Novel synthesis of 4-or 6-substituted indirubin derivatives

Article abstract of DOI:10.1080/00397910903318591

A simple and convenient route for synthesis of a series of 4-or 6-substituted indirubin derivatives by oxidation and subsequent condensation of indoxyl and isatin is described. Acidic reaction conditions are crucial to the condensation of 4-substituted derivatives, whereas for the condensation of 6-substituted derivatives, both acidic and basic conditions work well. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910903318591

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Novel Synthesis of 4- or 6-Substituted
Indirubin Derivatives
Aiying Zhang a , Mingfeng Yu a , Tian Lan a , Zenglu Liu a & Zhenmin
Mao a
a School of Pharmacy, Shanghai Jiaotong University , Shanghai, China
Published online: 04 Oct 2010.
To cite this article: Aiying Zhang , Mingfeng Yu , Tian Lan , Zenglu Liu & Zhenmin Mao (2010)
Novel Synthesis of 4- or 6-Substituted Indirubin Derivatives, Synthetic Communications: An
International Journal for Rapid Communication of Synthetic Organic Chemistry, 40:21, 3125-3134,
DOI: 10.1080/00397910903318591
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Synthetic Communications1, 40: 3125–3134, 2010
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910903318591
NOVEL SYNTHESIS OF 4- OR 6-SUBSTITUTED
INDIRUBIN DERIVATIVES
Aiying Zhang, Mingfeng Yu, Tian Lan, Zenglu Liu, and
Zhenmin Mao
School of Pharmacy, Shanghai Jiaotong University, Shanghai, China
A simple and convenient route for synthesis of a series of 4- or 6-substituted indirubin
derivatives by oxidation and subsequent condensation of indoxyl and isatin is described. Acidic
reaction conditions are crucial to the condensation of 4-substituted derivatives, whereas for the
condensation of 6-substituted derivatives, both acidic and basic conditions work well.
Keywords: Acid catalyzed; indirubin; steric hindrance; 4-substituted
INTRODUCTION
Dang gui long hui wan, a traditional Chinese medicine composed of 11 herbs,
has been used to treat chronic myelocytic leukemia (CML).[1] Indirubin (Fig. 1) is the
active ingredient of qingdai, one active constituent of dang gui long hui wan.[2]
Indirubin derivatives exhibit high activity against a broad spectrum of
experimental solid tumors.[3–7] For instance, E804 (Fig. 1) can directly inhibit Src
kinase activity in human cancer cells by preventing the phosphorylation of the
transcription factor Stat3 and down-regulating the expression of the antiapoptotic
proteins Mc1–1 and survivin.[4] 5-Substitued indirubin derivatives, designed and
tested by Kim et al., display excellent activity against various human cancer cells
(A549, SNU-638, and HT-1080) and oncogenic RK3E-ras rat kidney cells, with
IC50 values ranging from 1 to 12 mM.[5] Current evidence indicates that indirubin
derivatives inhibit cyclin-dependent kinases (CDKs).[8–11] CDKs play important
roles in several diseases, including cancers. Mechanism studies reveal that the activity
of indirubin derivatives against CDKs results in cell cycle arrest in the G2=M
phase.[12] Indirubin derivatives can also act as glycogen synthase kinases-3
(GSK-3) inhibitors.[13–15] GSK-3 is critical in cellular signal transduction pathway.
The 6-bromo- and the 5, 6-disubstitued indirubin-30-oximes show high activity
against GSK-3, with efficacy in nanomolar IC50 values.[9]
Although several substituent groups have been introduced in different
positions of indirubin, 4-substituted derivatives rarely have been achieved because
of the steric effect in their synthesis. So far, in the coupling step of the two bicyclic
Received March 30, 2009.
Address correspondence to Zhenmin Mao, School of Pharmacy, Shanghai Jiaotong University,
Shanghai, China. E-mail: pharmacy_411@yahoo.com.cn
3125

3126
A. ZHANG ET AL.
Figure 1. The structures of indirubin and E804.
rings, only 4-chloroindirubin has been reported with poor yield (5%).[9] A novel
method for synthesis of the 4-substitued compounds is explored and discussed in this
article. The same method also worked well for the synthesis of 6-substituted
indirubin derivatives.
