RU2486184C2 - Azaindole-indole derivatives, methods for preparing and using - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to azaindole-indole derivatives and pharmaceutically acceptable salts thereof of general formula: Y=Z (G), wherein Y means a group of azaindole of formula (Yl)

Z means a group of indole of formula (Z1)

or (Z2)

wherein the values "=", R, R¹, R^1', R², R³, R⁴, R^2', R³, R^4', R^5' are presented in cl. 1 of the patent claim.

EFFECT: compounds inhibit cycline-dependent kinase that enables using them in a pharmaceutical composition.

6 cl, 3 dwg, 9 tbl, 8 ex

Description

Technical field

The present invention relates to one group of azaindole-indole derivatives of a completely new structure. The molecular structure of these derivatives is characterized by the fact that they are connected by azaindoles with bimolecular indoles at different positions, large π-conjugated heterocyclic systems are formed. These derivatives through various mechanisms inhibit cell growth and proliferation disorders. The present invention also includes methods for the manufacture of derivatives, including pharmaceutical compositions of the described derivatives and their use.

State of the art

The present invention patent is a patent following the following patents:

1) “Specific indole compounds, the method of their manufacture and use in the treatment and prevention of cancer and other diseases”, CN 02138518, 1A, published 04.05.2005);

2) “An emulsion for use in insoluble preparations and a method for its manufacture”, CN 200410052816, 5A, priority July 14, 2004);

3) “A method of manufacturing derivatives of N (1) -hydrocarbyl-3 'oxime-indirubin (I) and their medical use”, CN 200510094482.2 A, published on 01.08.2007;

4) "Dispersion agent for the manufacture of insoluble preparations", CN 200610038112.1 A, priority 01/17/2006.

Monomeric compounds of plants are leading among drugs in the fight against cancer, such as Kamptothecin, isolated from the Kamptoteka genital, and Paclitaxel, obtained from Golovchatotissa, which are two well-known drugs.

Modern pharmaceutical studies of traditional herbal medicines have shown that indigo naturalis diindole derivatives - indirubins (1, indirubin, 2 ', 3-diindole, purple) are especially effective for the treatment of chronic myeloid leukemia (CML), they are characterized by rapid action, the use of a small amount less side effect, lower price, etc. Subsequently, structural treatments and extensive scientific studies of the biological activity of the compounds of diindole containing indigo naturalis [including indigo (2), 2,2'-diindole, blue; isoindigo (3), 3,3'-diindole, brown color], while it was noted that H-1-methyl-isoindigo have a better therapeutic effect than indirubins and low toxicity.

Further studies showed that the pharmacodynamic mechanism of indirubin and its derivatives prevent the spread of the tumor by inhibiting cyclin-dependent kinase, the so-called cyclin kinases (CZKs).

The family of cyclin-dependent kinases (CZKs) is a typical serine / threonine kinase, and signal growth molecules of cells that act mainly on different phases of the cell cycle (the cell cycle process is divided into four phases G1, S, G2 and M), which allows cells to grow and proliferate (DNA replication and chromosome segregation), dormancy (cells break away from the separation cycle, enter the dormant growth and separation phase, known as the G0 stage) or enter apoptosis. CZK also have the function of regulating nerves and thymus. CZKs differ from other kinases; they play the role of a catalyst and a regulator only if they are bound to the corresponding cyclins and a complex dimer complex is formed. In human cells, at least there are nine members of the CZK family (CZK 1 ~ 9) and 11 cyclins (A ~ J). Different CZKs are combined with different cyclins or their subradicals, as follows: CZK1 binds to cyclins A and B1-B3; CZK2 binds to cyclins A, D1-D3 and E; CZK4, CZK5 and CZK6 bind to cyclins D1-D3; CZK5 also binds mainly to pk35; CZK7 binds to cyclin H; CZK6 binds to cyclin K associated with D.

According to the results of biomedical research of the Nobel Prize in 2001 - "The relationship of cell proliferation and cancer", almost all tumor cells have various abnormalities of cell cyclin kinase ^[1-2] , cancer cells continuously enter phases S, G2 and M, and unlimited proliferated. For example, more than 85% of patients with breast cancer with abnormal cyclin E / CZK4 / 6 ^[3] . By inhibiting cyclin-dependent kinases, one can effectively block cell proliferation (but not kill cells), thereby either promoting cell differentiation and maturation, or promoting apoptosis; which allows to achieve the effect of treating various tumors. There is reason to believe that cell cyclin kinase, as an inhibitor, is a new type of broad-spectrum anticancer drug. In addition, these drugs inhibit cell reproduction, but do not kill cells; they are characterized by high selectivity, good efficiency and low toxicity.

Studies have shown that cyclin-dependent kinases as inhibitors can effectively inhibit breast cancer, colon cancer, prostate cancer, brain cancer, and other cancers. More significant is that they inhibit cell proliferation, these compounds also have good effects of inhibiting hormone-dependent prostate cancer cells (PC-3, DU-145), a number of hormone-resistant and chemotherapy-resistant cells of late metastatic prostate cancers. Thus, the search for a cyclin-dependent kinase inhibitor has already become a new rational strategy for the research and development of new anti-cancer drugs ^[4-6] .

To date, about 10 types of inhibitors and / or control agents of CKM, which are small in molecular structure, have attracted attention and have been studied; these are mainly CKK inhibitors directed to the ATP- region ^[7] . Indirubin and N-1-methyl-isoindigo used in clinical treatment are one of these species ^[8–9] . In addition, two more compounds entered the period of clinical trials: UCN-01 and flavopiridol, developed by the National Cancer Institute (NIR) of the USA ^[10] .

To summarize, indirubin-type diindole compounds are an important inhibitor of CZK and have low toxic side effects, but this type of compound is poorly soluble in water and fat, which interferes with their use in clinical treatment. In recent years, many foreign research institutes and pharmaceutical companies have carried out extensive structural changes in compounds of this type, however, the efficacy of indirubin-type diindole compounds against tumors is still far from satisfactory.

In short, in this area it is urgent to develop new inhibitors having excellent ability to inhibit the activity of cyclin-dependent kinases (CZK).

Disclosure of invention

The purpose of the present invention is to provide derivatives of azaindole-indole as an inhibitor of the central circulatory system; these compounds should have high inhibitory activity, dissolve well in water and other advantages.

Another objective of the present invention is to provide methods for the manufacture of these compounds, their pharmaceutical compositions and their use.

According to the first invention, derivatives of azaindole-indole and their pharmaceutically acceptable salts, according to the formula,

,

,

where Y represents an azaindole group according to the formula (Y1) or (Y2);

Z represents an indole group according to the formula (Z1) or (Z2);

"=" means a double bond located at the 3-position of the azaindole group Y and between the 2'- and 3'-positions of the indole-Z group;

in the above formulas, Y1, Y2, Z1 and Z2, R ¹ and R ^{1 '} independently represent H or the following groups which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ ~ C ₆ alkyl, aryl, aralkyl, acyl, aroyl, glycosyl or biosil protected by acyl, glycosyl or biosil; wherein the substituents described are selected from: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino;

R ² , R ³ , R ⁴ , R ^{2 '} , R ^3' , R ^{4 '} and R ^5' independently represent H, halogen, hydroxyl, sulfhydryl or the following groups, which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ ~ C ₄ alkyl, nitro, amino, amido, amide, C ₁ ~ C ₄ alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonic group, sulfamoyl, isocyanate or alkyl isocyanate, when the substituents described herein are selected from: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino;

R is oxygen, sulfur, selenium, or an NR ⁶ or NOR ⁶ group in which R ⁶ is H or the following groups which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ ~ C ₆ straight-chain alkyl or a branched bond, aryl, aralkyl, C ₃ ~ C ₆ alicyclic group, acyl, aroyl, sulfonyl or phosphoryl; wherein the substituents described are selected from: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino.

In another optimal example, the compounds described are shown in formulas (I), (II), (III) or (IV) in which formula (I) is 5-azaindirubin derivatives, formula (II) is 5-azaisoindigo derivatives, formula (III) - derivatives of 7-azaindirubin, and formula (IV) - derivatives of 7-azaisoindigo;

where R, R ¹ , R ² , R ³ , R ⁴ , R ^{1 '} , R ^2' , R ^{3 '} , R ^4' and R ^{5 'are} designated as described above.

In another optimal example, R ¹ and R ^{1 ′} independently are H, C ₁ ~ C ₆ alkyl, aryl, aralkyl, acyl, aroyl, glycosyl, protected by alkyl or glycosyl;

R ² , R ³ , R ⁴ , R ^{2 '} , R ^3' , R ^{4 '} and R ^5' independently represent H, halogen, hydroxyl, sulfhydryl, C ₁ ~ C ₄ alkyl, amino, amido, amide, C ₁ ~ C ₄ alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonic group or isocyanate;

the aforementioned glycosyls are arabinose, xylose, ribose, mannose or glucose;

R is oxygen, sulfur, selenium, or an NR ⁶ or NOR ⁶ group in which R ⁶ is H, a C ₁ ~ C ₆ straight chain or branched bond, aryl, aralkyl, a C ₃ ~ C ₆ alicyclic group, acyl , aroyl, sulfonyl or phosphoryl.

In another optimal example, the compounds described are selected from the following groups: 5-azaindirubin derivatives; (table 1: compounds 1-59), 5-azaisoindigo derivatives (table 2: compounds 60-89), 7-azaindirubin derivatives (table 3: compounds 92-150) and 7-azaisoindigo derivatives (table 4: compounds 151-180 )

In another optimal example, the aforementioned pharmaceutically acceptable salts include salts formed with inorganic acids or organic acids, wherein inorganic acids include the following: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid and sulfuric acid; organic acids include the following: formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1, 5), Asian acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, butyl carboxylic acid, diethylacetic acid, malonic acid , succinic acid, fumaric acid, pimelic acid, hexanediol acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, glyconic acid, ascorbic acid and, nicotinic acid, isonicotinic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid and amino acids.

Secondly, the present invention provides a Pharmaceutical composition comprising: (a) compounds or their pharmaceutically acceptable salts, according to claim 1 of the formula; (b) pharmaceutically acceptable carriers.

In another optimal example, the form of execution of the composition is as follows: injection for small, medium and large volumes, powder for injection, emulsion for injection, tablets, pills, capsules, ointments, creams, plasters, liniment, powder, aerosols, implants, drops, suppositories, ointments; various types of nano-drugs; or liposomes.

In another optimal example, the described pharmaceutical compositions can be used alone as a monomer or in combination with other drugs (e.g., joint use in surgery, use in combination with one or more European medicine or herbal medicines of Chinese medicine, or in combination with radioactive treatment and gene therapy, or combined use with bio-regulators).

Thirdly, the present invention provides a method of manufacturing a pharmaceutical composition comprising the following steps: (a) mixing the compounds or their pharmaceutically acceptable salts according to claim 1 and (b) pharmaceutically acceptable carriers, thereby forming a pharmaceutical composition.

