), 171.6, 171.2, 171.2, 171.1 (4 ꢃ OCOCH3),
160.8, 141.1, 136.2, 135.2, 134.6 (6 ꢃ CQu), 131.5 (C–H), 130.0 (C– 161.0, 160.7, 142.3, 136.5, 135.0, 134.8 (6 ꢃ CQu), 131.5 (2 ꢃ C–
H), 127.8 (C–H), 124.8 (C–H), 113.7 (C–H), 115.5 (C–H), 115.4 (C– H), 131.1 (2 ꢃ C–H), 129.4 (C–H), 127.4 (C–H), 124.5 (C–H),
H), 114.6 (C–H), 81.1 (C-10), 76.1 (C-50), 75.0 (C-30), 69.8 (C-40), 69.0 116.4 (C–H), 115.4 (2 ꢃ C–H), 82.4 (C-10), 76.7 (C-50), 72.4 (C-30),
(C-20), 63.5 (C-60), 62.6 (C-3), 56.5 (2 ꢃ NMe2), 21.6, 21.6, 21.4, 20.9 71.6 (C-40), 66.9 (C-20), 63.8 (C-3), 63.8 (C-60), 62.4 (C-3), 56.4 (2 ꢃ
(4 ꢃ OCOCH3). MS (EI, 70 eV): m/z (%) ¼ 675 (M+, 30.42), 344 (99), OMe), 21.6, 21.6, 21.6, 21.4 (4 ꢃ OCOCH3). MS (EI, 70 eV): m/z
331 (29), 169 (100), 127 (11), 109 (34). HRMS (EI): calcd. for (%) ¼ 675 (M+, 28), 344 (100), 331 (12), 169 (47), 109 (15). HRMS
C36H37NO12 [M]+ 675.23103, found 675.23139. Anal. calc. for (EI): calcd. for C36H37NO12 [M]+ 675.23103. Found 675.23126.
C36H37NO12 (675.68): C, 63.99; H, 5.52; N, 2.07. Found: C, 64.17; H, Anal. calc. for C36H37NO12 (675.68): C, 63.99, H; 5.52; N, 2.07.
0
0
5.33; N, 1.87.
Found: C, 64.02; H, 5.44; N, 2.13.
1-(2,3,4,6-Tetra-O-acetyl-b-D-mannopyranosyl)-3,3-diphenyl-
1-(2,3,4,6-Tetra-O-acetyl-b-D-mannopyranosyl)-3,3-bis[4-
indolin-2-one (5f). Starting with 4c (200 mg, 0.42 mmol), (dimethylamino)phenyl]-indolin-2-one (5i). Starting with 4c
benzene (20 mL) and AlCl3 (220 mg, 1.65 mmol) 5f was isolated (200 mg, 0.42 mmol), N,N-dimethylaniline (10 mL) and AlCl3
as a colorless solid (152 mg, 59%), mp ¼ 95–97 ꢁC. 1H NMR (220 mg, 1.65 mmol) 5i was isolated as a colorless solid (235 mg,
1
(250 MHz, acetone-d6): d ¼ 7.81–7.77, (m, 1H, Ar), 7.38–7.20 80%), mp ¼ 102–104 ꢁC. H NMR (300 MHz, CDCl3): d ¼ 7.56–
(m, 10H, Ar), 7.13–7.07 (m, 3H, Ar), 6.14 (d, 3J1 ,2 ¼ 1.5 Hz, 1H, 7.53 (m, 1H, Ar), 7.20–7.10 (m, 3H, Ar), 7.03–6.95 (m, 4H, Ar),
0
0
3
3
H-10), 5.67 (dd, J2 ,1 ¼ 1.7 Hz, J2 ,3 ¼ 3.0 Hz, 1H, H-20), 5.56– 6.64–6.61 (m, 4H, Ar), 5.90 (d, 3J2 ,1 ¼ 1.5 Hz, 1H, H-1 ), 5.68 (dd,
0
0
0
0
0
0
0
3
3
5.45 (m, 2H, H-30, H-40), 4.35–4.23 (m, 2H, H-50, H-60), 2.08, 2.08,
J1 ,2 ¼ 1.5 Hz, 3J3 ,2 ¼ 3.4 Hz, 1H, H-20), 5.43 (t, J3 ,4 ¼ 10.0 Hz
0
0
0
0
0
0
1.95, 1.78 (4 s, 4 ꢃ OCOCH3). 13C NMR (63 MHz, acetone-d6): d ¼ 1H, H-40), 5.30 (dd, J4 ,3 ¼ 10.0 Hz, J2 ,3 ¼ 3.4 Hz, 1H, H-30),
178.0 (C-2), 171.6, 171.2, 171.2, 171.1 (4 ꢃ OCOCH3), 144.5, 4.33–4.18 (m, 2H, H-60), 3.85–3.79 (m, 1H, H-50), 2.90, 2.89 (2 s,
142.8, 142.4, 134.3 (4 ꢃ CQu), 130.4 (2 ꢃ C–H), 130.2 (2 ꢃ C–H), 12H, 2 ꢃ NMe2), 2.12, 2.07, 1.97, 1.81 (4 s, 4 ꢃ OCOCH3). 13C
130.1 (2 ꢃ C–H), 130.0 (2 ꢃ C–H), 129.7 (C–H), 129.4 (C–H), NMR (75 MHz, CDCl3): d ¼ 177.7 (C-2), 170.5, 169.7, 169.4, 169.4
129.0 (C–H), 127.6 (C–H), 124.6 (C–H), 116.4 (C–H), 82.5 (C-10), (4 ꢃ OCOCH3) 149.6, 149.3, 139.9, 133.9 (4 ꢃ CQu), 129.4 (C–H),
76.7 (C-50), 72.4 (C-30), 71.6 (C-40), 66.9 (C-20), 63.8 (C-3), 63.7 128.5 (C–H), 128.4 (C–H), 126.8 (C–H), 125.5 (C–H), 122.7 (C–H),
(C-60), 21.6, 21.6, 21.6, 21.4 (4 ꢃ OCOCH3). MS (EI, 70 eV): m/z 113.7 (C–H), 112.3 (C–H), 112.0 (C–H) 80.7 (C-10), 75.3 (C-20),
(%) ¼ 615 (M+, 11.19), 331 (23), 285 (24), 256 (14), 169 (97), 109 70.5 (C-30), 70.0 (C-40), 65.4 (C-50), 62.2 (C-60), 60.6 (C-3), 40.4 (4
(68). HRMS (EI): calcd. for C34H33NO10 [M]+ 615.20990. Found ꢃ NMe2), 20.7, 20.7, 20.6, 20.4 (4 ꢃ OCOCH3). MS (EI, 70 eV):
615.210250. Anal. calc. for C34H33NO10 (615.63): C, 66.33; H, m/z (%) ¼ 701 (M+, 31.94), 370 (14), 281 (10), 231 (12), 181 (21),
3
3
0
0
0
0
5.40; N, 2.28. Found: C, 66.86; H, 5.74; N, 2.28.