RESULTS AND DISCUSSION
Indirubin, which has no substitution on its phenyl rings, can be synthesized by
chemosynthesis from coupling between acetoxyindole and isatin with sodium
carbonate as base[9,16,17] and biosynthesis from oxidation of indole by P450.[18,19]
Indirubins analogs can also be obtained from acidic conditions such as those that
use HCl and HOAc.[20,21]
Similarly, 6-substitued indirubins could be obtained by reaction of 6-substitued
isatins and 3-acetoxyindoles using sodium carbonate as reported.[9] However, with
the same reaction condition, 4-substitued indirubins cannot be synthesized, except
4-chloroindirubin, which is obtained in very poor yield (5%).[9] The steric hindrance
between the 4-substitution group in isatin and the 3-carbonyl group in acetoxyindole
during the coupling may cause the difficulty.
A modified reaction condition was explored to overcome this disadvantage.
Instead of sodium carbonate, tosic acid[22] was used in the coupling reaction, and
the targeted 4-substitued indirubins were able to be synthesized.
The key isatin intermediates were prepared through a two-step procedure.
Commercially available 3-substituted anilines (3a–e) as starting materials were
reacted with chloral hydrate and hydroxylamine hydrochloride to give the corre-
sponding isonitrosoacetanilides (4a–e). The isonitrosoacetanilides formed were then
heated in sulfuric acid to give a mixture of the cyclized compounds (5a–e and 6b–d)
in which isatins (5a–e) have substituents in the 4-position and the isatins (6a–d) have
substituents in the 6-position. These isomers were used for their corresponding 4- or
6-substituted final products (Scheme 1).
There was one exception occurred in the cyclization of isonitrosoacetanilide 4e,
which has a nitro substitution group. Because of the strong election-withdrawing
effect by the substitution of the nitro group, the isonitrosoacetanilide 4e was not
cyclized in aqueous sulfuric acid. A stronger reaction condition must be used to effect
the cyclization. Several reaction conditions were explored. Finally, the cyclization
occurred with concentrate sulfuric acid at 50 ꢀC over a prolonged reaction period

SYNTHESIS OF 4- OR 6-SUBSTITUTED INDIRUBIN DERIVATIVES
3127
Scheme 1. Preparation of indirubin derivatives.
of 20 h, with a brief temperature rise to 80 ꢀC for 10 min. The 4-nitroisatin (5e) was
obtained in a poor yield of 26%. There was no 6-nitroisatin observed.
Separation of 4- and 6-substituted isatin isomers formed during their
cyclizations was achieved depending upon their substituents. Chloroisomers (5b/
6b) and bromoisomers (5c/6c) can be separated by recrystallization, while the
fluroisomers (5a/6a) and the trifluromethylisomers (5d/6d) can be separated by
column chromatography.
The 4-substituted isatins did not react with acetoxyindole to form the desired
4-substituted indirubins under basic conditions, the most common coupling
condition. This was reasonable because of the steric effect between the 4-substitution
group in isatins and 3-carbonyl group in acetoxyindole. Under acidic conditions,
the 4-substituted isatins were able to react with acetoxyindole, and the desired
4-substituted indirubins were obtained in relatively poor yields. The possible
explanation was that during the coupling, proton from the acid may coordinate
the two oxygen atoms from the isatin and the 3-carbonyl group of the acetoxyindole
through hydrogen bonding, which could lead to the 4-substituted indirubin
(Scheme 2). In the basic reaction conditions, the coordination by the hydrogen bond
would not exist. The steric effect, however, still played a role to the poor reaction
yields of the 4-substituted indirubins.

3128
A. ZHANG ET AL.
Scheme 2. Reaction mechanism under acidic condition.
The 6-substitued indirubins were obtained both in acidic and basic reaction
conditions using the corresponding 6-substituted isatins. There was no steric effect
in this coupling, and very good reaction yields were obtained.
The coupling usually takes place at the 2, 3 sites to form indirubin, not at the 2,
2 sites, which leads to indigo.[9,16,17] It has been reported that the maximum absorp-
tion wavelengths (kmax) of most indirubins are less than 600 nm except those of 6, 60-
dinitroindirubin (kmax 624 nm), due to strong election-withdrawing effects by two
nitro groups, whereas the maximum absorption wavelengths (kmax) of indigos are
generally more than 600 nm.[18] The maximum absorption wavelength data of our
products are less than 600 nm (Table 1), which confirms that the products are
indirubins, not indigos.
