EP1231916A2 - Use of indirubine derivatives for making medicines - Google Patents

Abstract

The invention concerns the use of indirubine derivatives for making medicines inhibiting GSK-3 beta . The invention is useful for treating pathologies involving GSK-3 beta and CDK5.

Description

 use of indirubin derivatives for the manufacture of medicaments.

The subject of the invention is a new use in therapy of indirubin derivatives. indirubin belongs to the indigoids family. The term indigoide is used as the generic name for the dyes in the indigo group. These are bis-indoles, derived from various natural sources by fermentation, oxidation and dimerization in the presence of light.

Indirubin (or isoindigotine), corresponds to formula A

It is the active ingredient in Danggui Longhui an, used in traditional Chinese medicine to treat chronic diseases, such as leukemia.

In application EP 98 109 854.2, in which some of the inventors of the application are co-inventors, it is reported that derivatives of indigoids, among which indirubin and its derivatives, are powerful inhibitors of cyclin-dependent kinases (CDKs for short), with IC ₅₀ (50% inhibitory dose) from 50 to 200 nM (see also Hoessel et al., Nature Cell Biology, vol 1, no 1, May 1999). These kinases are key regulators of the cell cycle. Inhibitors compete with ATP to bind to the catalytic kinase subunit.

These CDK inhibitory properties, which cause a cell cycle arrest, give these derivatives of ¹ indigoids an interest in the treatment of pathologies linked to the loss of proliferation control such as cancers, psoriasis, cardiovascular diseases, infectious diseases, nephrology, neurodegenerative diseases and viral infections.

Surprisingly, the inventors have now demonstrated that, among these indigoids derivatives, only the indirubin derivatives also exert an inhibitory effect on another enzymatic target, constituted by glycogen synthase kinase-3β or GSK- 3β for short.

This kinase is an essential part of the WNT signal pathway. It is involved in multiple physiological processes: regulation of the cell cycle by controlling the levels of cyclin Dl and β-catenin, dorsoventral formation during development, action of insulin on glycogen synthesis, axonal outgrowth, neurotoxicity of HIV-1 mediated by Tat, et al.

In addition, it is known that GSK-3β and CDK5 are responsible for a good part of the abnormal hyperphosphorylation of the tau protein binding microtubules as observed in the helically paired filaments in Alzheimer's disease.

We also measure the interest of having derivatives inhibiting the activity of GSK-3β to promote cell division.

However, the only inhibitors of GSK-3β known to date consist of lithium and certain purine derivatives.

The selectivity of lithium has not been reported, but given the atomic nature of the product, it is likely to be very low. In addition, lithium only acts at considerable doses (IC ₅₀ around 10 M). It is the same with the purine derivatives described in application WO 98/16528, which are not very selective and whose IC ₅₀ are around 10 μM.

The invention provides a solution to these problems with the use of high-efficiency indirubins with IC _{50 of} less than 10 μM, and more generally of the order of 5 to 50 nM, for the manufacture of GSK inhibitor drugs. -3β.

In accordance with the invention, for the manufacture of the said drugs, indirubin derivatives corresponding to the general formula I are used:

.R ¹

in which R and R, identical or different, represent a hydrogen atom, a halogen atom; a hydroxy group; a methylenehydroxy group; an alkyl or alkyloxy or methylenealkoxy radical, with straight or branched chain, from C1 to C18; a cycloalkyl radical having 3 to 7 carbon atoms, optionally comprising one or more heteroatornes; a substituted or unsubstituted aryl, aralkyl or aryloxy radical, optionally comprising one or more heteroatoms; a mono-, di- or trialkylsilyl group having 1 to 6 carbon atoms, independently of each other, in each case, in the straight or branched chain alkyl group; a mono-, di- or triarylsilyl group, with substituted or unsubstituted aryl groups, independently of each other, in each case; a trifluoromethyl group; a -COM group; -COOM; or -CH ₂ COOM, with M representing a hydrogen atom, a C1 to C18 alkyl group, with straight or branched chain, optionally substituted by one or more groups hydroxy and / or amino, or an aryl group optionally comprising one or more heteroatoms, which may be substituted by one or more halogen atoms, alkyl radicals or alkoxy group; a group -NR ¹ R, in which R ¹¹ and R ¹ , which are identical or different, represent a hydrogen atom, an alkyl radical with a straight or branched chain, from C1 to C18, optionally also substituted by one or more hydroxy and / or amino groups, an aryl group, substituted or not, optionally comprising one or more heteroatoms; an acyl group; a methyleneamino group -CH ₂ -NR ^{ι: ι} R ¹² , in which R ¹¹ and R have the above meanings; a benzyl group, in which the benzene ring comprises, where appropriate, one or more heteroatoms; a methylenecycloalkyl group with 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a physiological amino acid residue bound to nitrogen, such as an amide; an O-glycoside or an N-glycoside, in which the glycoside is chosen from monosaccharides or disaccharides; or a methylenesulfonate group; R, R, R ⁴ , R ⁵ , R 7, R8, R9 and R10, identical or different, represent a hydrogen atom; halogen; a hydroxy group; a nitroso group; a nitro group; an alkoxy group; a C1 to C18 straight or branched chain alkyl group, optionally substituted by one or more hydroxy and / or amino groups; an aryl group, substituted or not, optionally comprising one or more heteroatoms; an aralkyl group, substituted or not, optionally comprising one or more heteroatoms; an aryloxy group, substituted or not, optionally comprising one or more heteroatoms; a methylenearyloxy group, substituted or not, optionally comprising one or more heteroatoms; a cycloalkyl group, with 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a methylenecycloalkyl group, with 3 to 7 carbon atoms, comprising if necessary one or more heteroatoms; a trifluoromethyl group; a -COM group; -COOM; or CH ₂ COOM, with M representing a hydrogen atom, a C1 to C8 alkyl group, with straight or branched chain, optionally further substituted by one or more hydroxy and / or amino groups, or an aryl group comprising if necessary one or more heteroatoms, which may be substituted by one or more halogen atoms, alkyl groups or alkoxy groups; a group -NR ¹¹ R ¹² , in which

R ¹¹ and R, identical or different, represent a hydrogen atom, an alkyl radical with a straight or branched chain, from C1 to C18, optionally substituted further by one or more hydroxy and / or amino groups, an aryl group substituted or unsubstituted, optionally comprising one or more heteroatoms, an acyl group, or in which the nitrogen atom is part of a cycloalkyl group having 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a group -CONR ^{1: L} R ¹² , in which R ¹¹ and R have the above meanings; a hydroxylamino group; a phosphate group; a phosphonate group; a sulfate group; a sulfonate group; a sulfonamide group; a group -S0 ₂ NR ^1X R ¹² , in which R ¹¹ and R ¹² have the meanings given above; an azo group - N = NR, in which R represents an aromatic group, optionally substituted by one or more carboxyl, phosphoryl or sulfonate groups, or an O-glycoside or N-glycoside group, in which the glycoside is chosen from monosaccharides or dissacharides; or R and R ⁵ , and R and R, respectively, together form, independently of one another, a ring having 1 to 4 CH ₂ groups, optionally substituted; and X and Y, identical or different, represent an oxygen atom; sulfur; selenium; tellurium; a group -NR ⁴ , in which R ⁴ represents a hydrogen atom, an alkyl group, with a straight or branched chain, from C1 to C18, optionally substituted by one or more carboxyl, phosphoryl or sulfonate, a substituted or unsubstituted aryl group, optionally comprising one or more heteroatoms, an aralkyl group, or a sulfonate group; or -NOR ¹⁴ , in which the group R ⁴ has the meanings given above, and the salts of these physiologically acceptable derivatives.

In one arrangement of the invention, one or more atoms of one or more benzene rings are replaced by nitrogen atoms.

In another arrangement, one or more aromatic or non-aromatic ring systems, which may include one or more heteroatoms independently of one another, are condensed to the indirubin system.

In yet another arrangement, the indirubin derivatives of formula (I) are linked to a polyethylene glycol ester or to a polyethylene glycol ether by bonds, respectively, ester or ether.

The invention relates more specifically to the indirubin derivatives having an IC ₅₀ with respect to GSK-3β of less than 10 μM and preferably of 1 μM, and in particular those with an IC _{50 of} less than 50 nM.

Preferably, in such derivatives, X and Y, identical or different, represent a group = O or = NOH.

In the corresponding families, the substituents R ¹ , R ³ , R ⁴ and R advantageously have the following meanings:

R: a hydrogen atom, halogen, alkyl, -S0 ₃ H, -S0 ₂ NH _2, -S0 ₂ -N (CH _{3) 2,} -S0 ₂ -NC ₂ H ₅ - OH, - S0 ₂ -N- (C ₂ H ₅ -OH) ₂ , -S0 ₂ -NH-CH ₃ ,

- R ⁴ and R ⁸ , independently of one another: a hydrogen or halogen atom,

R: an alkyl or aryl radical, the other substituents given in formula (I) representing a hydrogen atom. In a preferred family according to the invention,

X = Y, these two substituents representing a group = O.

Derivatives of this family constituting high-efficiency GSK-3β inhibitors, with IC _{50 of} less than 5 μM, most generally of 1 μM, or even 50 nM, advantageously include substituents R ¹ , R ³ , R ⁴ and R ⁸ with the following meanings:

- R ¹ : alkyl, in particular methyl, and phenyl,

- R: hydrogen, halogen (F, Cl, Br, I), N0 _2, - S0 ₃ H, -S0 ₂ NH _2, -S0 ₂ -N (CH _{3) 2,} -S0 ₂ -NC ₂ H ₅ - 0H, -S0 ₂ -N- (C ₂ H ₅ -0H) ₂ , -S0 ₂ -NHCH ₃ ,

-R ⁴ and R: halogen, in particular I or Br, the other substituents given in formula (I) representing a hydrogen atom. Particularly preferred derivatives are chosen from indirubin, 5-iodo-indirubin, 5-bromo-indirubin, 5-chloro-indirubin, 5-fluoro-indirubin, 5-methyl-indirubin, 5-nitro -indirubin, 5-SO ₃ H-indirubin, 5'-bromo-indirubin, 5-5 '-dibromo-indirubin or 5'-bromo-indirubin 5-sulfonic acid.

In another preferred family according to the invention, X represents a group = NOH and Y a group = O.