Fourthly, the present invention provides the use of compounds or their pharmaceutically acceptable salts for the treatment of the following diseases: diseases caused by abnormalities of the cyclin-dependent kinase, cell growth and proliferation disorder, or insulin resistance.

In another optimal example, the diseases described include malignant cancers, psoriasis, viral skin diseases, HIV, degeneration and disorder, and other diseases of the nervous system, as well as type 2 diabetes.

Fifthly, the present invention provides a composition that contains compounds or their pharmaceutically acceptable salts of the formula IG as an inhibitor of cyclin-dependent kinase.

In another optimal example, the compositions described are a pharmaceutical composition (containing carriers acceptable to pharmaceutical products), a food composition (containing carriers acceptable to food products), and a cosmetic composition (containing carrier acceptable to cosmetics).

Sixthly, the present invention provides methods for the internal and external inhibition of cyclin-dependent kinase of mammalian animals, or methods of treating diseases caused by excessively high activity of the cyclin-dependent kinase, including measures for administering to subjects requiring treatment, compounds of formula IG, or pharmaceutically acceptable salts, or pharmaceutical compositions containing compounds of the formula IG or pharmaceutically acceptable salts.

In another optimal example, the described diseases caused by too high cyclin-dependent kinase activity include: malignant tumors, psoriasis, viral skin diseases, HIV, neurodegeneration, disorder and other diseases of the nervous system.

Brief Description of the Drawings

Figure 1 shows the general structural formula of derivatives of 5- and 7-azaindirubin and derivatives of 5- and 7-azaisoindigo.

Figure 2 shows the degree of inhibition of the 5- and 7-azaindirubin derivative and the 5- and 7-azaisoindigo derivative with respect to the growth of androgen-independent human prostate cancer cells DU145. Using various concentrations of compounds No. 107, 108, 112, 115, the cancer cells DU145 are treated with a logarithmic period of human prostate growth for 72 hours, and the cell growth rate is measured by MTT.

Figure 3 shows the inhibition of the derivative of 5- and 7-azaindirubin and 5- and 7-azaisoindigo of the CK androgen-independent human prostate cancer cells DU145; using compounds No. 124, 126 of various concentrations, they process cancer cells DU145 of the logarithmic period of human prostate growth for 24 hours, the total protein volume of the cells is removed, measured using the Western blot method of phosphor-CZK2Thr160, p27 and cyclin-D1, taking β-Actin as an internal standard.

The best embodiment of the invention

After extensive and in-depth studies, the inventor for the first time created one group of azaindole-indole derivatives as inhibitors of cerebral palsy; these compounds are formed by azaindoles in connection with indole paramolecules at various positions; large π-conjugated heterocyclic systems are formed in them. Tests have shown that this group of azaindole indole derivatives can, through various mechanisms, exert biological activity, inhibit cell growth and proliferation, suppress cyclin-dependent kinases, induce endogenous cyclin kinase inhibitors (ICCs), and also restore the conversion of insulin signals and other functions, thereby treat various diseases caused by cell growth disorder, including cancer, psoriasis, viral skin diseases, HIV and neurodegeneration, nervous system disorder s, also type 2 diabetes caused by insulin resistance.

Compounds of the present invention

As used herein, the terms “compounds of the present invention” or “azaindole-indole derivatives” can be used interchangeably to refer to compounds or pharmaceutically acceptable salts shown in the general formula IG.

Specifically, in the present invention, a substantial structural transformation of the indirubin and isoindigo compounds is carried out, the solubility and bioavailability are increased, the effect in drug therapy is enhanced, the doses of the drug are reduced, the negative reactions of the drug are reduced. Compared to the mother nuclei of the existing indirubin and isoindigo compounds, large π-conjugated heterocyclic systems are formed in the mother nuclei of the compounds of the present invention, thereby improving the solubility of the compounds of the present invention in water.

One group of the optimal compound is shown in the general formula (I), the general formula (II), the general formula (III) and the general formula (IV), among them in the general formula (I) are derivatives of 5-azaindirubin, in the general formula (II) - 5-azaisoindigo derivatives, in the general formula (III) - 7-azaindirubin derivatives, in the general formula (IV) - 7-azaisoindigo derivatives.

R ¹ and R ^{1 ′} independently represent H or the following groups which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ ~ C ₆ alkyl, aryl, aralkyl, acyl, aroyl, glucosyl or biosil protected by acyl, glycosyl or biosil; wherein the substituents described are selected from: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino;

R ² , R ³ , R ⁴ , R ^{2 '} , R ^3' , R ^{4 '} and R ⁵ ' independently represent H, halogen, hydroxyl, sulfhydryl or the following groups, which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ ~ C ₄ alkyl, nitro, amino, amido, amide, C ₁ ~ C ₄ alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonic group, sulfamoyl, isocyanate or alkyl isocyanate, when the substituents described herein are selected from: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino;

R is oxygen, sulfur, selenium, or an NR ⁶ or NOR ⁶ group in which R ⁶ is H or the following groups which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ ~ C ₆ straight-chain or branched alkyl bonds, aryl, aralkyl, C ₃ ~ C ₆ alicyclic group, acyl, aroyl, sulfonyl or phosphoryl; wherein the substituents described are selected from: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino.

In the above general formulas (I), (II), (III) and (IV), there are better compounds, in particular: R ¹ and R ^{1 '} independently represent H, C ₁ ~ C ₆ alkyl, aryl, aralkyl, acyl , aroyl, glycosyl protected by alkyl or glycosyl;

R ² , R ³ , R ⁴ , R ^{2 '} , R ^3' , R ^{4 '} and R ^5' independently represent H, halogen, hydroxyl, sulfhydryl, C ₁ ~ C ₄ alkyl, amino, amido, amide, C ₁ ~ C ₄ alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonic group or isocyanate;

the aforementioned glycosyls are arabinose, xylose, ribose, mannose or glucose;

R is oxygen, sulfur, selenium, or an NR ⁶ or NOR ⁶ group in which R ⁶ is H, a C ₁ ~ C ₆ straight chain or branched bond, aryl, aralkyl, a C ₃ ~ C ₆ alicyclic group, acyl , aroyl, sulfonyl or phosphoryl.

More preferred types of compounds are derivatives of azaindolene-indole prepared in examples of implementation, namely in the following table:

Connection number type of 1-59 5-azaindirubin derivatives 60-89 5-azaisoindigo derivatives 92-150 7-azaindirubin derivatives 151-180 7-azaisoindigo derivatives

Pharmaceutically Acceptable Salts

Compounds of the present invention and their pharmaceutically acceptable salts formed with inorganic or organic acids are also included in the present invention. Inorganic acids include the following: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid. Organic acids include the following: formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1, 5), Asian acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, butyl carboxylic acid, diethylacetic acid, malonic acid , succinic acid, fumaric acid, pimelic acid, hexanediol acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, glyconic acid, ascorbic acid and, nicotinic acid, isonicotinic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid and amino acids.

In the present invention, the physical and chemical properties of the described salts are improved, the cell permeability, the ability to access the cells are increased, thereby increasing the effectiveness of the drug.

The activity of the compounds of the present invention

The compounds of the present invention and their salts are cyclin-dependent kinase inhibitors, and they can induce endogenous cyclin-dependent kinase inhibitors, thereby inhibiting cell growth and proliferation, and promote apoptosis of tumor cells. The compounds of the present invention and their salts by restoring the conversion of insulin signals increase the sensitivity of peripheral tissues in the use of insulin, reduce the effect of insulin resistance. Thus, the compounds and their salts indicated in the present invention can be used in the preparation of drugs for treating diseases, abnormal cyclin-dependent kinase, cell growth and proliferation disorder, or insulin resistance. These diseases include cancer, psoriasis, viral skin diseases, HIV, as well as neurodegeneration, a nervous system disorder, and type 2 diabetes.

To facilitate understanding of the present invention, the inventor has provided a description of the mechanism of action of the compounds of the present invention. It should be understood, however, that the scope of protection of the present invention is not limited to the described mechanism.

In the present invention, for example, azaindirubin is essentially the product of the binding of the azaindole molecule to the indole molecule at the 3.2 'position; for example, azaisoindigo, it is the product of the cohesion of the azaindole molecule with the indole molecule at the 3.3'-position. Replacing the nitrogen atom of one of the carbon atoms of the benzene ring allows one to obtain the following four types of isomers:

Purine (a specific example below) is one of the 10 known types of chemical structure inhibitors of the CHA, as well as a inhibitor of the CHA ^[7] in the first studies. In terms of structure, azaindoles and purines have many similarities, they have 6-membered aromatic heterocycles and 5-membered heterocycles.

The test results of the present invention showed that the compounds of the present invention have a similar activity of a CKK inhibitor.

Non-insulin-dependent diabetes mellitus (type 2 diabetes) is one of the main diseases in the world that is dangerous for human health and leads to its death, its pathogenesis mechanism is insulin resistance. Studies have shown that indirubin derivatives affect the activity of PI3K components (phosphoinositide-3-kinase) on the insulin signaling pathway by activation (protein kinase) and inhibition (the target for rapamycin in mammals), restore the conversion of insulin signals, thereby increasing the sensitivity of peripheral tissues to insulin, reduce the effect of insulin resistance.

Thus, the derivatives of the present invention have an effect against tumors, psoriasis, viral skin diseases, HIV, neurodegeneration, disorders of the nervous system, as well as type 2 diabetes.

Compositions and methods of use

The present invention also provides pharmaceutical compositions comprising the compounds of the present invention. The described compositions can be used to inhibit the activity of CZK, the induction of ILC, restore the conversion of the signal of insulin. The composition of the present invention can be a pharmaceutical combination (containing pharmaceutically acceptable carriers), a combination of health care products (containing pharmaceutically acceptable carriers), a combination of food products (containing acceptable food carriers), and a cosmetic combination (containing cosmetic acceptable carriers).

Preferably, if the composition of this invention is a pharmaceutical composition containing the compounds of the present invention (or their pharmaceutically acceptable salts), as well as a number of pharmaceutically acceptable carriers or excipients.

The embodiments of the pharmaceutical composition of the present invention are not limited, they can be in any clinically acceptable form. The forms of execution of the composition are as follows: injection for small, medium and large volumes, powder for injection, emulsion for injection, tablets, pills, capsules, ointments, creams, plasters, liniment, powder, aerosols, implants, drops, suppositories, ointments; various types of nano-drugs; of the corresponding liposomes, the aforementioned injections are mainly made. As a rule, various dosage forms must meet the methods of drug administration.

Preferably, the pharmaceutical compositions of this invention are made in the form of injections, liquid agents, solid agents. Such solid drug combinations can be made using conventional methods. Medicinal combinations, for example, injection, liquid preparations, solid preparations should be made under sterilization conditions or appropriate cleaning.