169 (23), 131 (28), 119 (29). HRMS (ESI): calcd for C23H44N3O10
1-(2,3,4,6-Tetra-O-acetyl-b-D-mannopyranosyl)-3,3-bis(4-methyl- [M + H]+ 702.30212 and for C23H43N3NaO10 [M + Na]+ 724.28407.
phenyl)indolin-2-one (5g). Starting with 4c (200 mg, 0.42 mmol), Found 702.30336 and 724.28526. Anal. calc. for C38H43N3O10
toluene (20 mL) and AlCl3 (230 mg, 1.72 mmol) 5g was isolated as a (701.76): C, 65.04; H, 6.18; N, 5.99. Found: C, 64.91; H, 6.12;
1
ꢁ
colorless solid (148 mg, 55%), mp ¼ 107–109 C. H NMR (300 N, 5.39.
MHz, acetone-d6): d ¼ 7.79–7.76, (m, 1H, Ar), 7.35–7.30 (m, 8H, Ar),
1-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-3,3-diphenyl-
7.19–7.05 (m, 1H, Ar), 7.00–6.96 (m, 2H, Ar), 6.11 (d, 3J1 ,2 ¼ 1.5 Hz, indolin-2-one (5j). Starting with 4d (200 mg, 0.42 mmol),
0
0
1H,H-10), 5.65 (dd, J2 ,1 ¼ 1.5 Hz, 3J2 ,3 ¼ 3.0 Hz, 1H, H-20), 5.55– benzene (20 mL) and AlCl3 (400 mg, 3.00 mmol) 5j was isolated
3
0
0
0
0
1
5.44 (m, 2H, H-30, H-40), 4.35–4.22 (m, 2H, H-50, H-60), 2.29, 2.27 (2 as a colorless solid (170 mg, 66%), mp ¼ 110–112 C. H NMR
s, 2 ꢃ Me), 2.08, 2.07, 1.95, 1.78 (4 s, 4 ꢃ OCOCH3). 13C NMR (63 (300 MHz, acetone-d6): d ¼ 7.64–7.61 (m, 1H, Ar), 7.44–7.28
MHz, acetone-d6): d ¼ 178.3 (C-2), 171.6, 171.2, 171.2, 171.1 (4 ꢃ (m, 8H, Ar), 7.23–7.14 (m, 5H, Ar), 5.95–5.86 (m, 2H, H-1, H-2),
ꢁ
3
3
OCOCH3), 142.3, 141.6, 140.0, 138.9, 138.5, 134.7 (6 ꢃ CQu), 130.8 5.58 (dd, J3 ,4 ¼ 3.4 Hz, J5 ,4 ¼ 0.9 Hz, 1H, H-40), 5.41–5.37
0
0
0
0
(2 ꢃ C–H), 130.7 (2 ꢃ C–H), 130.3 (2 ꢃ C–H), 129.9 (C–H), 129.5 (m, 1H, H-30), 4.56 (dt, J6 ,5 ¼ 6.4 Hz, 3J4 ,5 ¼ 0.8 Hz, 1H, H-50),
(C–H), 127.4 (C–H), 124.5 (C–H), 116.3 (C–H), 82.4 (C-10), 76.7 4.31–4.11 (m, 2H, H-60), 2.30, 1.98, 1.92, 1.36 (4 s, 4 ꢃ OCOCH3).
(C-50), 72.4 (C-30), 71.6 (C-40), 66.9 (C-20), 63.8 (C-60), 63.2 (C-3), 21.9, 13C NMR (63 MHz, acetone-d6): d ¼ 178.5 (C-2), 171.5, 171.5,
21.6, 21.6, 21.4 (4 ꢃ OCOCH3). MS (EI, 70 eV): m/z (%) ¼ 643 (M+, 171.1, 170.4 (4 ꢃ OCOCH3), 144.4, 143.0, 141.5, 134.0 (4 ꢃ CQu),
31), 331 (22), 312 (57), 284 (12), 222 (10), 169 (100), 109 (33). HRMS 130.6 (C–H), 130.3 (C–H), 130.2 (C–H), 130.0 (C–H), 130.0 (C–H),
3
0
0
0
0
(EI): calcd. for C36H37NO10 [M]+ 643.24120. Found 643.24124.