In a preferred group of this family, R ³ represents a halogen atom, in particular I, or a group -SO ₃ Na.

The corresponding derivatives advantageously have an IC ₅₀ with respect to GSK-3β of less than 100 nM, and even for many of them less than 50 nM.

Preferred derivatives of this group are chosen from indirubin-3 '-monoxime, 5-iodo-indirubin-3' -monoxime and 5-SO ₃ Na-indirubin-3 '-monoxime.

As indicated above, the derivatives defined above have already been described as inhibitors of CDKs, as well as other indigoids derived, for example, from indigo or isoindigo. However, surprisingly, only the indirubin derivatives also exert an inhibitory effect vis-à-vis GSK-3β. This effect is most generally of the same order of magnitude with respect to CDKs and GSK-3β.

The drugs produced in accordance with the invention using the indirubin derivatives defined above can be used for the treatment of pathologies in which GSK-3β is involved.

This is the case, for example, with diabetes, where GSK-3β inhibitors can be used as insulin mimetics. It is recalled that insulin acts by a cascade of biochemical events leading to an inhibition of GSK-3β and that this inhibition is responsible for the response of cells to 1 insulin.

Likewise, these drugs are of great interest for the treatment of neurodegenerative diseases. As demonstrated in the examples, the hyperphosphorylation of the tau protein caused by CDK5 and GSK-3β can indeed be inhibited by the indirubin derivatives. By administering the drugs produced according to the invention, it is then possible, thanks to their inhibitory effect on both CDK5 and GSK-3β, to prevent the hyperphosphorylation of the tau protein in Alzheimer's patients and to fight against neurodegeneration.

These drugs also find an application of great interest for the treatment of manic-depressive illnesses.

Mention will also be made of their use for the treatment of cancers where their inhibitory effect on both GSK-3β and CDK5, which results in apoptosis of the tumor cell, is advantageously used. These drugs are also effective for the treatment of diseases caused by single-celled parasites such as malaria, trypanosomes, leishmanias, toxoplasmas, pneumocystis and others, or multi-cellular parasites, such as fungi and worms.

During the preparation of the drugs, the active ingredients, used in therapeutically effective amounts, are mixed with the pharmaceutically acceptable vehicles for the chosen mode of administration. Thus for oral administration, the drugs are prepared in the form of capsules, tablets, dragees, capsules, pills, drops and the like. Such drugs may contain from 1 to 100 mg of active ingredient per unit. For injectable administration

(intravenous, subcutaneous, intramuscular), the drugs come in the form of sterile or sterilizable solutions.

The doses per unit of intake can vary from 1 to 50 mg of active ingredient. The daily dosage is chosen so as to obtain a final concentration of at most 100 μM of derivative of ¹ indirubin in the blood of the treated patient.

In order to illustrate the invention, without however limiting its scope, other characteristics and advantages are reported in the examples which follow. In these examples, reference will be made to FIGS. 1 to 3, which respectively represent,

- Figures 1A to 1C, the inhibitory effects of indirubins used according to the invention with respect to GSK-3β, CDK5 / p25 and CDKl / cyclin B, - Figure 2, the competitive effect with ATP for fixation with GSK-3β, and

- Figure 3, the inhibitory effect by indirubins, in vitro, of tau phosphorylation by GSK-3β. . Characterization of indirubins

The elementary analyzes were carried out using an elementary analysis apparatus of CHN Perkin-Elmer 2400. The spectra of RM ^ H were recorded at 400 MHz, of NMR ¹³ C at 100 MHz on a Bruker AMX apparatus. 400, with internal reference of tetramethylsilane. s denotes a singlet, d, a doublet and m, a multiplet. The mass spectra were taken according to the positive ion mode under electronic impact (El 70) and with a Finingan MAT 90 device.

. Examples of indirubin syntheses indirubin

Indirubin is prepared according to Russell et al,

J.Am.Chem. Soc. 1969, 91, 3851-3859 (modified method) using 1.76 g (12.0 mmol) of isatin and 2.00 g (11.4 mmol) of indoxylacetate. After filtration, the residue is washed 2 times with methanol, and several times with cold water, until the filtrate is neutral. The product is dried over KOH. 2.42 g (81.0%) of dark violet crystals are obtained Ry = 0.64 (ethyl acetate hexane 1 / lv / v); Mp 341-343 ° C: Hî-NMR (DMSO-d ₆ ) δ 6.91 (d, J = 8.1, C7H), 7.02 (m, C5H and C5'H), 7.26 (m , C6H), 7.42 (d, J = 8.1, C7'H), 7.58 (m C6'H), 7.66 (d, J ^" = C4'H), 8.77 (d , J = 7.7, C4H), 11.01 (s) and 10.88 (s) (N1H and NI Η); ¹³ C-NMR (DMSO -d ₆ , 90 ° C) δ 106.97 (s , C3), 109.57 (d, J = 162.2, C7), 113.15 (d, J = 168.7, C7 '), 119.37 (s, C3a'), 121.21 (d , J = 162.1, Hz) and 121.19 (d, J = 162.1 Hz) (C5 and C5 '), 121.58 (s, C3a), 124.62 (d, J = 164.6 , Hz) and 124.20 (d, J = 163.8, Hz) (C6 and C4 '), 129.15 {d, J = 159.8, C4), 136.85 (d, J ^" = 161 , 4, C6 '), 138.48 (s, C7a), 141.07 (s, C7a'), 152.35 (s, C2 *), 171.11 (s, C2), 188.27 (s , C3 '); MS m / e 262 (M ^* , 100), 234 (43), 205 (25), 131 (4). Anal. (C ₁₆ H ₁₀ N ₂ O ₂ ) C, H, N. 5-iodoindirubine The procedure is as described for one indirubin. Starting from 655 mg of 5-iodoisatin (2.40 mmol) and 399 mg of indoxylacetate

(2.28 mmol, the reaction leads to 720 mg of crude product. By recrystallization from ethanol, 58 mg (6.2%) of dark purple crystals are obtained; R _f = 0.68 (ethyl acetate / hexane

1/1 v / v); Mp 334-335 ° C: ^X H-NMR (DMSO-d ₆ ) δ 6.75 (d, J = 8.1, C7H), 7.04; 1H (m, C5'H), 7.42 ( d, "7 = 8.1, C7'H), 7.57 (m, C6H and C6'H), 7.65 (d, J = 7.5, C4'H), 9.11 (s, C4H), 11.00 (s) and 11.09 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO -d ₆ , 90 ° C) δ 83.73 (d, J = 16.9, C5), 105.18 (s, C3), 111.85 (d, J = 164.6 , C7), 113.43

(d, J = 167.2, C7 '), 119.27 (s, C3a'), 121.65 (d, J = 163.6, C5 '),

124.03 (s, C3a), 124.44 (d, J = 163.9, Hz, C4 '), 132.49 (d, J =

171.2, C6), 137.00 (d, J = 166.9, Hz) and 137.15 (d, J = 160.1

Hz) (C4 and C6 '), 139.26 (s, C7a'), 140.44 (s, C7a), 152.41 (s, C2 '), 170.48 (s, C2), 188.54 (s, C3 '); MS m / e 388 (M ⁺ , 100), 360 (3), 261 (6), 233 (16), 205 (16). Anal. (C ₁₆ H ₉ IN ₂ 0 ₂ ) C, H, N. 5-bromoindirubine

The procedure is as described for one indirubin. Starting from 1.30 g of 5-bromoisatin (5.75 mmol) and 1.00 g of indoxylacetate (5.71 mmol), the crude product is recrystallized from pyridine and leads to 1.16 g (59 , 3%) of black crystals with a violet hue; R _j = 0.49 (ethyl acetate hexane l / lv / v): ^ -RMN (DMSO-d ₆ ) δ 6.86 (d, J = 8.4, C7H), 7.05 (pt, C5'H), 7.41- 7.31 (m, C6H and C7'H), 7.59 (m, C6'H), 7.66 (d, J ≈ 7.5, C4'H), 8.94 (s, C4H), 11.10 (s) and 11.00 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO -d ₆ , 90 ° C) δ 104.92 (s, C3), 111.24 (d, J = 165.1, C7), 112.89 (s, C5), 113, 53 (d, J = 167.8, C7 '), 118.92 (s, C3a'), 121.64 (d, ^" = 164.4, C5 '), 123.41 (s, C3a), 124 , 49 (d, J = 164.0, C4 '), 126.60 (d, J = 171.3, C6), 131.03 (d, 167.1, C4), 137.29 (d, J ^" = 160.9, C6 '), 139.16 (s, C7a'), 139.74 (s, C7a), 152.42 (s, C2 '), 170.50 (s, C2), 188, 77 (s, C3 '); MS m / e 342 (M ^* _f 100), 340 (M0 100) 314 (18), 312 (18), 261 (7). Anal. (C ₁₆ H ₉ BrN ₂ 0 ₂ ) C, H, N. 5-chloroindirubine

The procedure is as described for one indirubin. Starting from 500 mg of

5-chloroisatin (2.75 mmol) and 480 mg of indoxylacetate (2.73 mmol), 766 mg (94.6%) of a purple powder are obtained, R, =

0.60 (ethyl acetate / hexane 1/1 v / v): Hî-NMR (DMSO-d ₆ ) δ 6.89

(d, J = 8.3, C7H), 7.04 (m, C5'H), 7.27 (d, J = 8.3, C6'H), 7.42

(d, J = 7.8, C7'H), 7.58 (m, C6 Η), 7.65 (d, J = 7.6, C4'H), 8.78

(s, C4H), 10.99 (s) and 11.09 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO-d ₆ ) δ

104.95 (s, C3), 110.61 (d, "7 = 164.6, C7), 113.50 (d, J = 168.6,

C7 '), 118.85 (s, C3a'), 121.54 (d, J ≈ 165.4, C5 '), 122.85 (s,

C3a), 123.76 (d, J = 170.2 Hz) and 124.41 (d, J ≈ 163.8 Hz) (C6 and C4 '), 125.00 (s, C5), 128.16 ( d, ^" = 167.0, C4), 137.21 (d, J =

159.8, C6 '), 139.10 (s) and 139.31 (s) (C7a and C7a'), 152.39 (s,

C2 '), 170.52 (s, C2), 188.72 (s, C3'); MS m / e 296 (M ⁺ , 100), 268 (39), 233 (35), 205 (50). Anal. (C ₁₆ H ₉ CIN ₂ 0 ₂ ) C, H, N.

