Identification of a Water-Soluble Indirubin Derivative as Potent Inhibitor of Insulin-like Growth Factor 1 Receptor through Structural Modification of the Parent Natural Molecule

Article abstract of DOI:10.1021/acs.jmedchem.7b00324

Indirubins have been identified as potent ATP-competitive protein kinase inhibitors. Structural modifications in the 5-and 3′-position have been extensively investigated, but the impact of substituents in 5′-position is not equally well-studied. Here, we report the synthesis of new indirubin 3′-and 5′-derivatives in the search of water-soluble indirubins by introducing basic centers. Antiproliferative activity of all compounds in tumor cells was evaluated along with kinase inhibition of selected compounds. The results show the 3′-position to tolerate large substituents without compromising activity, whereas bulk and rigid substituents in 5′-position appear unfavorable. Screening molecular targets of water-soluble 3′-oxime ethers revealed 6ha as preferential inhibitor of insulin-like growth factor 1 receptor (IGF-1R) in a panel of 22 protein kinases and in cells. Consistently, 6ha inhibited tumor cell growth in the NCI 60 cell line panel and induced apoptosis. The results indicate that the 5′-position provides limited space for chemical modifications and identify 6ha as a potent water-soluble indirubin-based IGF-1R inhibitor.

Full text of DOI:10.1021/acs.jmedchem.7b00324

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Identification of a water-soluble indirubin derivative as potent
inhibitor of insulin-like growth factor 1 receptor through
structural modification of the parent natural molecule
Xinlai Cheng, Karl-Heinz Merz, Sandra Vatter, Jochen Zeller, Stephan Müehlbeyer,
Andrea Thommet, Jochen Christ, Stefan Wölfl, and Gerhard Eisenbrand
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017
Downloaded from http://pubs.acs.org on May 30, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Identification of a waterꢀsoluble indirubin derivative
as potent inhibitor of insulinꢀlike growth factor 1
receptor through structural modification of the
parent natural molecule
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1,2 *
1
1
1
1
Xinlai Cheng ‡ , Karl-Heinz Merz ‡, Sandra Vatter , Jochen Zeller , Stephan Muehlbeyer ,
1
1
2
1
Andrea Thommet , Jochen Christ , Stefan Wölfl , Gerhard Eisenbrand
1
Department of Chemistry, Division of Food Chemistry and Toxicology, University of
Kaiserslautern, ErwinꢀSchrödingerꢀStr. 52, Dꢀ67663 Kaiserslautern, Germany
2
Department of Pharmacy and Molecular Biotechnology, Division of Pharmaceutical
Biology, University of Heidelberg, Im Neuenheimer Feld 364, Dꢀ69120 Heidelberg,
Germany
KEYWORDS. Indirubins; Traditional Chinese Medicine, Insulinꢀlike growth factor; NCI 60;
IGFꢀ1R inhibitor; Apoptosis; E738; E804; antiꢀcancer
ABSTRACT. Indirubins have been identified as potent ATPꢀcompetitive protein kinase
inhibitors. Structural modifications in the 5ꢀ and 3'ꢀposition have been extensively investigated,
but the impact of substituents in 5'ꢀposition is not equally wellꢀstudied. Here, we report the
synthesis of new indirubinꢀ3’ꢀ and 5’ꢀderivatives in the search of waterꢀsoluble indirubins by
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
introducing basic centres. Antiꢀproliferative activity of all compounds in tumor cells was
evaluated along with kinase inhibition of selected compounds. The results show the 3'ꢀposition to
tolerate large substituents without compromising activity, whereas bulk and rigid substituents in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5'ꢀposition appear unfavorable. Screening molecular targets of water soluble 3'ꢀoxime ethers
revealed 6ha as preferential inhibitor of insulinꢀlike growth factor 1 receptor (IGFꢀ1R) in a panel
of 22 protein kinases and in cells. Consistently, 6ha inhibited tumor cell growth in the NCI 60
cell line panel and induced apoptosis. The results indicate that the 5'ꢀposition provides limited
space for chemical modifications and identify 6ha as a potent waterꢀsoluble indirubinꢀbased
IGFꢀ1R inhibitor.
ACS Paragon Plus Environment
2

Page 3 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
The indigoid bisindoles (Figure 1) indirubin, indigo and isoindigo originate from two indole
1
moieties connected in 3,2’ꢀ, 2,2’ꢀ, or 3,3’ꢀposition. The darkꢀred indirubin is a minor constituent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
of the blue natural dye indigo and has been recognized as the main active agent of the traditional
1
formulation Dangui Luhui Wan in Traditional Chinese Medicine (TCM). Indirubin has been
used in China for treatment of a series of diseases including Chronic Myelogenous Leukemia
1
(
CML). Indirubin and its derivatives raised sustained scientific interest due to the discovery of
their high affinity binding to the ATPꢀbinding site of protein kinases involved in tumorigenesis,
2
3
4
5ꢀ7
8
e.g. CDKs and GSK3ß. VEGFRꢀ2, cꢀSrc, and CK2. Recently, our group revealed that
indirubins are able to inhibit TGFß/BMP signaling by chemically knocking down Smad protein
9
expression. Moreover, Nꢀmethylisoindigo has been found to reduce the population of CD133
positive cancer stem cells in primary pancreatic cancer cells through interference with cellular
1
0
metabolic signaling pathways. These new developments provide promising perspectives for
clinical application of indigoid bisindoles.
The main obstacle for clinical application of indirubin is very poor solubility in most solvents,
1
1
probably caused by strong binding forces in the crystal lattice. Studies on crystal structures of
12
12
3
3
indirubins coꢀcrystallized with CDK2, CDK2/CyclinA, CDK5/p25 and GSK3ß, revealed
positions 5 and 3’ to best tolerate structural modifications to increase solubility and
1
3
cytotoxicity. Recently, we reported the synthesis of indirubinꢀ5ꢀcarboxamides with basic
centers in the amide substituent leading to enhanced antiꢀproliferative effects and target
1
4
specificity against GSK3ß activity.
Their corresponding quaternary ammonium and
hydrochloride salts provide compounds with significantly improved waterꢀsolubility, while
14
maintaining cytotoxicity in cancer cells. The solubility of 6ꢀbromo substituted indirubins
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
15
designed as GSK3ß inhibitors was improved by chemical modifications in 3’ꢀposition. The 5’ꢀ
position opening out into the solvent canal should provide further options to increase solubility
1
2
and cytotoxicity. Molecular modeling binding studies of 5ꢀnitroꢀ5'ꢀhydroxyindirubinꢀ3'ꢀoxime
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with CDK2 revealed an additional Hꢀbond between the hydroxy group and Asp86 of the enzyme
1
6
affording improved CDK2 inhibitory activity, inhibition of tumor cell growth and induction of
17
apoptosis. 5'ꢀMethoxyindirubin was shown to induce apoptotic cell death in human
1
8
neuroblastoma cells. Synthesis and biological activity of 5'ꢀcarboxamido substituted indirubins
with increased water solubility supposedly acting as structural analogues of sunitinib have been
19
reported by Wang et al. Indirubins with 7ꢀbromo or 7ꢀtrifluoromethyl substituent and polar
acidic substituents in 5'ꢀ or 6'ꢀposition were shown to bind in an inverse manner to the binding
2
0
site of DYRK kinases. Additionally, selfꢀemulsifying drug formulations have been developed,
21ꢀ23
which significantly increased the bioavailability of indirubin derivatives in vitro and in vivo.
Here we concentrated on chemical modifications of substituents in 3’ꢀ and 5’ꢀposition of the
1
9
indirubin core. With one exception in the literature, merely indirubins with smallꢀsized 5'ꢀ
substituents have been synthesized and investigated. We modified 5'ꢀcarboxy substituted
1
4
indirubins in a manner previously described for the 5ꢀposition. For the 3'ꢀposition which is
directed towards the ribose/triphosphate canal, hence providing copious space for structural
modification, an extended range of substituents, varying length and functionality, was
synthesized. All compounds were tested for antiꢀproliferative activity in cancer cells. Promising
compounds were selected for evaluating in vitro the inhibitory effect on diverse kinases.
Solubility data were collated and structure activity relationship was discussed.
Results and Discussion
Chemistry
ACS Paragon Plus Environment
4

