Synthesis and antiproliferative activity of 7-azaindirubin-3′-oxime, a 7-aza isostere of the natural indirubin pharmacophore

Article abstract of DOI:10.1021/np9003905

The bis-indole alkaloid indirubin and its analogues bear a very interesting natural pharmacophore. They are recognized mainly as kinase inhibitors, but several other activities make them possible candidates for preclinical studies. Based on the previously reported activity of 7-bromoindirubin-3-oxime and its derivatives, the synthesis of indirubins bearing a heterocyclic nitrogen atom at position 7 was carried out. Herein, we report the first synthesis of 7-azaindirubin-3-oxime (12) as well as its antiproliferative activity against 57 cancer cell lines and its inhibitory activity against a series of kinases. 7-Azaindirubin (10) and its 3-oxime derivative (12) showed reduced activity as kinase inhibitors in comparison with other known indirubin derivatives, but antiproliferative activity with a best GI₅₀ value of 0.77 μM.

Full text of DOI:10.1021/np9003905

J. Nat. Prod. 2009, 72, 2199–2202
2199
Synthesis and Antiproliferative Activity of 7-Azaindirubin-3′-oxime, a 7-Aza Isostere of the
Natural Indirubin Pharmacophore
Marina Kritsanida,† Prokopios Magiatis,† Alexios-Leandros Skaltsounis,*,† Youyi Peng,‡ Peng Li,‡ and Lawrence P. Wennogle‡
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, UniVersity of Athens, Panepistimiopolis Zografou,
15771, Athens, Greece, and Intra-Cellular Therapies Inc., 3960 Broadway, New York, New York 10032
ReceiVed June 26, 2009
The bis-indole alkaloid indirubin and its analogues bear a very interesting natural pharmacophore. They are recognized
mainly as kinase inhibitors, but several other activities make them possible candidates for preclinical studies. Based on
the previously reported activity of 7-bromoindirubin-3′-oxime and its derivatives, the synthesis of indirubins bearing a
heterocyclic nitrogen atom at position 7 was carried out. Herein, we report the first synthesis of 7-azaindirubin-3′-
oxime (12) as well as its antiproliferative activity against 57 cancer cell lines and its inhibitory activity against a series
of kinases. 7-Azaindirubin (10) and its 3′-oxime derivative (12) showed reduced activity as kinase inhibitors in comparison
with other known indirubin derivatives, but antiproliferative activity with a best GI50 value of 0.77 µM.
The bis-indole alkaloid indirubin and its analogues (collectively
referred to as indirubins) were among the early cyclin-dependent
kinase (CDK) inhibitors to be discovered.1 The red-purple indirubin
(Figure 1) is an isomer of the blue indigo. Indirubins can be found
in various indigo dye-producing plants (more than 200 species).2
They are also present in the historic “Tyrean purple” dye extracted
from several Muricidae mollusks.3,4 Indirubins have also been found
in various wild-type and recombinant bacteria.5,6 Finally, indirubin
and indigo are occasionally present in human urine.7 Moreover,
indirubin is the active ingredient of a Traditional Chinese Medicine
recipe, “Danggui Longhui Wan”, used to treat various diseases
including chronic myelocytic leukemia.8 Besides CDKs, indirubins
were found to target glycogen synthase kinase-3 (GSK-3),9 aurora
kinases,10 and the aryl hydrocarbon receptor (AhR), also known
as the dioxin receptor.7,11 Experimental evidence suggests that the
antiproliferative effects of indirubins derive from their ability to
inhibit CDKs.12,13 However, interaction with the AhR contributes
to a marked arrest in the G1 phase of the cell cycle.14 Finally, it
was recently shown that some indirubins are able to prevent the
phosphorylation and subsequent activation of the transcription factor
STAT3, leading to a down-regulation of survival factors such as
survivin and Mcl-1 and leading to the subsequent induction of cell
death.15
Figure 1. Structure of natural indirubin.