5-f luroroindirubine

The procedure is as described for one indirubin. Starting from 500 mg of 5-fluoroisatin (3.03 mmol) and 530 mg of indoxylacetate

(3.00 mmol), 776 mg (92.4%) of a purple powder are obtained, Rt = 0.32 (ethyl acetate / hexane 1/2 v / v): 1H-NMR (DMSO-d6 ) δ 6.87 (dd, J _HιS = 8.3, J _HιIt = 4.7 C7H), 7.10-7.01 (m, C6H and C5'H), 7.42 (d, J = 8 , 2, C7'H), 7.58 (pt, C6'H), 7.65 (d, J = 7.4, C4 Η), 8.56 (d, J _FfH ≈ 10.6, C4H) , 11.00 (b, NlH and Nl'H); ¹³ C-NMR (DMSO- d ₆ ) δ 105.93 (s, C3), 110.13 (dd, J _CtH = 163.5, J _CF = 8.7, C7), 111.34 (dd, J _CιlI = 169.3, J _CιF = 27.6, C6), 113.57 (d, J = 162.0, C7 '), 115.24 (dd, J- _{C # H} = 188.9, J _CιF = 24.7, C4) 118.98 (s, C3a '), 121.64 (d, J = 164.2, C5 ^• ), 122.41 (d, J _{C, F} = 10.8, C3a) , 124.56 (d, J = 163.5, C4 '), 137.31 (s, C7a) 137.36

(d, J = 162.8, C6 '), 139.12 (s, C7a') 152.60 (s, C2 '), 157.40 (d, J _{C <F} = 233.2, C5), 171.02 (s, C2), 188.96 (s, C3 '); MS m / e 80 (M ⁺ , 100), 252 (73), 223 (32). Anal. (C ₁₆ H ₉ FN ₂ 0 ₂ ) C, H, N. -methylindirubin The procedure is as described for one indirubin. Starting from 500 mg of

5-methylisatin (3.10 mmol) and 540 mg of indoxylacetate (3.07 mmol), 781 mg (92.0%) of a purple powder are obtained, R, =

0.44 (ethyl acetate / hexane 1/2 v / v): ^ Η-NMR (DMSO-d ₆ ) δ 2.33 (s, CH ₃ ), 7.08-7.0 (m, C6H and C5'H), 6.79 (d, J = 7.9, C7H), 7.42

(d, J = 7.9, C7'H), 7.58 (pt, C6 Η), 7.64 (d, J = 7.6, C4'H),

8.63 (s, C4H), 10.79 (s) and 11.00 (s) (N1H and Nl'H); ¹³ C-NMR

(DMSO-d ₆ ) δ 20.99 (q, J = 126.5, CH3), 106.79 (s, C3), 109.17 (d,

J = 161.3, C7), 113.33 (d, J = 168.5, C7 '), 119.94 (s, C3a'), 121.09 (d, J = 164.4 C5 ') 121 , 45 (s, C3a), 124.19 (d, J = 163.7,

C4 '), 125.11 (d, J = 164.4, C6), 129.72 (d, J = 157.4, C4),

129.72 (s, C5) 136.96 (d, J = 161.6, C6 '), 138.08 (s) and 138.65

(s) (C7a and C7a '), 152.38 (s, C2'), 171.93 (s, C2), 188.49 (s,

C3 '); MS m / e 276 (M ⁺ , 100), 261 (10), 248 (47), 247 (53). Anal. (C ₁₇ H ₁₂ N ₂ 0 ₂ ) C, H, N.

5-nitroindirubine

The procedure is as described for one indirubin. Starting from 1.00 g of 5-nitroisatin (5.20 mmol) and 900 mg of indoxylacetate (5.14 mmol), 1.39 g (88.2%) of a purple powder are obtained, R _f = 0.16 (ethyl acetate / hexane v / v): ^X H-NMR (DMSO-d ₆ ) δ 7.01 (d,

J = 8.7, C7H), 7.05 (pt, C5'H), 7.40 (d, J ≈ 8.1, C7 '), 7.58

(pt, C6-H), 7.64 (d, J = 7.5, C4'H), 8.13 (d, J = 8.7, C6H),

9.60 (s, C4H), 11.15 (s) and 11.49 (s) (N1H and Nl'H); ¹³ C-NMR

(DMSO-d ₆ ) δ 103.51 (s, C3), 109.27 (d, J = 167.8, C7), 113.68 (d, J = 167.8, C7 '), 118.83 (s, C3a '), 119.48 (d, J = 167.8 C6)

121.59 (s, C3a), 121.97 (d, J = 163.0, C5 '), 124.60 (d) and 124.69

(d) (C4 and C4 '), 137.37 (d, J = 161.6, C6 •), 139.97 (s, C5)

136.96 (d, J = 161.6, C6 '), 138, (s) and 138.65 (s, C7a'),

141.63 (s, C5), 145.71 (s, C7a), 152.38 (s, C2 '), 170.93 (s, C2), 188.78 (s, C3'); MS m / e 307 (M ⁺ , 100), 276 (10), 262 (100), 234

(23). Anal. (C ₁₆ H ₉ N ₃ 0 ₄ ) C, H, N. indirubin-5-sulfonic acid as (sodium salt) The procedure is as described for one indirubin. Starting with 250 mg of the sodium salt dihydrate of isatin-5-sulfonic acid (1.00 mmol) and 170 mg of indoxylacetate (0.971 mmol), we obtain

258 mg (70.8%) of a purple solid, R _f = 0.73 (ethanol): Hl-NMR (DMSO-d ₆ ) δ 6.84 (d, J = 8.0, C7H), 7 , 04 (m, C5 Η), 7.43 (d, J =

8.0, C7'H), 7.54-7.61 (m, C4 'H and C6 * H), 7.67 (d, J = 7.4, C6H),

9.13 (s, C4H), 10.99 (s) and 11.05 (s) (N1H and Nl'H); ¹³ C-NMR

(DMSO-d ₆ ) δ 106.25 (s, C3), 108.20 (d, J = 163.3, C7), 113.39 (d,

J = 168.6, C7 '), 120.50 (s) and 118.98 (s) (C3a and C3a'), 121.28 (d, J = 157.9 C5 ') 122.57

(d, J = 169, 4, C6), 124.27 (d, J = 165.6, C4 '), 126.83

(d, J = 163.3, C4), 137.02 (d, J = 161.0, C6 '), 138.45 (s, C5),

140.89 (s) and 141.64 (s) (C7a and C7a '), 152.39 (s, C2'), 171.14

(s, C2), 188.33 (s, C3 '). Anal. (C ₁₆ H ₉ N ₂ Na0 ₅ S 1.5 H ₂ 0) C, H, N. 5 '-bromoindirubin

The procedure is as described for one indirubin. Starting with 59 mg of isatin (0.40 mmol) and 100 mg of 5-bromo-indoxylacetate

(0.394 mmol), 123 mg (92.6%) of a purple powder are obtained,

R _f = 0.59 (ethyl acetate / hexane 1/1 v / v): ^ -RMN (DMSO-d ₆ ) δ 6.89 (d, J = 7.8, C7H), 7.02 ( pt, C5H), 7.26 (pt, C6H), 7.39 (d,

J = 8.5, C7'H), 7.71 (d, J = 8.5, C6'H), 7.76 (s, C4'H), 8.73 (d,

J = 7.8, C4H), 10.89 (s) and 11.08 (s) (N1H and Nl'H); ¹³ C-NMR

(DMSO-d ₆ ) δ 107.43 (s, C3), 109.54 (d, J = 161.4, C7), 112.63 (s,

C5 '), 115.49 (d, J = 169.4, C7'), 120.64 (s) and 121.20 (s) (C3a and C3a '), 121.20 (d, J = 160, 0 C5) 124.69 (d, J = 154.5, C6),

126.31 (d, J = 163.0, C4 '), 129.56 (d, J ^" = 159.0, C4), 137.69

(s, C7a ') 138.87 (d, J = 166.1, C6'), 141.05 (s, C7a), 151.23

(s, C2 ¹ ), 170.64 (s, C2), 187.15 (s, C3 '); MS m / e 342 (M \ 100),

340 (M ⁺ , 100), 314 (16), 312 (16), 261 (1), 233 (44). Anal. (C ₁₆ H ₉ BrN ₂ 0 ₂ ) H, N, C: wedge, 56.3; found, 55.7.

5.5 '-dibromoindirubin

The procedure is as described for one indirubin. Starting with 89 mg of 5-bromoisatin (0.39 mmol) and 100 mg of 5-bromoisatin indoxylacetate (0.394 mmol), 154 mg (94.0%) of a purple powder are obtained: hi-NMR (DMSO-d ₆ ) δ 6.84 (d, J = 8.1, C7H), 7.42 -

7.36 (m, C6H and C7'H), 7.71 (d, J ≈ 8.5, C6 Η), 7.76 (s, C4'H),

8.88 (s, C4H), 11.01 (s) and 11.16 (s) (N1H and Nl'H); MS / e 422 (M \ 47), 420 (M ^* , 100), 418 (M048), 394 (3), 392 (7), 390 (3),

342 (25), 340 (25), 313 (15), 311 (15). Anal. (C ₁₆ H ₈ Br ₂ N ₂ 0 ₂ ) C, H,

N. 5 '-bromoindirubin-5-sulfonic acid (sodium salt)

The procedure is as described for one indirubin. Starting from 224 mg of sodium salt isatin-5-sulfonic acid dihydrate

(0.787 mmol) and 190 mg of 5-bromo-indoxylacetate (0.748 mmol), 204 mg (56.9%) of a purple powder are obtained: ^X H-NMR

(DMSO-d ₆ ) ,8 6.82 (d, J = 8.0, C7H), 7.40 (d, J = 8.5, C7 Η), 7.55

(d, J = 8.0, C6H), 7.73 (d, J = 8.5, C6 Η), 7.79 (s, C4'H), 9.09 (s, C4H), 11, 11 (b, NlH and Nl'H). Anal. (C ₁₆ H ₈ Br ₂ N ₂ Na0 ₅ S ₂ H ₂ 0) H, N,