Page 5 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of indirubin-3’-derivatives
For the synthesis of the indirubinꢀ3’ꢀoxime derivatives (Scheme 1) we first followed the process
2
4
25
described by Baeyer, modified by Russell and Kaupp, consisting of basic condensation of
isatins (2a-2d) with 3ꢀacetylindoxyl (3) to obtain indirubins (4a-4d; Scheme 1, reaction a). In a
recent paper, Saito et al. reported a series of methoxyindirubins and their antiꢀproliferative
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8
effects in human neuroblastoma cells, in which however the synthetic method was not
completely described. We prepared 5ꢀmethoxyisatin (2b) as well as 5ꢀhydroxyisatin (2d) by
direct ortho metalation of Bocꢀprotected pꢀaniline (8b, 8c) using tꢀBuLi, subsequent reaction
with diethyl oxalate, deprotection and cyclization via adding HCl (Scheme 2). In the case of 2d,
OH had to be initially protected with a triisopropylsilyl group to avoid side reactions (Scheme 2,
reaction a). Heating with hydroxylamine hydrochloride in pyridine converted the indirubins (4a-
4d) into their corresponding oximes (5a-5d; Scheme 1, reaction b), which in turn were
substituted with a set of halogenides using 1,1,3,3ꢀtetramethylꢀguanidine (TMG) as base to get
1
4
the products in high yields (6a-6q, Scheme 1, reaction c). For the compounds 6d and 6e,
butaneꢀtriol (9) and pentaneꢀtriol (12) were first protected with acetone (Scheme 3, reaction a).
Compound 12 is commercially unavailable and was prepared in a cyclization of Lꢀglutamic acid
(10) with sodium nitrite, followed by reduction of 5ꢀoxotetrahydrofuranꢀ2ꢀcarboxylic acid (11)
26
using LiAlH (Scheme 3, reaction c and d). The resulting dioxolanes (13a, 13b) were
4
halogenated by perbromomethane and triphenylphosphine in bromoform (14a, 14b; Scheme 3,
27
reaction b). Afterwards halogenides (14a, 14b) were introduced into the indoxyl moiety and the
protecting group was hydrolyzed in 1 N HCl (Scheme 1, reaction c).
To achieve enhanced water solubility, a polar substituent, Nꢀmethylpiperazine was connected to
indirubinꢀ3’ꢀ(2ꢀbromoethyl)ꢀoximether
(6o)
or
5ꢀmethoxyꢀindirubinꢀ3’ꢀ(2ꢀbromoethyl)ꢀ
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
oximether (6r; Scheme 1, reaction d). Treatment with THF containing saturated HCl converted
6h and 6k to the corresponding hydrochloride (6ha and 6ka; Scheme 1, reaction e).
Synthesis of indirubin-5’-derivatives
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Our approach to the synthesis of indirubinꢀ5’ꢀderivatives (23) is outlined in Scheme 4. These
indirubins (23) were prepared by the acidic condensation of various 1ꢀacetylꢀ3ꢀindoxyl acetates
8
, 13
(3, 22) with isatins (2) in moderate to good yields (50 ꢀ 90%; Scheme 4, reaction j).
Substituted 3ꢀindoxyl acetates (22) were synthesized from Nꢀphenylꢀglycineꢀoꢀcarboxylic acids
2
8
(21) by heating with sodium acetate in acetic anhydride (Scheme 4, reaction i). The obtained
5’ꢀindirubins (23) could be readily converted into 5’ꢀsubstituted indirubinꢀ3’ꢀoximes (24) using
hydroxylꢀamine hydrochloride in pyridine as mentioned above (Scheme 4, reaction k).
The synthetic route to 21 (Scheme 4) depended on the substituent R . 4ꢀAminoisophthalic acid
1
(16) was prepared via oxidation of 2,4ꢀdimethylꢀ1ꢀnitrobenzene in an aqueous solution of
KMnO at 85°C followed by catalytic hydrogenation with palladium on carbon (Pd/C; Scheme 4
4
2
9
I, reaction a and b). Hydrogenation of 5ꢀmethoxyꢀ2ꢀnitrobenzoic acid afforded 2ꢀaminoꢀ5ꢀ
methoxybenzoic acid (17; Scheme 4 II, reaction b). The reaction of 16 or 17 with chloroacetic
2
8
acid and sodium carbonate at 100°C provided the required 21a (Scheme 4 I and II, reaction c).
Acetylꢀprotected Nꢀphenylglycineꢀo,pꢀdicarboxylic acid 21b was synthesized by reaction of 2,4ꢀ
1
4
dimethylaniline (18) with chloroacetic acid, acetylation and subsequent oxidation with
permanganate (Scheme 4 III, reaction d). This compound is applicable for the cyclization, too.
Alternatively, 2ꢀchlorobenzoic acids (19, 20) were transformed into 21a via copper catalyzed
3
0
nucleophilic aromatic displacement (Scheme 4 IV and V, reaction f). For the synthesis of 5’ꢀ
hydroxyindirubin, 3ꢀaminoꢀ5ꢀchloroꢀbenzoic acid was diazotized to produce 5ꢀchloroꢀ3ꢀ
ACS Paragon Plus Environment
6

Page 7 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
31
hydroxybenzoic acid that was subsequently protected by a benzyl group to yield 20 (Scheme 4
V, reaction g and h).
To improve solubility and bioactivity, we placed a basic center in the amide group of indirubinꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
’ꢀcarboxamides (23e-g; Scheme 5). Activation of 5’ꢀcarboxyindirubin (23d) with
pentafluorophenyl trifluoroacetate and subsequent amidation were performed analogously to the
1
4
method reported for the synthesis of indirubinꢀ5ꢀcarboxamides. 5’ꢀAminoindirubin (23b) was
synthesized by Pd/C hydrogenation of 23a (Scheme 6).
Biological activity
Antiproliferative activity in human tumor cell lines
Recently, several compounds with distinct chemical backbones have been identified as panꢀassay
interference compounds (PAINS), which often give false positive signals in various biological
32ꢀ34
assays in highꢀthroughput screening.
We recruited a recently developed software, called
cAPP, and performed in silico screening of our indirubin derivatives using available PAINS
3
5
filters. The results confirmed that the basic structure of 3,2’ꢀbisindole is not related to any
reported PAINS. The result was listed in supporting information (SI. Table 1).
As next, we determined the antiꢀproliferative effects of compounds in LXFL529L (large cell
lung carcinoma cell line) and/or MCF7 (human breast adenocarcinoma cells) in the
sulforhodamine B (SRB) assay. The IC ꢀvalue, structure and solubility were summarized in
50
Table 1. Indirubinꢀ3’ꢀethyloxime ether (6a) showed an IC value of 7.6 ꢁM. Replacing the ethyl
5
0
21, 23
group with propaneꢀ1,2ꢀdiol resulted in 10ꢀfold higher cytotoxicity (6c, also known as E804,
0.9 µM). Interestingly, a positive relationship between toxicity and length of alkyl chain was
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
observed for compounds carrying an alkane diolꢀgroup in 3’ꢀposition (6d, 0.65 µM and 6e, 0.56
µM), while removal of one hydroxyl group from 6d reduced toxicity (6b, 2.6 µM). Replacing
diol of 6c with ßꢀDꢀglucopyranose gave the compound 6f with a comparable activity (1.1 ꢁM),
but 15ꢀfold higher solubility. Introducing basic substituents into 3’ꢀposition led to indirubinꢀ3’ꢀ
{2ꢀ[pꢀ(N,Nꢀdimethyl)aminoanilino]ꢀethyl}ꢀoxime ether (6g, 5.1 µM) which showed 6ꢀfold less
inhibition, as compared to 6c while 6h (0.9 µM) with a methylpiperazine substituent showed a
similar IC value.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
In view of the fact that 5ꢀ and 3’ꢀ position share the ribose/triphosphate canal, using two large
14
substituents at both positions could significantly reduce bioactivity. We therefore incorporated
small groups including methylꢀ, hydroxylꢀ and methoxyꢀgroup into the 5ꢀposition. In comparison
to 6f lacking a substituent in 5ꢀposition, additional methyl (6m, 2.0 µM) or hydroxyl (6n, 1.2
µM) substituents attenuated solubility, whereas a methoxy group (6j) slightly increased the
7
, 9
solubility (1.5ꢀfold). Remarkably, the compounds 6l (also known as E738) and 6k carrying
additional basic substituent in 3’ꢀposition exhibited similar cytotoxicity as their respective
counterparts 6c and 6h, but showed 25ꢀ and 200ꢀfold higher aqueous solubility, respectively.
Transformation 6h and 6k into hydrochloride salts achieved two well water soluble indirubins
6ha and 6ka (solubility >25 g/L) without loss of cytotoxicity.
In comparison to 3’ꢀ and 5ꢀindirubins, modifications in 5’ꢀposition did not notably improve
cytotoxicity or solubility (Table 2). The solubility of 5’ꢀnitroindirubin (23a) was too low (<< 0.1
mg/L) to determine an IC value using the SRBꢀassay, although the antiꢀcancer activity of its 5ꢀ
50
3
6
analogue (5ꢀnitroindirubin) was reported in cells and animal model. Appreciable cytotoxicity
was achieved by hydrogenation of 23a. The compound 23b exhibited an IC of 7.6 µM. Our
5
0
result showed that introduction of basic centers in 3’ꢀposition (compounds 6g, 6h and 6k)
ACS Paragon Plus Environment
8