respectively), 7-bromoindirubin potently induced a rapid nonapo-
ptotic, caspase-independent, cell death.18
On the basis of the activity of 7-bromoindirubin-3′-oxime and
its derivatives17 we envisaged the synthesis of new indirubins
bearing a heterocyclic nitrogen atom at position 7. Herein, we report
the first synthesis of 7-azaindirubin and its 3′-oxime as well as their
antiproliferative activity and their inhibitory activity against a series
of kinases. Recently, a series of N-substituted azaindirubins was
synthesized and tested for their cytotoxicity21 as well as a series
of other indirubin derivatives.22
The synthesis of the aza-heterocyclic skeleton of indirubins 10
and 11 was achieved through a dimerization reaction of the
appropriate 7-azaisatin (2) or N-Me-7-azaisatin (6) with 3-acetoxy-
indole (9) under conditions similar to those previously employed
in the synthesis of indirubins.16 The key nucleus of 7-azaisatin (2)
was synthesized by treatment of commercial 7-azaindole with NBS/
DMSO following a previously reported method for N-substituted
azaindoles.23 The yield of the above reaction was relatively low,
but it increased greatly in the case of 1-methyl-7-azaindole (4),
leading to 1-methyl-7-azaisatin (6) and subsequently to 1-methyl-
7-azaindirubin (11). In the same reaction, two byproducts with an
isoindigo skeleton (7 and 8) were obtained. The two synthesized
azaindirubins were transformed into the corresponding 3′-oximes,
12 and 13.
All test compounds showed IC50 > 300 nM against the four
kinases CDK1, CDK2, CDK5, and GSK3 beta (Table S1, Sup-
porting Information). The activity was significantly weaker than
that of indirubin-3′-oxime.3 Although molecular modeling studies
indicated that 7-azaindirubin and its 3′-oxime derivative show the
same placement into the ATP pocket of the kinases investigated
and the same number of hydrogen bonds as indirubin-3′-oxime
(Figure 2), they exhibited reduced activity as kinase inhibitors,
probably due to electrostatic reasons. In contrast, they showed
interesting antiproliferative activity, probably via a mechanism not
related to kinase inhibition.
During the past few years, we have isolated or synthesized more
than 150 indirubin derivatives.3,14,16-19 Among these derivatives,
the most effective is 6-bromoindirubin-3′-oxime, showing potent
inhibitory activity on GSK-3, CDK1, and CDK5 with IC50 values
of 0.005, 0.320, and 0.083 µM, respectively.3 This agent has been
used as a pharmacological tool in numerous studies in order to
investigate the physiological role of GSK-3 in various cellular
settings and control the fate of embryonic stem cells.20 Very recently
we developed a new series of highly potent, water-soluble deriva-
tives of 6-bromoindirubin-3′-oxime with activity on circadian
rhythms due to inhibition of GSK-3.19
Another group of indirubins is the 7-substituted family. While
synthesizing and testing the biological activity of new indirubins,
we discovered randomly that 7-bromoindirubin-3′-oxime displayed
potent cell-death-inducing properties. Despite weak or insignificant
inhibitory activity on various classical kinase targets of indirubins
(IC50 values for GSK-3, CDK1, and CDK5 32, 22, and 33 µM,
* To whom correspondence should be addressed. Tel: +30 210 727 4598.
Fax: +30 210 727 4594. E-mail: skaltsounis@pharm.uoa.gr.
† University of Athens.
Among the synthesized 7-azaindirubin derivatives, compound
12 gave 81% CDK5 inhibition at 3 µM and demonstrated slightly
better selectivity than 6-bromoindirubin-3′-oxime. Compound 13,
‡ Intra-Cellular Therapies, Inc.
10.1021/np9003905  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/08/2009

2200 Journal of Natural Products, 2009, Vol. 72, No. 12
Notes
Experimental Section
General Experimental Procedures. All chemicals were purchased
from Aldrich Chemical Co. Melting points were determined with a
Sanyo Gallenkamp apparatus. Infrared spectra (IR) were recorded on
a Perkin-Elmer Paragon 500 FT-IR spectrometer using multiple internal
reflectance (MIR) on a KRS-5 crystal at 45°. NMR spectra were
recorded on Bruker DRX 400 and Bruker AC 200 spectrometers [1H
(400 MHz) and 13C (50 MHz)]. Chemical shifts are expressed in ppm
downfield from TMS. CIMS spectra were determined on a Finnigan
GCQ Plus mass spectrometer using CH4 as the CI ionization reagent.