129.3 (C–H), 129.1 (C–H), 128.0 (C–H), 124.9 (C–H), 114.3 (C–H),
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81.4 (C-10), 74.7 (C-20), 73.1 (C-30), 69.5 (C-40), 66.7 (C-50), 64.0 remaining residue solved in ethyl acetate and the solution
(C-60), 63.3 (C-3), 21.7, 21.5, 21.4, 20.8 (4 ꢃ OCOCH3). MS (EI, 70 washed three times with water. Aer separation of the organic
eV): m/z (%) ¼ 615 (M+, 28.01), 331 (61), 285 (25), 169 (100), 127 layer and evaporation of the solvent the solid product 6 was
(14), 109 (32). HRMS (EI): calcd for C34H33NO10 [M]+ 615.20990. obtained.
Found 615.21111. Anal. calc. for C34H33NO10 (615.63): C, 66.33;
H, 5.40; N, 2.28. Found: C, 66.33; H, 5.05; N, 1.94.
3,3-Diphenyl-1-(b-L-rhamnopyranosyl)indolin-2-one
Starting with 5a (100 mg, 0.18 mmol), dray methanol (4 mL) and
(6a).
1-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-3,3-bis(4- 0.1% solution of NaOMe (0.49 mL, 5 mol%) 6a was isolated as a
1
ꢁ
methylphenyl)indolin-2-one (5k). Starting with 4d (200 mg, 0.42 colorless solid (62 mg, 80%), mp ¼ 123–125 C. H NMR (300
mmol), toluene (10 mL) and AlCl3 (220 mg, 1.65 mmol) 5k was MHz, CDCl3): d ¼ 7.75–7.72 (m, 1H, Ar), 7.34–7.20 (m, 12H, Ar),
1
3
isolated as a colorless solid (157 mg, 62%), mp ¼ 111–113 ꢁC. H 7.07–7.02 (m, 1H, Ar), 5.63 (d, J1 ,2 ¼ 1.1 Hz, 1H, H-10), 4.53
0
0
NMR (300 MHz, acetone-d6): d ¼ 7.62–7.59 (m, 1H, Ar), 7.42– (brs, 1H, OH), 4.22–4.02 (m, 3H, H-20, 2 ꢃ OH), 3.70–3.47 (m,
7.36 (m, 1H, Ar), 7.27–7.24 (m, 1H, Ar), 7.16–7.05 (m, 9H, Ar), 3H, H-30, H-40, H-50), 1.35 (d, J6 ,5 ¼ 5.8 Hz, 3H, H-60). 13C NMR
3
0
0
5.94–5.86 (m, 2H, H-1, H-2), 5.58 (dd, 3J3 ,4 ¼ 3.4 Hz, J5 ,4 ¼ 0.9 (63 MHz, CDCl3): d ¼ 178.4 (C-2), 144.5, 144.0, 143.7, 134.0 (4 ꢃ
3
0
0
0
0
3
Hz, 1H, H-40), 5.40–5.36 (m, 1H, H-30), 4.54 (dt, J6 ,5 ¼ 6.6 Hz,
C
Qu), 130.4 (2 ꢃ C–H), 130.2 (2 ꢃ C–H), 130.1 (2 ꢃ C–H), 130.0
0
0
3
0
0
0
0
J4 ,5 ¼ 0.9 Hz, 1H, H-5 ), 4.30–4.10 (m, 2H, H-6 ), 2.29, 2.28 (2 s, (2 ꢃ C–H), 129.3 (C–H), 129.1 (C–H), 128.9 (C–H), 127.0 (C–H),
2 ꢃ Me), 2.30, 1.98, 1.92, 1.36 (4 s, 4 ꢃ OCOCH3). 13C NMR (75 124.1 (C–H), 117.2 (C–H), 113.8 (C–H), 84.6 (C-10), 77.3 (C-20),
MHz, acetone-d6): d ¼ 178.8 (C-2), 171.5, 171.5, 171.1, 170.3 (4 ꢃ 75.8 (C-30), 74.2 (C-40), 73.8 (C-50), 63.9 (C-3), 19.3 (C-60). MS (EI,
OCOCH3), 141.4, 140.3, 138.8, 138.6, 134.4 (5 ꢃ CQu), 130.8 (C– 70 eV): m/z (%) ¼ 431 (M+, 25), 285 (100), 256 (32), 208 (21), 165
H), 130.6 (C–H), 130.0 (C–H), 130.0 (C–H), 129.9 (C–H), 127.9 (10). HRMS (EI): calcd for C26H25NO5 [M]+ 431.17272. Found
(C–H), 124.8 (C–H), 114.1 (C–H), 81.3 (C-10), 74.7 (C-20), 73.2 431.17278.
(C-30), 69.5 (C-40), 66.7 (C-50), 63.4 (C-60), 63.3 (C-3), 21.9 (2 ꢃ Me)
3,3-Bis(4-methylphenyl)-1-(b-L-rhamnopyranosyl)indolin-2-
21.7, 21.5, 21.4, 20.8 (4 ꢃ OCOCH3). MS (EI, 70 eV): m/z (%) ¼ one (6b). Starting with 5b (100 mg, 0.17 mmol), dray methanol
643 (M+, 52.00), 331 (77), 312 (52), 284 (10), 169 (100), 127 (8), (4 mL) and 0.1% solution of NaOMe (0.46 mL, 5 mol%) 6b was
109 (20). HRMS (EI): calcd for C36H37NO10 [M]+ 643.24120. isolated as a colorless solid (60 mg, 77%), mp ¼ 128–129 ꢁC. 1H
Found 643.24204. Anal. calc. for C36H37NO10 (643.68): C, 67.17; NMR (300 MHz, acetone-d6): d ¼ 7.71–7.68 (m, 1H, Ar), 7.24–
0
0
0
H, 5.79; N, 2.18. Found: C, 67.49; H, 5.75; N, 1.80.