In another optimal example, the present invention provides injections of a compound (or pharmaceutically acceptable salts thereof) of the present invention, i.e. emulsions, sub-micro-emulsions, nano-emulsions prepared using surfactants and / or a solvent and / or oil components and / or other auxiliary materials.

In another optimal example, the present invention provides a solid dispersion agent of a compound (or pharmaceutically acceptable salts thereof) of the present invention, i.e. high concentration preparations are dispersed in water-soluble, non-water-soluble, enteric-soluble inert carriers, a dispersion system is formed in the form of a solid, and by traditional manufacturing methods: capsules, tablets, pills, ointments, suppositories, as well as injectables, etc. As a result of this compound, they not only retain a high degree of dispersion, but are also stably stored.

Drug Administration

When using a drug combination, a safe and effective amount of a compound of the present invention is administered in mammals; the so-called safe and effective amount, as a rule, is at least about 1 mg / day, and in most cases no more than 10 mg / kg of body weight. And the best dose is about 1 mg / day or about 3 mg / kg of body weight. Of course, the specific dose is also determined depending on the methods of drug administration and the patient’s health status and other factors, these issues should be within the qualifications of experienced doctors.

The compounds (or their pharmaceutically acceptable salts) of the present invention can be used separately as a monomer or in combination with other drugs. The selected combined use includes: joint use with surgery, use in combination with one or more European medicine, in combination with herbal medicines of Chinese medicine, in combination with radioactive treatment and gene therapy, in combination with bio-regulators.

Methods of administering the pharmaceutical compositions of the present invention are not limited to specific conditions, including: oral administration, injection, intratumoral administration, implant administration, intracavitary administration, anal administration, skin-tight administration, internal and external application.

Optimal injections include: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary injection.

Manufacturing methods

The compound of the present invention described in the general formulas IG can be prepared in accordance with the process according to the following formula, as well as taking into account known synthetic methods in the field of this technology. In general, in the next preparation process, all reactions should be carried out at a temperature of -10 ° to the recirculation temperature, usually at room temperature (about 25 °) to the recirculation temperature. It is better if the reaction temperature is at a temperature of 5-100 °, even better at temperatures of 20-80 °. The reaction time, as a rule, without special restrictions, is usually 1 minute - 24 hours, preferably 1-20 hours. Solvents should usually be polar, such as water, the master file of the drug, alcohols (e.g. methanol, ethanol, isopropanol, etc.). Physicochemical methods such as ¹ H NMR spectroscopy ( ¹ H-NMR), mass spectrometry (MS), and elemental analysis for structural identification can be used in the synthesis of chemical compounds.

1. Intermediate and target compounds of 5-azaindirubin (general formula I)

(1) Synthesis of intermediate: 1-hydrocarbyl-5-azaindole-2,3-dione (A):

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , acyl protected glycosyl, etc.

5-azaindole with hydrocarbyl is taken at the N-1 position, and then, as a result of the oxidation reaction with CrO ₃ and СН ₃ COOH, product (A) is obtained: 1-hydrocarbyl-5-azaindole-2,3-dione ^[11] .

(2) Synthesis of intermediate: 1-acetyl-3-hydroxyindole derivatives (B)

Where R ^{3 ′} = H, Cl, Br, F, CH ₃ , OCH ₃ , SCH ₃ , Ph, etc.

Product (B) is prepared as follows: substituted derivatives of 2-aminobenzoic acid with chloroacetic acid are taken as a substituent, acylation and ring closure are performed in the presence of acetic anhydride and sodium acetate, and reduced.

(3) Synthesis of the target compound: derivatives of 5-azaindirubin (I)

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , glycosyl protected by acyl, etc .; R ^{3 ′} = H, Cl, Br, F, CH ₃ , OCH ₃ , SCH ₃ and Ph, etc.

1-hydrocarbyl-5-azaindole-2,3-dione and 1-acetyl-3-hydroxyindole or 5-halogen-containing-1-acetyl-3-hydroxyindole are heated under acidic conditions, irrigating N ₂ , respectively, to obtain 1-hydrocarbyl-5 derivatives α-azindirubin (I).

(4) Synthesis of the target compound: 3'-oximido-5-azaindirubin derivatives

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , glycosyl protected by acyl, etc .; R ^{3 ′} = H, Cl, Br, F, CH ₃ , OCH ₃ , SCH ₃ , Ph, etc.

(5) Synthesis of the target compound: 5-azainindirubin-3'-oxime ether, etc.

Where R = CH ₃ ON, EtON, R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , etc., R ^{3 '} = H, Cl, Br and F, and so on.

2. The target compound 5-azaisoindigo (General formula II)

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , glycosyl protected by acyl, etc., R ^{3 '} = H, Cl, Br, F, OH and OCH ₃ , etc.

Derivatives of 1-hydrocarbyl-5'-substituted 5-azaisoindigo (II) are prepared by reacting 1-hydrocarbyl-5-azaindole-2,3-dione (A) with 5-substituted 2-hydroxyindole under alkaline conditions.

3. Intermediate and target compounds of 7-azaindirubin (general formula III)

(1) Synthesis of intermediate: 1-hydrocarbyl-7-azaindole-2,3-dione (C):

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ glycosyl protected by acyl, etc.

Take 7-azaindole with an alkyl group in the N-1 position, and then, as a result of the oxidation reaction with CrO ₃ and CH ₃ COOH ^[11] , product (C) is obtained: 1-hydrocarbyl-7-azaindole-2,3-dione (III )

(2) Synthesis of the target compound: 7-azaindirubin (III)

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , acyl protected glycosyl, etc .; R ^{3 ′} = H, Cl, Br, F, CH ₃ and OCH ₃ , SCH ₃ and Ph, etc.

1-hydrocarbyl-7-azaindole-2,3-dione and 1-acetyl-3-hydroxyindole or 5-halogen-containing-1-acetyl-3-hydroxyindole are heated under acidic conditions, irrigating N ₂ , respectively, to give 1-hydrocarbyl-7 derivatives α-azindirubin (III).

(3) Synthesis of the target compound: 3'-oxime-7-azaindirubin.

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ ; acyl protected glycosyl, etc .; R ^{3 ′} = H, Cl, Br, F, CH ₃ and OCH ₃ , SCH ₃ and Ph, etc.

(4) Synthesis of the target compound: 7-azainindirubin-3'-oxime ether, etc.

Where R = CH ₃ ON, EtON, R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , etc., R ^{3 '} = H, Cl, Br and etc.

4. Target compound: 7-azaisoindigo (general formula IV)

Where R ¹ = CH ₃ , C ₂ H ₅ , nC ₃ H ₇ , nC ₄ H ₉ , Ph-CH ₂ , acyl protected glycosyl, etc .; R ^{3 '} = H, Cl, Br, F, OH and OCH ₃ , etc.

Derivatives of 1-hydrocarbyl-5'-substituted 7-azaisoindigo (IV) are prepared by reacting 1-hydrocarbyl-7-azaindole-2,3-dione (C) with 5-substituted 2-hydroxyindole under alkaline conditions.

The main advantages of this invention are:

1) The present invention completely changed the atomic structure of the molecule of the parent nucleus of indirubin and isoindigo, thus forming a class of compounds with a completely new structure, and improved the electrical properties of the original molecule. Given that pyridine is soluble in water, and benzene is almost insoluble in water, the present invention improved the solubility of the proposed compound in water, thereby increasing bioavailability.

2) The compounds of the present invention are cyclin-dependent kinase inhibitors, they induce endogenous cyclin kinase inhibitors, thereby inhibiting cell growth and proliferation, promoting apoptosis of tumor cells.

3) The compounds of the present invention, by restoring the conversion of insulin signals, make peripheral tissues insulin sensitive and reduce the role of insulin resistance.

4) The compounds of the present invention have improved physicochemical properties and increased cell permeability, they can easily enter the cells, thereby increasing the effectiveness of the drug.

Specific Implementation Examples

The invention is further explained in the detailed description of examples of its implementation. These examples only illustrate the invention; they do not limit its essence to the given specific options. In the experimental methods used in the following examples, all procedures are performed under standard conditions or according to the manufacturer’s instructions, and all parts, percentages and fractions are indicated by weight, unless expressly stated otherwise.

Example 1. Preparation of compounds

The melting point of the derivatives of 5- or 7-azaindirubin and 5- or 7-isoindigo obtained in this embodiment was measured by a Mel-TEMP melting point measuring instrument without calibration. Mass spectrum (MS) was determined using an HP1100LC / MSD mass spectrometer.

Thin layer chromatographic plates (TLCs) were made from GF254 silica gel (Qingdao Haiyang Chemical Co., Ltd) and a 0.8% solution of CMC-Na in distilled water; the components were mixed, activated for 1 hour at a temperature of 100-110 °, then stored in a dryer and brought up under ultraviolet light (wavelengths 254 nm and 365 nm). Chromatographic columns were packed with silica gel (200-300 mesh or 100-200 mesh) (Qingdao Haiyang Chemical Co., Ltd) using the dry method. The 1H-NMR (1H-NMR) hydrogen spectrum was determined using a Bruck AV-300 type nuclear magnetic resonance apparatus, internal standard TMS. Elemental analysis was performed using Elementar ario EL III equipment.

Reagents were commercially available chemically pure and analytically pure variety products. Unless otherwise indicated, reagents were used directly without additional treatments.

Example 1-1 Preparation of Intermediates

(1) 1-methyl-5-azaindole-2,3-dione

1-methyl-5 azaindole (2.0 g, 15 mmol) was dissolved in 70 ml of AcOH, 3.2 g of CrO _{3 were} previously suspended in 20 ml of water, acetic acid was poured into the above solution, the reaction was carried out for 0.5 hour at room temperature, the mixture diluted with water, extracted 3 times with chloroform; organic substances are dissolved in chloroform, organic phases are integrated and washed, after dehydration the composition is enriched. The orange intermediates of 1-methyl-5-azaindole-2,3-dione (1.5 g, yield 62%; mp 140-142 ° C.) are obtained.

2) 2- (N-carboxymethylamine) -5-chlorobenzoic acid

2-amino-5-chlorobenzoic acid (2.0 g, 11.6 mmol) was dissolved in 15 ml of a solution of 2 mol / L Na ₂ CO ₃ ; chloroacetic acid (0.69 g, 7.3 mmol) was dissolved in 7.5 ml of a solution of 2 mol / L Na ₂ CO ₃ , the latter solution was slowly added dropwise to the previous solution. Then the mixture was stirred at 80 ° C for 20 hours, cooled to room temperature, 50 ml of ether and 8 ml of 2 mol / L hydrochloric acid were added. The organic phases were separated and dried with MgSO ₄ . After concentration, a light brown solid was obtained. Purification by silica gel column chromatography (ethyl acetate / methanol, 1/1; v / v) gave a white solid (2- (N-carboxymethylamino) -5-chlorobenzoic acid) (1.58 g, 59% yield; mp: 182 -183 ° C).