Column chromatography was performed using Merck flash silica gel
60 (40-63 µm), with an overpressure of 300 mbar. Elemental analysis
was performed using a Perkin-Elmer 2400 CHN elemental analyzer.
7-Azaindole was purchased from AcrosOrganics.
Preparation of 7-Azaisatin (2). To a solution of 7-azaindole (1)
(300 mg, 2.54 mmol) in anhydrous DMSO (21 mL) was added NBS
(905 mg, 5.08 mmol), and the resulting solution was stirred at 60 °C
for 6 h. Then, the mixture was stirred under vacuum in a water pump
(in order to remove the HBr produced) at 80 °C for 20 h. Water (17
mL) was then added, and the product was extracted with CH2Cl2. The
CH2Cl2 extract was washed with H2O, dried over anhydrous Na2SO4,
and carefully evaporated using a high vacuum pump (N2 trap, under
40 °C). The solid residue was submitted to flash chromatography with
CH2Cl2-MeOH (100:0 to 90:10) to afford 2 (38 mg, 0.26 mmol, 10%)
and 3 (90 mg, 0.32 mmol, 13%).
To a solution of 3 (16 mg, 0.058 mmol) in anhydrous DMSO (1.12
mL) was added NBS (10 mg, 0.058 mmol), and the mixture was stirred
at 80 °C for 1.5 h under vacuum in a water pump. After purification
following the procedure mentioned above, 2 was obtained quantitatively.
Storage of 2 under humidity and in the atmosphere caused degrada-
tion of the product.
Figure 2. Binding of 7-azaindirubin-3′-oxime (12) into the ATP
pocket of GSK3ꢀ.
Table
1. In
Vitro
Antiproliferative
Activity
of
7-Azaindirubin-3′-oxime (12) against 10 Sensitive Cancer Cell
LinesR
cell line
GI50 (µM)
KM12 (colon)
NCI-H226 (non-small cell lung)
RXF393 (renal)
NCI-H322 M (non-small cell lung)
UO-31 (renal)
IGROV1 (ovarian)
SK-OV-3 (ovarian)
RPMI (leukemia)
0.77
1.1
1.2
1.3
1.6
1.6
1.8
1.9
1.9
2.0
7-Azaisatin (2): yellowish, amorphous solid; 1H NMR (CDCl3, 400
MHz) δ 9.34 (1H, brs, NH), 8.42 (1H, dd, J ) 5.5, 1.5 Hz, H-6), 7.86
(1H, dd, J ) 7.5, 1.5 Hz, H-4), 7.10 (1H, dd, J ) 7.5, 5.5 Hz, H-5);
13C NMR (CDCl3, 50 MHz) δ 181.6 (C-3), 163.8 (C-7a), 158.1 (C-2),
155.3 (C-6), 133.8 (C-4), 119.7 (C-5), 112.2 (C-3a); CIMS m/z 149
(M + H)+; anal. C 56.76%, H 2.72%, N 18.91%, calcd for C7H4N2O2,
C 56.71%, H 2.73%, N 18.85%.
2,3-Dibromo-3H-pyrrole-[2,3-b]pyridine (3): orange-red, amor-
phous solid; 1H NMR (CDCl3, 400 MHz) δ 8.23 (1H, dd, J ) 5.3, 1.4
Hz, H-6), 7.85 (1H, dd, J ) 7.6, 1.4 Hz, H-4), 7.13 (1H, dd, J ) 7.6,
5.3 Hz, H-5), 2.70 (1H, s, H-3); 13C NMR (CDCl3, 50 MHz) δ 151.6
(C-7a), 148.9 (C-6), 133.9 (C-4), 126.3 (C-3a), 118.9 (C-5), 44.7 (C-
2), 42.9 (C-3); CIMS m/z 276, 278, 280 (M + H)+; anal. C 30.47%,
H 1.46%, N 10.15%, calcd for C7H4Br2N2, C 30.41%, H 1.46%, N
10.13%.