Page 9 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
remarkably improved cytotoxicity and/or water solubility. Moreover, we previously reported that
indirubinꢀ5ꢀcaboxamides with similar basic centers showed enhanced solubility and antiꢀtumor
1
4
activity. Nevertheless, similar modifications in 5’ꢀposition failed to increase solubility and
activity (23e-g). Additionally, 5’ꢀcarboxyindirubin (23d) was inactive regardless of further
adding a carboxyl group in 5ꢀposition (23h) or conversion into oxime (24d). Derivatives carrying
small substituents, like amino, hydroxy and methoxy in 5’ꢀposition (23b, 23c, 23i), illustrated
good antiꢀproliferative effect, while converting 23c into its 3'ꢀoxime 24c further increased
cytoxicity by about 3 times.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Inhibition of protein kinases in vitro
On the basis of chemical structure and cytotoxicity, several compounds were selected for
evaluating inhibitory effects on recombinant kinases including CDK1/CyclinB, CDK2/CyclinA,
CDK2/CyclinE, CDK6/CyclinD, and GSK3ß. The IC values were listed in Table 3. Focusing
5
0
on CDK2/CycE, 6c and 6d showed similar effects (IC : 0.21 ꢁM and 0.23 ꢁM). A methoxy
5
0
group in 5ꢀposition significantly increased activity (5b: 0.054 µM; 6j: 0.09 µM and 6l: 0.043
M), while dramatic ablation of activity was observed by replacement or withdrawal of the
ꢁ
methoxy group (6f and 6m). Analysis from molecule modeling suggested that the hydrochloride
product of 6h (6ha) should not be expected to effectively inhibit CDKs and GSK3ß (SI. Figure
1). We profiled 6ha and 6l against a panel of 22 protein kinases at 1 µM as previously described
1
4
(Figure 2A). As expected (Figure 2B), 6l was the potent multiꢀkinase inhibitor and inhibited 12
out of 22 protein kinases (residual activity < 50%). The result also confirmed that CDKs and
GSK3ß was not targeted by 6ha, although its 6ꢀbromoꢀsubstitueted counterpart had shown
GSK3ß, CDK1 and CDK5 inhibition with IC50 values of 0.005 µM, 0.3 µM and 0.5 µM
15
respectively. Intriguingly, we observed potent an inhibitory effect (~30% residual activity) on
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
insulinꢀlike growth factor receptor1 (IGFꢀ1R). This was confirmed by using the ADP Glo®
assay (Promega, Germany), showing an IC value of 169 nM (Figure 2C).
50
In comparison to indirubin, which was previously reported to inhibit activity of various
CDK/Cyclin complexes at around 10 µM (IC50ꢀCDK1/CycB1: 10 µM; IC50ꢀCDK2/CycA: 7.5 µM; IC50ꢀ
CDK2/CycE: 2.2 µM; IC50ꢀCDK4/CycD1: 12 µM and IC IC50ꢀCDK5/p35NCK: 5.5 µM), but not IGFꢀ1R (
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
IC50ꢀIGFꢀ1R: >1000 µM), 6ha showed highly selective inhibitory effect on IGFꢀ1R. Recently,
Anastassiadis et al. systematically analyzed the activity of 178 commercially available protein
3
7
kinase inhibitors on a panel of 300 protein kinases. To get more insight into the selectivity of
our compounds, we generated the heat map (Figure 2B) to compare inhibitory effects of 6ha and
6l with relevant compounds provided in this study, which included three indirubin derivatives
(25, 6c and 26), previously characterized IGFꢀ1Rꢀrelated inhibitors (27-31), and compounds
37
inhibiting 50% of IGFꢀ1R activity (32-37) in that study, whose structures were listed in figure
2D. We found that indirubin derivatives generally targeted Auroras, CDKs, CHK2, FGFꢀR1 and
GSK3ß with exception of 6ha (Figure 2B). Of note, the reported IGFꢀ1R or Akt inhibitors could
not block the activity of IGFꢀ1R in their study. Indeed, potent antagonists of IGFꢀ1R were CDK
inhibitors (Figure 2B). Taken together, 6ha possessed a distinct pattern of protein kinase
profiling as compared to other compounds in our analysis and showed the unique inhibitory
activity.
6ha inhibited IGF-1R mediated signaling in various cell lines
IGF is a growth factor whose effects are transmitted by its receptor IGFꢀ1R. Ligand binding
activates the phosphatidylinositol 3ꢀkinase (PI3K)—protein kinase B (PKB/Akt) signaling
3
8
pathway. In tumor cells, this signal cascade is normally hyperactivated to meet the special
ACS Paragon Plus Environment
10

Page 11 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
38
requirement of tumor cells in metabolism, proliferation, survival, and motility. The cells
originated from IGFꢀ1R knockꢀout embryos do not undergo transformation to malignancy
3
9
implicating inhibition of activated IGFꢀ1R as a prerequisite in tumorigenesis. Therefore, the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
development of IGFꢀ1R inhibitors may offer novel perspectives for basic cancer research and
4
0
clinical therapy. To evaluate the effect of 6ha in cells, we performed immunocytochemistry in
MCF7 cells (breast cancer cells) treated with 6ha for 1 h. A specific antibody against
phosphorylated IGFꢀ1R at Tyr 1161 (pꢀIGFꢀ1R) was used, whose activity is required for fully
active IGFꢀ1R and related downstream signaling pathway. As showed in figure 3A, the majority
of IGFꢀ1R detected by the antibody was localized on the membrane (mock treatment). Upon
4
1
treatment the signal was completely dismissed. Ly294002 (38, Figure 2D), a PI3K inhibitor,
was used as positive control (10 µM) and showed mild inhibitory effect. As next, we performed
immunoblot and further confirmed that phosphorylation of IGFꢀ1R was sufficiently blocked by
6ha even at a concentration of 0.5 µM for 1 h treatment in MCF7 cells (Figure 3B).
Moreover, we evaluated effects of 6ha on the downstream pathway in various cancer cells
treated for 1 h by detecting the level of phosphorylated Akt (S473), a wellꢀknown target
3
8
molecule of the IGFꢀPI3Kꢀrelated signaling pathway. Doseꢀdependent inhibition was observed
in all four cell lines tested, including MCF7, HeLa (cervical cancer cells), MDA MB231 (breast
cancer cells) and Panc1 (pancreatic cancer cells). In comparison to the positive control, 38 at a
concentration of 10 µM, the effect of 6ha was comparable to better in certain cells (Figure 4A).
In timeꢀdependent study the activity of Akt was shut down by 6ha as early as 15 min after
incubation of MCF7 cells (Figure 4B).
6ha induced PARP cleavage, cell apoptosis and cell arrest
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We found cleaved PARP in MCF7 treated with 6ha for 6 h, implicating the induction of cell
1
0
apoptosis (Figure 4C). Here, a commercial Src inhibitor (Srci, 4ꢀ(4’ꢀphenoxyanilino)ꢀ6,7ꢀ
dimethoxyquinazoline, 10 µM) was used as the positive control. In good agreement, apoptotic
cells were visualized by FITC conjugated annexin v and propidium iodide (PI), and quantified by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
fluorescence activated cell sorting (FACS) as previously described. Bortezomib (Bz, [(1R)ꢀ3ꢀ
methylꢀ1ꢀ({(2S)ꢀ3ꢀphenylꢀ2ꢀ[(pyrazinꢀ2ꢀylcarbonyl)amino]propanoyl}amino)butyl]boronic
4
2
acid), a proteasome inhibitor, was used as positive control. The result confirmed that 6ha
induced cellular apoptosis from a concentration of 0.5 µM (Figure 5). Analysis of DNA content
revealed gradually elevated cell cycle arrest majorly at G2/M phase in MCF7 cells treated with
6ha for 24 h and 48 h (Figure 6), consistent with a previous report that inhibition of IGFꢀ1R
4
3
caused cell apoptosis and cell arrest in G2/M.
Activity of 6ha was further evaluated by measuring the antiꢀproliferative effect on the NCI 60
panel of cell lines. A high activity of 6ha in all NCI 60 cell lines (Figure 7 and supplementary
information) with GI value (50% growth inhibition) between 3.19 µM (BTꢀ549) to 0.08 µM
5
0
(
KM12) was observed. Of note, GI values in MCF7 and MDA MB231 are 0.59 µM and 2.14
50
µM respectively, in consistent with the premise of 6ha blocking Akt phosphorylation similarly in
these two cell lines (Figure 4A).
Conclusion
Taken together, the results show that bulky and rigid substituents are not suitable for the 5’ꢀ
position, bringing about reduced toxicity and water solubility. The conversion of indirubinꢀ3'ꢀ
derivatives to stable and waterꢀsoluble hydrochloride salts was achieved. Compound 6ha
surprisingly effected potent inhibition of IGFꢀ1R without notably inhibiting CDKs and GSK3ß in
ACS Paragon Plus Environment
12

Page 13 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a panel of 22 protein kinases. Further analyses identified that 6ha inactivated IGF und related
signalling in various tumor cells, and induced cell apoptosis and cell arrest at G2/M phases in
MCF7 cells. In the NCI60 cell line panel, the compound generally inhibited the growth of
malignant tumor cells at low micromolar concentrations.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental Section
Cell culture
LXFL529L cells were grown at 37°C in RPMI 1640 containing 10% heatꢀinactivated fetal
bovine serum (FBS). MCF7, Panc1, HeLa and MDA MB231 cells were cultivated in DMEM
supplemented with 10% FBS in a humidified atmosphere of 5% CO (cell culture media and
2
supplements were obtained from Invitrogen, Karlsruhe, Germany). Cells were tested routinely
for absence of mycoplasm contamination. 38 was purchased from Calbiochem (Germany) and
Srci from Merck (Germany). Compounds were dissolved in DMSO to obtain 10 mM stock
solution and further dilutions were prepared in respective medium.
Sulforhodamine B assay
4
4
Effects on cell growth were determined according to the method of Skehan et al. with slight
modifications. Briefly, cells were seeded into 24ꢀwell plates with a density of 10,000 cells/well
and allowed to grow for 24 h before treatment. Thereafter, cells were incubated with the
respective drug for 3 days in serum containing medium. Incubation was stopped by addition of
trichloroacetic acid (50% solution). After 1 h at 4°C, plates were washed four times with water.
The dried plates were stained with a 0.4% solution of sulforhodamine B. The dye was eluted
with Trisꢀbuffer (10 mM, pH 10.5) and quantified photometrically at 570 nm. Cytotoxicity was
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
calculated as percent survival, determined by the number of treated over control cells. GI of
5
0
NCI60 was performed by the US National Cancer Institute.
Solubility in water
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Standard solutions of the indirubin compounds were prepared in ethanol and maxima of
absorbance were defined. Solubility was determined spectrophotometrically at maximum of
1
4
absorbance by calibration method.
Immunoblot
MCF7 cells were cultured in DMEM with 10% FBS for 24 h and then incubated with
compounds as indicated in the text. Cells were lysed and proteins were analyzed by western blot
3
3
with ECL detection as described before. For each sample 40 µg of total protein were resolved
with 10% SDSꢀPAGE and immunoblotted with antibodies specific for pꢀIGFꢀ1R (Y1161), pꢀAkt
(
S473), Akt and PARP (Cell Signaling Technologies, Germany) and ßꢀActin antibody for control
(Sigma Aldrich, Germany). Primary antibodies were incubated at a 1:1000 dilution in TBS (pH
.5) with 0.1% Tweenꢀ20 and 5% BSA/milk with gentle agitation overnight at 4°C. Secondary
7
antibodies (Sigma Aldrich) were incubated in TBS (pH 7.5) with 5% BSA/Milk and 0.1%
Tweenꢀ20 at a 1:10000 dilution for 1 h at room temperature.
Immunocytochemistry
4
5ꢀ46
Immunocytochemistry was performed as previously described.
Briefly, MCF7 cells were
incubated with 6ha (1 µM) for 1 h. 0.1% DMSO was used as mock and 38 (10 µM) as positive
control. Cells were fixed with 4% PFA at RT for 15 min, permeabilized and blocked with
blocking buffer (5% goat serum and 0.3% Triton Xꢀ100 in PBS) for 1 hr. Cells were incubated
ACS Paragon Plus Environment
14