It was reported the Z configuration resulted from the stable six-member ring
formed from the hydrogen bond between 10-imido-H and the 2-carbonyl-O, which
was the only configuration found in indirubins (Scheme 3).[9]
The 6-substituted oximes (20Z, 30E) were prepared from the classical reaction
between the corresponding indirubins and hydroxylamine hydrochloride.[9,16] How-
ever, the 4-substituted indirubins did not react with hydroxylamine hydrochloride to
form their oxime derivatives because of the steric hindrance (Scheme 4).
Table 1. Maximum absorption wavelength data of products
Product
kmax (DMSO)
7a
7b
7c
7d
7e
546 nm
555 nm
554 nm
565 nm
580 nm

SYNTHESIS OF 4- OR 6-SUBSTITUTED INDIRUBIN DERIVATIVES
3129
Scheme 3. Z configuration of indirubins caused by hydrogen band.
Scheme 4. Preparation of indirubin-30-oxime derivatives.
EXPERIMENTAL
Melting points were uncorrected. 1H NMR and 13C NMR spectra were
obtained using a Varian 300-MHz spectrophotometer in dimethylsulfoxide
(DMSO-d6) solutions, and chemical shift values are expressed in d values (ppm) rela-
tive to tetramethylsilane (TMS) as internal standard. Mass spectra (MS) were carried
out on a MicroMass GCT. Reaction progress was monitored by analytical thin-layer
chromatography (TLC) on precoated glass plates (silica gel 60 F254 plate), and the
products were visualized by ultraviolet (UV) light. The structures of novel com-
1
pounds were identified by infrared (IR), MS, elemental analysis, H NMR, and 13C
NMR spectra, except for 8d, which was identified without 13C NMR because of its
poor solubility. Other structures of known compounds were identified by 1H NMR.
General Procedure for Synthesis of Isatins (5a–e, 6a–d)
Chloral hydrate (3.73 g, 22.6 mmol) and Na2SO4(25.52 g, 179.2 mmol) were
dissolved in water (50 mL) in a 250-mL beaker and warmed to 35 ꢀC. A warm mixed

3130
A. ZHANG ET AL.
solution of the appropriate commercial 3-fluoroanilines 3a (20.4 mmol) in water
(15 mL) and an aqueous solution of concentrated HCl (2 mL) were added, followed
by a warm solution of hydroxylamine hydrochloride (4.48 g, 69.5 mmol) in water
(20 mL). The mixture was stirred and heated at 80–90 ꢀC for 2 h and then allowed
to cool for 1 h, by which time the temperature had fallen to 50 ꢀC; it was then filtered.
The pale cream product was washed by stirring with water and filtered to give
3-fluoroisonitrosoacetanilide (4a) with 83% yield; compounds 4b–e were prepared
in the same way with yields of 85%, 92%, 80%, and 88% respectively.
Sulfuric acid (86%, 5 mL) was heated in a 10-mL beaker to 60 ꢀC. The dry
3-fluoro-isonitrosoacetanilide (4a) (5 mmol) was added in portions with stirring over
30 min at a controlled temperature of less than 65 ꢀC. The mixture was then heated to
85 ꢀC for 15 min and cooled in an ice batch for 1 h. The solution was poured onto
water (20 mL) and left to stand for 1 h before filtering the orange-red precipitate.
The product was washed by water (50 mL) and filtered to give a mixture of 5a
and 6a.
The filtrate was extracted with acetic ether. The drying acetic ether layer
(MgSO4) and the precipitate were purified by chromatography on silica gel with
cyclohexane–acetic ether (5:1) to give 5a and 6a, with yields of 6% and 88%
respectively.
Compounds 5d and 6d were prepared in the same method with yields of 26%
and 11%, respectively.
The chloroisatins (5b and 6b) were prepared with the same method as the fluor-
oisatins, but the method of separation was somewhat different. The crude product
was dissolved in a solution of NaOH (25 g) in water (150 mL) at 60 ꢀC and then acidi-
fied with acetic acid (75 mL). After standing for 30 min and cooling in an ice bath for
30 min, the precipitate was filtered and washed with water (50 mL) to give 5b with a
yield of 53%. The combined filtrate and washing were acidified with an aqueous
solution of concentrated HCl (80 mL) in an ice batch for 2 h. The precipitate was
filtered off and washed with water (50 mL) to give 6b with a yield of 32%.
The bromoisatins (5c and 6c) were prepared with the same method as chloroi-
satins with yields of 41% and 28% respectively.