C: wedge, 40.1; found 39.6. indirubin-3 '-monoxime

This compound is prepared as described Farb erke vorm. Meister

Lucius & Brùning in Hoechst a.M. Verfahren zur Herstellung von Derivaten der Indirubine. DRP 283726. The crude product is recrystallized from ethanol / water (7/2, v / v). The desired compound is obtained in the form of dark red crystals, R, = 0.55

(ethyl acetate / hexane 1/1 v / v): ^ -RMN (DMSO-d ₆ ) δ 6.90 (d, J

= 7.9, C7H), 7.42 (d, J = 7.9, C7'H), 7.58 (pt, C6'H), 7.64 (d, J = 7.6, C7H) , 6.95 (m, C5H), 7.03 (m ,, C5'H), 7.13 (m, C6H),

7.41 (m, C6'H and C7 Η), 8.24 (d, J = 7.2, C4H), 8.65 (d, J =

7.2, C4H) 10.72 (s) and 11.73 (s) (N1H and Nl'H), 13.48 (s, NOH);

¹³ C-NMR (DMSO-d ₆ ) δ 98.88 (s, C3), 108.91 (d, J = 162.3, C7),

111.52 (d, J = 165.8, C7 '), 116.54 (s, C3a'), 120.43 (d, 160.2, C5 '), 121, 49 (d, J = 161, 6 C5) 122.66 (s, C3a), 123.09 (d, J ≈

165.1, C6), 125.92 (d, J = 154.0, C4), 127.95 (d, J = 164.4,

C4-), 132.02 (d, J = 159.5, C6 '), 138.34 (s, C7a), 144.83 (s,

C7a '), 145.32 (s, C2'), 151.22 (s, C3 '), 170.95 (s, C2); MS m / e 277 (M \ 100%), 260 (87%), 247 (24%), 220 (14%), 205 (11).

Anal. (C ₁₆ H ₁₁ N ₃ 0 ₂ 0.25 H ₀ O) C, H, N.

5-iodoindirubin-3 '-oxime

The procedure is as described for 1 'indirubin-3' -monoxime. Starting from 250 mg of indirubin-3 '-monoxime (0.644 mmol) and 175 mg of hydroxylamine hydrochloride (2.52 mmol) in 7.5 ml of pyridine, a precipitate is obtained. The crude product is washed with water and recrystallized from ethanol. We get 119 mg

(45.9%) of red crystals, R _f = 8.50 (ethyl acetate / hexane 1/1 v / v): Hi-NMR (DMSO-d ₆ ) 6.73 (d, J = 6, 7, C7H), 7.09-7.01 (m,

C5'H), 7.44-7.40 (m, C6, C6'H, and C7'H), 8.26 (d, J = 7.6,

C4'H), 8.90 (s, C4H), 10.79 (s) and 11.88 (s) (N1H and Nl'H),

13.68 (s, NOH); ¹³ C-NMR (DMSO-d6) δ 83.69 (s, C5), 96.59 (s, C3),

110.89 (d, C7), 111.51 (d, C7 '), 116.44 (s, C3a'), 121.77 (d, C5 ') 125.40 (s, C3a), 127.42 (d, C4 '), 130.00 (d, C6), 131.52 (d,

C6 ¹ ), 133.40 (d, C4), 137.20 (s, C7a), 144.01 (s, C7a '), 146.68

(s, C2 '), 151.52 (s, C3'), 170.25 (s, C2); MS m / e 403 (M ⁺ , 100),

387 (9), 373 (10), 276 (5), 260 (46). Anal. (C ₁₇ H ₁₂ N ₂ 0 ₂ ) C, H, N.

6 -iodoindirubin The procedure is as described for one indirubin. Starting from 250 mg of 6-iodoisatin (0.92 mmol) and 120 mg of indoxylacetate (0.69 mmol), one obtains 182 mg (68.0%) of a purple powder, Rf = 0.47 (ethyl acetate / hexane l / lv / v): ^X H-NMR (DMSO-d ₆ ) δ 7.01 (pt, J = 7.5, C5'H), 7.19 (s, C7H) , 7.38 (m, C5H and C7 Η), 7.56 (pt, J = 7.3, C6'H), 7.62 (d, J = 7.6, C4'H), 8.49 (d, J = 8.3, C4H), 10.93 (s) and 11.03 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO-d6) δ 94.60 (d, J = 10.4, C6), 105.57 (s, C3), 113.71 (d, J = 169.5, C7 '), 118.05 (d, J = 167.1, C4 '), 119.17 (s, C3a'), 121.24 (s, C3a), 121.65 (d, J = 163.4, C7), 124.56 (d, J = 163.4, C5 '), 126.17 (d, J = 167.1, C4), 129.99

(d, J ^" = 167.5, C5), 137.29 (d, J ≈ 161.0, C6 '), 139.07 (s, C7a'), 142.12 (s, C7a), 152, 56 (s, C2 '), 170.70 (s, C2), 188.83 (s, C3'); MS m / e 388 (M ⁺ , 100), 360 (9), 261 (15), 233 (53), 205 (53), 127 (2). Anal. (C ₁₆ H ₉ IN ₂ 0 ₂ ) H, N. C: wedge, 49.5%; find

48.5%.

1-méthylindirubine

The procedure is as described for one indirubin. Starting from 200 mg of 1-methylisatin (1.24 mmol) and 217 mg of ¹ indoxylacetate (1.24 mmol), the crude product is recrystallized from pyridine and sublimed (140 ° C., under vacuum), one obtains 195 mg (56.9%) of slightly purple violet clear beads; R _f = 0.88 (ethyl acetate / hexane 3/2 v / v: ^X H-NMR (DMSO-d ₆ ) δ 3.29 (s, CH ₃ ), 7.02- 7.12 (m , C5H, C7H, and C5'H), 7.35 (pt, C6H), 7.43 (d, J = 8.3,

C7'H), 7.59 (pt, C6'H), 7.67 (d, J = 7.6, C4'H), 8.81 (d, J = 7.3,

C4H), 11.07 (s, Nl'H); MS m / e 276 (M0 100), 248 (25), 247 (54).

Anal. (C ₁₇ H ₁₂ N ₂ 0 ₂ ) C, H, N.

1-phenylindirubin The procedure is as described for indirubin. Starting from 500 mg of 1-phenylisatin (2.24 mmol) and 334 mg of indoxylacetate

(1.90 mmol), 597 mg (92.6%) of a purple powder are obtained,

R _f = 0.92 (ethyl acetate / hexane l / lv / v): ^ Η-NMR (DMSO-d ₆ ) δ

6.83 (pt, J = 7.3, C7H), 7.05 (pt, C5'H), 7.16 (pt, C5H), 7.29 (pt, C6H), 7.42 (d, J = 8.0, C7'H), 7.53-7.48 (m) and 7.64-7.58 (m) (C6'h and phenyl-H, 7.69 (d, J = 7.3, C4 Η), 8.92 (d, J 7.6, C4H), 11.17 (s, Nl'H); ¹³ C-NMR (DMSO-d ₆ ) δ 104.96 (s, C3), 108.71 (s, C7), 113.48 (s, C7 '), 118.96 (s, C3a'), 120.80 (s, C3a), 121.50 (d, J = 159 , 1, Hz), and 122.34 (d, J _{C, H} = 160.8, Hz), (C5 and C5 '), 124.44 (d) and 124.56 (d) (C6 and C4' ), 126.75 (d, J = 161.5, C3 "and C5"), 127.93 (d, J = 161.5, Hz) and 128.99 (d, J = 160.8 Hz) ( C4 and C4 ^" ), 129.46 (d, J = 161.5, C2" and C6 "), 137.18 (d, J ≈ 161.6, C6 '), 139.11 (s) and 133, 97 (s) (C7a 'and Cl "), 141.34 (s, C7a), 152.32 (s, C2'), 168.36 (s, C2), 188.44 (s, C3 ^! ); MS m / e 388 (M ^* , 100), 311 (13), 310 (58), 309 (39) Anal. (C ₂₂ H ₁₄ N ₂ 0 ₂ ) C, H, N. 3 'acid-hydroxyiminoidirubin- 5-sulfonic (sodium salt) We operate according to Lucius et al above (modified method) with

500 mg sodium salt of indirubin-5-sulfonic acid

(0.644 mmol) and 400 mg of ¹ hydroxylamine hydrochloride (5.76 mmol) in 15 ml of pyridine. The precipitate produced by adding 100 ml of hydrochloric acid and 20 ml of a saturated sodium chloride solution to the reaction mixture is combined with the precipitate obtained from the filtrate, which was produced by adding 15 ml of a saturated solution of sodium chloride. This crude product is recrystallized from water. 170 mg (34.0%) of black scales with scales, with a red shade are obtained, R _j =

0.76 (ethanol): hî-NMR (DMSO-d ₆ ) δ 6.82 (d, J = 8.1, C7H), 7.10-

6.97 (m, C5'H), 7.41-7.39 (m, C6 'and C7'), 7.48 (d, J = 7.9,

C6H), 8.26 (d, J = 7.7, C4'H), 8.92 (s, C4H), 10.82 (s) and 11.80

(s) (N1H and Nl'H), 13.79 (s, NOH); ¹³ C-NMR (DMSO-d ₆ ) δ 98.64 (s, C3), 107.32 (d, J = 162.3, C7), 111.36 (d, J = 165.8, C7 ') , 116.59 (s, C3a '), 120.55 (d, J = 167.2, C5'), 121.42 (d, J = 159.5, C6), 121.65 (s, C3a) , 123.74 (d, J = 163.0, C4), 128.02 (d, J = 167.8, C4 '), 131.84 (d, J = 158.8, C6'), 138, 21 (s, C5), 140.87 (s, C7a), 144.53 (s, C7a '), 145.52 (s, C2'), 151.32 (s, C3 '), 171.17 ( s, C2) Anal. (C ₁₆ H ₁₀ N ₃ NaO ₂ S) C, H, N. indirubin-5-suifonamide