Preparation of Isatins 6 and 9. In order to prepare the 1-methylated
derivatives of 7-azaisatin, 1-methylation of 7-azaindole was carried out
first. To a solution of 1 (350 mg, 2.96 mmol) in anhydrous DMF (10
mL) was added NaH (100 mg, 4.35 mmol), under N2, and the mixture
was stirred for 30 min at room temperature. A solution of CH3I (218
µL, 3.50 mmol) in anhydrous DMF (2 mL) was added slowly, and
stirring was continued for 12 h at room temperature. Water (20 mL)
was added, and the product was extracted with CH2Cl2. The CH2Cl2
extract was washed with H2O, dried over anhydrous Na2SO4, and
carefully evaporated using a high vacuum pump (N2 trap, under 40
°C). The solid residue was submitted to flash chromatography with
CH2Cl2 to give the yellowish oil 4 (377 mg, 2.86 mmol, 96%). Product
4 showed identical spectroscopic data with those described in the
literature.23
Isatin 6 (74 mg, 0.46 mmol, 50%) was prepared by a procedure
analogous to that of isatin 2, and the spectroscopic data coincided with
those described in the literature.23 Purification by flash chromatography
with CH2Cl2-MeOH (100:0 to 98:2) afforded also compounds 5 (15
mg, 0.052 mmol, 6%, spectroscopic data identical with those described
in the literature),23 7 (12 mg, 0.041 mmol, 4.5%), and 8 (0.5 mg, 0.0013
mmol, 0.15%).
A-498 (renal)
T-47D (breast)
R Tested by the Developmental Therapeutics Program, National
Cancer Institute, Frederick, MD.
the N-1-methylated derivative of 12, did not show any CDK
inhibitory activity at the test concentrations used. According to our
previous study, the N1-H group in 6-bromoindirubin-3′-oxime forms
a hydrogen bond with the backbone carbonyl group of Glu81 in
CDK5.3 Capping the N1-H with a methyl group abolished this key
H-bond and consequently diminished compound inhibitory activity.
7-Azaindirubin (10), 7-azaindirubin-3′-oxime (12), and diaza-
isoindigo (7) were submitted for in vitro biological testing against
the 57 cancer cell line screen provided by the U.S. National Cancer
Institute, Developmental Therapeutics Program. The results obtained
at a concentration of 10 µM are given in Tables S2-S4, Supporting
Information.
7-Azaindirubin-3′-oxime (12) presented the most potent anti-
proliferative activity and was further evaluated for growth inhibition
(GI50). The GI50 values (Table 1; Table S5, Supporting Information)
ranged from 100 to 0.77 µM, showing the most potent activity
against a colon cancer cell line (KM12). Other cell lines showing
GI50 values of less than 2 µM included three renal, two ovarian,
and two non-small cell lung cancer cell lines. The fact that
compound 10 had a similar antiproliferative profile (Table S2,
Supporting Information), yet was weakly active versus CDK1, -2,
and -5 and GSK3, suggests that inhibition of these kinases is not
responsible for the observed antiproliferative activity of these
compounds. The mechanism of action of indirubins with reduced
kinase inhibitory activity like 10 and 12 may be attributed to their
activity on the AhR receptor.14 However, it is clear that the
inhibition of the commonly screened kinases (CDK1, CDK2,
CDK5, GSK3) is not enough to explain the antiproliferative activity
of indirubins.
(3E)-7,7′-Diaza-1,1′-dimethylisoindigo (7): orange-red crystals; mp
278 °C; 1H NMR (DMSO, 400 MHz) δ 9.39 (2H, dd, J ) 8.0, 1.2 Hz,
H-4,4′), 8.31 (2H, dd, J ) 5.1, 1.2 Hz,, H-6, 6′), 7.15 (1H, dd, J ) 8.0,
5.1 Hz, H-5, 5′), 3.32 (6H, s, N-CH3, N′-CH3); 13C NMR (DMSO, 50
MHz) δ 167.7 (C-2, 2′), 157.7 (C-7a, 7a′), 150.2 (C-6, 6′), 137.5 (C-4,
4′), 132.2 (C-3, 3′), 118.6 (C-5, 5′), 116.1 (C-3a, 3a′), 25.4 (2 × CH3);

Notes
Journal of Natural Products, 2009, Vol. 72, No. 12 2201
Scheme 1. Oxidation of 7-Azaindole Reagentsa
a Reagents and conditions: (i) NBS (2 equiv), anh. DMSO, H2O; (ii) NBS (1 equiv), anh. DMSO, H2O; (iii) NaH, CH3I.