7.00 (m, 13H, Ar), 5.60 (d, 3J1 ,2 ¼ 1.3 Hz, 1H, H-1 ), 4.53 (brs, 1H,
1-(2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-3,3-bis(4- OH), 4.25–4.01 (m, 3H, H-20, 2 ꢃ OH), 3.70–3.46 (m, 3H, H-30, H-
3
methoxyphenyl)indolin-2-one (5l). Starting with 4d (200 mg, 40, H-50), 2.29, 2.28 (2 s, 2 ꢃ OMe), 1.35 (d, J6 ,5 ¼ 5.6 Hz, 3H, H-
0.42 mmol), anisole (10 mL) and AlCl3 (220 mg, 1.65 mmol) 5l 60). 13C NMR (63 MHz, acetone-d6): d ¼ 178.8 (C-2), 143.9, 141.6,
was isolated as a colorless solid (260 mg, 92%), mp ¼ 104–106 140.9, 138.6, 138.4, 134.5 (6 ꢃ CQu), 130.7 (2 ꢃ CH), 130.6 (2 ꢃ
ꢁC. 1H NMR (250 MHz, CDCl3): d ¼ 7.44–7.41 (m, 1H, Ar), 7.33– CH), 130.3 (2 ꢃ CH), 129.2 (2 ꢃ CH), 126.8 (2 ꢃ CH), 124.0 (CH),
0
0
7.25 (m, 1H, Ar), 7.18–7.06 (m, 6H, Ar), 6.83–6.77 (m, 4H, Ar), 117.0 (CH), 84.6 (C-10), 77.3 (C-50), 75.8 (C-40), 74.3 (C-30), 73.8 (C-
5.84 (“t”, 3J1 ,2 ¼ 9.5 Hz, 3J3 ,2 ¼ 9.8 Hz, 1H, H-20), 5.73 (d, J2 ,1 20), 63.3 (C-3), 21.9 (2 ꢃ Me), 19.3 (C-60). MS (EI, 70 eV): m/z (%)
3
0
0
0
0
0
0
¼ 9.5 Hz, 1H, H-10), 5.55 (d, J3 ,4 ¼ 3.2 Hz, 1H, H-40), 5.16 (dd, ¼ 459 (M+, 49), 313 (100), 298 (20), 284 (31), 270 (29), 222 (43),
3
0
0
J2 ,3 ¼ 9.8 Hz, J4 ,3 ¼ 3.2 Hz, 1H, H-3 ), 4.25–4.09 (m, 3H, H-5 , 194 (13). HRMS (EI): calcd for C28H29NO5 [M]+ 459.20402. Found
3
3
0
0
0
0
0
0
H-60), 3.76 (s, 2 ꢃ OMe), 2.29, 2.06, 1.97, 1.33 (4 s, 4 ꢃ OCOCH3). 459.20427.
13C NMR (63 MHz, CDCl3): d ¼ 178.1, 170.3, 169.9, 169.8 (4 ꢃ
3,3-Bis(4-methoxyphenyl)-1-(b-L-rhamnopyranosyl)indolin-
OCOCH3), 168.9 (C-2), 158.9, 158.6 (2 ꢃ C-400), 139.0 (C-7a), 2-one (6c). Starting with 5c (200 mg, 0.32 mmol), dray methanol
134.5 (C-100), 132.8 (C-3a), 132.7 (C-5), 129.9, 129.2 (2 ꢃ C-200), (4 mL) and 0.1% solution of NaOMe (0.87 mL, 5 mol%) 6c was
128.1 (C-4), 126.0 (C-6), 123.3 (C-5), 113.8, 113.6 (2 ꢃ C-300), isolated as a colorless solid (129 mg, 81%), mp ¼ 125–127 ꢁC.1H
112.0 (C-7), 79.7 (C-10), 73.1 (C-20), 71.5 (C-30), 67.3 (C-40), 64.6 (C- NMR (300 MHz, acetone-d6): d ¼ 7.71–7.68 (m, 1H, Ar), 7.24–
50), 61.3 (C-60), 61.0 (C-3), 55.2, 55.2 (2 ꢃ OMe), 20.7, 20.6, 20.5, 7.12 (m, 6H, Ar), 7.06–7.01 (m, 1H, Ar), 6.88–6.82 (m, 4H, Ar),
19.6 (4 ꢃ OCOCH3). MS (EI, 70 eV): m/z (%) ¼ 675 (M+, 27), 344 5.60 (d, 3J1 ,2 ¼ 1.1 Hz, 1H, H-10), 4.54 (brs, 1H, OH), 4.15–3.99
0
0
(85), 331 (74), 169 (75), 127 (12), 109 (28). HRMS (EI): calcd for (m, 3H, H-20, 2 ꢃ OH), 3.79–3.46 (m, 9H, H-30, H-40, H-50, 2 ꢃ
3
0
0
C
C
36H37NO12 [M]+ 675.23103. Found 675.23040. Anal. calc. for OMe), 1.35 (d, J6 ,5 ¼ 5.6 Hz, 3H, H-60). 13C NMR (63 MHz,
36H37NO12 (675.68): C, 63.99; H, 5.52; N, 2.07. Found: C, 64.03; acetone-d6): d ¼ 178.8 (C-2), 160.6, 160.4, 143.6, 136.2, 135.5,
H, 5.59; N, 2.06.