The procedure is as described above for 1 indirubin. Starting from 120 mg of isatin-5-sulfonamide (0.530 mmol) and 79 mg of indoxylacetate (0.045 mmol), 79 mg (92.0%) of 5-methyl indirubin are obtained in the form of black powder containing 8 mass% of indigo (indigo was identified by DC and hî-NMR); R _f = 0.79 (ethyl acetate): ^ -RMN (DMSO-d ₆ ) δ 7.04-7.08 (m, C7H and C5'H), 7.23 (s, NH ₂ ), 7.44 (d, J = 7.9, C7'H), 7.60 (pt, C6'H), 7.69 (d, J = 7.4, C6H), 7.74 (d, J = 8.1, C4'H), 9.31 (s, C4H), 11.15 (s) and 11.25 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO-d ₆ ) δ 104.92 (s, C3), 113.56 (d, J = 159.9, C7 '), 119.01 (s, C3a'), 121.13 ( s, C3a), 121.72 (d, J = 165.2, C5 '), 122.27 (d, = 169.8, C6), 124.44 (d, J = 164.5, C4 ^• ) , 126.98 (d, J = 159.2, C4), 109.22 (d, J = 165.2, C7), 137.24 (s, C5), 137.24 (d, J = 160.5, C6 '), 139.32

(s, C7a '), 142.99 (s, C7a), 152.45 (s, C2'), 170.98 (s, C2),

188.52 (s, C3 '). Indirubin-5-sulfonic acid dimethylamide The procedure is as described above for 1 indirubin. Starting from

397 mg of dimethylamide of isatin 5-sulfonic acid (1.56 mmol) and 246 mg of indoxylacetate (1.40 mmol), one obtains

240 mg (46.5%) of the desired acid in the form of a purple powder, R _f = 0.39 (ethyl acetate / hexane 3/2 v / v): ¹ H-NMR (DMSO-d ₆ ) δ 2.65 (s, 2 CH ₃ ), 7.02-7.13 (m, C7H and C5'H), 7.44 (d,

J = 8.1, C7'H), 7.57-7.72 (m, C6H, C4'H, and C6'H), 9.21 (s, C4H),

11.18 (s) and 11.33 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO-d6) δ 37.20

(q, J = 139, 4, CH3,) 104.16 (s, C3), 109.11 (d, J = 165.8, C7),

113.22 (d, J = 167.2, C7 '), 118.71 (s, C3 a'), 121.36 (d, J = 165.1, C5 '), 121.48 (s, C3a ), 123.18 (d, J = 172.0, C6), 124.22

(d, J = 163.7, C4 '), 127.64 (s, C5), 128.06 (d, J = 166.5, C49,

136.93 (d, J = 164.4, C6 '), 139.34 (s, C7a'), 143.53 (s, C7a),

152.10 (s, C2 '), 170.51 (s, C2), 188.37 (s, C3'); MS m / e 369

(M +, 83), 326 (6), 262 (84), 261 (100). Anal. (C ₁₈ H ₁₅ N ₃ 0 ₄ S) H, N; C: wedge, 58.5%; found, 57.8%.

(2-hydroxyethyl) -amide of indirubin-5 sulfonic acid

The procedure is as described for one indirubin. Starting from 150 mg of the

(2-hydroxyethyl) -amide of 5-isatin-sulfonic acid (0.630 mmol) and of 83 mg of indoxylacetate (0.47 mmol), 143 mg (78.9%) of the desired compound are obtained in the form of purple powder;

R _f = 0.62 (methanol / ethyl acetate 1/20 v / v): hî-NMR (DMSO-d ₆ ) δ 2.84 (t, J {CH2, CH21} = 6.5, visible in adding D ₂ 0, -N-CH ₂ -),

3.40 (m, -CH ₂ -0), 4.67 (t, J = 4.6, OH), 7.07 (m, C7H and C5'H),

7.43 -7.46 (m, C7 'H and S0 ₂ -NH-; adding D ₂ 0: 7.44, d, J ^" = 8.1, C7'H), 7.61 (pt, C6, H), 7.69-7.71 (m, C6H and C4 •), 9.27 (s,

C4H), 11.18 (s) and 11.29 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO-d ₆ ) δ

45.00 (t, J = 138.0, -NH-CH ₂ ), 59.85 (t, J = 141.1, -CH ₂ -OH),

104.52 (s, C3), 109.44 (d, J = 165.9, C7), 113.60 (d, J = 168.5, C7 '), 118.92 (s, C3a'), 121.36 (s, C3a), 121.74 (d, J = 164.0,

C5 '), 122.95 (d, J = 162,, C6), 124.52 (d, J = 159.6, C4 •),

127.53 (d,

163.4, C6 '), 139.44 (s, C7a'), 143.32 (s, C7a), 152.46 (s, C2 •), 170.87 (S, C2), 188.70 ( s, C3 '), MS m / e 385 (M ⁺ , 40), 355 (48),

325 (29), 262 (35), 261 (100). Anal. (C ₁₈ H ₁₅ N ₃ 0 ₅ S) C, H, N. bis- (2-hydroxyethyl) -amide of indirubin-5-sulfonic acid

The procedure is as described for one indirubin. Starting with 400 mg of bis- (2-hydroxyethyl) amide of isatin-5-sulfonic acid bis- (2-hydroxyethyl) amide (1.27 mmol) and 167 mg of indoxyl acetate

(0.953 mmol), 355 mg (86.7%) of the above-mentioned amide are obtained in the form of a purple powder, Rf = 0.51 (methanol / ethyl acetate 1/20 v / v): ^ Η-NMR (DMSO-d ₆ ) δ 3.20 (t, J = 6.5,

2 -N-CH ₂ ), 3.56 (m, 2 -CH ₂ 0-), 4.84 (t, J = 5.5, 2 -OH), 7.09- 7.05 (m, C7H and C5'H), 7.72-7.68 (m, C6H and C4 '), 7.45 (d, J = 8.0, C7'H), 7.61 (m, C6'), 9 , 26 (s, C4H), 11.18 (s) and 11.32 (s) (N1H and Nl'H); ¹³ C-NMR (DMSO-d ₆ ) δ 51.32 (t, J = 135.0, 2 -NH-CH ₂ ), 60.01 (t, = 141.0, 2 -CH ₂ -OH), 104.31 (s, C3), 109.48 (d, J = 165.3, C7), 113.62 (d, J = 168.5, C7 '), 118.91 (s, C3a'), 121.69 (s, C3a), 121.79 (d, J = 164.0, C5 '), 122.88 (d, J = 162.4, C6),

124.64 (d, J = 162.7, C4 '), 127, 98 (d, J = 165.2, C4), 131.51

(s, C5), 137.32 (d, J = 160.1 Hz, C6 '), 139.61 (s, C7a'), 143.59

(s, C7a), 152.48 (s, C2 '), 170.81 (s, C2), 188.87 (s, C3'); MS m / e 429 (M ^* , 9), 369 (22), 365 (22), 355 (63), 261 (31), 43 (100). Anal. (C ₂₀ H ₁₉ N ₃ O ₆ S) C, H, N. methylamide of indirubin-5-sulfonic acid

The procedure is as described above for 1 indirubin. Starting from 380 mg of isatin-5-sulfonic acid methylamide (1.58 mmol) and 227 mg of indoxylacetate (1.30 mmol), 370 mg (80.1%) of the amide are obtained. above in the form of a purple powder, R _f = 0.72 (ethyl acetate): Hî-NMR (DMSO-d ₆ ) δ 2.46 (d, J = 5.0, CH ₃ ), 7.05-7.09 (m, C7H and C5 Η), 7.32 (q, NH-CH ₃ ), 7.45 (d, J = 8.3, C7'H), 7.61 (m , C6'H), 7.67-7.71 (m, C6H and C4 '), 9.27 (s, C4H), 11.18 (s) and 11.30 (s) (N1H and Nl'H); ¹³ C-

NMR (DMSO-dg) δ 28.65 (q, J = 13 8.7, CH3), 104.48 (s, C3),

109.41 (d, J = 165.7, C7), 113.58 (d, J = 165.7, C7 '), 118.90

(s, C3a '), 121.42 (s, C3a), 121.73 (d, J = 171.0, C5'), 123.13 (d, J = 164.5, C6), 124.50 (d, J = 159.8, C4 '), 127.61 (d, J =

165.7, C4), 131.93 (s, C5), 137.28 (d, J = 160.6, C6 '), 139.43 (s,

C7a '), 143.36 (s, C7a), 152.45 (s, C2'), 170.85 (s, C2), 188.68

(s, C3 '); MS m / e 255 (M +, 15), 263 (18), 262 (100), 261 (7).

Anal. (C ₁₇ H ₁₃ N ₃ 0 ₄ S) C, H, N. 5-iodoisatine

The procedure is as described by Roedig, Mueller E. (Hrsg.), Methoden der organishen Chemie (Houbeyn-Weyl), Stuttgart, Georg Thieme Verlag 1960,5 / 4, 586, and Borsche et al Chem.Ber. 1924, 1770- 1775, R _f = 0.60 (acetone / petroleum ether 40-70 l / lv / v); Mp 262 ° C: Hl-NMR (DMSO-d ₆ ) δ 6.75 (d, J = 8.3, C7H), 7.75 (s, C4H), 7.78 (d, J = 8.2 , C6H), 11.09 (b, N1H); ¹³ C-NMR (DMSO-d ₆ ) δ 85.39 (s, C5), 114.59 (d, = 167, 1, C7), 119.89 (d, J = 7.2, C3a), 132 , 38 (d, J- = 170.3, C4), 145.76 (d, J = 167.1, C6), 145.76 (d, J = 167.1, C6), 149.95 (b , C7a), 158.66 (s, C2), 183.06 (s, C3); MS m / e 262 (M *, 100), 234 (43).

isatin-5-sulfonic acid dimethylamide

The procedure is as described by Haller, patent DE 715760, 1938,

R _f = 0.18 (ethyl acetate / hexane 1 / lv / v): ^X H-NMR (DMSO-d ₆ ) δ 2.62 (s, 2 CH ₃ ), 7.12 (d, J 8 , 2, C7H), 7.71 (s, C4H), 7.94 (d, J = 8.2, C6H), 11.47 (s, N1H); ¹³ C-NMR (DMSO-d ₆ ) δ 182.89 (s, C3), 159.37 (s, C2), 153.60 (s, C7a), 136.98

(d, J = 167.32, C6), 128.62 (s, C5), 123.27 (d, "J = 169.2, C4), 118.05 (s, C3a), 112.68 ( d, J = 169.2, C7), 37.48 (q, J = 140.1, 2 CH ₃ ). Anal. (C ₁₀ H ₁₀ N ₂ O ₄ S) C, H, N. bis- (2-hydroxyethyl) -amide of isatin-5-sulfonic acid