(3) 1-acetyl-5-chloro-3-acetoxyindole

2- (N-carboxymethylamino) -5-chlorobenzoic acid (1.20 g, 5.2 mmol) and anhydrous sodium acetate (0.6 g, 7.3 mmol) were dissolved in 8 ml of acetic anhydride. After stirring for 5 hours at 60 ° C., the reaction mixture was cooled to room temperature, and sodium acetate was filtered off. After concentration, 100 ml of ethyl acetate was added to the concentrated filtrate to dissolve, another 100 ml of water and 20 ml of saturated sodium bicarbonate were added, and the organic layers were separated. Organic matter in the aqueous layer was extracted with ethyl acetate (50 ml × 2). The combined organic phases were washed with saturated sodium bicarbonate (100 ml × 2), dried and evaporated to give a white body (1-acetyl-5-chloro-3-acetoxyindole) (0.84 g, yield: 64%).

(4) 1-acetyl-5-chloro-3-carboxyindole

1-acetyl-5-chloro-3-acetoxyindole (1.0 g, 3.97 mmol) was mixed in 20 ml of water with sodium sulfite (1.0 g, 7.94 mmol), the mixture was heated at 80 ° C for 3 hours, cooled to room temperature, extracted with ethyl acetate (100 ml × 2). The combined organic phases were dried. After concentration, a white needle-shaped solid (1-acetyl-5-chloro-3-carboxyindole) was obtained (0.84 g, 66% yield; mp: 186-188 ° C).

(5) 1-methyl-7-azaindole 2,3-dione

1-methyl-7-azaindole (2 g, 15 mmol) was dissolved in 70 ml of acetic acid, 3.2 g of CrO _{3 was} suspended in 20 ml of water. The reaction mixture was stirred for 0.5 h at room temperature and diluted with water. The mixture was extracted with trichloromethane three times. The combined organic phases were washed with water, dried and evaporated. An orange intermediate was obtained (1-methyl-7-azaindole-2,3-dione) (1.73 g, yield: 71.3%; mp: 162-163 ° C).

(6) 2- (N-carboxymethylamino) -5-bromobenzoic acid

2-amino-5-bromobenzoic acid (2 g, 9 mmol) was dissolved in 15 ml of a solution of 2 mol / L Na ₂ CO ₃ ; chloroacetic acid (0.69 g, 7.3 mmol) was dissolved in 7.5 ml of a solution of 2 mol / L Na ₂ CO ₃ .

Then, after stirring for 20 hours at a temperature of 80 ° C, the reaction mixture was cooled to room temperature. 50 ml of ether and 8 ml of 2 mol of hydrochloric acid were added to the mixture. The organic phase was separated and dried with MgSO ₄ . After evaporation, a light brown solid was obtained.

Purification by silica gel column chromatography (ethyl acetate / methanol, volume per volume, 1/1) gave a white solid (2- (N-carboxymethylamine) -5-bromobenzoic acid) (1.55 g, yield: 60%,; t .p .: 178-180 ° C).

(7) 1-acetyl-5-bromo-3-acetoxyindole

2- (N-carboxymethylamino) -5-bromobenzoic acid (0.84 g, 3.4 mmol) and anhydrous sodium acetate (0.6 g, 7.3 mmol) were dissolved in 8 ml of acetic anhydride. After stirring for 5 hours at 60 ° C., the reaction mixture was cooled to room temperature, and sodium acetate was filtered off. After concentration, 100 ml of ethyl acetate was added to the concentrated filtrate to dissolve, another 100 ml of water and 20 ml of saturated sodium bicarbonate were added, and the organic layers were separated. Organic matter in the aqueous layer was extracted with ethyl acetate (50 ml × 2). The combined organic phases were washed with saturated sodium bicarbonate (100 ml × 2), dried and evaporated to give a white body (1-acetyl-5-bromo-3-acetoxyindole) (1.3 g, yield: 25.4%).

(8) 1-acetyl-5-bromo-3-carboxyindole

1-acetyl-5-bromo-3-acetoxyindole (1.0 g, 3.38 mmol) and sodium sulfite (1.0 g, 7.94 mmol) were mixed in 20 ml of water. After heating at 80 ° C for 3 hours, the reaction mixture was cooled to room temperature, extracted with ethyl acetate (50 ml × 2). The combined organic phases were dried. After concentration, a white needle-shaped solid (1-acetyl-5-bromo-3-carboxyindole) was obtained (0.7 g, 82% yield; mp: 180-182 ° C).

Example 1-2. Synthesis of target compounds

(1) 1-methyl-5-azaindirubin (2)

To 1-methyl-5-azaindole-2,3-dione (0.2 g, 1.23 mmol) was added 1-acetyl-3-hydroxyindole (0.21 g, 1.2 mmol), 20 ml of water and 0.02 g of paratoluenesulfonic acid. The reaction mixture was stirred and boiled under nitrogen for 1 hour to obtain a purplish-red solution. After cooling, the mixture was extracted with trichloromethane, washed with water and evaporated. Got a purple solid. After purification by silica gel column chromatography (trichloromethane / petroleum ether, v / v, 3/1; v / v), it was recrystallized with ethyl acetate to obtain red needle-shaped crystals of 1-methyl-5-azaindirubin (2) (0.15 g, 44% yield; 114-116 ° C).

ESI-MS: 278.1 [M + H] ⁺ , C ₁₆ H ₁₁ N ₃ O ₂ (277.2);

¹ H NMR (AV-300, CDCl ₃ , ppm) δ: 3.48 (s, 3H, -CH ₃ ), 7.08 (m, 1H, 5'-H), 7.09 (m, 1H, 6'-H), 7.16 (dd, 1H, J = 7.6 Hz, 4'-H), 7.76 (d, J = 7.6 Hz, 1H, 7'-H), 8.10 (s, 1H, 4-H), 8.26 (dd, J = 5.5 Hz; 1H. 6-H), 9.10 (dd, J = 5.5 Hz, 1H, 7-H), 10.4 (bs, 1H, NH);

Similarly for: C ₁₆ H ₁₁ N ₃ O ₂ : C, 69.31 H, 3.97 N, 15.16;

Received: C, 69.15 N, 4.09 N, 15.29.

(2) 1-benzyl-5'-chloro-5-azaindirubin (19)

The same method was used as (1), 1-benzyl-5-azaindole-2,3-dione and 1-acetyl-5-chloro-3-hydroxyindole in an amount as in (1), and 0.02 g of paratoluene-sulfonic acids were dissolved in 20 ml of water. The reaction mixture was stirred and boiled under nitrogen for 1 hour to obtain a purplish-red solution. After cooling, the mixture was extracted with trichloromethane, washed with water and evaporated. A purple solid was obtained. After purification by silica gel column chromatography (trichloromethane / petroleum ether, v / v, 3/1; v / v), it was recrystallized with ethyl acetate to obtain red needle-shaped crystals of 1-benzyl-5'-chloro-5-azaindiribine (19) (0.18 g, 39% yield; mp: 110-112 ° C).

ESI-MS: 389 [M + H] ⁺ , C ₂₂ H ₁₄ ClN ₃ O ₂ (387.9);

¹ H NMR (AV-300, CDCl ₃ , ppm) δ: 5.21 (s, 2H, N-CH ₂ ), 10.44 (s, 1H, NH), 6.91 ~ 9.02 (m, 11H, Ar-Hs);

Similarly for C ₂₂ H ₁₄ ClN ₃ O ₂ : C, 68.13 H, 3.64 N, 10.83;

Received: C, 68.42 N, 3.59 N, 10.89.

(3) 1-butyl-5-azainindirubin-3'-oxime (40)

1-Butyl-5-azainindirubin (0.4 g, 1.25 mmol, dissolved by method (1)) was dissolved in 12 ml of methanol, adding 6 ml of anhydrous pyridine and 0.15 g of hydroxylamine hydrochloride (2.2 mmol).

The reaction mixture was stirred and boiled for 1 hour, cooled and concentrated to remove most of the solvent. The residue was discarded in 100 ml of crushed ice, stirring vigorously, then filtered, resulting in an orange solid. Purification by silica gel column chromatography (trichloromethane / petroleum ether, 3/1; v / v) yielded an orange crystalline powder of 1-butyl-5'-5-azaindirubin-3'-oxime (40) (0.32 g, 90% yield; t .p .: 250-252 ° C).

ESI-MS: 335.1 [M + H] ⁺ , C ₁₉ H ₁₈ N ₄ O ₂ (334.3);

¹ H NMR (AV-300, D6-DMSO, ppm) δ: 0.91 (t, 3H, -CH ₃ ), 1.3l (m, 2H, -CH ₂ ), 2.28 (m, 2H, -CH ₂ ), 3.28 (m, 2H, N-CH ₂ ), 7.10 ~ 8.81 (m, 7H, Ar-Hs), 11.71 (s, 1H, NH), 13.70 (s, 1H, N-OH);

Similarly for C ₁₉ H ₁₈ N ₄ O ₂ : C, 68.25 H, 5.43 N, 16.76;

Received: C, 68.33 H, 5.56 N, 16.66.

(4) 1-butyl-5-aza -indirubin-3'-monoxide O-methyl (53)

1-butyl-5-azainindirubin-3'-oxime (1.5 g, 4.5 mmol) was added to 50 ml of 5% potassium hydroxide in anhydrous ethanol, dissolved at warm temperature, and filtered. 5 ml of CH31 was added dropwise to the filtrate with constant stirring. During the reaction, heat was generated, a dark red precipitate was obtained. After stirring for 0.5 hours, the precipitate was filtered and washed with water until a neutral solution was obtained, dried and a dark red crude product was obtained, which was recrystallized with acetone, and dark red crystals of 1-butyl-5-aza -indirubin-3'-monoxime O- were obtained methyl (53) (1.20 g, 77% yield; mp: 209-211 ° C).

ESI-MS: 349.1 [M + H] ⁺ , C ₂₀ H ₂₀ N ₄ O (348.2);

¹ H-NMR (AV-300, D6-DMSO, ppm) δ: 0.98 (t, 3H, -CH ₃ ), 1.46 (m, 2H, -CH ₂ ), 2.08 (m, 2H, -CH ₂ ), 3.86 (m, 2H, N-CH ₂ ), 4.16 (s, 3H, O-CH ₃ ), 7.10 ~ 9.19 (m, 7H, Ar-Hs), 10.86 (bs, 1H, NH);

Similarly for C ₂₀ H ₂₀ N ₄ O ₂ : C, 68.95 N, 5.79 N, 16.08

Received: C, 68.81 N, 5.62 N, 15.85.