134.5 (6 ꢃ CQu), 131.2 (2 ꢃ CH), 131.0 (2 ꢃ CH), 128.8 (CH),
126.6 (CH), 123.7 (CH), 116.7 (CH), 115.1 (2 ꢃ CH), 115.1 (2 ꢃ
CH), 84.3 (C-10), 77.1 (C-50), 75.6 (C-40), 74.0 (C-30), 73.5 (C-20),
62.3 (C-3), 56.2 (2 ꢃ NMe2), 19.1 (C-60). MS (EI, 70 eV): m/z (%) ¼
491 (M+, 19), 344 (100), 298 (12), 316 (22), 302 (11), 238 (18).
General procedure B: synthesis of the deprotected
N-glycosylated 3,3-diarylindolin-2-ones 6a–l
One equivalent of the acetyl-protected N-glycosylated 3,3-diary- HRMS (EI): calcd for C28H29NO7 [M]+ 491.19385. Found
lindolin-2-one 5 was solved in dry MeOH. To the stirred solution 491.19386. Anal. calc. for C28H29NO7 (491.53): C, 68.42; H, 5.95;
5 mol% NaOMe (0.1% solution) was added and the mixture N, 2.85. Found: C, 68.07; H, 6.16; N, 2.29.
stirred for 12 h. When the reaction was complete (TLC-control)
3,3-Bis[4-(dimethylamino)phenyl]-1-(b-L-rhamnopyranosyl)
the reaction mixture was neutralized by addition of acetic acid. indolin-2-one (6d). Starting with 5d (200 mg, 0.31 mmol), dray
The solvent was removed under reduced pressure, the methanol (4 mL) and 0.1% solution of NaOMe (0.84 mL,
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5 mol%) 6d was isolated as a colorless solid (138 mg, 86%), mp 69.3 (C-20), 63.7 (C-60), 63.3 (C-3), 21.9 (2 ꢃ Me). MS (EI, 70 eV):
¼ 156–158 ꢁC. 1H NMR (300 MHz, CDCl3): d ¼ 7.65–7.62 (m, 1H, m/z (%) ¼ 475 (M+, 23), 313 (100), 298 (17), 284 (21), 270 (21), 222
Ar), 7.21–6.98 (m, 7H, Ar), 6.67–6.63 (m, 4H, Ar), 5.56 (d, 3J1 ,2
¼
(20). HRMS (EI): calcd for C28H29NO6 [M]+ 475.19894. Found
0
0
1.1 Hz, 1H, H-10), 4.64 (brs, 1H, OH), 4.15–3.94 (m, 3H, H-20, 2 ꢃ 475.199822.
OH), 3.69–3.45 (m, 3H, H-30, H-40, H-50), 2.89, 2.88 (2 ꢃ NMe2),
1-(b-D-Mannopyranosyl)-3,3-bis(4-methoxyphenyl)indolin-2-
1.34 (d, 3J6 ,5 ¼ 5.8 Hz, 3H, H-6 ). 13C NMR (63 MHz, CDCl3): d ¼ one (6h). Starting with 5h (200 mg, 0.30 mmol), dray methanol
179.8 (C-2), 151.6, 151.5, 143.9, 135.7, 132.2, 131.5 (6 ꢃ CQu), (8 mL)/dray THF (2 mL) and 0.1% solution of NaOMe (0.80 mL,
130.9 (2 ꢃ CH), 130.7 (2 ꢃ CH), 128.7 (CH), 126.8 (CH), 123.7 5 mol%) 6h was isolated as a colorless solid (126 mg, 84%), mp
(CH), 116.5 (CH), 113.8 (C–H), 84.6 (C-10), 77.3 (C-50), 75.9 (C-40), ¼ 135–137 ꢁC. 1H NMR (300 MHz, acetone-d6): d ¼ 7.72–7.69 (m,
0
0
0
75.9 (C-30), 74.4 (C-20), 62.4 (C-3), 41.5 (2 ꢃ NMe2), 19.3 (C-60). 1H, Ar), 7.22–7.00 (m, 7H, Ar), 6.87–6.83 (m, 4H, Ar), 5.63 (d,
3
MS (EI, 70 eV): m/z (%) ¼ 517 (M+, 73), 370 (100), 342 (50), 262
J1 ,2 ¼ 1.5 Hz, 1H, H-10), 4.54–4.53 (m, 1H, OH), 4.18–3.72 (m,
0
0
(9), 223 (9), 183 (8). HRMS (EI): calcd for C30H35N3O5 [M]+ 7H, H-20, H-30, H-40, H-60, 2 ꢃ OMe, 2 ꢃ OH), 3.52–3.46 (m, 1H,
517.25712. Found 517.25699. Anal. calc. for C30H35N3O5 H-50).13C NMR (63 MHz, acetone-d6): d ¼ 179.1 (C-2), 160.8,
(517.62): C, 69.61; H, 6.82. Found: C, 69.47; H, 6.96%.