Scheme 2. Synthesis of 7-Azaindirubins and Their Corresponding 3′-Oxime Derivativesa
a Reagents and conditions: (i) Na2CO3, MeOH, 25 °C; (ii) H2NOH · HCl, pyr, 120 °C.
CIMS m/z 293 (M + H)+; anal. C 65.75%, H 4.14%, N 19.17%, calcd
for C16H12N4O2, C 65.62%, H 4.15%, N 19.21%.
CIMS m/z 281 (M + H)+; anal. C 69.31%, H 4.00%, N 15.15%, calcd
for C16H11N3O2, C 69.37%, H 4.00%, N 15.17%.
(3E)-5-Bromo-7,7′-diaza-1,1′-dimethylisoindigo (8): orange-red,
amorphous solid; 1H NMR (CDCl3, 400 MHz) δ 9.67 (1H, d, J ) 2.4
Hz, H-4), 9.46 (1H, dd, J ) 8.0, 1.4 Hz, H-4′), 8.31 (1H, d, J ) 2.4
Hz, H-6), 8.26 (1H, dd, J ) 5.1, 1.4 Hz, H-6′), 7.03 (1H, dd, J ) 8.0,
5.1 Hz, H-5′), 3.38 (3H, s, N-CH3), 3.35 (3H, s, N′-CH3); CIMS m/z
371, 373 (M + H)+; anal. C 51.77%, H 2.99%, N 15.09%, calcd for
C16H11BrN4O2, C 51.82%, H 3.01%, N 15.06%.
Preparation of Indirubins 10 and 11. Methanol (10 mL) was stirred
vigorously under N2 for 20 min, and then 7-azaisatin (2) (50 mg, 0.34
mmol) and 3-acetoxyindole (42 mg, 0.24 mmol) were added; stirring
was continued for 5 min. Anhydrous Na2CO3 (76 mg) was added, and
the stirring was continued for 3 h. The dark precipitate was filtered
and washed with aqueous methanol (1:1, 20 mL). Purification was
achieved by flash chromatography with EtOAc-THF (95:5) to afford
10 (146 mg, 0.17 mmol, 73%).
Preparation of Oximes 12 and 13. Indirubin derivative 10 (26 mg,
0.099 mmol) was dissolved in pyridine (5 mL). With magnetic stirring,
hydroxylamine hydrochloride (70 mg, 1 mmol) was added and the
mixture was heated under N2 and refluxed (120 °C) for 24 h. Water
(20 mL) was added, and the red precipitate obtained was filtered and
washed successively with H2O and cyclohexane. Purification was
achieved by flash chromatography with CH2Cl2-THF (95:5) to afford
12 (26 mg, 0.09 mmol, 95%).
Oxime 13 was prepared by a procedure analogous to that used for
oxime 12, but no further purification was necessary. After washing
extensively with H2O and cyclohexane, 13 was obtained quantitatively.
(2′Z,3′E)-7-Azaindirubin-3′-oxime (12): red crystals; mp >300 °C;
IR 3090 (br), 1673, 1615, 1560, 1460, 1442, 1337, 1315, 1231, 1185
1
cm-1; H NMR (DMSO, 400 MHz) δ 13.64 (1H, brs, N-OH), 11.71
(1H, s, N′-H), 11.15 (1H, s, N-H), 8.78 (1H, dd, J ) 6.3, 1.0 Hz, H-4),
8.23 (1H, d, J ) 7.5 Hz, H-4′), 7.97 (1H, d, J ) 6.3 Hz, H-6), 7.44
(1H, d, J ) 7.5 Hz, H-7′), 7.38 (1H, t, J ) 7.5 Hz, H-6′), 7.05 (1H, t,
J ) 7.5 Hz, H-5′), 6.94 (1H, t, J ) 6.3 Hz, H-5); 13C NMR (DMSO,
50 MHz) δ 169.9 (C-2), 152.0 (C-7a), 150.8 (C-3′), 146.0 (C-2′), 144.4
(C-7a′), 143.4 (C-6), 131.7 (C-6′), 128.4 (C-4′), 127.3 (C-4), 121.5
(C-5′), 116.7 (C-3a), 116.1 (C-5), 115.9 (C-3a′), 111.5 (C-7′), 95.9 (C-
3); CIMS m/z 279 (M + H)+; anal. C 64.74%, H 3.62%, N 20.13%,
calcd for C15H10N4O2, C 64.53%, H 3.63%, N 20.19%.