Synthesis of 4-Nitroisatins (5e)
The dry 3-nitroisonitrosoacetanilide (4e) (5 mmol) was added to concentrated
sulfuric acid (10 mL) in portions with stirring for 20 h at 50 ꢀC. The mixture was then
heated to 80 ꢀC for 10 min and cooled in an ice batch for 1 h. The solution was
poured into water (50 mL) and extracted with acetic ether. After drying the acetic
ether layer (MgSO4) and removing the solvent under reduced pressure, the residue
was purified by chromatography on silica gel with cyclohexane–acetic ether (5:1)
to give 5e (yield 26%).
General Procedure for Synthesis of Indirubins (7a–e, 8a–d)
4-Fluoroisatin (5a) (1 mmol) and 3-acetoxyindole (1 mmol) were dissolved in
alcohol (15 mL) in a 50-mL beaker at 70 ꢀC. The solution was stirred with tosic acid
(0.24 mmol) at 65–70 ꢀC for 3 h and then allowed to cool. After removal of the solvent

SYNTHESIS OF 4- OR 6-SUBSTITUTED INDIRUBIN DERIVATIVES
3131
under reduced pressure, the residue was purified by chromatography on silica gel with
cyclohexane–acetic ether (5:1) and dichloromethane–methanol (100:1) to give 7a.
The method of synthesis of 7b–e and 8a–d was the same.
4-Fluoroindirubin (7a). Yield: 3%; mp > 300 ꢀC; IR (cmꢁ1, KBr): 3224, 1677,
1
1619, 1578, 1466, 1303, 1248, 1139; H NMR (300 MHz, DMSO-d6) d (ppm) 11.17
(1H, s, 10-NH), 11.01 (1H, s, 1-NH), 7.62–7.65 (1H, d, J ¼ 7.7 Hz, 40-H), 7.55–7.61
(1H, m, 60-H), 7.37–7.40 (1H, d, J ¼ 8.0 Hz, 70-H), 7.22–7.29 (1H, m, 6-H),
7.02–7.07 (1H, t, J ¼ 6.9 Hz, 50-H), 6.78–6.87 (1H, m, 5-H), 6.73–6.76 (1H, d,
J ¼ 7.7 Hz, 7-H); 13C NMR (75 MHz, DMSO-d6) d (ppm) 183.2, 171.0, 152.1,
141.9, 138.7, 137.4, 130.8, 130.3, 124.9, 122.4, 120.4, 119.8, 114.0, 109.6, 109.4,
106.3; MS (ESI) m=z: 282 [M]þ. Anal. calcd. for C16H9FN2O2 (%): C, 68.57; H,
3.24; N, 10.00. Found: C, 68.59; N, 17.70.
4-Chloroindirubin (7b). Yield: 49%; mp > 300 ꢀC; IR (cmꢁ1, KBr): 3325,
1
1664, 1614, 1585, 1465, 1301, 1240, 1138, 1004; H NMR (300 MHz, DMSO-d6) d
(ppm) 11.25 (1H, s, 10-NH), 10.95 (1H, s, 1–NH), 7.64–7.66 (1H, d, J ¼ 5.7 Hz,
40-H), 7.56–7.60 (1H, t, J ¼ 6.3 Hz, 60-H), 7.35–7.37 (1H, d, J ¼ 6.0 Hz, 70-H),
7.19–7.23 (1H, t, J ¼ 6.0 Hz, 6-H), 7.01–7.07 (2H, m, 5-H, 50-H), 6.83–6.85 (1H, d,
J ¼ 5.1 Hz, 7-H)[9]; 13C NMR (75 MHz, DMSO-d6) d (ppm) 186.3, 170.7, 151.1,
142.7, 137.9, 136.6, 130.0, 129.2, 124.3, 122.8, 121.9, 120.7, 119.2, 113.5, 107.8,
103.3; MS (ESI) m=z: 298[M]þ. Anal. calcd. for C16H9ClN2O2 (%): C, 64.77; H,
3.06; N, 9.44. Found: C, 65.97; N, 10.48.