160.6 (2 ꢃ C-400), 143.9, 136.4, 135.7, 134.7 (4 ꢃ CQu), 131.4 (2 ꢃ
1-(b-D-Glucopyranosyl)-3,3-bis(4-methoxyphenyl)indolin-2- C–H), 131.3 (2 ꢃ C–H), 129.2 (C–H), 126.8 (C–H), 123.9 (C–H),
one (6e). Starting with 5e (100 mg, 0.15 mmol), dray methanol (4 117.3 (C–H), 115.4 (2 ꢃ C–H), 115.3 (2 ꢃ C–H), 84.8 (C-10), 82.2
mL) and 0.1% solution of NaOMe (0.40 mL, 5 mol%) 6e was (C-50), 76.0 (C-30), 73.6 (C-40), 69.4 (C-20), 63.8 (C-60), 62.6 (C-3),
isolated as a colorless solid (61 mg, 82%), mp ¼ 145–147 ꢁC. 1H 56.4 (2 ꢃ OMe). MS (EI, 70 eV): m/z (%) ¼ 507 (M+, 30), 344 (100),
NMR (300 MHz, acetone-d6): d 7.36–7.33 (m, 1H, Ar), 7.28–7.16 330 (13), 316 (36), 302 (17), 238 (26), 210 (9). HRMS (EI): calcd for
(m, 6H, Ar), 7.10–7.05 (m, 1H, Ar), 6.89–6.78 (m, 4H, Ar), 5.49 (d,
C
28H29NO8 [M]+ 507.18877. Found 507.18870.
3
J1 ,2 ¼ 9.4 Hz, 1H, H-10), 4.18–3.49 (m, 16H, H-20, H-30, H-40, H-
1-(b-D-Mannopyranosyl)-3,3-bis[4-(dimethylamino)phenyl]-
0
0
50, H-60, 4 ꢃ OH, 2 ꢃ OMe). 13C NMR (75 MHz, acetone-d6): d ¼ indolin-2-one (6i). Starting with 5i (200 mg, 0.28 mmol), dray
179.2 (C-2), 160.9, 160.6, 142.4, 137.1, 135.4, 135.2 (6 ꢃ CQu), methanol (6 mL) and 0.1% solution of NaOMe (0.76 mL, 5
131.6 (2 ꢃ C–H), 131.4 (2 ꢃ C–H), 129.6 (C–H), 127.7 (C–H), mol%) 6i was isolated as a colorless solid (122 mg, 80%), mp ¼
1
ꢁ
124.2 (C–H), 115.3 (2 ꢃ C–H), 114.2 (C–H), 84.3 (C-10), 81.7 (C- 149–151 C. H NMR (300 MHz, acetone-d6): d ¼ 7.65–7.62 (m,
50), 80.3 (C-30), 72.3 (C-20), 70.9 (C-40), 63.5 (C-60), 62.7 (C-3), 56.5, 1H, Ar), 7.19–6.98 (m, 7H, Ar), 6.67–6.63 (m, 4H, Ar), 5.59 (d,
3
56.4 (2 ꢃ OMe). MS (EI, 70 eV): m/z (%) ¼ 507 (M+, 30), 344 (100),
J1 ,2 ¼ 1.5 Hz, 1H, H-1 ), 4.61–4.14 (m, 3H, H-2 , 2 ꢃ OH), 3.95–
0
0
0
0
330 (11), 316 (35), 302 (16), 238 (26), 210 (9). HRMS (EI): calcd for 3.40 (m, 7H, H-30, H-40, H-50, H-60, 2 ꢃ OH), 2.89 (s, 2 ꢃ NMe2).
C
C
28H29NO8 [M]+ 507.18877. Found 507.18861. Anal. calc. for 13C NMR (63 MHz, acetone-d6): d ¼ 179.8 (C-2), 151.6, 151.5,
28H29NO8 (507.53): C, 66.26; H, 5.76. Found: C, 65.76; H, 5.82. 143.9, 135.6, 132.1, 131.5, (6 ꢃ CQu), 130.9 (2 ꢃ C–H), 130.8 (2 ꢃ
1-(b-D-Mannopyranosyl)-3,3-diphenylindolin-2-one
(6f). C–H), 128.8 (C–H), 126.8 (C–H), 123.7 (C–H), 116.3 (C–H), 113.9
Starting with 5f (100 mg, 0.16 mmol), dray methanol (8 mL)/ (2 ꢃ C–H), 113.8 (2 ꢃ C–H), 84.8 (C-10), 82.1 (C-50), 76.1 (C-30),
dray THF (2 mL) and 0.1% solution of NaOMe (0.43 mL, 5 73.5 (C-40), 69.5 (C-20), 63.8 (C-60), 62.4 (C-3), 41.5 (2 ꢃ OMe). MS
mol%) 6f was isolated as a colorless solid (55 mg, 76%), mp ¼ (EI, 70 eV): m/z (%) ¼ 277 (M+, 53), 413 (19), 370 (100), 342 (65),
1
ꢁ
133–134 C. H NMR (300 MHz, acetone-d6): d ¼ 7.76–7.73 (m, 326 (17), 281 (12), 251 (13), 223 (17), 207 (16). HRMS (EI): calcd
1H, Ar), 7.35–7.18 (m, 7H, Ar), 7.07–7.01 (m, 1H, Ar), 5.67 (d, for C30H35N3O6 [M]+ 533.25204. Found 533.25108. Anal. calc. for
3
J1 ,2 ¼ 1.1 Hz, 1H, H-10), 4.54 (brs, 1H, OH), 4.23–3.47 (m, 9H,
C
30H35NO6 (533.62): C, 67.52; H, 6.61; N, 7.87. Found: C, 67.46;
0
0
H-20, H-30, H-40, H-50, H-60, 3 ꢃ OH). 13C NMR (63 MHz, acetone- H, 6.40; N, 7.10.
d6): d ¼ 178.5 (C-2), 144.4, 144.0, 143.7, 134.0 (4 ꢃ CQu), 130.4 (2
1-(b-D-Galaktopyranosyl)-3,3-diphenylindolin-2-one
(6j).