Indirubin 11 was prepared by a procedure analogous to that of 10,
but no further purification was necessary. After washing with aqueous
methanol (1:1, 20 mL) and extensively with water, 11 was obtained
quantitatively.
(2′Z)-7-Azaindirubin (10): purple crystals; mp >300 °C; IR 3355
1
(br), 1670, 1615, 1595, 1460, 1306, 1210 cm-1; H NMR (DMSO,
400 MHz) δ 11.43 (1H, s, N-H), 11.09 (1H, s, N′-H), 8.91 (1H, dd, J
) 7.5, 1.0 Hz, H-4), 8.11 (1H, dd, J ) 5.1, 1.0 Hz, H-6), 7.67 (1H, dd,
J ) 7.5, 1.0 Hz, H-4′), 7.60 (1H, t, J ) 7.8 Hz, H-6′), 7.43 (1H, d, J
) 7.8, Hz, H-7′), 7.05 (2H, overlapped, H-5, 5′); 13C NMR (DMSO,
50 MHz) δ 188.4 (C-3′), 169.9 (C-2), 154.4 (C-7a), 152.3 (C-7a′), 146.3
(C-6), 138.8 (C-2′), 136.9 (C-6′), 130.6 (C-4), 124.1 (C-4′), 121.3 (C-
5′), 118.4 (C-3a′), 117.1 (C-5), 115.5 (C-3a), 113.2 (C-7′), 103.4 (C-
3); CIMS m/z 264 (M + H)+; anal. C 68.44%, H 3.45%, N 15.96%,
calcd for C15H9N3O2, C 68.52%, H 3.45%, N 15.91%.
(2′Z)-7-Aza-1-methylindirubin (11): purple crystals; mp 235 °C;
IR 3320 (br), 1655, 1618, 1590, 1475, 1449, 1335, 1110 cm-1; 1H NMR
(DMSO, 400 MHz) δ 11.13 (1H, s, N′-H), 8.92 (1H, dd, J ) 7.5, 1.4
Hz, H-4), 8.19 (1H, dd, J ) 5.1, 1.4 Hz, H-6), 7.67 (1H, d, J ) 7.7
Hz, H-4′), 7.61 (1H, t, J ) 7.7 Hz, H-6′), 7.43 (1H, d, J ) 7.7, Hz,
H-7′), 7.12 (1H, dd, J ) 7.5, 5.1 Hz, H-5), 7.05 (1H, t, J ) 7.7 Hz,
H-5′) 3.31 (3H, s, N-CH3); 13C NMR (DMSO, 50 MHz) δ 188.7 (C-
3′), 168.7 (C-2), 154.4 (C-7a), 152.7 (C-7a′), 146.5 (C-6), 139.5 (C-
2′), 137.5 (C-6′), 130.9 (C-4), 124.6 (C-4′), 121.8 (C-5′), 119.0 (C-
3a′), 118.0 (C-5), 115.6 (C-3a), 113.7 (C-7′), 102.7 (C-3), 24.9 (CH3);
(2′Z,3′E)-7-Aza-1-methylindirubin-3′-oxime (13): red crystals; mp
295 °C; IR 3183 (br), 1655, 1611, 1564, 1450, 1345, 1245, 1136 cm-1
;
1H NMR (DMSO, 400 MHz) δ 13.68 (1H, brs, N-OH), 11.67 (1H, s,
N′-H), 8.81 (1H, dd, J ) 7.5, 1.0 Hz, H-4), 8.22 (1H, d, J ) 7.8 Hz,
H-4′), 8.09 (1H, dd, J ) 5.1, 1.0 Hz, H-6), 7.46 (1H, d, J ) 7.8 Hz,
H-7′), 7.42 (1H, t, J ) 7.8 Hz, H-6′), 7.07 (1H, t, J ) 7.8 Hz, H-5′),
7.02 (1H, dd, J ) 7.5, 5.1 Hz, H-5), 3.31 (3H, s, N-CH3); 13C NMR
(DMSO, 50 MHz) δ 168.7 (C-2), 151.9 (C-7a), 151.3 (C-3′), 147.1
(C-2′), 144.6 (C-7a′), 143.4 (C-6), 132.0 (C-6′), 128.7 (C-4′), 127.5
(C-4), 122.2 (C-5′), 117.1 (C-3a), 116.9 (C-5), 116.4 (C-3a′), 112.0
(C-7′), 94.3 (C-3), 24.8 (CH3); CIMS m/z 293 (M + H)+; anal. C
65.75%, H 4.14%, N 19.17%, calcd for C16H12N4O2, C 65.72%, H
4.15%, N 19.23%.