4-Bromoindirubin (7c). Yield: 8%; mp > 300 ꢀC; IR (cmꢁ1, KBr): 3293, 1671,
1
1610, 1583, 1462, 1293, 1199, 1137, 1000; H NMR (300 MHz, DMSO-d6) d (ppm)
11.24 (1H, s, 10-NH), 10.95 (1H, s, 1-NH), 7.67–7.69 (1H, d, J ¼ 7.7 Hz, 40-H),
7.57–7.63 (1H, t, J ¼ 8.0 Hz, 60-H), 7.37–7.37 (1H, d, J ¼ 8.0 Hz, 70-H), 7.20–7.23
(1H, dd, J ¼ 8.2 Hz, 5-H), 7.11–7.16 (1H, t, J ¼ 7.7 Hz, 6-H), 7.05–7.10 (1H, t,
J ¼ 7.4 Hz, 50-H), 6.89–6.92 (1H, dd, J ¼ 7.4 Hz, 7-H); 13C NMR (75 MHz,
DMSO-d6) d (ppm) 186.2, 170.4, 151.2, 142.9, 137.7, 136.8, 129.1, 125.9, 124.4,
122.1, 122.1, 119.4, 119.1, 113.5, 108.3, 104.7; MS (ESI) m=z: 342 [M]þ. Anal. calcd.
for C16H9BrN2O2 (%): C, 56.33; H, 2.66; N, 8.21. Found: C, 57.21; N, 7.87.
4-Trifluoromethylindirubin (7d). Yield: 19%; mp > 300 ꢀC; IR (cmꢁ1, KBr):
1
3316, 1672, 1593, 1468, 1310, 1291, 1214, 1111, 1006; H NMR (300 MHz, DMSO-
d6) d (ppm) 11.22 (1H, br, 10-NH), 11.02 (1H, s, 1-NH), 7.54–7.61 (2H, m, 40-H,
60-H), 7.33–7.38 (2H, m, 6-H, 70-H), 7.19–7.21 (1H, d, J ¼ 7.6 Hz, 5-H), 7.00–7.07
(2H, m, 50-H, 7-H); 13C NMR (75 MHz, DMSO-d6) d (ppm) 187.1, 170.6, 152.4,
142.5, 140.2, 138.0, 130.3, 128.9, 125.5, 123.0, 120.5, 119.7, 119.1, 114.3, 113.0,
103.6, 29.8; MS (ESI) m=z: 331 [M]þ. Anal. calcd. for C17H9F3N2O2 (%): C,
61.82; H, 2.75; N, 8.48. Found: C, 61.97; N, 9.15.
4-Nitroindirubin (7e). Yield: 15%; mp > 300 ꢀC; IR (cmꢁ1, KBr): 3364, 1675,
1
1593, 1462, 1354, 1289, 1193, 1142; H NMR (300 MHz, DMSO-d6) d (ppm) 11.42
(1H, s, 10-NH), 11.23 (1H, s, 1-NH), 7.57–7.62 (2H, m, 40-H, 60-H), 7.51–7.54 (1H,
dd, J ¼ 6.3 Hz, 70-H), 7.36–7.40 (2H, m, 5-H, 6-H), 7.19–7.21 (1H, dd, J ¼ 5.7 Hz,
7-H), 7.04–7.07 (1H, t, J ¼ 5.4 Hz, 50-H); 13C NMR (75 MHz, DMSO-d6) d (ppm)
188.1, 170.4, 152.4, 145.6, 143.3, 139.9, 138.3, 128.9, 125.7, 123.3, 119.4, 119.4,

3132
A. ZHANG ET AL.
117.2, 114.9, 114.5, 101.5. MS (ESI) m=z: 308 [M]þ. Anal. calcd. for C16H9N3O4 (%):
C, 62.54; H, 2.95; N, 13.68. Found: C, 61.47; N, 13.82.
6-Fluoroindirubin (8a). Yield: 72%; mp > 300 ꢀC; 1H NMR (300 MHz,
DMSO-d6) d (ppm) 11.07 (1H, s, 10-NH), 10.97 (1H, s, 1-NH), 8.76–8.81 (1H, m,
4-H), 7.63–7.65 (1H, d, J ¼ 7.2 Hz, 40-H), 7.54–7.59 (1H, t, J ¼ 7.8 Hz, 60-H),
7.39–7.42 (1H, d, J ¼ 7.5 Hz, 70-H), 6.98–7.03 (1H, t, J ¼ 7.2 Hz, 5-H), 6.80–6.90
(1H, m, 50-H), 6.69–6.72 (1H, dd, J ¼ 9.0 Hz, 7-H).
6-Chloroindirubin (8b). Yield: 81%; mp > 300 ꢀC; 1H NMR (300 MHz,
DMSO-d6) d (ppm) 11.05 (1H, s, 10-NH), 11.02 (1H, s, 1-NH), 8.74–8.76 (1H, d,
J ¼ 8.5 Hz, 4-H), 7.65–7.67 (1H, d, J ¼ 7.4 Hz, 40-H), 7.56–7.62 (1H, t, J ¼ 8.5 Hz,
60-H), 7.41–7.44 (1H, d, J ¼ 8.0 Hz, 70-H), 7.01–7.10 (2H, m, 5-H, 50-H), 6.92 (1H,
s, 7-H).