Page 15 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
with pꢀIGFꢀ1R antibody (1:200) in the antibody dilution buffer (1% BSA and 0.2% Triton Xꢀ100
in PBS) at 4°C overnight. The secondary antibody (Goat antiꢀrabbit Alexa Flour® 594) Dianova,
Germany) was added and incubated for 1 hr. Hoechst 33258 (1 µg/mL in PBS) was used to
visualize nuclei.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Apoptosis assay and cell cycle analysis
MCF7 cells were cultured in DMEM containing 10% FBS and 1% PS for 24 h and then
incubated with indicated concentrations of 6ha for 24h and harvested. Cells were collected by
centrifugation and supernatant was discarded. For apoptosis assay, cells were resuspended in a
solution of 5 µL FITCꢀconjugated annexin v (BD Bioscience, Germany) in 50 µL annexin v
binding buffer (10 mM HEPES pH7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl and 1.8 mM
2
CaCl ) and incubated for 15 min in the dark at room temperature. Afterwards, a solution of 1.25
2
µL propidium iodide (PI, 1 mg/mL) in 450 µL annexin v binding buffer were added, incubated
for 10 min in the dark at room temperature. Then cells were immediately analyzed by flow
cytometry (FACS, Fluorescenceꢀactivated cell sorting) using FACSCalibur (Becton Dickinson)
and CellQuest Pro (BD) analysis software. For cell cycle analysis, the cell pellets were fixed in
70% Ethanol for at least 24 h, washed twice with iceꢀcold PBS and resuspended in 500 µL PBS.
The suspension was incubated with RNase A (50 µg/mL) for 1 h at 37 °C and subsequently
stained with PI (50 µg/mL) for 5 min and analyzed by FACS.
In Vitro Kinase assay
In vitro inhibition of isolated or recombinant kinases, CDK1/CyclinB, CDK2/CyclinA,
CDK2/CyclinE, CDK6/CyclinD, and GSKꢀ3ß (Upstate, Lake Placid, USA , # 14ꢀ475), was
analyzed following the manufacturer’s protocol with 0.4 µCi [gꢀ32P] ATP per sample vial.
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Briefly, kinase assay mixtures were incubated for 10 min at 30 °C. Thereafter, reaction solutions
(20 µL) were spotted on P81 phosphocellulose squares. After washing (3x, 5min with 0.75%
phosphoric acid) phosphocellulose sheets were rinsed with acetone and dried. The squares were
then transferred into scintillation vials and substrate phosphorylation was measured by βꢀ
counting. The IC values were determined by linear regression of the data points comparing
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
drugꢀtreated samples with solvent control. Each measurement was done in duplicate in at least
three independent experiments. Kinase profiling assay for 6ha at 1 µM was performed by
1
4
ProQinase (Germany) as previously reported. IGFꢀ1R activity was determined in ADP Glo
assay (Promega, Germany) according to the manufacturers protocol.
Synthesis
The syntheses of intermediates were described in supporting information
Reagents
Solvents and reagents obtained from commercial suppliers were at least of reagent grade and
were distilled or dried according to prevailing methods prior to use, if necessary. The syntheses
were done under argon atmosphere, when required. Argon 4.8 was purchased from Air Liquide
and was dried over phosphorus pentoxide. For monitoring the reactions, Alugram SIL G/UV254
sheets for TLC (Macherey &Nagel) were used. Column chromatography was accomplished
using silica gel 60 (Macherey & Nagel, 0.063 – 0.200 mm), for flash chromatography silica gel
60 (Macherey & Nagel, 0.040ꢀ0.063 mm) was used.
Analytical Methods
ACS Paragon Plus Environment
16

Page 17 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
13
1
13
H and C NMR spectra were recorded on a Bruker AMXꢀ400 ( H NMR: 400 MHz, C
1
13
NMR: 100 MHz) or on a Bruker AMXꢀ600 ( H NMR: 600 MHz, C NMR: 150 MHz).
Chemical shifts are reported in ppm from tetramethylsilane with solvent as the internal standard
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13
(
H DMSOꢀd : δ 2.49; C DMSOꢀd : δ 39.5).
6 6
Elemental analyses were performed on an Element Analyzer Perkin Elmer EA 240 or 2400
CHN in the University of Kaiserslautern, Dept. of Chemistry.
HPLC was employed to confirm the purity of compounds >96%.
General procedure for the synthesis of indirubin derivatives (4)
Under argon atmosphere at room temperature, a suspension of 3ꢀindoxyl acetate (1.1 mmol),
isatin (1.2 mmol) and sodium carbonate (2.5 mmol) in degassed methanol (25 mL) was stirred
for 5 h. The reddish precipitate was filtered off, washed with methanol and water and dried to
afford the indirubin.
5-Methoxyindirubin (4b) (92.4%)
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ) 3.77 (s, 3H), 6.79 (d, 1H, J = 8.4 Hz), 6.85 (dd, 1H, J = 8.4
6
4
3
3
3
Hz, J = 2.4 Hz), 7.01 (t, 1H, J = 7.7 Hz), 7.41 (d, 1H, J = 8.1 Hz), 7.56 (t, 1H, J = 8.4 Hz),
3
4
13
1
7.65 (d, 1H, J = 7.7 Hz), 8.48 (d, 1H, J = 2.6 Hz), 10.69 (s, 1H), 11.01 (s, 1H). Cꢀ{ H}ꢀNMR
(150 MHz; DMSOꢀd ) 55.4, 107.2, 109.8, 110.5, 113.4, 115.1, 119.0, 121.3, 122.2, 124.4, 134.8,
6
137.1, 138.4, 152.5, 154.3, 171.0, 181.8. Anal. Calcd for C H N O : C 69.9, H 4.1, N 9.6;
17 12 2 3
Found: C 69.7, H 3.7, N 10.0.
5
-Methylindirubin (4c) (92.0%)
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
HꢀNMR (400 MHz; DMSOꢀd ) 2.33 (s, 3H), 6.79 (d, 1H, J = 7.9 Hz), 7.00ꢀ7.08 (m, 2H),
6
3
3
7
1
1
.42 (d, 1H, J = 7.9 Hz), 7.58 (m, 1H), 7.64 (d, 1H, J = 7.6 Hz), 8.63 (s, 1H), 10.79 (s, 1H),
13
1
1.00 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 21.0, 106.8, 109.2, 113.3, 119.9, 121.1,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21.5, 124.2, 125.1, 129.7, 129.7, 137.0, 138.1, 138.7, 152.4, 171.9, 188.5. Anal. Calcd for
C H N O : C 73.9, H 4.4, N 10.1; Found: C 73.8, H 4.3, N 10.2.
17
12
2
2
5
-Hydroxyindirubin (4d) (94.3%)
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ) 6.67 (m, 2H), 7.00 (t, 1H, J = 7.3 Hz), 7.40 (d, 1H, J = 8.1
6
3
Hz), 7.56 (t, 1H, J = 7.3 Hz), 7.64 (d, 1H, 3J = 7.3 Hz), 8.31 (s, 1H), 9.02 (s, 1H), 10.57 (s, 1H),
13
1
1
0.98 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 107.5, 109.8, 112.2, 113.4, 116.2, 119.0,
6
1
21.1, 122.1, 124.3, 133.6, 137.1, 138.2, 152.1, 152.5, 170.2, 188.6. Anal. Calcd for
C H N O : C 69.1, H 3.6, N 10.1; Found: C 69.0, H 3.8, N 9.9.
16
10
2
3
General procedure for the synthesis of indirubin-3’-oxime (5) (yield) A mixture of
indirubin(1, 4b-d) (1.1 mmol) and hydroxylamine hydrochloride (24.4 mmol) in pyridine (7 mL)
was refluxed for 4 h. The mixture was diluted with 2 N HCl (50 mL) to yield the corresponding 5
as a reddish solid.
Indirubin-3’-oxime (5a) (78.9%)
1
3
HꢀNMR (400 MHz; DMSOꢀd ) 6.90 (d, 1H, J = 7.6 Hz), 6.95 (m, 1H), 7.01ꢀ7.05 (m, 1H),
6
3
3
7
1
1
.13 (m, 1H), 7.40ꢀ7.41 (m, 2H), 8.24 (d, 1H, J = 7.6 Hz), 8.65 (d, 1H, J = 7.2 Hz), 10.72 (s,
1
3
1
H), 11.73 (s, 1H), 13.48 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 98.9, 108.9, 111.5,
6
16.5, 120.4, 121.5, 122.7, 123.1, 125.9, 128.0, 132.0, 138.3, 144.8, 145.3, 151.2, 171.0. Anal.
.
Calcd for C H N O 0.25 H O: C 68.2, H 4.1, N 14.9; Found: C 68.4, H 4.1, N 14.8.
1
6
11
3
2
2
ACS Paragon Plus Environment
18