The procedure is as described by Haller above, starting from 526 mg of bis- (2-hydroxyethyl) amino (5.00 mmol) and 1.00 g of 3.3- dichloro-2-oxo-2.3-dihydroindol-5-sulfonyl (see Haller above) (3.34 mmol). The precipitate which forms is oily and muddy. The supernatant is decanted, then the precipitate and the supernatant are mixed with 5 ml of water. The mixture is brought to reflux for 2 h. On cooling, a precipitate of red crystals is obtained. Yield: 476 mg (45.3%), R _f = 0.51 (methanol / ethyl acetate 1/20 v / v): 1H-NMR (DMSO-d6) δ 2.78 (t, J = 6 , 2, 2 -N-CH ₂ -), 3.38 (t, J = 6.2, 2-CH ₂ -0-), 7, 08 (d, J = 8.2, C7), 7, 78 (s, C4), 7.96 (d, = 8.2, C6), 11.43 (s, N1H); ¹³ C-NMR (DMSO-d ₆ ) Ô 50.73 (t, J = 138.8.2 -N-CH ₂ -), 59.64 (t, J = 141.7, 2 -CH ₂ 0H- ), 112, 57 (d, J = 168.6, C7), 117.91 (s, C3a), 122.78 (d, J = 169.3, C4), 133.29 (S, C5), 136.44 (d, J = 166.4, C6), 153.28 (s, C7a), 159.37 (s, C2), 182.95 (s, C3). Anal. (C ₁₂ H ₁₄ N ₂ 0 ₆ S) C, H, N. 6-lodoisatine The procedure is as described by Marvel et al, Organic Syntheses, Coll. Vol. 1, 2 ^nd ed .; Gilman, H Ed .; Wiley & Sons: New York, 1941, 327-330, using 1.50 g of 2-hydroxyimino-N- (3-iodophenyl) acetamide (5.17 mmol) and 3.9 ml of concentrated sulfuric acid. After filtration, the crude product, formed from a mixture of 4-and 6-iodoisatin and by-products, is purified according to Sadler, J. Org. Chem. 1956, 21, 169-170 and Holt et al, Proc.R.Soc. London B 1958, 148, 481-494. 216 mg (15.0%) of orange crystals are obtained; Mp 255-258 ° C; R _f = 0.76 (ethyl acetate / hexane l / lv / v): ^ -RMN (DMSO-d6) δ 7.25 (m, C4H and C7H), 7.45 (d, J≈ 7, 6, C5H), 11.07 (s, N1H); 13C-NMR (DMSO-d6) δ 107.28 (d, J = 10.4, C6), 117.41 (s, C3a), 120.83 (d, J = 170.3, C7), 125, 84 (d, J ^" = 173.7, C4), 131.82 (d, J = 167.9, C5), 151.30 (s, C7a), 159.35 (s, C2), 183.79 (s, C3) Anal. (C ₈ H ₄ IN0 ₂ ) C, H, N. isoindigo The procedure is as described by Wahl et al, Weekly reports of the sessions of the Academy of Sciences, 1909, 716-19 and 4 (5), 1039 -43: R _f = 0.67 (ethyl acetate / hexane l / lv / v): ^ -RMN (DMSO-d ₆ ) δ 6.85 (d, J = 1, 1 , C7H and C7'H), 6.97 (pt, C5'H and C5'H), 7.34 (pt, C6H and C6'H), 9.07 (d, J = 8.0, C4H and C4'H),

10.90 (s, N1H and Nl'H); ¹³ C-NMR (DMSO-d6) 109.42 (d, J ≈ 162.5,

C7 and C7 '), 121.04 (d, J = 161.2, C5 and C5'), 121.58 (s, C3a and

C3a '), 129.21 (d, J = 166.5, C6 and C6'), 132.52 (d, J = 159.9, C4 and C4 '), 133.24 (s, C3 and C3' ), 143.98 (s, C7a and C7a '), 168.88

(s, C2 and C2 '); MS m / e 262 (M +, 100), 234 (85), 205 (18). Anal.

(C ₁₆ H ₁₂ N ₂ 0 ₂ ) C, H, N.

2,2 '- bisindole

The procedure is as described by Bergman et al, Tetrahedron 1995, 51 (19), 563-42, R _f = 0.82 (UV, ethyl acetate / hexane l / l): GC / MS

R _t = 14.1 min, m / e 232 (M ⁺ , 100); λ ^, (ethanol) 221 (4.78), 271

(4.00), 333 (4.80), 351 (4.79). Anal. (C ₁₆ H ₁₂ N ₂ ) C, H, N.

3,3 '-diphenyl-2.2' -bisindole

The procedure is as described by Fύrstner et al, Angewandte Chemie, 1995, 107 (6), 725-8. Recrystallization from ethyl acetate / petroleum ether (40-70) 115 v / v leads to colorless crystals (55.8%), R _f = 0.50 (UV, ethyl acetate / petroleum ether (40-70) 115 v / v): GC / MS R _t = 19.0 min, m / e 384

(M ^* , 100). isatin-5-sulfonamide

A 25% aqueous ammonia solution (5.0 ml) is cooled beforehand to 0-5 ° C before adding a sample of

0.50 g of 3, 3-dichloro-2-oxo-2, 3-dihydroindol-5-sulfonyl chloride (see Haller above), (1.7 mmol), in portions, stirring and cooling to new. After 2 hours, a small amount of crushed ice and 20% hydrochloric acid are added until the mixture has an acid reaction. The solvent is removed in vacuo and the orange residue is dried over KOH. The yellow powder obtained is extracted 3 times with 35 ml of acetone. The acetone is removed under vacuum, which leads to 140 mg (36.4%) of orange powder, Rf≈ 0.73 (ethyl acetate): ^X H-NMR (DMSO-d ₆ ) δ 7.05 (d,

J = 8.2, Cl), 7.41 (s, NH ₂ ), 7.85 (s, C4), 7.98 (d, J = 8.2, C6),

11.39 (s, N1H); ¹³ C-NMR (DMSO-d ₆ ) δ 112.12 (d, J = 168.3, C7), 117.73 (s, C3a), 121.67 (d, J≈ 166.8, C4), 134.97 (d, J = 165.4,

C6), 138.36 (s, C5), 152.56 (s, C7a), 159.50 (s, C2), 184.22 (s,

C3); MS m / e 226 (M ⁺ , 28), 198 (100).

(2-hydroxyethyl) -amide of isatin-5-sulfonic acid The operation is carried out according to Haller above, starting from 295 mg of 2-aminoethanol (4.83 mmol, 0.300 ml), 5.1 ml of ethanol and 0.900 mg of chloride 3.3-dichloro-2-oxo-2, 3-dihydroindol-5-sulfonyl

(Haller) (3.01 mmol). After 30 min of stirring, 0.100 ml of 2-aminoethanol is again added, and 15 min later 20 g of crushed ice and 0.900 ml of 20% hydrochloric acid. By warming up to room temperature, an oily mass is obtained which is deposited at the bottom of the container. The supernatant is left to settle and the remaining solvent is removed in vacuo.

The yellow oil obtained is brought to reflux in water for 2 h. While cooling, the orange solution obtained precipitates 374 mg (45.8%) of yellow crystals, R _f = 0.52 (methanol / ethyl acetate 1/20 v / v): ^X H-NMR (DMSO-d ₆ ) δ 2.73- 2.82 (m, N-CH ₂ -), 3.38

(t, J = 6.1 Hz, -CH ₂ -0-), 7.07 (d, J = 7.6, C7), 7.66 (t, J = 5.7

Hz, -S0 ₂ -NH-), 7.81 (s, C4), 7.96 (d, J = 8.4, C6), 11.42 (s, NI H). isatin-5-sulfonic acid methylamide

The operation is carried out according to Haller above, starting from 0.572 ml of a 40% aqueous solution of methylamine (6.62 mmol), 5.6 ml of ethanol and 1.00 g of 3, 3-dichloro chloride. -2-oxo-2, 3-dihydroindol-5-sulfonyl (Haller) (3.34 mmol). The precipitate which forms is removed by filtration and the mixture is brought to reflux in water for 2 h. By cooling, the solution precipitates

(yellow crystals). Yield: 420 mg (52.3 i), R _f = 0.29 (ethyl acetate / hexane 2/1 v / v): ^X H-NMR (DMSO-d ₆ ) δ 2.41 (d, J = 5.0, CH3), 7.08 (d, J = 8.3, C7), 7.48 (q, J = 5.0, NH ₂ ), 7.77 (s, C4),

7.95 (d, J = 8.3, C6), 11.42 (s, N1H); ¹³ C-NMR (DMSO-d ₆ ) δ 28.52

(q, J = 139.2, CH ₃ ), 112.40 (d, J = 168.9, C7), 118.04 (s, C3a), 122.48 (d, J = 169.2, C4), 133.13 (s, C5), 136.15 (d, J = 166.0,

C6), 153.44 (S, C7a), 159.44 (s, C2), 183.04 (s, C3).

2-hydroxyimino-N- (3-iodophenyl) -acetamide

The procedure is as described by Marvel and Hiers above and the product obtained is purified by proceeding according to Holt and Sadler above; Mp 152-154 ° C; R _f = 0.29 (ethyl acetate / hexane l / lv / v): Hi-NMR (DMSO-d _ff ) δ 7.12 (pt, J = 8.1, C5 Η), 7.44 ( pt, J =

7.9, C4'H or C6'H), 7.63 (s, C2H), 7.65 (pt, J = 8.2, C4 'H or

C6'H), 8.16 (Pt, J ≈ 1.7, C2'H), 10.27 (s, NH), 12.27 (s, C2NOH); ¹³ C-NMR (DMSO-d ₆ ) δ 94.69 (d, J = 11.7, C3 •), 119.32 (d, J

= 165.7, C6 '), 128.22 (d, J ≈ 168.0, C2'), 130.95 (d, J = 162.8,

C5), 132.57 (d, J = 168.1, C4 '), 140.06 (s, Cl'), 144.05 (d, J =

171.5, C2), 160.67 (s, Cl).