6-Bromoindirubin (8c). Yield: 87%; mp > 300 ꢀC; 1H NMR (300 MHz,
DMSO-d6) d (ppm) 11.06 (1H, s, 10-NH), 11.00 (1H, s, 1-NH), 8.67–8.69 (1H, d,
J ¼ 8.2 Hz, 4-H), 7.64–7.67 (1H, d, J ¼ 7.4 Hz, 40-H), 7.56–7.61 (1H, t, J ¼ 8.2 Hz,
60-H), 7.41–7.44 (1H, d, J ¼ 8.0 Hz, 70-H), 7.20–7.24 (2H, dd, J ¼ 8.2 Hz, 5-H),
7.01–7.06 (1H, m, 50-H), 7.02 (1H, s, 7-H).
6-Trifluoromethylindirubin (8d). Yield: 91%; mp > 300 ꢀC; IR (cmꢁ1, KBr):
1
3344, 1665, 1622, 1481, 1381, 1300, 1176, 1002; H NMR (300 MHz, DMSO-d6) d
(ppm) 11.22 (1H, s, 10-NH), 11.12 (1H, s, 1-NH), 8.85–8.87 (1H, d, J ¼ 6.3 Hz,
4-H), 7.63–7.65 (1H, d, J ¼ 6.3 Hz, 40-H), 7.55–7.59 (1H, t, J ¼ 8.2 Hz, 60-H),
7.40–7.42 (1H, d, J ¼ 8.0 Hz, 70-H), 7.34–7.36 (2H, dd, J ¼ 8.2 Hz, 5-H), 7.07 (1H,
s, 7-H), 7.00–7.04 (1H, t, J ¼ 7.2 Hz, 50-H); MS (ESI) m=z: 331 [M]þ. Anal. calcd.
for C17H9F3N2O2 (%): C, 61.82; H, 2.75; N, 8.48. Found: C, 63.09; N, 8.90.
General Procedure for Synthesis of 6-Substituted Indirubin-3’-
oximes (9a–d)
The appropriate 4-fluoroindirubin (9a) (1 mmol) was dissolved in pyridine
(10 mL). Hydroxylamine hydrochloride (10 equiv) was added with stirring, and the
mixture was heated under reflux (120 ꢀC) for 3 h. Then the solvent was evaporated
under reduced pressure, and the residue was washed with water and cyclohexane
to give 6-fluoroindirubin-30-oxime (9a).
The synthesis of 9b–d used the same method.
6-Fluoroindirubin-3’-oxime (9a). Yield: 78%; mp > 300 ꢀC; 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.50 (1H, s, 30-NOH), 11.62 (1H, s, 10-NH), 10.87
(1H, s, 1-NH), 8.61–8.66 (1H, m, 4-H), 8.19–8.22 (1H, d, J ¼ 7.5 Hz, 40-H),
7.38–7.40 (2H, m, 60-H, 70-H), 6.98–7.04 (2H, m, 5-H, 50-H), 6.62–6.74 (1H, m, 7- H).
6-Chloroindirubin-3’-oxime (9b). Yield: 75%; mp > 300 ꢀC; 1H NMR
(300 MHz, DMSO-d6) d (ppm) 12.06 (1H, s, 10-NH), 10.59 (1H, s, 1-NH), 8.66–8.69
(1H, d, J ¼ 8.2 Hz, 4-H), 8.34–8.36 (1H, d, J ¼ 7.4 Hz, 40-H), 7.34–7.36 (1H, d,
J ¼ 7.7 Hz, 70-H), 7.24–7.30 (1H, t, J ¼ 8.5 Hz, 60-H), 6.98–7.03 (1H, t, J ¼ 8.2 Hz,
50-H), 6.81–6.85 (2H, m, 5-H,7- H).

SYNTHESIS OF 4- OR 6-SUBSTITUTED INDIRUBIN DERIVATIVES
3133
6-Bromoindirubin-3’-oxime (9c). Yield: 73%; mp > 300 ꢀC; 1H NMR
(300 MHz, DMSO-d6) d (ppm) 13.59 (1H, s, 30-NOH), 11.75 (1H, s, 10-NH), 10.84
(1H, s, 1-NH), 8.55–8.58 (1H, d, J ¼ 8.4 Hz, 4-H), 8.22–8.24 (1H, d, J ¼ 7.3 Hz,
40-H), 7.41–7.43 (2H, m, 60-H, 70-H), 7.03–7.12 (3H, m, 5-H, 50-H, 7-H).