Page 19 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
-Methoxy-indirubin-3’-oxime (5b) (95.0%)
1HꢀNMR (400 MHz; DMSOꢀd6) 3.76 (s, 3H), 6.72 (dd, 1H, 3J = 7.6 Hz, 4J = 2.5 Hz), 6.73 (d,
1
H, 3J = 8.3 Hz), 7.00ꢀ7.04 (m, 1H), 7.38ꢀ.739 (m, 2H), 8.24 (d, 1H, 3J = 7.4 Hz), 8.33 (d, 1H,
J = 2.1 Hz), 10.52 (s, 1H), 11.76 (s, 1H), 13.49 (s, 1H). 13Cꢀ{1H}ꢀNMR (150 MHz; DMSOꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
d6) 55.5, 99.5, 108.8, 109.6, 111.2, 111.5, 116.5, 121.4, 123.5, 128.0, 132.0, 132.3, 144.8, 145.3,
.
1
51.4, 154.2, 171.1. Anal. Calcd for C H N O H O: C 62.8, H 4.7, N 12.9; Found: C 63.0, H
1
7
13
3
3
2
4
.4, N 13.2.
5-Methyl-indirubin-3’-oxime (5c) (56.0%)
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ) 2.33 (s, 3H), 6.76 (d, 1H, J = 7.6 Hz), 6.93 (d, 1H, J = 7.6
6
3
Hz), 7.01 (m, 1H), 7.38 (m, 2H), 8.23 (d, 1H, J = 7.4 Hz), 8.48 (s, 1H), 10.59 (s, 1H), 11.71 (s,
1
3
1
1
H), 13.40 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 21.0, 99.4, 108.7, 111.7 116.8, 121.6,
6
1
22.9, 124.0, 126.6, 128.2, 129.2, 132.2, 136.4, 145.1, 145.3, 151.5, 171.3. Anal. Calcd for
.
C H N O 0.25 H O: C 69.0, H 4.6, N 14.2; Found: C 69.3, H 4.5, N 14.2.
17
13
3
2
2
5-Hydroxy-indirubin-3’-oxime (5d) (88.2%)
1
1
4
3
HꢀNMR (400 MHz; DMSOꢀd ) 6.59 (dd, H, 3J = 7.6 Hz, J = 2.5 Hz), 6.65 (d, 1H, J = 8.3
6
4
3
Hz), 7.00 (m, 1H), 7.40ꢀ7.35 (m, 2H), 8.12 (d, 1H, J = 2.1 Hz), 8.22 (d, 1H, J = 7.5 Hz), 8.71
13
1
(
s, 1H), 10.41 (m, 2H), 13.45 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 99.8, 108.6, 111.1,
6
1
11.3, 112.6, 116.6, 121.3, 123.6, 128.1, 131.2, 132.0, 144.8, 145.0, 151.4, 151.6, 171.1. Anal.
Calcd for C H N O . H O: C 61.7, H 4.2, N 13.5; Found: C 61.7, H 4.1, N 13.9.
1
6
11
3
3
2
General procedure for the synthesis of indirubin-3’-oxime ethers (6, except 6d, 6e)
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
,1,3,3ꢀTetramethylguanidine (582 µL) and alkyl halogenide (5.4 mmol) were added to
indirubinꢀ3’ꢀoxime (0.5 mmol) in ethanol (7 mL) with stirring. The mixture was refluxed for 2 h,
cooled to 0°C and diluted with water (20 mL) and 1N HCl (20 mL). The precipitate was filtered,
washed with water and dried to afford 6 except for 6d and 6e.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Indirubin-3’-ethyl-oxime ether (6a) (86.0%)
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ) 1.50 (t, 3H, J = 7.1 Hz), 4.63 (m, 2H), 6.89 (d, 1H, J = 7.6
6
3
4
3
Hz), 7.00 (m, 2H), 7.14 (dt, 1H, J = 7.6 Hz, J = 1.0 Hz), 7.40 (m, 2H), 8.13 (d, 1H, J = 7.6 Hz),
3
13
1
8.61 (d, 1H, J = 7.7 Hz), 10.74 (s, 1H), 11.97 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd )
6
1
1
6
4.4, 72.1, 100.0, 108.9, 111.7, 116.1, 120.6, 121.4, 122.3, 123.1, 126.4, 128.2, 132.7, 138.6,
.
44.2, 145.4, 151.0, 170.8. Anal. Calcd for C H N O H O: C 66.9, H 5.3, N 13.0;. Found: C
18 15
3
2
2
7.3, H 4.9, N 13.0.
Indirubin-3’-(4-hydroxybutyl)-oxime ether (6b) (68.0%)
1
HꢀNMR (400 MHz; DMSOꢀd ) 1.62 (m, 2H), 1.93 (m, 2H), 3.48 (m, 2H), 4.50 (m, 1H), 4.61
6
3
(
m, 2H), 6.90 (d, 1H, J = 7.6 Hz), 6.96ꢀ7.05 (m, 2H), 7.12ꢀ7.16 (m, 1H). 7.38ꢀ7.44 (m, 2H),
3
3
13
1
8
.11 (d, 1H, J = 7.6 Hz), 8.61 (d, 1H, J = 7.8 Hz), 10.74 (s, 1H), 11.68 (s, 1H). Cꢀ{ H}ꢀNMR
(150 MHz; DMSOꢀd ) 25.7, 29.2, 60.7, 76.7, 100.2, 109.2, 112.0, 116.4, 120.9, 121.7, 122.6,
6
1
23.5, 126.6, 128.3, 133.0, 138.9, 144.2, 145.6, 151.3, 171.1. Anal. Calcd for C H N O : C
20 19 3 3
6
8.8, H 5.5, N 12.0;. Found: C 68.5, H 5.4, N 11.8.
Indirubin-3’-(2,3-dihydroxypropyl)-oxime ether (E804, 6c) (44.0%)
1
HꢀNMR (400 MHz; DMSOꢀd ) 3.52 (m, 2H), 3.97ꢀ4.04 (m, 1H), 4.49ꢀ4.53 (m, 1H), 4.63ꢀ
6
3
3
4.68 (m, 1H), 4.80 (m, 1H), 5.11 (d, 1H, J = 5.1 Hz), 6.90 (d, 1H, J = 7.8 Hz), 6.96ꢀ7.05 (m,
ACS Paragon Plus Environment
20