(5) 1-isopropyl-5-azaisoindigo (73)

1-Isopropyl-5 azaindole-2,3-dione (0.4 g, 2.1 mmol) and 2-hydroxyindole (0.28 g, 2.1 mmol) were added in 10 ml of ethanol, the pH was adjusted to 9 with 1 mol / L NaOH, the reaction was carried out at 70 ° C for two hours and got a brown body. After cooling, it was filtered, washed with water and ethanol, dried in a vacuum, and the result was a red-brown solid of 1-isopropyl-5-azaisoindigo (73) (0.44 g, 66% yield; mp: 128 ~ 130 ° С )

ESI-MS: 306.1 [M + H] ⁺ , 304.2 [M-H] ^- , C ₁₈ H ₁₅ N ₃ O ₂ (305.3);

¹ H NMR (AV-300, D6-DMSO, ppm) δ: 1.52 (d, 6H, -CH (CH ₃ ) ₂ ), 4.81 (m, 1H, -CH (CH ₃ ) ₂ ), 6.86-9.32 ( m, 7H, Ar-Hs), 10.96 (bs, 1H, NH);

Similarly for C ₁₈ H ₁₅ N ₃ O ₂ : C, 70.81 N, 4.95 N, 13.76

Received: C, 70.62 N, 5.10 N, 13.58.

(6) 1-methyl-7-azainindirubin (93)

To 1-methyl-7-azaindole 2,3-dione (0.2 g, 1.23 mmol) was added 1-acetyl-3-hydroxyindole (0.21 g, 1.2 mmol), 20 ml of water and 0.02 g of paratoluenesulfonic acid. The reaction mixture was recycled with nitrogen with stirring for 1 hour, and a purplish-red solution was obtained. After cooling, the solution was extracted with chloroform, washed with water, concentrated, and a purple body was obtained. After purification by silica gel column chromatography (trichloromethane / petroleum ether, 3/1 v / v), it was recrystallized with ethyl acetate to obtain red needle-shaped crystals of 1-methyl-7-aza -indiribine (93) (0.14 g, 41.1% yield; etc.). : 116 ~ 118 ° C).

ESI-MS: 278.1 [M + H] ⁺ , C ₁₆ H ₁₁ N ₃ O ₂ (277.2);

¹ H NMR (AV-300, CDCl ₃ , ppm) δ: 3.59 (s, 3H, -CH ₃ ), 7.08 (m, 1H, 5'-H), 7.09 (m, 1H, 6'-H), 7.16 (dd, 1H, J = 7.6 Hz, 4-H), 7.58 (m, 1H, 5-H), 7.76 (d, J = 7.6 Hz, 1H, 7'-H), 8.21 (dd, J = 5.5 Hz, 1H, 4-H), 9.13 (dd, J = 5.5 Hz, 1H, 6-H), 10.4 (bs, 1H, NH);

Similarly for C ₁₆ H ₁₁ N ₃ O ₂ : C, 69.31 H, 3.97 N, 15.16;

Received: C, 69.05 N, 4.18 N, 15.34.

(7) 1-benzyl-5'-bromo-7-azaindirubin (109)

By the same method as in (6), 20 ml of water and 0.02 g were added to 1-benzyl-7-azaindole 2,3-dione and 1-acetyl-5-bromo-3-hydroxyindole in an amount as in (6) paratoluenesulfonic acid. The reaction mixture was recycled with nitrogen with stirring for 1 hour, and a purplish-red solution was obtained. After cooling, the solution was extracted with chloroform, washed with water, concentrated, and a purple body was obtained. After purification by silica gel column chromatography (trichloromethane / petroleum ether, 3/1 v / v), it was recrystallized with ethyl acetate to obtain red needle-shaped crystals of 1-benzyl-5'-bromo-7-aza -indirubin (109) (0.14 g, 27% yield; t mp: 112 ~ 114 ° C).

ESI-MS: 433 [M + H] ⁺ , C ₂₂ H ₁₄ BrN ₃ O ₂ (432.2);

¹ H NMR (AV-300, CDCl ₃ , ppm) δ: 5.24 (s, 2H, N-CH ₂ ), 10.44 (s, 1H, NH), 6.91 ~ 9.0 (m, 11H, Ar-Hs);

Similarly for C ₂₂ H ₁₄ BrN ₃ O ₂ : C, 61.13 H, 3.26 N, 9.72;

Received: C, 60.72 N, 3.57 N, 9.38.

(8) 1-butyl-7-azainindirubin-3'-oxime (131)

1-butyl-7-azainindirubin (0.4 g, 1.25 mmol, prepared by method (6)) was dissolved in 12 ml of methanol by adding 6 ml of anhydrous pyridine and 0.15 g of hydroxylamine hydrochloride (2.2 mmol). The reaction mixture was heated and recycled for 1 hour, cooled, concentrated to remove most of the solvent, the residues were dropped into 100 ml of crushed ice, stirring vigorously, filtered, resulting in an orange body. After purification by silica gel column chromatography (trichloromethane / petroleum ether, 3/1 v / v), it was recrystallized with ethyl acetate to give an orange solid 1-butyl-7-aza -indirubin-3'-oxime (131) (0.31 g, 87.7% yield) ; mp: 254 ~ 256 ° C).

ESI-MS: 335.1 [M + H] ⁺ , C ₁₉ H ₁₈ N ₄ O ₂ (334.3);

¹ H NMR (AV-300, D6-DMSO, ppm) δ: 0.92 (t, 3H, -CH ₃ ), 1.31 (m, 2H, -CH ₂ ), 2.28 (m, 2H, -CH ₂ ), 3.32 (m, 2H, N-CH ₂ ), 7.03 ~ 8.81 (m, 7H, Ar-Hs), 11.7 (s, 1H, NH), 13.7 (s, 1H, N-OH);

Similarly for C ₁₉ H ₁₈ N ₄ O ₂ : C, 68.25 N, 5.43 N, 16.76;

Received: C, 68.09 N, 5.60 N, 16.58.

(9) 1-butyl-7-azaindirubin-3'-monooxy O-methyl ether (144)

1-butyl-7-azainindirubin-3'-oxime (1.5 g, 4.5 mmol) was added to 50 ml of 5% potassium hydroxide in anhydrous ethanol, dissolved at warm temperature, and filtered. 5 ml of CH31 was added dropwise to the filtrate with constant stirring. During the reaction, heat was generated, a dark red precipitate was obtained. After stirring for 0.5 hours, the precipitate was filtered and washed with water until a neutral solution was obtained, dried and a dark red crude product was obtained, which was recrystallized with acetone, and dark red crystals of 1-butyl-7-azaindirubin-3'-monoxime O- were obtained methyl (144) (1.26 g, yield 80.5%; mp: 212-214 ° C).

ESI-MS: 349.1 [M + H] ⁺ , C ₂₀ H ₂₀ N ₄ O (348.2);

¹ H-NMR (AV-300, D6-DMSO, ppm) δ: 0.98 (t, 3H, -CH ₃ ), 1.46 (m, 2H, -CH ₂ ), 2.08 (m, 2H, -CH ₂ ), 3.88 (m, 2H, N-CH ₂ ), 4.16 (s, 3H, O-CH ₃ ), 7.06 ~ 9.19 (m, 7H, Ar-Hs), 10.86 (bs, 1H, NH);

Similarly for C ₂₀ H ₂₀ N ₄ O ₂ : C, 68.95 N, 5.79 N, 16.08

Received: C, 68.79 N, 5.59 N, 15.88.

(10) 1-isopropyl-7-azaisoindigo (164)

1-isopropyl-7 azaindole-2,3-dione (0.4 g, 2.1 mmol) and 2-hydroxyindole (0.28 g, 2.1 mmol) were added to 10 ml of ethanol, pH was adjusted to 9 with 1 mol / L NaOH, the reaction was carried out at 70 ° C for two hours and got a brown body. After cooling, it was filtered, washed with water and ethanol, dried in a vacuum, and the result was a red-brown solid of 1-isopropyl-7-azaisoindigo (164) (0.42 g, 63.2% yield; mp: 132 ~ 134 ° C).

ESI-MS: 306.1 [M + H] ⁺ , 304.2 [M-H] ^- , C ₁₈ H ₁₅ N ₃ O ₂ (305.3);

¹ H NMR (AV-300, D6-DMSO, ppm) δ: 1.51 (d, 6H, -CH (CH ₃ ) ₂ ), 4.78 (m, 1H, -CH (CH ₃ ) ₂ ), 6.86-9.3 ( m, 7H, Ar-Hs), 10.99 (bs, 1H, NH);

Similarly for C ₁₈ H ₁₅ N ₃ O ₂ : C, 70.81 N, 4.95 N, 13.76

Received: C, 70.53 N, 5.04 N, 13.52.

In accordance with the above method, 59 compounds of 5-azaindirubin (1) were prepared, in addition, 5-azaindirubin derivatives of types 2, 19, 40 and 53 were synthesized. Their structures are shown in Table 1, all structures of these new compounds were confirmed by IR spectrometry, ultraviolet spectrometry (UV / VIS), mass spectrometry (ESI-MS), NMR (1H-NMR) and elemental analysis.

In the formula 1, R ² ~ R ⁴ , R ^{1 '} , R ^2' , R ^{4 '} and R ^5' respectively represents H, the rest, see table 1:

Table 1 The structure of compounds of 5-azaindirubin (I) No. R ¹ R ^{3 '} R one 2 3 four one N N ABOUT 2 CH ₃ H ABOUT 3 CH ₃ OH ABOUT four CH ₃ OCH ₃ ABOUT 5 CH ₃ SCH ₃ ABOUT 6 C ₂ H ₅ H ABOUT 7 i-C ₃ H ₇ H ABOUT 8 n-C ₄ H ₉ H ABOUT 9 CH ₂ -Ph H ABOUT 10 CH ₃ Br ABOUT eleven CH ₃ Cl ABOUT

Continuation of table 1 one 2 3 four 12 C ₂ H ₅ Br O 13 C ₂ H ₅ Cl O fourteen iC ₃ H ₇ Br O fifteen iC ₃ H ₇ Cl O 16 n-C ₄ H ₉ Br O 17 n-C ₄ H ₉ Cl O eighteen CH ₂ -Ph Br O 19 CH ₂ -Ph Cl O twenty CH ₂ -Ph F O 21 Triacetyl ribosyl H O 22 Phco Och ₃ S 23 CH ₃ CH ₂ CO Och ₃ S 24 CH ₃ Br N-OH 25 CH ₃ Cl N-OH 26 CH ₃ Ph N-OH 27 CH ₃ SCH ₃ N-OH 28 C ₂ H ₅ Br N-OH 29th C ₂ H ₅ Cl N-OH thirty iC ₃ H ₇ Br N-OH 31 i-C ₃ H ₇ Cl N-OH 32 n-C ₄ H ₉ Br N-OH 33 n-C ₄ H ₉ Cl N-OH 34 CH ₂ -Ph Br N-OH 35 CH ₂ -Ph Cl N-OH 36 CH ₂ -Ph F N-OH 37 CH ₃ H N-OH 38 C ₂ H ₅ H N-OH

Continuation of table 1 one 2 3 four 39 iC ₃ H ₇ H N-OH 40 n-C ₄ H ₉ H N-OH 41 CH ₂ -Ph H N-OH 42 ribosyl H N-OH 43 Glycosyl H N-OH 44 CH ₃ H N-OCH ₃ 45 CH ₃ Br N-OCH ₃ 46 CH ₃ Cl N-OCH ₃ 47 C ₂ H ₅ H N-OCH ₃ 48 C ₂ H ₅ Br N-OCH ₃ 49 C ₂ H ₅ Cl N-OCH ₃ fifty i-C ₃ H ₇ H N-OCH ₃ 51 i-C ₃ H ₇ Br N-OCH ₃ 52 iC ₃ H ₇ Cl N-OCH ₃ 53 n-C ₄ H ₉ H N-OCH ₃ 54 n-C ₄ H ₉ Br N-OCH ₃ 55 nC ₄ H ₉ Cl N-OCH ₃ 56 CH ₂ -Ph H N-OCH ₃ 57 CH ₂ -Ph F N-OCH ₃ 58 CH ₂ -Ph Br N-OCH ₃ 59 CH ₂ -Ph Cl N-OCH ₃

In accordance with the preparation method of 1-Isopropyl-5-azaisoindigo (73), a total of 30 compounds of 5-azaisoindigo (73) were synthesized, their structure is shown in Table 2. The structures of all these new compounds were confirmed by IR spectrometry and ultraviolet spectrometry (UV / VIS) , mass spectrometry (ESI-MS), NMR (1H-NMR) and elemental analysis.