6-Trifluoromethylindirubin-3’-oxime (9d). Yield: 50%; mp 251–255 ꢀC; IR
1
(cmꢁ1, KBr): 3559, 1674, 1617, 1464, 1315, 1250, 1148, 1058; H NMR (300 MHz,
DMSO-d6) d (ppm) 13.78 (1H, s, 30-NOH), 11.95 (1H, s, 10-NH), 10.10 (1H, s,
1-NH), 8.74–8.76 (1H, d, J ¼ 8.4 Hz, 4-H), 8.21–8.23 (1H, d, J ¼ 7.6 Hz, 40-H),
7.40–7.47 (2H, m, 60-H, 70-H), 7.22–7.24 (1H, d, J ¼ 8.8 Hz, 5-H), 7.10 (1H, s,
7-H), 7.05–7.09 (1H, t, J ¼ 7.2 Hz, 50-H); 13C NMR (75 MHz, DMSO-d6) d (ppm)
171.4, 152.0, 148.2, 145.2, 145.2, 138.8, 132.9, 128.6, 123.2, 123.2, 123.0, 117.6,
117.1, 112.8, 105.5, 105.5, 97.8; MS (ESI) m=z: 331 [M]þ. Anal. calcd. for
C17H10F3N3O2 (%): C, 59.14; H, 2.92; N, 12.17. Found: C, 68.59; N, 13.31.
ACKNOWLEDGMENTS
The authors are indebted to Shanghai Zhongke Qiaochang Crop Protection
Inc. for the NMR spectral measurements and Shanghai Pharmaceutical Group
New Hualian pharmaceutical factory for the IR spectral measurements.
REFERENCES
1. Xiao, Z. J.; Hao, Y. S.; Liu, B. C.; Qian, L. S. Indirubin and meisoindigo in the treatment
of chromic myelogenous leukemia in China. Leuk. Lymph. 2002, 43, 1763–1768.
2. Ji, X. J.; Zhang, F. R.; Lei, J. L.; Xu, Y. T. Studies on the antineoplastic action and
toxicity of synthetic indirubin. Acta Pharmaceutica Sinica. 1981, 16, 146–148.
3. Ji, X. J.; Liu, X. M.; Li, K.; Chen, R. H.; Wang, L. G. Pharmacological studies of
meisoindigo: Absorption and mechanism of action. Biomed. Environ. Sci. 1991, 4,
332–337.
4. Yu, H.; Jove, R. The STATs of cancer—New molecular targets come of age. Nat. Rev.
Cancer 2004, 4, 97–105.
5. Kim, S.-A.; Kim, Y.-C.; Kim, S.-W.; Lee, S.-H.; Min, J.-J.; Ahn, S.-G.; Yoon, J.-H.
Antitumor activity of novel indirubin derivatives in rat tumor model. Clin. Cancer Res.
2007, 13, 253–259.
6. Tang, W.; Hemm, I.; Bertram, B. Recent development of antitumor agents from Chinese
herbal medicines, part I: Low molecular compounds. Planta Med. 2003, 69, 97–108.
7. Libnow, S.; Methling, K.; Hein, M.; Michalik, D.; Harms, M.; Wende, K.; Flemming, A.;
Ko¨ckerling, M.; Reinke, H.; Bednarski, P. J.; Lalk, M.; Langer, P. Synthesis of
indirubin-N0-glycosides and their anti-proliferative activity against human cancer cell
lines. Bioorgan. Med. Chem. 2008, 16, 5570–5583.
8. Perabo, F. G. E.; Fro¨ssler, C.; Landwehrs, G.; Schmidt, D. H.; Von Rucker, A.; Wirger,
¨
A.; Muller, S. C. Indirubin-30-monoxime, a CDK inhibitor induces growth inhibition and
¨
apoptosis-independent up-regulation of survivin in transitional cell cancer. Anticancer
Res. 2006, 26(3A), 2129–2135.
9. Polychronopoulos, P.; Magiatis, P.; Skaltsounis, A. L.; Myrianthopoulos, V.; Mikros, E.;
Tarricone, A.; Musacchio, A.; Roe, S. M.; Pearl, L.; Leost, M.; Greengard, P.; Meijer, L.