Page 21 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
3
2
1
1
1
H), 7.12ꢀ7.16 (m, 1H), 7.39ꢀ7.45 (m, 2H), 8.18 (d, 1H, J = 7.6 Hz), 8.62 (d, 1H, J = 7.8 Hz),
1
3
1
0.75 (s, 1H), 11.68 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 63.0, 70.2, 78.9, 100.3,
6
09.1, 111.9, 116.5, 120.9, 121.6, 122.5, 123.6, 126.5, 128.6, 133.0, 138.8, 144.2, 145.6, 151.4,
71.1. Anal. Calcd for C H N O : C 65.0, H 4.9, N 12.0;. Found: C 64.8, H 4.8, N 12.4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
9
17
3
4
General procedure for the synthesis of (6d, 6e)
A suspension of 6p oder 6q (2.0 mmol) in 1N HCl (100 mL) was stirred at room temperature
over night. The mixture was filtered and dried to afford 6d or 6e.
Indirubin-3’-(3,4-dihydroxybutyl)-oxime ether (6d) (62.6% two steps)
1
2
3
HꢀNMR (400 MHz; DMSOꢀd ): 1.81ꢀ2.15 (m, 2H), 3.32 (dd, 1H, J = 10.7 Hz, J = 5.8 Hz),
6
2
3
2
7
1
.48ꢀ2.50 (m, 2H), 3.40 (dd, 1H, J = 10.7 Hz, J = 5.8 Hz), 3.69ꢀ4.7 (m, 3H), 6.89 (d, 1H, 3J =
3
.4 Hz), 6.97ꢀ7.04 (m, 2H), 7.14 (dt, 1H, J = 7.8 Hz, 4J = 1,2 Hz), 7.39ꢀ7.42 (m, 2H), 8.13 (d,
3
3
13
1
H, J = 7.4 Hz), 8.63 (d, 1H, J = 7.9 Hz), 10.58 (s, 1H), 11.76 (s, 1H). C{ H}ꢀNMR (600
MHz; DMSOꢀd ): 33.0, 65.9, 68.0, 73.4, 99.9, 108.9, 111.7, 116.1, 120.6, 121.4, 122.3, 123.2,
6
.
1
26.3, 128.1, 132.7, 138.6, 143.9, 146.4, 151.0, 170.8. Anal. Calcd for C H N O H O: C 62.7,
20
19
3
4
2
H 5.5, N 11.0; Found: C 63.0, H 5.1, N 10.8.
Indirubin-3’-(4,5-dihydroxypentyl)-oxime ether (6e) (13.7%, two steps)
1
3
HꢀNMR (400 MHz; DMSOꢀd ): 1.24ꢀ5.05 (m, 11H), 6.89 (d, 1H, J = 7.7 Hz), 6.97ꢀ7.03 (m,
6
3
2
1
1
H), 7.14 (t, 1H, J = 7.6 Hz), 7.39ꢀ7.43 (m, 2H), 8.11ꢀ8.18 (m, 1H), 8.59ꢀ8.62 (m, 1H), 10.75 (s,
1
3
1
H), 11.69 (s, 1H). C{ H}ꢀNMR (600 MHz; DMSOꢀd ): 25.1, 33.0, 65.9, 68.0, 73.4, 100.0,
6
09.0, 111.8, 116.3, 120.7, 121.5, 122.4, 123.3, 126.4, 128.5, 132.8, 138.6, 144.4, 145.4, 151.1,
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
.
1
70.9. Anal. Calcd for C H N O 0.33 H O: C 65.4, H 5.7, N 10.9; Found: C 65.0, H 5.3, N
21
21
3
4
2
10.9.
Indirubin-3’-(2-ß-D-glucopyranosylethyl)-oxime ether (6f) (54.0%)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
HꢀNMR (400 MHz; DMSOꢀd ) 2.96ꢀ3.18 (m, 4H), 3.43ꢀ3.45 (m, 1H), 3.66ꢀ3.68 (m, 1H),
6
3
3
4.02ꢀ4.03 (m, 1H), 4.22ꢀ4.24 (m, 1H), 4.30 (d, 1H, J = 7.7 Hz), 4.49 (t, 1H, J = 5.8 Hz), 4.75 (t,
3
3
3
3
2
H, J = 4.9 Hz), 4.90 (d, 1H, J = 5.0 Hz), 4.93 (1H, J = 4.9 Hz), 5.04 (d, 1H, J = 4.9 Hz), 6.89
3
3
(
d, 1H, J = 7.4 Hz), 6.97ꢀ7.03 (m, 2H), 7.14 (t, 1H, J = 7.4 Hz), 7.37ꢀ7.45 (m, 2H), 8.21 (d, 1H,
3
3
13
1
J = 7.7 Hz), 8.59 (d, 1H, J = 7.7 Hz), 10.75 (s, 1H); 11.68 (s, 1H). Cꢀ{ H}ꢀNMR (150 MHz;
DMSOꢀd ) 61.1, 61.3, 67.1, 70.0, 72.4, 73.6, 76.2, 77.2, 100.4, 103.4, 109.1, 111.9, 116.4, 120.9,
6
1
22.6, 123.5, 126.5, 126.6, 132.8, 138.9, 144.1, 145.7, 151.7, 171.1. Anal. Calcd for
.
C H N O H O: C 57.5, H 5.4, N 8.4;. Found: C 57.7, H 5.6, N 8.5.
24
25
3
8
2
Indirubin-3’-{2-[p-(N,N-dimethyl)-amino-anilino]-ethyl}-oxime ether (6g) (84.6%)
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ): 2.67 (s, 6H), 3.53 (t, 2H, J = 5.8 Hz), 4.69 (t, 2H, J = 5.8
6
3
Hz), 6.57ꢀ6.63 (m, 4H), 6.70ꢀ6.89 (m, 4H), 7.14 (t, 1H, J = 7.4 Hz), 7.42ꢀ7.34 (m, 2H), 8.13 (d,
3
3
13
1
1
H J = 7.4 Hz), 8.61 (d, 1H, J = 7.9 Hz), 10.78 (s, 1H), 11.69 (s, 1H). Cꢀ{ H}ꢀNMR (150
MHz, DMSOꢀd ): 39.4ꢀ39.9, 100.2, 109.0, 111.7, 113.5, 115.4, 116.3, 120.1, 121.5, 122.4,
6
.
1
23.3, 126.4, 128.7, 132.8, 138.7, 140.1, 144.0, 145.5, 151.5, 170.9. Anal. Calcd for C H N O
26 25 5 2
0
.5H O: C 69.6, H 5.8, N 15.6; Found: C 69.9, H 6.1, N 15.4.
2
Indirubin-3’-[2-(4-methylpiperazino)-ethyl]-oxime ether (6h) (94.2%)
1
3
HꢀNMR (400 MHz; DMSOꢀd ): 2.12 (s, 3H), 2.48ꢀ2.50 (m, 8H), 2.87 (t, 2H, J = 3.9 Hz),
6
3
3
4.67 (t, 2H, J = 3.9 Hz), 6.89 (d, 1H, 3J = 4.8 Hz), 6.96ꢀ7.02 (m, 2H), 7.14 (dd, 1H, J = 5.0 Hz,
ACS Paragon Plus Environment
22

Page 23 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
3
3
J = 0.6 Hz), 7.40ꢀ7.42 (m, 2H), 8.16 (d, 1H, J = 5.0 Hz), 8.60 (d, 1H, J = 5.2 Hz), 10.76 (s,
1
3
1
1
1
1
H), 11.68 (s, 1H). C{ H}ꢀNMR (150 MHz; DMSOꢀd ): 45.3, 53.7, 54.5, 56.1, 73.9, 100.1,
6
08.7, 111.2, 116.1, 120.2, 121.0, 122.2, 122.9, 126.0, 128.0, 132.4, 138.5, 143.8, 145.3, 151.1,
70.7. Anal. Calcd for C H N O : C 68.5, H 6.3, N 17.4; Found: C 68.2, H 6.2, N 17.2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
3
25
5
2
Indirubin-3’-[2-(4-methylpiperazino)-ethyl]-oxime ether hydrochlorid (6ha) (82.2%)
1
HꢀNMR (400 MHz; D O/DMSOꢀd ): 2.81 (s, 3H), 3.44ꢀ3.64 (m, 10H), 4.88 (s, 2H), 6.93 (d,
2
6
3
3
3
1
H, J = 4.8 Hz), 6.94ꢀ7.04 (m, 2H), 7.15 (t, 1H, J = 7.4 Hz), 7.22 (d, 1H, J = 7.8 Hz), 7.41 (t,
3
3
3
13
1
1H, J = 7.4 Hz), 8.15 (d, 1H, J = 7.4 Hz ), 8.47 (d, 1H, J = 7.8 Hz). C{ H}ꢀNMR (150 MHz;
DMSOꢀd ): 44.4, 51.6, 50.8, 56.8, 71.2, 100.4, 110.9, 112.2, 117.1, 122.7, 123.4, 123.4, 124.8,
6
.
1
27.8, 129.9, 134.5, 138.7, 145.9, 146.0, 153.7, 172.7. C H N O 2HCl: C 58.0, H 5.7, N 14.7;
23 25
5
2
Found: C 57.7, H 5.8, N 14.3.
5-Methoxy-indirubin-3’-(2-ß-D-glucopyranosylethyl)-oxime ether (6j)
1
HꢀNMR (400 MHz; DMSOꢀd ) 2.99ꢀ3.13 (m, 4H), 3.42ꢀ3.45 (m, 1H), 3.65ꢀ3.69 (m, 1H),
6
3
3
3
.78 (s, 3H), 4.01ꢀ4.05 (m, 1H), 4.21ꢀ4.24 (m, 1H), 4.29 (d, 1H, J = 7.7 Hz), 4.48 (t, 1H, J = 5.5
3
3
3
3
Hz), 4.74 (t, 2H, J = 4.8 Hz), 4.91 (d, 1H, J = 4.8 Hz), 4.94 (d, 1H, J = 4.0 Hz), 5.03 (d, 1H, J
3
4
3
3
= 4.4 Hz), 6.74 (dd, 1H, J = 7.4 Hz, J = 2.2 Hz), 6.79 (d, 1H, J = 8.1 Hz), 7.01 (t, 1H, J = 7.3
3
4
Hz), 7.45ꢀ7.38 (m, 2H), 8.23 (d, 1H, J = 7.7 Hz), 8.27 (d, 1H, J = 2.2 Hz), 10.59 (s, 1H), 11.75
1
3
1
(
s, 1H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 55.4, 61.1, 61.2, 66.6, 70.0, 73.4, 76.0, 76.7,
6
7
7.0, 100.9, 103.1, 108.7, 109.3, 111.2, 111.6, 121.5, 123.0, 128.9, 132.5, 132.9, 143.8, 145.4,
.
9
1
51.8, 154.1, 171.0. Anal. Calcd for C H N O 0.5H O: C 57.5, H 5.4, N 8.0;. Found: C 57.3,
2
5
27
3
2
H 5.2, N 8.0.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
-Methoxy-indirubin-3’-[2-(4-methylpiperazino)-ethyl]-oxime ether (6k) (76.1%)
1
3
HꢀNMR (400 MHz; DMSOꢀd ): 2.12 (s, 3H), 2.48ꢀ2.50 (m, 8H), 2.87 (t, 2H, J = 3.9 Hz),
6
3
3
4
3
3
.77 (s, 3H), 4.68 (t, 2H, J = 3.9 Hz), 6.74 (dd, 1H, J = 5.5 Hz, J = 1.8 Hz), 6.79 (d, 1H, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
5
.5 Hz), 7.02 (m, 1H), 7.39ꢀ7.43 (m, 2H), 8.18 (d, 1H, J = 5.0 Hz), 8.29 (d, 1H, J = 1.8 Hz),
10.59 (s, 1H), 11.74 (s, 1H). Anal. Calcd for C H N O : C 66.5, H 6.3, N 16.2; Found: C 66.4,
2
4
27
5
3
H 6.2, N 16.0.
5-Methoxy-indirubin-3’-[2-(4-methyl-piperazino)-ethyl]-oxime ether hydrochloride (6ka)
(88.6%)
1HꢀNMR (400 MHz; D2O/DMSOꢀd6): 2.78 (s, 3H), 3.31ꢀ3.71 (m, 10H), 3.79 (s, 3H), 4.93 (s,
2
H), 6.74ꢀ6.82 (m, 2H), 7.02 (m, 1H), 7.40ꢀ7.45 (m, 2H), 8.23ꢀ8.26 (m, 2H), 10.64 (s, 1H), 11.76
.
.
(
s, 1H). Anal. Calcd for C H N O 2HCl H2O: C 55.0, H 6.0, N 13.4; Found: C 54.6, H 5.9, N
24 27 5 3
1
3.0.
5-Methoxy-indirubin-3’-(2,3-dihydroxypropyl)-oxime ether (6l) (81.3%, E738)
1
3
HꢀNMR (400 MHz; DMSOꢀd ) 3.51 (t, 2H, J = 5.5 Hz), 3,79 (s, 3H), 3.97 (m, 1H), 4.53 (dd,
6
2
3
2
3
3
1
H, J = 10.6 Hz, J = 4.3 Hz), 4.64 (dd, 1H, J = 10.6 Hz, J = 4.3 Hz), 4.77 (t, 1H, J = 5.9 Hz),
3
3
4
3
5.10 (d, 1H, J = 5.5 Hz), 6.73 (dd, 1H, J = 8.2 Hz, J = 2.4 Hz), 6.79 (d, 1H, J = 8.6 Hz), 7.03
3
4
(
m, 1H), 7.42 (m, 2H), 8.19 (d, 1H, J = 7.4 Hz), 8.30 (d, 1H, J = 2.7 Hz), 10.58 (s, 1H), 11.76
1
3
1
(
s, 1H). C{ H}ꢀNMR (600 MHz; DMSOꢀd ): 55.5, 62.8, 69.8, 78.5, 100.8, 108.6, 109.3, 111.7,
6
1
12.9, 116.3, 121.5, 123.0, 128.6, 132.5, 132.9, 144.0, 145.4, 151.5, 154.2, 171.0. Anal. Calcd
for C H N O : C 63.0, H 5.0, N 11.0; Found: C 62.8, H 5.0, N 10.9.
2
0
19
3
5
5-Methyl-indirubin-3’-(2-ß-D-glucopyranosylethyl)-oxime ether (6m)
ACS Paragon Plus Environment
24