In formula II, R ² ~ R ⁴ , R ^{2 '} , R ^4' and R ^{5 '} , respectively, are H, the rest see Table 2:

table 2 The structure of compounds 5-azaisoindigo (II) No. R ¹ R ^{1 '} R ^{3 '} one 2 3 four 60 CH ₃ H H 61 CH ₃ H Br 62 CH ₃ H Cl 63 CH ₃ H F 64 CH ₃ CH ₃ IT 65 CH ₃ CH ₂ CO H OCH ₃ 66 Phco H OCH ₃ 67 Triacetyl ribosyl H N 68 C ₂ H ₅ H N 69 C ₂ H ₅ C ₂ H ₅ Br 70 C ₂ H ₅ H Cl 71 C ₂ H ₅ H F 72 C ₂ H ₅ C ₂ H ₅ IT 73 iC ₃ H ₇ H N 74 iC ₃ H ₇ H Br 75 iC ₃ H ₇ H Cl 76 iC ₃ H ₇ H F 77 iC ₃ H ₇ iC ₃ H ₇ IT 78 n-C ₄ H ₉ H N

Continuation of table 2 one 2 3 four 79 n-C ₄ H ₉ H Br 80 n-C ₄ H ₉ H Cl 81 n-C ₄ H ₉ n-C ₄ H ₉ OH 82 nC ₄ H ₉ H F 83 nC ₄ H ₉ n-C ₄ H ₉ Och ₃ 84 CH ₂ -Ph H H 85 CH ₂ -Ph H Br 86 CH ₂ -Ph CH ₂ -Ph Cl 87 CH ₂ -Ph H F 88 CH ₂ -Ph CH ₂ -Ph OH 89 ribosyl H H

In accordance with the above methods for the preparation of 7-azaindirubin derivatives 93, 109, 131 and 144, a total of 59 compounds of the 7-azaindirubin (III) type were synthesized, their structures are shown in Table 3. The structures of all these new compounds were confirmed by IR spectrometry and ultraviolet spectrometry ( UV / VIS), mass spectrometry (ESI-MS), NMR (1H-NMR) and elemental analysis.

In the formula III, R ² , R ⁴ , R ^{1 '} , R ^2' , R ^{4 '} and R ^{5' are} respectively H, the rest see Table 3:

The structure of compounds 7-azaindirubin (III)

Table 3 No. R ¹ R ³ R ^{3 '} R one 2 3 four 5 92 N N N O 93 CH ₃ N N O 94 CH ₃ CH ₃ IT O 95 CH ₃ CH ₃ OCH ₃ O 96 CH ₃ CH ₃ SCH ₃ O 97 C ₂ H ₅ N H O 98 iC ₃ H ₇ N H O 99 n-C ₄ H ₉ N H O one hundred CH ₂ -Ph N H O 101 CH ₃ N Br O 102 CH ₃ N Cl O 103 C ₂ H ₅ N Br O 104 C ₂ H ₅ H Cl O 105 i-C ₃ H ₇ N Br O 106 i-C ₃ H ₇ H Cl O 107 n-C ₄ H ₉ N Br O 108 n-C ₄ H ₉ N Cl O 109 CH ₂ -Ph N Br O 110 CH ₂ -Ph N Cl O 111 CH ₂ -Ph NHCH ₃ F O 112 Triacetyl ribosyl CH ₃ N O 113 Phco CH ₃ OCH ₃ S 114 CH ₃ CH ₂ CO F OCH ₃ S 115 CH ₃ N Br N-OH 116 CH ₃ N Cl N-OH 117 CH ₃ CF ₃ Ph N-OH

Continuation of table 3 one 2 3 four 5 118 CH ₃ CH ₃ SCH ₃ N-OH 119 C ₂ H ₅ H Br N-OH 120 C ₂ H ₅ H Cl N-OH 121 i-C ₃ H ₇ H Br N-OH 122 i-C ₃ H ₇ H Cl N-OH 123 n-C ₄ H ₉ H Br N-OH 124 n-C ₄ H ₉ H Cl N-OH 125 CH ₂ -Ph H Br N-OH 126 CH ₂ -Ph H Cl N-OH 127 CH ₂ -Ph H F N-OH 128 CH ₃ H H N-OH 129 C ₂ H ₅ H H N-OH 130 i-C ₃ H ₇ H H N-OH 131 nC ₄ H ₉ H H N-OH 132 CH ₂ -Ph H H N-OH 133 ribosyl CH ₃ H N-OH 134 glucosyl CH ₃ H N-OH 135 CH ₃ H H N-OCH ₃ 136 CH ₃ H Br N-OCH ₃ 137 CH ₃ H Cl N-OCH ₃ 138 C ₂ H ₅ H H N-OCH ₃ 139 C ₂ H ₅ H Br N-OCH ₃ 140 C ₂ H ₅ H Cl N-OCH ₃ 141 iC ₃ H ₇ H H N-OCH ₃ 142 iC ₃ H ₇ H Br N-OCH ₃ 143 iC ₃ H ₇ H Cl N-OCH ₃ 144 n-C ₄ H ₉ H H N-OCH ₃

Continuation of table 3 one 2 3 four 5 145 n-C ₄ H ₉ H Br N-OCH ₃ 146 n-C ₄ H ₉ H Cl N-OCH ₃ 147 CH ₂ -Ph H N N-OCH ₃ 148 CH ₂ -Ph H F N-OCH ₃ 149 CH ₂ -Ph H Br N-OCH ₃ 150 CH ₂ -Ph H Cl N-OCH ₃

In accordance with the above methods for the preparation of 1-isopropyl-7-azaisoidigo (164), a total of 30 compounds of 7-azaisoindigo (IV) were synthesized, their structures are shown in Table 4. The structures of all these new compounds were confirmed by IR spectrometry and ultraviolet spectrometry (UV (VIS), mass spectrometry (ESI-MS), NMR (1 H-NMR) and elemental analysis.

In the formula IV, R ² , R ⁴ , R ^{2 '} , R ^4' and R ^{5 'are} respectively H, the rest see table 4.

Table 4 Structures of 7-azaindigo (IV) compounds No. R ¹ R ^{1 '} R ³ R ^{3 '} one 2 3 four 5 151 CH ₃ N N N 152 CH ₃ H N Br 153 CH ₃ H H Cl 154 CH ₃ H H F 155 CH ₃ CH ₃ CH ₃ IT

Continuation of table 4 one 2 3 four 5 156 CH ₃ CH ₂ CO H F OCH ₃ 157 Phco H CH ₃ OCH ₃ 158 Triacetyl ribosyl H CH ₃ N 159 C ₂ H ₅ H N N 160 C ₂ H ₅ C ₂ H ₅ N Br 161 C ₂ H ₅ H N Cl 162 C ₂ H ₅ H N F 163 C ₂ H ₅ C ₂ H ₅ CF ₃ IT 164 iC ₃ H ₇ H N H 165 iC ₃ H ₇ H N Br 166 i-C ₃ H ₇ H N Cl 167 iC ₃ H ₇ H OCH ₃ F 168 i-C ₃ H ₇ iC ₃ H ₇ N IT 169 n-C ₄ H ₉ H N H 170 n-C ₄ H ₉ H N Br 171 n-C ₄ H ₉ H H Cl 172 n-C ₄ H ₉ n-C ₄ H ₉ SCH ₃ IT 173 n-C ₄ H ₉ H H F 174 n-C ₄ H ₉ n-C ₄ H ₉ OCH ₃ OCH ₃ 175 CH ₂ -Ph H H N 176 CH ₂ -Ph H H Br 177 CH ₂ -Ph CH ₂ -Ph SCH ₃ Cl 178 CH ₂ -Ph H OCH ₃ F 179 CH ₂ -Ph CH ₂ -Ph H IT 180 ribosyl H CH ₃ N

Example 2. Antitumor activity test (1)

1. Materials and devices

(i) Cell lines: androgen-independent prostate cancer cells DU145, purchased from the United States, American Type Culture Collection.

(ii) reagents RPM1 Medium 1640 (USA company GIBCOBRL), calf serum (Hangzhou bio-engineering company Syjitsin), MTT (company Sigma), HEPES (LLC Shanghai biotechnological company Lizhu-dongfeng), L-glutamine (imported from Japan), dimethyl sulfoxide (DMSO, analytical reagent).

Test samples: 20 of the new compounds 5-azaindirubin and 5-azaisoindigo (in-house production), 38 of the new compounds 7-azaindirubin and 7-azaisoindigo (in-house production) were selected.

Control substances: 1-ethyl-indirubin (90), 1-ethyl-indirubin-3'-oxime (91), (own production, structures identified, treated with retinoic acid).

(iii) Preparation of reagents

A. Cell culture medium: 10.4 g of 1640 medium powders, 2.1 g of sodium bicarbonate, 0.3 g of glutamine, 5.95 g of HEPES, 100,000 units of penicillin and 100,000 units of streptomycin were added to 1000 ml of double distilled water. The mixture was sterilized by filtration using a microporous membrane filter; after packaging, it was stored at a temperature of -20 ° C. Prior to use, inactivated calf serum was added to the medium.

B. Calf serum: was inactivated in a water bath at 56 ° C for 30 minutes, after packaging was stored at -20 ° C.

C. MTT: dissolved in 5 mg / ml PBS, stored in a dark place at 4 ° C, valid for two weeks.

D. PBS: 8.00 g of sodium chloride, 0.20 g of potassium chloride, 3.4 g of twelve-hydrate disodium hydrogen phosphate, 0.20 g of dipotassium hydrogen phosphate were dissolved in a double distilled water bath at 37 ° C, packaged in 1000 ml containers, after packaging they were wounded at 4 ° C.