Kinase Inhibition Assays. The kinase inhibition assays were
performed using γ-33P-ATP as the radioligand. The targeted kinases
were human GSK3ꢀ, CDK1, CDK2, and CDK5. The concentration of

2202 Journal of Natural Products, 2009, Vol. 72, No. 12
Notes
(6) Wu, Z. L.; Aryal, P.; Lozach, O.; Meijer, L.; Guengerich, F. P. Chem.
BiodiVersity 2005, 2, 51–65.
ATP used in the assays was within 15 µM of the apparent Km for ATP
on the corresponding kinase. All experiments were performed as
duplicates.
(7) Adachi, J.; Mori, Y.; Matsui, S.; Takigami, H.; Fujino, J.; Kitagawa,
H.; Miller, C. A., 3rd; Kato, T.; Saeki, K.; Matsuda, T. J. Biol. Chem.
2001, 276, 31475–31478.
GSK3ꢀ Assay. In a final reaction volume of 25 µL, GSK3R (h)
(5-10 mU) was incubated with 8 mM 3-(N-morpholino)propane-
sulfonic acid (MOPS) at pH 7.0, 0.2 mM EDTA, 20 µM YR-
RAAVPPSPSLSRHSSPHQS(p)EDEEE (phospho GS2 peptide), 10
mM Mg acetate, and [γ-33P-ATP] (specific activity, ca. 500 cpm/pmol,
concentration as required). The reaction was initiated by the addition
of the MgATP mix. After incubation for 40 min at room temperature,
the reaction was stopped by the addition of 5 µL of a 3% phosphoric
acid solution. Then 10 µL of the reaction was spotted onto a P30
filtermat and washed three times for 5 min in 50 mM phosphoric acid
and once in methanol prior to drying and scintillation counting.
CDK1, CDK2, and CDK5 Assays. In a final reaction volume of
25 µL, CDK1/cyclinB (h) (5-10 mU), CDK2/cyclinA (h) (5-10 mU),
or CDK5/p35 (h) (5-10 mU) was incubated with 8 mM MOPS pH
7.0, 0.2 mM EDTA, 0.1 mg/mL histone H1, 10 mM Mg acetate, and
[γ-33P-ATP] (specific activity, ca. 500 cpm/pmol, concentration as
required). The reaction was initiated by the addition of the MgATP
mixture. After incubation for 40 min at room temperature, the reaction
was stopped by the addition of 5 µL of a 3% phosphoric acid solution.
Then 10 µL of the reaction was spotted onto a P30 filtermat and washed
three times for 5 min in 75 mM phosphoric acid and once in methanol
prior to drying and scintillation counting.
(8) Xiao, Z.; Hao, Y.; Liu, B.; Qian, L. Leuk. Lymphoma 2002, 43, 1763–
1768.
(9) Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder,
G. L.; Greengard, P.; Biernat, J.; Mandelkow, E. M.; Eisenbrand, G.;
Meijer, L. J. Biol. Chem. 2001, 276, 251–260.
(10) Myrianthopoulos, V.; Magiatis, P.; Ferandin, Y.; Skaltsounis, A. L.;
Meijer, L.; Mikros, E. J. Med. Chem. 2007, 50, 4027–4037.
(11) Kawanishi, M.; Sakamoto, M.; Ito, A.; Kishi, K.; Yagi, T. Mutat. Res.
2003, 540, 99–105.
(12) Marko, D.; Scha¨tzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer,
L.; Eisenbrand, G. Br. J. Cancer 2001, 84, 283–289.
(13) Damiens, E.; Baratte, B.; Marie, D.; Eisenbrand, G.; Meijer, L.
Oncogene 2001, 20, 3786–3797.