. Buffers The pads used have the following compositions:

Homogenization buffer: - 60 mM β-glycerophosphate, 15 mM p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 15 mM EGTA, 15 mM MgCl ₂ , 1 mM DTT, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylphosphate, 10 μg leupeptin / ml, 10 μg aprotinin / ml, 10 μg soybean trypsin inhibitor / ml and 100 μM benzamidine.

Buffer A: 10 mM MgCl ₂ , 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 μg heparin / ml. Buffer-C: homogenization buffer, but containing 5 mM of EGTA, and devoid of NaF and protease inhibitors.

Tween-20 salt tris buffer (TBST): 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween-20 ^R.

Hypotonic lysis buffer (HLB): 50 mM Tris-HCl ph 7.4, 120 mM NaCl, 10% glycerol, 1% Nonidet-P40, 5 mM DTT, 1 mM EGTA, 20 mM NaF, 1 mM orthovanadate, 5 μM microcystin, 100 μg / ml of each of the following products: leupeptin, aprotinin, pepstatin. Kinase preparations and activity determinations

The kinase activities were determined in buffer A or C (unless otherwise indicated), at 30 ° C, at a final ATP concentration of 15 μM. The values of the blank tests were subtracted and the activities calculated in pmoles of phosphate incorporated for a 10 min incubation. The values of the activities are generally expressed in% of the maximum activity, that is to say, in the absence of inhibitors. Control tests were carried out using appropriate dilutions of Me ₂ SO. In some cases, as indicated below, the phosphorylation of the substrates is determined by autoradiography after SDS-PAGE.

The GSK-3β used is either the enzyme purified from rabbit muscle or expressed and purified from Sf9 insect cells (Hughes et al, 1992, Eur. J. Biochem., 203: 305,311). The determinations were carried out with a 1/100 dilution in 1 mg of BSA / ml of 10 mM DTT, with 5 μl of 40 μM GS-1 as substrate, in buffer A, in the presence of 15 μM [γ ³² P ] ATP (3000 Ci / moles; 1 mCi / ml) in a final volume of 30 μl. After 30 minutes of incubation at 30 ° C, aliquots of 25 μl of supernatant were applied to 2.5 x 3 cm strips of Whatman P81 phosphocellulose paper, and 20 seconds later, the filters were washed. 5 times (for at least 5 min each time), in a solution of 10 ml of phosphoric acid / 1 of water. The wet filters were counted in the presence of 1 ml of ACS scintillation fluid (Amersham).

The CDK1 / cyclin B used was extracted with the aid of a homogenization buffer from oocytes of starfish (Marthasterias glacialis) and purified by affinity chromatography on p9 beads - Sepharose from

CKShs1 from which the product was eluted by free p9, as described by Meijer et al. , 1997, (Methods in Enzymology, vol 283

: 113-128), and Borgne et al. , 1999, J. Biol. Chem. 274: 11977-

11986.

The kinase activity was determined in buffer C, with 1 mg of histone Hl / ml, in the presence of 15 μM of [γ ³² P] ATP

((3000 Ci / mmol; 1 mCi / ml) in a final volume of 30 μl.

After 10 minutes of incubation at 30 ° C, aliquots of

25 μl of supernatant were deposited on phosphocellulose P81 papers and treated as described above. CDK5 / p25 was reconstituted by mixing equal amounts of recombinant mammalian CDKS and p25 expressed in E. coli as a GST fusion protein

(Glutathione-S-transferase) and purified by affinity chromatography on glutathione-agarose p25 is a truncated version of p35, the CDK5 activator of 35kDa. Its activity was determined in buffer C as described for CDKl / cyclin B.

. In vitro and in vivo phosphorylation of tau:

Cells and viruses: the Sf9 cells (InVitrogen, San Diego, CA) were cultured at 27 ° C. in a monolayer culture Grace medium (Gibco BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum and 50 μg of gentamycin / ml and 2.5 μg of amphotericin / ml. BaculoGold was obtained from PharMingen (San Diego, CA), and pVL1392 from InVitrogen.

Tau transfection: we excised, from a bacterial expression vector pNG2 (Biernat et al., 1993, Neuron 11: 153-163) and the gene coding for htau23, the shortest human tau isoform , with Xbal and BamHI. The gene was inserted into the baculovirus transfer vector pVL1392 cut with the same endonucleases. The BaculoGold system was used for the construction of the vector containing the baculovirus tau. BaculoGold DNA is a modified type of baculovirus containing a lethal deletion. Co-transfection of BaculoGold DNA with a complementary baculovirus transfer vector makes it possible to recover the lethal deletion of this viral DNA and to reconstitute viable virus particles carrying the sequence coding for htau23.

The plasmid DNA used for the transfections was purified using QIAGEN cartridges (Hilden,

Germany).

Sf9 cells cultured in monolayers (2 × 10 ⁶ cells in a 60 mm cell culture vessel) were co-transfected with baculovirus DNA (0.5 μg of BaculoGold DNA) and with derivatives of pVL1392 ( 2 μg) using the calcium phosphate co-precipitation method. The presence of recombinant protein was examined in infected cells 5 days after infection with SDS-

PAGE and Western blot.

Phosphorylation of tau in Sf9 cells.

To determine the effects of kihase inhibitors on tau phosphorylation, the Sf9 cells infected with the baculovirus expressing htau23 were treated 36 hours after infection, with 50 μM of indirubin-3 '-monoxime for 5 hours before to be collected. To obtain control tau samples with higher phosphorylation, Sf9 cells expressing htau23 were treated with 0.2 μM okadaic acid for 5 hours before harvest.

Western blot de tau:

Sf9 cells were infected with a recombinant virus at an MOI of 1 to 5.

Cell lysates were prepared in hypotonic lysis buffer (HLB).

After 15 minutes of centrifugation at 16000 g, the supernatant was recovered and its NaCl concentration increased to 500 mM. The supernatant was then boiled for 10 min. and recentrifuged at 16000 g for 15 min. The proteins (3 μg) were resolved by SDS-PAGE, transferred to a PVDF membrane and studied with a Western blot with the following antibodies: AT-8 (1: 2000), AT-180 (1: 1000), AT -100 (1: 500), PHF-1 (1: 600) and the anti-tau polyclonal antibody K9JA.

Phosphorylation of tau in vi tro was performed using purified GSK-3β and the recombinant human tau-32 protein as a substrate. After 30 minutes of incubation in the presence of different concentrations of indirubin-3 '- monoxime, under the conditions for studying the GSK-3β described above, the kinase reaction was stopped by adding Laemmli buffer. The tau protein was resolved in SDS-PAGE at 10% and its phosphorylation rate visualized by autoradiography. Example 1: Study of the inhibition of GSK-3β, CDK5 / p25 and CDKl / cyclin B by the indirubins.

The kinase activities were determined with an appropriate substrate (GSK-3β: GS1 peptide; CDKs: histone Hl) in the presence of 15 μM of ATP and at increasing concentrations of the derivatives tested. The IC ₅₀ values were calculated from the dose / response curves and are given in Table 1.

Table 1

with other indigoids, such as isatin or indigo derivatives, give the results reported in Table 2. Table 2

Examination of these two tables shows that, among the indigoids, only the indirubin derivatives exert both an inhibitory effect with respect to GSK-3β and CDKs. It comes out in clear effect of these results that neither isatin, nor indigo or their derivatives, exert a significant effect on one of these 3 kinases.

Figures 1A to 1D give the dose-response curves for 5-iodo-indirubin-3 '-monoxime (A), 5,5'- dibromoindirubine (B), 5-sulfonic acid indirubine-3' - monoxime (C) and 1 'indirubin- 3' -monoxime (D). Inhibition of

GSK-3β and CDKs is determined as indicated above.

The activity is expressed in% of the maximum activity (without inhibitors).

Example 2 Study of the mechanism of action of indirubins

Kinetic experiments are carried out for this purpose by varying both the ATP levels and the indirubin-3 '-monoxime concentrations. The results obtained are given in FIG. 2 (l / V as a function of l / ATP).

ATP concentrations in the reaction mixture vary from 0 to 2 μM, the concentration of GS-1 being kept constant at 6.7 μM. It is found that indirubin-3 '-monoxime acts in competition with ATP to bind.

The linearity of the slope in the box in the figure shows that it is a linear inhibitor.

The apparent inhibition constant K _± is 50 nM. Example 3 Study of the inhibition by indirubins, in vitro and in vivo, of the phosphorylation of tau by GSK-3β

The results obtained with 1 'indirubin-3 are reported.

-monoxime on the phosphorylation of a physiological substrate, constituted by the tau protein binding microtubules. A recombinant human tau protein expressed in a bacterium is used. The protein is phosphorylated in vi tro by GSK-3β in the presence of increasing concentrations of indirubin-3 '-monoxime. We then proceed to a resolution by

SDS-PAGE, followed by autoradiography.

FIG. 3A gives the results obtained and shows that phosphorylation is inhibited in a dose-dependent manner by 1 'indirubin-3' -monoxime, with an IC ₅₀ around 100 nM. A quantification of these results is given in FIG. 3B which gives the% of phosphorylation of tau as a function of the concentration of indirubin-3 '-monoxine (nM).

In vivo studies have also been performed. The results are illustrated in Figure 3C.

The Sf9 cells expressing htau23 or have not undergone any treatment (-) or have been exposed to 0.2 μM of okadaic acid (OA), or to 50 μM of indirubin-3 '-monoxime or to NG- 97 for 5h. The cell lysates (3 μg of htau23) were resolved by SDS-PAGE, stained with Coomassie blue or reacted with various antibodies. K9JA (pan-tau antibody) recognizes all preparations containing tau. AT8, AT 180 and PHF1 are specific for various phosphorylated units SP or TP, namely, respectively, Ser 202; Thr 205, Thr 231; Ser 235 and Ser 396; Ser 404 (numbering in htau 40, which corresponds to the longest isoform of the human tau protein). AT 100 recognizes the tau protein phosphorylee at T 212 and S214 (very specific reaction of the tau protein in Alzheimer's disease, but which also occurs in Sf9 cells, if the 2 sites are phosphorylated).