Structural basis for the synthesis of indirubins as potent and selective inhibitors of glyco-
gen synthase kinase-3 and cyclin-dependent kinases. J. Med. Chem. 2004, 47, 935–946.

3134
A. ZHANG ET AL.
10. Hoessel, R.; Leclerc, S.; Endicott, J. A.; Nobel, M. E. M.; Lawrie, A.; Tunnah, P.; Leost,
M.; Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.; Tang, W.; Eisenbrand, G.;
Meijer, L. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits
cyclin-dependent kinases. Nat. Cell Biol. 1999, 1, 60–67.
11. Davies, T. G.; Tunnah, P.; Meijer, L.; Marko, D.; Eisenbrand, G.; Endicott, J. A.; Noble,
M. E. M. Inhibitor binding to active and inactive CDK2: The crystal structure of
CDK2-cyclin A=indirubin-5-sulphonate. Structure 2001, 9, 389–397.
12. Marko, D.; Scha¨tzle, S.; Friedel, A.; Genzlinger, A.; Zankl, H.; Meijer, L.; Eisenbrand, G.
Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human
tumour cells. Br. J. Cancer. 2001, 84(2), 283–289.
13. Hoessel, R.; Leclerc, S.; Endicott, J. A.; Nobel, M. E. M.; Lawrie, A.; Tunnah, P.; Leost,
M.; Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.; Tang, W.; Eisenbrand, G.;
Meijer, L. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits
cyclindependent kinases. Nat. Cell Biol. 1999, 1, 60–67.
14. Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder, G. L.; Greengard,
P.; Biernat, J.; Wu, Y.-Z.; Mandekowi, E.-M.; Eisenbrand, G.; Meijer, L. Indirubins
inhibit glycogen synthase kinase-3b and CDK5=p25, two kinases involved in abnormal
tau phosphorylation in Alzheimer’s disease, a property common to most CDK inhibitors.
J. Biol. Chem. 2001, 276(1), 251–260.
15. Meijer, L.; Skaltsounis, A.-L.; Magiantis, P.; Polychromopoulos, P.; Knockaert, M.;
Leost, M.; Ryan, X. P.; Vonica, C. A.; Brivanlou, A.; Dajani, R.; Crovace, C.; Tarricone,
C.; Musacchio, A.; Roe, S. M.; Pearl, L.; Greengard, P. GSK-3 selective inhibitors derived
from Tyrian purple indirubins. Chem. Biol. 2003, 10, 1255–1266.
16. Moon, M. J.; Lee, S. K.; Lee, J. W.; Song, W. K.; Kim, S. W.; Kim, J. I.; Cho, C.; Choi, S.
J.; Kim, Y. C. Synthesis and structure–activity relationships of novel indirubin derivatives
as potent anti-proliferative agents with CDK2 inhibitory activities. Bioorgan. Med. Chem.
2006, 14, 237–246.
17. Tanoue, Y.; Hara, A.; Kai, N.; Sakata, K.; Hashimoto, M.; Nagai, T. A revised synthetic
scheme of 6, 60-dibromoindirubin. J. Heterocycl. Chem. 2007, 44, 1135–1137.
18. Wu, Z.-L.; Aryal, P.; Lozach, O.; Meijer, L.; Guengerich, F. P. Biosynthesis of new
indigoid inhibitors of protein kinases using recombinant cytochrome P450 2A6. Chem.
Biodivers. 2005, 2, 51–65.
19. Guengerich, F. P.; Sorrells, J. L.; Schmitt, S.; Krauser, J. A.; Aryal, P.; Meijer, L.
Generation of new protein kinase inhibitors utilizing cytochrome P450 mutant enzymes
for indigoid synthesis. J. Med. Chem. 2004, 47, 3236–3241.
20. Guha, S. K.; Mitra, A. K. Indigoid vat dyes of the isatin series, part XIII: 3-Indole-20-
(60-bromo)thionaphthene-indoigos. J. Indian Chem. Soc. 1967, 44, 694–696.
21. Wang, Z. H.; Li, W. Y.; Li, F. L.; Zhang, L.; Hua, W. Y.; Cheng, J. C.; Yao, Q. Z.
Synthesis and antitumor activity of 7-azaindirubin. Chin. Chem. Lett. 2009, 20, 542–544.
22. Wu, K.-M.; Zhang, M.-Y.; Fang, Z.; Huang, L. Potential antileukemic agents: Synthesis
of derivatives of indirubin, indigo, and isoindigotin. Acta Pharm. Sin. 1985, 20, 821–826.