Page 25 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
HꢀNMR (400 MHz; DMSOꢀd ) 2.33 (s, 3H), 2.99ꢀ3.14 (m, 1H), 3.41ꢀ3.46 (m, 1H), 3.65ꢀ3.67
6
3
(
m, 1H), 3.78 (s, 3H), 4.01ꢀ4.05 (m, 1H), 4.21ꢀ4.24 (m, 1H), 4.29 (d, 1H, J = 7.7 Hz), 4.48 (t,
3
3
3
3
1
5
H, J = 5.5 Hz), 4.74 (t, 2H, J = 4.8 Hz), 4.91 (d, 1H, J = 4.8 Hz), 4.94 (d, 1H, J = 4.0 Hz),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
3
3
.03 (d, 1H, J = 4.4 Hz), 6.79 (d, 1H, J = 8.0 Hz), 7.00ꢀ7.08 (m, 2H), 7.42 (d, 1H, J = 7.9 Hz),
3
13
1
7.58 (m, 1H), 7.64 (d, 1H, J = 7.7 Hz), 8.63 (s, 1H), 10.78 (s, 1H), 11.00 (s, 1H). Cꢀ{ H}ꢀ
NMR (150 MHz; DMSOꢀd ) 21.1, 61.1, 61.2, 66.6, 70.0, 73.4, 76.0, 76.7, 77.0, 99.5, 109.0,
6
1
16.9, 121.8, 129.7, 124.2, 126.5, 128.2, 126.9, 129.5, 132.5, 136.7, 145.1. 145.2, 151.4, 171.2.
Anal. Calcd for C H N O : C 60.4, H 5.5, N 8.5;. Found: C 60.0, H 5.8, N 8.6.
2
5
27
3
8
5
-Hydroxy-indirubin-3’-(2-ß-D-glucopyranosylethyl)-oxime ether (6n)
1
HꢀNMR (400 MHz; DMSOꢀd ) 3.00ꢀ3.14 (m, 1H), 3.40ꢀ3.46 (m, 1H), 3.65ꢀ3.70 (m, 1H),
6
3
3
3
.79 (s, 3H), 4.00ꢀ4.05 (m, 1H), 4.21ꢀ4.25 (m, 1H), 4.30 (d, 1H, J = 7.7 Hz), 4.47 (t, 1H, J = 5.5
3
3
3
3
Hz), 4.75 (t, 2H, J = 4.8 Hz), 4.90 (d, 1H, J = 4.8 Hz), 4.94 (d, 1H, J = 4.0 Hz), 5.00 (d, 1H, J
3
= 4.4 Hz), 6.60 (d, 1H, 3J = 7.6 Hz), 6.65 (d, 1H, J = 8.3 Hz), 6.92ꢀ7.02 (m, 1H), 7.40ꢀ7.35 (m,
4
3
2
H), 8.13 (d, 1H, J = 2.1 Hz), 8.22 (d, 1H, J = 7.5 Hz), 8.71 (s, 1H), 10.38 (s, 1H), 10.41 (s,
1
3
1
1
H). Cꢀ{ H}ꢀNMR (150 MHz; DMSOꢀd ) 61.1, 61.2, 66.6, 70.0, 73.2, 76.1, 76.9, 77.2, 100.8,
6
109.6, 110.1, 111.3, 112.5, 118.6, 121.5, 122.6, 129.1, 131.5, 132.3, 145.0, 145.2, 152.4, 153.6,
71.3. Anal. Calcd for C H N O : C 57.7, H 5.1, N 8.4; Found: C 57.4, H 5.0, N 8.8.General
1
2
4
25
3
9
procedure for the synthesis of indirubin-5’-derivatives (23) (yield)
Under argon atmosphere at room temperature, a suspension of 3ꢀindoxyl acetate (1.1 mmol),
isatin (1.2 mmol) and sodium carbonate (2.5 mmol) in degassed methanol (25 mL) was stirred
for 5 h. The reddish precipitate was filtered off, washed with methanol and water and dried to
afford indirubine.
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
’-Nitroindirubin (23a) (87.5 %):
1
3
3
HꢀNMR (400 MHz, DMSOꢀd ): 6.89 (d, 1H, J = 7.9 Hz), 7.03 (t, 1H, J = 7.5 Hz), 7.29 (t,
6
3
3
4
3
1
H, J = 7.3 Hz), 7.58 (d, 1H, J = 9.0 Hz), 8.36 (d, 1H, J = 2.2 Hz), 8.41 (dd, 1H, J = 8.9 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
13
1
J = 2.2 Hz), 8.75 (d, 1H, J = 7.7 Hz), 10.98 (s, 1H, NH), 11.57 (s, 1H, NH). Cꢀ{ H}ꢀNMR
(600 MHz, DMSOꢀd ): 110.1. 110.2, 114.0, 119.1, 120.4, 121.2, 121.7, 125.6, 130.8, 132.0,
6
1
37.9, 141.4, 142.0, 156.0, 170.4, 187.0. Anal. Calcd for C H N O : C, 62.6; H, 3.0; N, 13.7.
16 9 3 4
Found: C, 62.2; H, 2.9; N, 13.5.
5’-Aminoindirubin (23b) (50.0 %):
1
3
3
4
HꢀNMR (400 MHz, DMSOꢀd ): 6.90 (d, 1H, J = 7.7 Hz), 7.01 (dt, 1H, J = 7.5 Hz, J = 0.7
6
3
4
3
3
Hz), 7.26 (dt, 1H, J = 7.7 Hz, J = 1.1 Hz), 7.48 (d, 1H, J = 8.8 Hz), 7.51 (dd, 1H, J = 8.6 Hz,
3
4
3
J = 1.8 Hz), 7.59 (d, 1H, J = 1.8 Hz), 8.74 (d, 1H, J = 7.7 Hz), 10.02 (s, 2H), 10.94 (s, 1H),
13
1
1
1.10 (s, 1H). Cꢀ{ H}ꢀNMR (600 MHz, DMSOꢀd ): 107.8, 109.9, 114.7, 118.6, 119.5, 121.5,
6
125.0, 126.0, 129.9, 131.6, 138.4, 141.4, 151.4, 151.6, 170.9, 188.0. Anal. Calcd for
C H N O : C, 69.3; H, 4.0; N, 15.2. Found: C, 69.2; H, 3.9; N, 15.3.
16
11
3
2
5’-Hydroxyindirubin (23c) (58.0 %):
1
3
4
HꢀNMR (400 MHz; DMSOꢀd ): 6.88 (d, 1H, J = 7.8 Hz), 6.97 (d, 1H, J = 2.7 Hz), 6.99 (t,
6
3
3
4
3
1
H, J = 7.8 Hz), 7.03 (dd, 1H, J = 8.5 Hz, J = 2.4 Hz), 7.22 (m, 1H), 7.23 (d, 1H, J = 8.5 Hz
3
13
1
)
, 8.71 (d, 1H, J = 7.5 Hz), 9.6, (s, 1H), 10.77 (s, 1H), 10.84 (s, 1H). Cꢀ{ H}ꢀNMR (600 MHz,
DMSOꢀd ): 105.7, 109.2, 109.7, 114.5, 119.7, 121.4, 121.8, 124.6, 125.3, 129.0, 139.4, 140.8,
6
1
46.1, 152.6, 171.2, 189.2. Anal. Calcd for C H N O : C, 69.1; H, 3.6; N, 10.1. Found: C, 69.0;
16 10 2 3
H, 3.7; N, 10.1.
ACS Paragon Plus Environment
26