The disappearance of the AT100 signal after treatment with 1 'indirubin-3' - monoxime shows that this derivative is well capable of inhibiting GSK-3β type activity in Sf9 cells. Figure 3D represents the diagram of tau isoforms, epitopes recognized by antibodies and preferred phosphorylation sites: (a) htau 23, (b) htau 40, the smallest and largest of the 6 isoforms generated by the assembly alternative (residues 352 and 441). The htau 23 protein lacks the N-terminal inserts and the second repeat. The repetitions are shown in gray and the adjacent regions are shaded. Some epitopes are indicated. Example 4 Study of the inhibition by indirubins, in vitro and in vivo, of the phosphorylation of DARPP-32 by CDK5 / p25

The neural protein DARPP-32 has been identified as a physiological substrate for CDK5 / p25. DARPP-32 becomes a cAMP-dependent kinase inhibitor (PKA) when it is phosphorylated by CDK5 / p25 on Thr 75.

This protein was used as a substrate for in vitro phosphorylation by CDK5 / p25.

Slices of striatum from the brain of an adult mouse are prepared by operating according to standard methodology. After equilibration in a pad of oxygenated Krebs bicarbonate by continuous aeration (95% 0 ₂ /5% C0 ₂ ), the sections are treated with different concentrations of indirubin-3 '-monoxime, or with 10 μm of roscovitine for 60 min. , or are left in the Krebs bicarbonate pad for the same time. The sections are homogenized by sonication in SDS

1% and 50 mM NaF at boiling point. Protein concentrations are determined by the BCA method using a standard BSA curve. Equal amounts of protein (80 μg) are subjected to an SDS-PAGE using a 15% acrylamide gel, then an electrophoresis transfer is carried out on a cellulose membrane, then immunoblotting with an antibody specific for a situation phosphorylation capable of specifically detecting DARPP-32 phosphorylates on Thr 75. The results are reported in FIG. 4. It can be seen that indirubin-3 '-monoxime inhibits this phosphorylation in a concentration-dependent manner, with an IC ₅₀ around 100 nM. The phosphorylation of DARPP-32 by CDK5 / p25 can be controlled with a phosphospecific antibody directed against

DARPP-32 phospho-Thr 75.

No phosphorylation is observed in vivo on this site in the tissue p35 ^~ / ^~ • There is an inhibition by

1 'indirubin-3' -monoxime, which shows that indirubins are capable of inhibiting CDK5 in vivo.

Claims

1. Use for the manufacture of GSK-3β inhibitor drugs of indirubin derivatives corresponding to the general formula I: in which R and R, identical or different, represent a hydrogen atom, a halogen atom; a hydroxy group; a methylenehydroxy group; an alkyl or alkyloxy or methylenealkoxy radical, with straight or branched chain, from C1 to Cl8; a cycloalkyl radical having 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a substituted or unsubstituted aryl, aralkyl or aryloxy radical, optionally comprising one or more heteroatoms; a mono-, di- or trialkylsilyl group having 1 to 6 carbon atoms, independently of each other, in each case, in the straight or branched chain alkyl group; a mono-, di- or triarylsilyl group, with substituted or unsubstituted aryl groups, independently of each other, in each case; a trifluoromethyl group; a -COM group; -COOM; or -CH ₂ COOM, with M representing a hydrogen atom, a C1 to C18 alkyl group, with straight or branched chain, optionally substituted by one or more hydroxy and / or amino groups, or an aryl group comprising if necessary one or more heteroatoms, which may be substituted by one or more halogen atoms, alkyl radicals or alkoxy group; a group -NR ^" "^" R, in which R ¹¹ and R ¹² , which are identical or different, represent a hydrogen atom, a C1 to C18 straight or branched chain alkyl radical, optionally further substituted by one or more hydroxy and / or amino groups, an aryl group, substituted or not, optionally comprising one or more heteroatoms; an acyl group; a methyleneamino -CH ₂ -NR R group, in which R ¹¹ and R ¹ have the above meanings; a benzyl group, in which the benzene ring comprises, where appropriate, one or more heteroatoms; a methylenecycloalkyl group with 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a physiological amino acid residue bound to nitrogen, such as an amide; an O-glycoside or an N-glycoside, in which the glycoside is chosen from monosaccharides or disaccharides; or a methylenesulfonate group; R, R, R, c -iggig R, R, R, R and R, identical or different, represent a hydrogen atom; halogen; a hydroxy group; a nitroso group; a nitro group; an alkoxy group; a C1 to C18 straight or branched chain alkyl group, optionally substituted by one or more hydroxy and / or amino groups; an aryl group, substituted or not, optionally comprising one or more heteroatoms; an aralkyl group, substituted or not, optionally comprising one or more heteroatoms; an aryloxy group, substituted or not, optionally comprising one or more heteroatoms; a methylenearyloxy group, substituted or not, optionally comprising one or more heteroatoms; a cycloalkyl group, with 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a methylenecycloalkyl group, with 3 to 7 carbon atoms, optionally comprising one or more heteroatoms; a trifluoromethyl group; a -COM group; -COOM; or CH ₂ COOM, with M representing a hydrogen atom, a C1 to C18 alkyl group, with straight or branched chain, optionally further substituted by one or more hydroxy and / or amino groups, or an aryl group comprising if necessary one or more heteroatoms, may be substituted by one or more halogen atoms, alkyl groups or alkoxy groups; a group -NR ¹ ^ ^{"1" 2} , in which R ¹¹ and R ¹ , identical or different, represent a hydrogen atom, an alkyl radical with a straight or branched chain, from C1 to C18, optionally also substituted by one or more hydroxy and / or amino groups, a group substituted or unsubstituted aryl, optionally comprising one or more heteroatoms, an acyl group, or in which the nitrogen atom is part of a cycloalkyl group having 3 to 7 carbon atoms, optionally comprising one or more heteroatoms ; a group -CONR ^{1: L} R ¹² , in which R ¹¹ and R ¹ have the above meanings; a hydroxylamino group; a phosphate group; a phosphonate group; a sulfate group; a sulfonate group; a sulfonamide group; a group -S0 ₂ NR ^{ι "} R ¹² , in which R ¹¹ and R ¹² have the meanings given above; an azo group - N = NR ¹³ , in which R ¹ represents an aromatic group, optionally substituted by a or more carboxyl, phosphoryl or sulfonate groups, or an O-glycoside or N-glycoside group, in which the glycoside is chosen from monosaccharides or dissacharides; or R ¹ and R ⁵ , and R ⁶ and R ¹⁰ , respectively, together form, independently of one another, a ring having 1 to 4 CH ₂ groups, optionally substituted; and X and Y, identical or different, represent an oxygen atom, sulfur, selenium, tellurium; a group -NR ¹⁴ , in which R ¹⁴ represents a hydrogen atom, an alkyl group, with a straight or branched chain, from C1 to C18, optionally substituted by one or more carboxyl, phosphoryl or sulfonate groups, a substituted or unsubstituted aryl group, optionally comprising one or more heteroatoms , an aralkyl group, or a sulfonate group; or -NOR ¹⁴ , in which the group R ¹⁴ has the meanings given above, and the salts of these physiologically acceptable derivatives. 2. Use according to claim 1, characterized in that, in said indirubin derivatives, one or more atoms of one or more benzene rings are replaced by nitrogen atoms. 3. Use according to claim 1 or 2, characterized in that in said indirubin derivatives, one or more aromatic or non-aromatic ring systems, which comprise, where appropriate, one or more heteroatoms independently of one another , are condensed to the indirubin system. 4. Use according to any one of claims 1 to 3, characterized in that the indirubin derivatives are linked to a polyethylene glycol ester or to a polyethylene glycol ether by bonds, respectively, ester or ether. 5. Use according to any one of claims 1 to 4, characterized in that, in said indirubin derivatives, X and Y, identical or different, represent a group = O or = NOH. 6. Use according to claim 5, characterized in that the substituents R, R, R and R of the said indirubin derivatives have the following meanings: - R ^3: a hydrogen atom, halogen, alkyl, -S0 ₃ H, -S0 ₂ NH _2, -S0 ₂ -N (CH _{3) 2,} -S0 ₂ -NC ₂ H ₅ -OH, -S0 ₂ -N- (C ₂ H ₅ -OH) ₂ , -S0 ₂ -NH-CH ₃ , - R ⁴ and R ⁸ , independently of one another: a hydrogen atom or halogen, R: an alkyl or aryl radical, the other substituents given in formula (I) representing a hydrogen atom. 7. Use according to any one of claims 1 to 4, characterized in that in said indirubin derivatives, X = Y, these two substituents representing a group = O. 8. Use according to claim 7, characterized in that the substituents R, R, R and R, in said indirubin derivatives, have the following meanings: - R: alkyl, in particular methyl, and phenyl - R ^3: hydrogen, halogen (F, Cl, Br, I), N0 _2, - S0 ₃ H, -S0 ₂ NH _2, -S0 ₂ -N (CH _{3) 2,} -S0 ₂ -NC ₂ H ₅ -OH, -S0 ₂ -N- (C ₂ H ₅ -OH) ₂ , - S0 ₂ -NHCH ₃ , -R ⁴ and R: halogen, in particular I or Br, the other substituents given in formula (I) representing a hydrogen atom. 9. Use according to claim 8, characterized in that said derivatives are chosen from indirubin, 5-iodo-indirubin, 5-bromo-indirubin, 5-chloro-indirubin, 5-fluoro-indirubin, 5-methyl-indirubin, 5-nitro-indirubin, 5-SO ₃ H-indirubin, 5 '-bromo-indirubin, 5- 5' -dibromo-indirubin or 5 '-bromo-indirubin acid 5 - sulfonic. 10. Use according to any one of claims 1 to 4, characterized in that, in said indirubin derivatives, X represents a group = NOH and Y a group = O. 11. Use according to claim 10, characterized in that R represents a halogen atom, in particular I, or a group -S0 ₃ Na. 12. Use according to claim 11, characterized in that said derivatives are chosen from 1 'indirubin-3' - monoxime, 5-iodo-indirubin-3 '-monoxime and 5-S0 ₃ Na-indirubin-3' - monoxime. 13. Use according to any one of claims 1 to 12, characterized in that the medicaments are prepared for oral administration, in the form of capsules, tablets, dragees or capsules. 14. Use according to any one of claims 1 to 12, characterized in that the medicaments are prepared for administration by injectable route, in the form of a solution. 15. Use according to any one of claims 1 to 14, for the treatment of pathologies involving GSK-3β and CDK5.