(14) Knockaert, M.; Blondel, M.; Bach, S.; Leost, M.; Elbi, C.; Hager, G.;
Naggy, S. R.; Han, D.; Denison, M.; Ffrench, M.; Ryan, X. P.;
Magiatis, P.; Polychronopoulos, P.; Greengard, P.; Skaltsounis, L.;
Meijer, L. Oncogene 2004, 23, 4400–4412.
(15) Nam, S.; Buettner, R.; Turkson, J.; Kim, D.; Cheng, J. Q.; Muehlbeyer,
S.; Hippe, F.; Vatter, S.; Merz, K. H.; Eisenbrand, G.; Jove, R. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 5998–6003.
(16) Polychronopoulos, P.; Magiatis, P.; Skaltsounis, A. L.; Myriantho-
poulos, V.; Mikros, E.; Tarricone, A.; Musacchio, A.; Roe, S. M.;
Pearl, L.; Leost, M.; Greengard, P.; Meijer, L. J. Med. Chem. 2004,
47, 935–946.
(17) Ferandin, Y.; Bettayeb, K.; Kritsanida, M.; Lozach, O.; Polychro-
nopoulos, P.; Magiatis, P.; Skaltsounis, A. L.; Meijer, L. J. Med. Chem.
2006, 49, 4638–4649.
Antiproliferative Activity Test. The Developmental Therapeutics
Program-National Cancer Institute screening procedure is described
in detail elsewhere.24,25
(18) Ribas, J.; Bettayeb, K.; Ferandin, Y.; Garrofe´-Ochoa, X.; Knockaert,
M.; Totzke, F.; Scha¨chtele, C.; Mester, J.; Polychronopoulos, P.;
Magiatis, P.; Skaltsounis, A. L.; Boix, J.; Meijer, L. Oncogene 2006,
25, 6304–6318.
(19) Vougogiannopoulou, K.; Ferandin, Y.; Bettayeb, K.; Myrianthopoulos,
V.; Lozach, O.; Fan, Y.; Johnson, C. H.; Magiatis, P.; Skaltsounis,
A.-L.; Mikros, E.; Meijer, L. J. Med. Chem. 2008, 51, 6421–6431.
(20) Sato, N.; Meijer, L.; Skaltsounis, A. L.; Greengard, P.; Brivanlou, A.
Nat. Med. 2004, 10, 55–63.
(21) Wang, Z. H.; Li, W. Y.; Li, F. L.; Zhang, L.; Hua, W. Y.; Cheng,
J. C.; Yao, Q. Z. Chin. Chem. Lett. 2009, 20, 542–544.
(22) Kim, S.-H.; Choi, S. J.; Kim, Y.-C.; Kuh, H.-J. Arch. Pharm. Res.
2009, 32, 915–922.
Supporting Information Available: Details about biological activity
of tested compounds as well as NMR spectra of the new synthesized
indirubins (10-13) are available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
(1) Hoessel, R.; Leclerc, S.; Endicott, J.; Noble, M.; Lawrie, A.; Tunnah,
P.; Leost, M.; Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.;
Tang, W.; Eisenbrand, G.; Meijer, L. Nat. Cell Biol. 1999, 1, 60–67.
(2) Balfour-Paul, J. Indigo; British Museum Press: London, 1998.
(3) Meijer, L.; Skaltsounis, A. L.; Magiatis, P.; Polychronopoulos, P.;
Knockaert, M.; Leost, M.; Ryan, X. P.; Vonica, C. D.; Brivanlou, A.;
Dajani, R.; Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.;
Greengard, P. Chem. Biol. 2003, 10, 1255–1266.
(23) Tatsugi, J.; Zwiwei, T.; Izawa, Y. ArkiVok 2001, 67–73.
(24) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski,
M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.;
Boyd, M. R. Cancer Res. 1988, 48, 589–601.
(4) Cooksey, C. J. Molecules 2001, 6, 736–769.
(5) Guengerich, F. P.; Sorrells, J. L.; Schmitt, S.; Krauser, J. A.; Aryal,
(25) Boyd, M. R.; Paull, K. D. Drug DeV. Res. 1995, 34, 91–109.
P.; Meijer, L. J. Med. Chem. 2004, 47, 3236–3241.
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