ꢃ C–H), 130.2 (2 ꢃ C–H), 130.1 (2 ꢃ C–H), 130.0 (2 ꢃ C–H), Starting with 5j (100 mg, 0.16 mmol), dray methanol (4 mL) and
129.4 (C–H), 128.9 (C–H), 126.9 (C–H), 124.0 (C–H), 117.5 (C–H), 0.1% solution of NaOMe (0.43 mL, 5 mol%) 6j was isolated as a
1
84.8 (C-10), 82.1 (C-50), 76.0 (C-30), 73.7 (C-40), 69.3 (C-20), 63.9 (C- colorless solid (54 mg, 75%), mp ¼ 149–151 C. H NMR (300
ꢁ
3), 63.7 (C-60). MS (EI, 70 eV): m/z (%) ¼ 447 (M+, 23), 285 (100), MHz, acetone-d6): d ¼ 7.58–7.56 (m, 1H, Ar), 7.35–7.22 (m, 12H,
3
270 (10), 256 (47), 208 (22), 180 (12), 165 (13). HRMS (EI): calcd Ar), 7.11–7.06 (m, 4H, Ar), 5.49 (d, J1 ,2 ¼ 1.5 Hz, 1H, H-10),
0
0
for C26H25NO6 [M]+ 447.16764. Found 447.16804.
4.48–3.74 (m, 10H, H-20, H-30, H-40, H-50, H-60, 4 ꢃ OH). 13C NMR
1-(b-D-M annopyranosyl)-3,3-bis(4-methylphenyl)indolin-2- (75 MHz, acetone-d6): d ¼ 178.4 (C-2), 145.2, 143.4, 142.5, 134.4
one (6g). Starting with 5g (100 mg, 0.16 mmol), dray methanol (4 (4 ꢃ CQu), 130.6 (2 ꢃ C–H), 130.3 (2 ꢃ C–H), 130.0 (2 ꢃ C–H),
mL) and 0.1% solution of NaOMe (0.43 mL, 5 mol%) 6g was 129.9 (2 ꢃ C–H), 129.7 (C–H), 129.1 (C–H), 128.7 (C–H), 127.7
isolated as a colorless solid (58 mg, 78%), mp ¼ 139–140 ꢁC. 1H (C–H), 124.2 (C–H), 114.6 (C–H), 84.3 (C-10), 79.6 (C-20), 76.7 (C-
NMR (300 MHz, acetone-d6): d ¼ 7.73–7.70 (m, 1H, Ar), 7.22– 30), 71.2 (C-40), 68.5 (C-50), 64.2 (C-3), 63.5 (C-60). MS (EI, 70 eV):
7.00 (m, 11H, Ar), 5.64 (d, 3J1 ,2 ¼ 1.3 Hz, 1H, H-1 ), 4.55 (brs, 1H, m/z (%) ¼ 447 (M+, 38), 285 (100), 256 (61), 208 (26), 180 (13), 165
OH), 4.23–3.72 (m, 9H, H-20, H-30, H-40, H-50, H-60, 3 ꢃ OH), (13). HRMS (EI): calcd. for C26H25NO6 [M]+ 447.16764. Found
3.52–3.46 (m, 1H, H-50), 2.29, 2.28 (2 s, 2 ꢃ Me). 13C NMR (63 447.16766.
0
0
0
MHz, acetone-d6): d ¼ 178.8 (C-2), 143.9, 141.5, 140.9, 138.6,
1-(b-D-Galactopyranosyl)-3,3-bis(4-methylphenyl)indolin-2-
138.4, 134.4 (6 ꢃ CQu), 130.7 (2 ꢃ C–H), 130.6 (2 ꢃ C–H), 130.3 one (6k). Starting with 5k (100 mg, 0.16 mmol), dray methanol
(2 ꢃ C–H), 130.1 (2 ꢃ C–H), 129.2 (C–H), 126.8 (C–H), 123.9 (C– (4 mL) and 0.1% solution of NaOMe (0.43 mL, 5 mol%) 6k was
H), 117.3 (C–H), 84.8 (C-10), 82.1 (C-50), 76.0 (C-30), 73.6 (C-40), isolated as a colorless solid (58 mg, 78%), mp ¼ 153–155 ꢁC.
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1H NMR (300 MHz, acetone-d6): d ¼ 7.56–7.53 (m, 1H, Ar), 7.28–