Page 27 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
’-Carboxyindirubin (23d) (70.9 %):
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ): 6.88 (d, 1H, J = 7.9 Hz), 7.02 (d, 1H, J = 7.8 Hz), 7.26 (d,
6
3
3
3
1
H, J = 7.8 Hz), 7.47 (d, 1H, J = 9.1 Hz), 8.09 (m, 2H), 8.76 (d, 1H, J = 7.8 Hz), 10.93 (s,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
1
1
H), 11.23 (s, 1H), 12.87 (s, 1H). Cꢀ{ H}ꢀNMR (600 MHz, DMSOꢀd ): 108.2, 109.6, 113.2,
6
118.9, 121.2, 121.3, 123.8, 125.0, 125.5, 129.8, 137.7, 138.1, 141.3, 154.9, 166.4, 170.6, 187.7.
.
Anal. Calcd for C H N O 0.5 H O: C, 64.8; H, 3.5; N, 8.9. Found: C, 64.9; H, 3.3; N, 8.8.
1
7
10
2
4
2
General procedure for the synthesis of indirubin-5’-carboxamides (23e, 23f and 23g)
(yield)
Under argon a solution of 23k (0.43 mmol), amino compound (0.6 mmol) and DMAP (0.49
mmol) in dried dioxane (12 mL) was refluxed for several hours till no starting material was
detected by TLC. After cooling the mixture was diluted with 0.1 N HCl (100 mL). The
precipitate was filtered and dried in vacuo to afford indirubinꢀ5’ꢀcarboxamides
5’-(4-Methylpiperazinocarbonyl)-indirubin (23e) (72.5 %):
1
3
HꢀNMR (400 MHz; DMSOꢀd ): 2.19 (s, 3H), 2.31 (s, 4H), 3.48 (m, 4H), 6.90 (d, 1H, J = 7.4
6
3
3
3
Hz), 7.02 (t, 1H, J = 7.4 Hz), 7.25 (d, 1H, J = 7.8 Hz), 7.46 (d, 1H, J = 8.2 Hz), 7.60ꢀ7.62 (m,
3
.
2H), 8.76 (d, 1H, J = 7.8 Hz), 10.92 (s, 1H), 11.12 (s, 1H). Anal. Calcd for C H N O HCl: C,
2
2
20
4
3
6
2.12; H, 5.0; N, 13.2. Found: C, 62.3; H, 5.0; N, 13.2.
5
’-{N-[2-(Dimethylamino)-ethyl]-aminocarbonyl}-indirubin (23f) (37.2 %):
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ): 2.16ꢀ2ꢀ40 (m, 10H), 6.90 (d, 1H, J = 7.4 Hz), 7.02 (t, 1H, J
6
3
3
3
=
7.4 Hz), 7.26 (t, 1H, J = 7.4 Hz), 7.45 (d, 1H, J = 8.7 Hz), 8.06 (d, 1H, J = 8.2 Hz), 8.17 (s,
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
13
1
1
H), 8.46 (s, 1H), 8.77 (d, 1H, J = 7.8 Hz), 11.15 (s, 2H). Cꢀ{ H}ꢀNMR (600 MHz, DMSOꢀ
d6): 37.4, 45.3, 58.3, 108.2, 109.6, 113.2, 118.9, 121.2, 121.3, 123.8, 125.0, 125.5, 129.8, 137.7,
.
1
38.1, 141.3, 154.9, 166.4, 170.6, 187.7. Anal. Calcd for C H N O HCl: C, 61.1; H, 5.1; N,
2
1
20
4
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3.6. Found: C, 61.3; H, 5.3; N, 13.5.
5’-{N-[4-(4-Methylpiperazino)-phenyl]-aminocarbonyl}-indirubin (23g) (57.9 %):
1
HꢀNMR (400 MHz; DMSOꢀd ): 2.23 (s, 3H), 2.54 (m, 4H), 3.30 (m, 4H), 6.97 (m, 3H), 7.09
6
3
3
3
3
(
t, 1H, J = 7.8 Hz), 7.33 (g, 1H, J = 8.2 Hz), 7.55 (d, 1H, J = 8.2 Hz), 7.66 (d, 2H, J = 9.0
3
3
Hz), 8.21 (d, 1H, J = 7.8 Hz), 8.38 (s, 1H), 8.84 (d, 1H, J = 7.8 Hz), 10.13 (s, 1H), 11.01 (s,
13
1
1
H), 11.31 (s, 1H). Cꢀ{ H}ꢀNMR (600 MHz, DMSOꢀd ): 45.8, 48.8, 54,6, 107.9, 109.7, 113.2,
6
1
15.4, 118.7, 121.3, 121.4, 121.5, 123.7, 125.0 , 127.7, 129.8, 131.0, 136.7, 138.4, 141.3, 147.5,
.
3
1
54.2, 163.7, 170.8, 188.2. Anal. Calcd for C H N O 3HCl: C, 57.1; H, 4.8; N, 11.9. Found:
2
8
25
5
C, 57.3; H, 4.6; N, 11.8.
5,5’-Dicarboxyindirubin (23h) (77.6 %):
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ): 6.96 (d, 1H, J = 7.9 Hz), 7.48 (d 1H, J = 8.8 Hz), 7.88 (dd,
6
3
4
3
1
H, J = 7.9 Hz, J = 1.3 Hz), 8.10ꢀ8.12 (m, 2H), 9.42 (d, 1H, J = 0.9 Hz), 11.27 (s, 1H), 11.36
1
3
1
(s, 1H), 12.56 (s, 2H). Cꢀ{ H}ꢀNMR (600 MHz, DMSOꢀd ): 107.0, 109.4, 113.5, 119.1, 121.2,
6
1
23.8, 123.9, 125.7, 126.6, 131.5, 138.0, 138.9, 144.7, 155.2, 166.5, 167.5, 172.1, 187.9. Anal.
.
Calcd for C H N O H O: C, 58.7; H, 3.3; N, 7.6. Found: C, 58.4; H, 3.5; N, 7.8.
18 10
2
6
2
5
,5’-Dimethoxyindirubin (23i) (88.1 %)
1
3
3
HꢀNMR (400 MHz; DMSOꢀd ): 3.76ꢀ3.77 (m, 6H), 6.78 (d, 1H, J = 8.6 Hz), 6.83 (d, 1H, J
6
3
3
= 2.7 Hz), 7.18ꢀ7.21 (m, 2H), 7.34 (d, 1H, J = 8.6 Hz), 8.47 (d, 1H, J = 2.4 Hz), 10.66 (s, 1H),
ACS Paragon Plus Environment
28

Page 29 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
.
1
0.84 (s, 1H). Anal. Calcd for C H N O 0.33H O: C, 65.9; H, 4.5; N, 8.5. Found: C, 65.9; H,
1
8
14
2
4
2
4.7; N, 8.3.
Pentafluorophenyl-indirubin-5’-carboxylate (23k) :
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Under argon atmosphere a solution of indirubinꢀ5’ꢀcarboxylic acid (450 mg, 0.96 mmol),
pentafluorophenyl trifluoroacetate (800 mg, 2.86 mmol), pyridine (0.25 mL) and catalytic
amount of dimethylaminopyridine (DMAP) in dried DMF (15 mL) was stirred at room
temperature for 4 h, diluted with ethyl acetate and washed with 0.1 N HCl (100 mL). After
removal of solvent, the residue was dried in vacuo to yield a reddish solid 23k (439.8 mg, 0.93
1
3
3
mmol, 97.0 %). HꢀNMR (400 MHz; DMSOꢀd ): 6.89 (d, 1H, J = 7.8 Hz), 7.02 (t, 1H, J = 7.4
6
3
3
3
4
Hz), 7.28 (t, 1H, J = 7.0 Hz), 7.62 (d, 1H, J = 8.7 Hz), 8.27 (dd, 1H, J = 8.7 Hz, J = 1.6 Hz),
4
3
8
.34 (d, 1H, J = 1.6 Hz), 8.75 (d, 1H, J = 7.8 Hz), 11.06 (s, 1H), 11.52 (s, 1H).
General procedure for the synthesis of 5’-indirubin-3’-oxime (24)
A mixture of indirubin (1.1 mmol) and hydroxylamine hydrochloride (24.4 mmol) in pyridine
(
7 mL) was refluxed for 4 h. The mixture was diluted with 0.1N HCl (50 mL) to yield a reddish
solid 24
5’-Hydroxyindirubin-3’-oxime (24c) (93.0 %):
1
4
4
HꢀNMR (400 MHz; DMSOꢀd ): 6.81 (d, 1H, J = 1.7 Hz), 6.93 (d, 1H, J = 1.7 Hz), 6.92 (dd,
6
3
4
3
3
1
H, J = 5.6 Hz, J = 0.8 Hz), 7.08 (m, 1H), 7.25 (d, 1H, J = 5.6 Hz), 7.72 (d, 1H, J = 1.5 Hz),
3
8
.58 (d, 1H, J = 5.1 Hz), 9.20 (s, 1H), 10.64 (s, 1H,), 11.58 (s, 1H), 13.44 (s, 1H). Anal. Calcd
.
for C H N O H O: C, 61.7; H, 4.2; N, 13.5. Found: C, 61.8; H, 4.5; N, 13.1.
1
6
11
3
3
2
ACS Paragon Plus Environment
29