E. Dimethyl sulfoxide solutions were prepared for 58 test samples, substances (90 and 91) and treated with retinoic acid and stored at -20 ° C.

(iv) Basic appliances:

CO ₂ incubator (type GB16, German company Heraeus): Cleaning table (SW-CJ-1F, Suzhou Antai Air Technology Co., Ltd); horizontal centrifuge (type LXJ-II, Shanghai Medical Instrument Plant No. 3); enzyme immunoassay analyzer (BIO RAD Model 550, United States of America); inverted biological microscope (XSZ-D2, Chongqing Optical Instrument Factory); device for quick mixing (type SK-1, Changzhou Electrical Appliance Company, Guohua LLC); thermostatic electric tank (type DK-8D, Shanghai Medical Thermostat Equipment Factory); flow cytometer (FACSCalibur, American company BD); platform vibrator (type 752-A, Shanghai Factory of Medical Analytical Instruments); electronic scales (type BS110S, German company Sartorius).

2. Methods.

(i) Cell Culture

DU145 cells are inoculated in RPM11640 medium with 10% fetal calf serum, delivered in an incubator at 5% CO ₂ , 37 ° C, transferred every 2-3 days, during the experiment, cells are taken in a logarithmic phase.

(ii) Experimental groups

In the experiments, cells are taken in the logarithmic phase, a cell suspension is created, after uniform mixing, the viability of the cells is determined by trypan blue staining, the number of viable cells should be more than 98%. The cell suspension is divided equally into several groups: 1: empty control groups (cell suspension); 2: experimental groups (cell suspension + drug).

(iii) IC ₅₀ values are determined by MTT (half the amount of inhibition)

Prepare a backup solution using dimethyl sulfoxide with a concentration of 20 mmol (experiment is carried out for 4 hours). The experiments are carried out under sterile conditions, in a working solution based on RPMI1640 medium with 10% fetal calf serum, a working solution of the tested drug with a concentration of 80 μmol is prepared, the concentration of the drug is gradually increased by 2 times (1.25-20 μmol).

DU145 cells were taken in a logarithmic phase, centrifuged, the suspension with a density of 2.5 × 104 / ml was prepared from RPMI1460 medium containing 10% calf serum. The suspension was inoculated into a 96-hole plate, with 5,000 cells / 200 ml in each hole, cultured for 24 hours at 37 ° C in 5% CO ₂ medium. Then, according to the above concentration, they were inoculated into 6 groups (including the control group), 8 bottom holes were created in each group. After elimination for 72 hours, cell viability was measured by MTT colorimetry. The absorption value (A) was measured with a detection wavelength of 540 nm, a reference wavelength of 630 nm. The degree of inhibition is calculated in accordance with the following formula:

The inhibition coefficient (I) was calculated by the following equation, where T is the spectral absorbance of the experimental groups, and C is the spectral absorbance of the pure control group:

I = (1-T / C) × 100%.

In accordance with the inhibition concentration curve, the regression equation was calculated to obtain a 50% and 90% inhibition concentration (IC ₅₀ and IC ₉₀ μM). The results are presented in table 5 and table 6 and figure 2.

Table 5 Derivatives of 5-azaindirubin and 5-azaindigotine: IC ₅₀ and IC ₉₀ , inhibiting the growth of tumor cells DU145 No. IC ₅₀ (µM) IC ₉₀ (µM) No. IC ₅₀ (µM) IC ₉₀ (µM) 7 > 100 not tested 38 6.4 22.0 8 11.5 39.0 63 48.0 > 100 9 12.0 85.0 67 8.0 44.0 12 8.8 28.0 71 17.0 87.0 fifteen 9.0 48.0 75 9.7 29.0 eighteen 6.6 23.5 78 16.0 51.0 25 1.7 12.2 79 14.5 77.0 28 4.5 14.0 82 > 100 > 100 29th 2.2 6.8 thirty 3.3 7.0 90 7.2 31.0 31 3.7 11.5 91 4.2 21.8 35 7.3 20.0 Tretinoin > 50

Table 6 Derivatives of 7-azaindirubin and azaisoidigotine: IC ₅₀ and IC ₉₀ , inhibiting the growth of tumor cells DU145 No. IC ₅₀ (µM) IC ₉₀ (µM) No. IC ₅₀ (μM) IC ₉₀ (μM) 95 40.0 > 100 126 7.1 19.0 98 > 100 not tested 128 2.7 7.1 99 11.5 87.0 129 3.8 12.0 one hundred 9.5 31.5 130 7.05 17.0 101 11.5 60.0 131 16.0 80.0 102 8.0 18.0 154 43.0 > 100 103 10.0 32.2 158 10.5 49.0 104 8.5 45.0 160 14.0 61.0 105 8.1 28.5 161 14.0 45.0 106 18.5 100.0 162 17.5 89.0 107 14.0 26.0 164 9.05 19.0 108 11.0 42.0 165 8.3 28.0 109 7.0 24.0 166 10.0 31.5 111 7.4 34.0 169 14.0 47.0 116 6.9 16.0 170 15.0 90.0 119 4.0 12.0 171 9.0 46.0 120 5.4 61.0 173 > 100 > 100 121 3.1 29.0 122 8.0 25.0 90 7.0 30.0 123 3.7 5.0 91 4.1 22.0 124 1.8 12.0 Tretinoin > 50 Note: in the table, the molecular structures of the control substances 90 and 91 are as follows:

3. Discussion

(i) From the results of the MTT test it is easy to see that the vast majority of derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigotine have strong anti-tumor activity, the inhibitory effects against the growth of tumor cells are much stronger than when treated with retinoic acid as an induced differentiated cell agent. Even more important, the vast majority of derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigotine have good inhibitory effects on androgen-independent prostate cancer cells DU-145, which there is nothing to fight with in current clinical practice ;

(ii) many of the derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigo have an IC ₅₀ value close to or less than the IC _{50 of the} control substances (90 and 91), and composition 91 is a known inhibitor of CDKs ^[13]. The structures of compounds 38, 129 are very similar to 91, the difference between them is only in the types of atoms at 5 or 7 positions. It is also proposed that the new synthesized compositions of the present invention may have a similar mechanism for inhibiting the growth of tumor cells;

(iii) 3'-oximed and 5'-halogenated or 7-azaindirubinated derivatives show a more significant inhibitory effect on the growth of tumor cells, especially compounds 25, 30, 116, 121 and 124, and compounds 30, 121 and 124 have better safety .

Example 3. Test antitumor activity (2)

1. Tumor cells: human liver cancer cells 7701 QGY, HepG-2, human lung cancer cells A549, human chronic myeloid leukemia cells K562, human CEM leukemia cells, KIll mouse melanoma cells.

2. Using the methods described in example 2, the biological activity of part of the newly synthesized derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigo (20 compounds) on the activity of inhibiting the growth of various tumor cells was determined. The test results at 50% inhibitory concentration (IC ₅₀ , μm) are presented in table 7.

Table 7 Inhibitory concentrations (IC ₅₀ , μm) of 5- or 7-azaindirubin derivatives and 5- or 7-azaisoindigo tumor cell growth inhibition Cancer cells IC ₅₀ (µM) 7701QGY HepG-2 A549 K562 CEM Kiii one 2 3 four 5 6 7 95 23.9 50.1 35.2 8.1 9.0 15.6 99 12.4 29.3 18.4 6.7 7.1 10.1 101 15.0 28.8 21.6 7.8 12.0 23.0 102 39.5 41.5 22.8 9.5 7.0 18.2 105 28.0 18.0 19.6 20.4 10.2 14.0 106 28.0 18.0 19.6 20.4 10.2 14.0 109 10.0 21.2 15.8 12.5 9.0 18.6 111 28.5 17.0 24.3 15.2 11.0 15.1 116 11.1 20.5 12.5 8.2 5.6 6.3 119 13.5 8.1 8.5 7.9 9.0 8.1

Continuation of table 7 one 2 3 four 5 6 7 121 14.9 16.0 18.1 11.5 11.3 6.0 126 11.2 16.3 9.6 5.1 8.1 14.2 128 7.6 12.6 10.0 7.7 10.5 3.6 129 7.4 13.0 14.1 3.4 7.0 4.2 154 36.0 29.3 12.4 19.0 9.8 12.0 158 44.0 18.0 22.3 2.8 12.0 8.3 160 25.4 31.1 29.1 10.2 10.8 15.0 164 33.1 29.0 23.3 18.6 12.7 9.1 170 28.0 25.0 19.5 10.3 8.5 18.5 176 13.7 20.6 13.2 4.1 7.4 8.3

The results of the above tests for antitumor activity indicate that 5- or 7-azaindirubin derivatives and 5- or 7-azaisoindigo derivatives have diverse bioactivities of tumor cell growth inhibition, thereby opening a new direction in the study of indirubin antitumor compounds, providing the material basis for the development of new antitumor drugs.

Example 4. Inhibitory Effects of CDKs

Reagents: The sources of the test compounds are the same as in Example 2. Unless otherwise indicated, other chemicals were purchased from Sigma, an American company. Polyacrylamide gel for protein electrophoresis (SDS-PAGE), SDS, electrophoresis buffer, protein transfer buffer, nitrocellulose membranes, etc. were purchased from American Bio-Rad Life Science Company. Western bolt test kit and films were purchased from American GE Company. Phospho-CDK2 ^Thr160 antibody, p27 endogenous cyclinkinase inhibitor antibody, CyclinD1 and β-Actin antibodies were purchased respectively from American Cell Signaling Inc., DAKO and Santa Cruz Biochemical Technology Company.

Tumor cells and their cultivation: the same as in example 2.

Western blot tested Phospho-CDK2, p27, and cyclin D1:

The androgen-independent human prostate cancer cells were treated with DU145, in accordance with the concentration, as shown in FIG. 3, using derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigo No. 124 and No. 126 for 24 hours. Cells were collected, washed, in accordance with the methods described in the literature ^[14] , proteins were extracted, and the required amount was taken. 50 mg of protein was taken, SDS-polyacrylamide gel electrophoresis was performed. After electrophoresis, proteins were transferred to nitrocellulose membranes using a specific Phospho-CDK2 antibody, an endogenous cyclinkinase inhibitor p27 antibody and a CyclinDl antibody, a Western blot test was performed, the β-Actin antibody was chosen as the internal standard, and the test results were recorded on an ECI film.

Results and discussion.

According to testing, indirudin compounds have the function of inhibiting cancer cell CDKs. The above examples 2 and 3 showed that the derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigo of the present invention have a strong function of inhibiting cell growth. To further clarify that these compounds, by regulating the activity of cyclin kinase, act as inhibitors of cell growth, the Western blot method was used using a specific antibody of the phosphorylated protein Cdc2 and other antibodies of the important proteins p27 and cyclin D1p27 that regulate the cell cycle; observed the effect of activators of representative compounds No. 124 and No. 126 of the present invention on the expression of protein p27 and cyclin D1.