Antiproliferative activity by sulforhodamine B (SRB) assay
3
7.04 (m, 11H, Ar), 5.47 (d, J1 ,2 ¼ 9.2 Hz, 1H, H-10), 4.48–3.72
(m, 10H, H-20, H-30, H-40, H-50, H-60, 4 ꢃ OH), 2.29, 2.27 (2 ꢃ
Me). 13C NMR (75 MHz, acetone-d6): d ¼ 178.7 (C-2), 142.5,
142.3, 140.6, 138.6, 138.1, 134.8 (6 ꢃ CQu), 130.6 (2 ꢃ C–H),
130.5 (2 ꢃ C–H), 130.5 (2 ꢃ C–H), 130.2 (2 ꢃ C–H), 129.5 (C–H),
127.6 (C–H), 124.1 (C–H), 114.5 (C–H), 84.3 (C-10), 79.6 (C-20),
76.7 (C-30), 71.2 (C-40), 68.5 (C-50), 63.5 (C-3), 63.5 (C-60), 21.9 (2
ꢃ Me). MS (EI, 70 eV): m/z (%) ¼ 475 (M+, 15), 313 (100), 298 (12),
284 (17), 270 (18), 222 (16). HRMS (EI): calcd for C28H29NO6 [M]+
475.19894. Found 475.19925.
0
0
Antiproliferative activity was studied using the method reported
by Skehan et al.28 The cell monolayers were washed with Hank's
Balanced Salt Solution (HBSS) to remove non adherent cells. All
the three cell lines were incubated with a range of concentra-
tions of test compounds (1, 25, 50 and100 mM concentration) for
ꢁ
up to 24 h at 37 C in a 5% CO2 incubator. On completion of
incubation period, cells were xed with 50% ice cold TCA buffer
and placed for 1 h at 4 ꢁC. The excess TCA was removed by
washing plates at least 5 times with HBSS and then air dried.
Fixed cells were further treated with sulforhodamine B solution
(0.4%) and allowed to stain for 20–30 min. Aer staining cells
were rinsed with 1% acetic acid and plates were allowed to dry.
Plates were treated with 10 mM Tris for 5–10 min at room
temperature and their absorbance was recorded at 565 nm.
Blank background optical density was measured in wells incu-
bated with growth medium and without cells. Negative control
values were obtained from H157 incubated in RPMI medium,
HT-144 in MEM medium and HCEC in DMEM medium without
inhibitors. Vincristine and methotrexate was used as a positive
control. % growth inhibition of the compounds was calculated
3,3-Bis(4-methoxylphenyl)-1-(b-D-galactopyranosyl)indolin-
2-on (6l). Starting with 5l (100 mg, 0.15 mmol), dray methanol (4
mL) and 0.1% solution of NaOMe (0.41 mL, 5 mol%) 6l was
isolated as a colorless solid (59 mg, 79%), mp ¼ 147–149 ꢁC. 1H
NMR (300 MHz, acetone-d6): d ¼ 7.56–7.53 (m, 1H, Ar), 7.27–
7.15 (m, 11H, Ar), 7.09–7.04 (m, 11H, Ar), 6.88–6.78 (m, 11H, Ar),
5.47 (d, 3J1 ,2 ¼ 9.2 Hz, 1H, H-10), 4.48–4.40 (m, 1H, H-20), 4.33–
4.31 (m, 1H, H-30), 4.22–4.19 (m, 1H, H-40), 4.10–4.08 (m, 1H, H-
50), 3.93–3.72 (m, 12H, H-60, 3 ꢃ OH, 2 ꢃ OMe). 13C NMR (75
MHz, acetone-d6): d ¼ 179.0 (C-2), 160.8, 160.5 (2 ꢃ C-400), 142.4,
137.3, 135.4, 135.1 (4 ꢃ CQu), 131.6 (2 ꢃ C–H), 131.3 (2 ꢃ C–H),
129.5 (C–H), 127.6 (C–H), 124.1 (C–H), 114.5 (C–H), 84.3 (C-10),
79.6 (C-20), 76.7 (C-30), 71.2 (C-40), 68.5 (C-50), 63.5 (C-3), 62.8
(C-60), 56.5, 56.4 (2 ꢃ OMe). MS (EI, 70 eV): m/z (%) ¼ 507 (M+,
20), 344 (100), 316 (27), 302 (13), 238 (20), 210 (7). HRMS (EI):
calcd for C28H29NO8 [M]+ 507.18877. Found 507.18846.
0
0
using the formula: % growth inhibition ¼ 100 – [(ODsample
/
ODcontrol) ꢃ 100].
Acknowledgements
¨
Financial support by the BMBF “REMEDIS”-project “Hohere
¨
Lebensqualitat durch neuartige Mikroimplantate” (Grant no.
Antiproliferative activity
Cell lines and cell cultures
03IS2081A) and by the DAAD (Deutsch-Pakistanische Hoch-
schulzusammenarbeit) is gratefully acknowledged.
For the purpose of Antiproliferative activity, malignant
cutaneous melanoma cells HT-144 (ATCC HTB-63), Lung
carcinoma (H157) cell lines (ATCC CRL-5802) and human
corneal epithelial cells (HCEC) obtained from RIKEN Bio
Resource Center, Japan were used. These H157 were main-
tained in RPMI-1640 medium as described previously.27 The
medium was supplemented with heat-inactivated fetal
bovine serum (10%), L-glutamine (2 mM), pyruvate (1 mM),
penicillin (100 U mLꢀ1) and streptomycin (100 mg mLꢀ1). The
cultured was proceeded at 37 ꢁC in a 5% CO2 incubator,
horizontally in T75 cm2 sterile tissue culture asks. HT-144
(ATCC HTB-63) cells were cultured in minimal essential
medium (MEM) with 10% fetal calf serum, with the addition
of essential amino acids. HCEC were grown in DMEM
medium [containing 5% heat-inactivated fetal bovine serum,
insulin (5 mg mLꢀ1), epidermal growth factor human (10 ng
mLꢀ1) and DMSO (0.5%)]. Under these conditions, HCEC
exhibited corneal epithelial cell-specic properties. For
experiments, both cancerous cell lines (H157 and HT-144)
and normal cell line (HCEC) were grown in 96-well plates by
inoculating at a cell density of 104 and 5 ꢃ 104 cells per 100
mL per well respectively and these well plates were incubated
at 37 ꢁC in a 5% CO2 incubator in controlled humidity.
Conuent monolayers of all the three cell lines were formed
within 24 h, which were subsequently used for further
experiments.
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