As shown in FIG. 3, after treatment with compounds No. 124 and No. 126 for 24 hours, CDK2 activity (phosphorylation) in androgen-independent cancer cells of the human prostate DU145 was significantly reduced. At the same time, the expression of cyclin D1 protein also decreased significantly. In contrast, under the same experimental conditions, the effect of endogenous cyclin kinase inhibitors on p27 expression was significantly increased. As a result of changes in these signaling proteins, cell growth is inhibited. Of these, derivatives of 5- or 7-azaindirubin and 5- or 7-azaisoindigo induce the expression p27, perhaps by activating the AhR receptor pathway ^[15] .

Example 5. Research solid dispersants

Example 5-1.

Compound 110 in examples 1-2 5 mg Polyethylene glycol 400 50 mg

Process technology: polyethylene glycol 400 was melted at 50 ° C by adding compound 110 in examples 1-2, mixed until uniform, with stirring, cold vulcanization was performed with ice, dried in a desiccator for 24 hours, then in accordance with conventional methods made pills or capsules.

Example 5-2.

Compound 18 in examples 1-2 5 mg Polyethylene glycol 6000 50 mg Lactose: microcrystalline cellulose = 10: 1 1 g

Process technology: took compound 18 in examples 1-2, was dissolved in a moderate alkaline solution of ethanol, adding polyethylene glycol 6000, melted at 50 ° C; stirring until uniform, auxiliary materials were added (lactose / microcrystalline cellulose = 10/1), stirring to the state of a semi-dry powder, placed in a 60 ° C oven for 24 hours, tablets or capsules were made in accordance with conventional methods.

Example 5-3.

Compound 18 in examples 1-2 5 mg Polyvinylpyrrolidone K-25 50 mg Lactose / Microcrystalline Cellulose = 10/1 1 g

Process technology: they took compound 18 in examples 1-2, poured into a moderate alkaline ethanol solution, mixing until complete dissolution, adding polyvinylpyrrolidone K-25, mixed until dissolved, auxiliary materials (lactose / microcrystalline cellulose = 10/1) were added, mixing to uniformity, placed in a 60 ° C oven for 24 hours, tablets or capsules were made in accordance with conventional methods.

Example 5-4.

Compound 31 in examples 1-2 5 mg Polyoxyethylene (35) castor oil 350 mg Lactose / Microcrystalline Cellulose = 1/4 1 g

Technology process: They took compound 31 in examples 1-2, dissolved in a suitable amount of chloroform, added castor oil polyoxyethylene (35), mixing to dissolve, added auxiliary materials (lactose / microcrystalline cellulose = 1/4), mixing until uniform. The mixture was heated in a 80 ° C water bath to allow trichloromethane to evaporate, dried in a 60 ° C oven for 24 hours, and tablets or capsules were made in accordance with conventional methods.

Example 5-5.

Compound 116 in examples 1-2 5 mg Poloxamer 188 100 mg Microcrystalline cellulose 1 g

Process of the technology: compound 116 was taken in examples 1-2, dissolved in an appropriate alkaline ethanol solution, poloxamer 188 was added, mixed until dissolved, auxiliary materials (microcrystalline cellulose) were added, mixed until uniform, placed in a 60 ° C dryer for 24 hours In accordance with conventional methods, tablets or capsules were made.

Example 5-6.

Composition 18 in examples 1-2 5 mg Poloxamer 188 100 mg Polyethylene glycol 6000 50 mg Lactose / Microcrystalline Cellulose = 10/1 1 g

Process technology: took compound 18 in examples 1-2, was dissolved in a moderate alkaline solution of ethanol, poloxamer 188 and polyethylene glycol 6000 were added, mixed until dissolved, auxiliary materials (lactose / microcrystalline cellulose = 10/1) were added, mixed to uniformity, placed in 60 ° C oven for 24 hours, in accordance with conventional methods, tablets or capsules were made.

Example 5-7.

Compound 119 in examples 1-2 5 mg Polyvinylpyrrolidone K-25 50 mg Poloxamer 188 50 mg Polyethylene glycol 6000 50 mg Lactose 1 g

Process technology: compound 119 in examples 1-2 was dissolved in moderate alkaline ethanol, adding poloxamer 188, polyethylene glycol 6000 and polyvinylpyrrolidone K-25, stirring until dissolved, auxiliary materials were added, the oven was placed in a 60 ° C for 24 hours, then in accordance tablets or capsules were made with conventional methods.

Example 5-8.

Compound 18 in examples 1-2 5 mg Polyvinylpyrrolidone K-25 50 mg Poloxamer 188 100 mg Polyethylene glycol 6000 100 mg Lactose / Microcrystalline Cellulose = 10/1 1 g

Process: Compound 18 was taken in examples 1-2, placed in a moderate amount of an alkaline ethanol solution, poloxamer 188, polyethylene glycol 6000 and polyvinylpyrrolidone K-25 were added, stirring until dissolved, auxiliary materials (lactose / microcrystalline cellulose = 10/1) were added, stirring until uniform, and placed in a 60 ° C oven for 24 hours, tablets or capsules were made in accordance with conventional methods.

Example 5-9.

Compound 29 in examples 1-2 5 mg Polyoxyethylene (40) castor oil 750 mg Polyethylene glycol 4000 50 mg

Process technology: compound 29 in examples 1-2 was dissolved in polyoxyethylene (40) hydrogenated castor oil / ethanol solution, adding polyethylene glycol 4000, heated to 50 ° C, stirring until dissolved, the solvents were isolated, solidified with ice bags, placed in an oven at 60 ° C for 24 hours, in accordance with conventional methods, granules or capsules were made.

Example 5-10

Compound 121 in examples 1-2 5 mg Polyoxyethylene (40) castor oil 350 mg Polyvinylpyrrolidone K-25 50 mg Lactose / Microcrystalline Cellulose = 7/3 1 g

Process of the technology: compound 121 was taken in examples 1-2, polyoxyethylene (40) hydrogenated castor oil / ethanol solution was dissolved, polyvinylpyrrolidone K-25 was added, mixing until dissolved, auxiliary materials (lactose / microcrystalline cellulose = 7/3) were added, mixing until uniform, frozen and dried at -50 ° C for 24 hours, tablets or granules were made in accordance with conventional methods.

Example 5-11

Compound 18 in examples 1-2 5 mg Polyoxyethylene (40) castor oil 350 mg Polyvinylpyrrolidone K-25 50 mg Sodium dodecyl sulfate 10 mg Lactose / Microcrystalline Cellulose = 10/1 1 g

Process technology: castor oil and sodium dodecyl sulfate were dissolved in an appropriate volume of an ethanol solution of polyoxyethylene (40), after complete dissolution, compound 18 was added in examples 1-2, stirring until complete dissolution, polyvinylpyrrolidone K-25 was added, mixed until dissolved, auxiliary materials (lactose / microcrystalline cellulose = 10/1), mixing until uniform, and placed in a 60 ° C oven for 24 hours, pills were made in accordance with conventional methods.

Example 5-12

Compound 18 in examples 1-2 5 mg Polyoxyethylene (40) castor oil 350 mg Polyvinylpyrrolidone K 17 50 mg Lactose / Microcrystalline Cellulose = 10/1 1 g

Process technology: Compound 18 was taken in Examples 1-2, polyoxyethylene (40) hydrogenated castor oil / ethanol solution was dissolved, polyvinylpyrrolidone K17 was added, mixing until dissolved, auxiliary materials (lactose / microcrystalline cellulose = 10/1) were added, mixing until uniform , placed in a 60 ° C oven for 24 hours, tablets or capsules were made in accordance with conventional methods.

Example 5-13

Compounds 34 in examples 1-2 5 mg Vitamin E Polyethylene Glycol Succinate 350 mg Polyvinylpyrrolidone K-90 50 mg Lactose / Microcrystalline Cellulose = 5/5 1 g

Process of the technology: compound 34 was taken in examples 1-2, it was dissolved in vitamin E polyethylene glycol succinate / ethanol solution, adding polyvinylpyrrolidone K-90, stirring until dissolution, auxiliary materials were added (lactose / microcrystalline cellulose = 5: 5), mixing until uniform , and placed in a 60 ° C oven for 24 hours, tablets or capsules were made in accordance with conventional methods.

Example 6. Studies on the solubility of part of the solid dispersion preparations.

Instruments: RCZ-5A Intelligent solubility percent detector, manufactured by Tianjin University Precision Instruments Factory; UV1900 Ultraviolet Spectrophotometer, manufactured by Shanghai Yayan Electronic Science And Technology Co., Ltd.

Solubility test method: tested according to the solubility test method (third method) of Chinese Pharmacopeia)), 2005. According to this method, it was tested at a rotation speed of 100 rpm, a temperature of 37 ± 0.5 ° C, in 100 ml aqueous solution of 1% sodium dodecyl sulfate by ultrasonic degassing. Samples were taken after 45 minutes, immediately filtered through a 0.8 μm film, and the filtrate was diluted. Light absorption was measured using UV1900, the percentage of solubility was calculated.

Results and discussion:

In the above examples, the percent solubility of a portion of the solid dispersion preparations was investigated. The results are presented in table 8.

Table 8 Comparison of the solubility of part of the solid dispersion preparations Implementation examples The percentage of solubility (%) Compound 18 in examples 1-2 four 5-2 27 5-3 40 5-6 fifteen 5-8 56 5-11 71 5-12 92

From the above table it is seen that the solid dispersion preparations, which are made from the compounds of the present invention, overcome the disadvantages of these compounds, which are poor hydrophilicity and difficulty in creating the appropriate drugs. As a result of a significant increase in the solubility of the compounds, useful preparations can be obtained. Due to the use of solid dispersion preparations with joint carriers, under the interaction of more than one carrier, the compounds of the present invention have a better solubility than with one carrier, the solubility of the corresponding solid dispersion preparations will be much better.

Example 7. Injection studies

Example 7-1

Compounds 18 in Examples 1-2 10 mg Medium Chain Triglycerides 400 mg Lecithin 200 mg Glycerol 225 mg Poloxamer 188 200 mg

Technology process: all of the following processes are performed in a sterile room.

Oil phase: Compound 18 was taken in Examples 1-2, introduced into dimethyl sulfoxide, heated at low temperature to dissolve, adding medium chain triglycerides. The mixture was heated at low temperature, stirred, mixed evenly.

The aqueous phase: glycerol, Poloxamer 188, adding water for injection, was heated at low temperature with vigorous stirring, slowly poured into the oil phase and continued stirring for 3 minutes. Evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-2

Compound 25 in examples 1-2 10 mg Oleic Acid Monoglycerides 500 mg Phospholipids 300 mg Glycerol 225 mg Poloxamer 188 200 mg

Technology process: all of the following processes are performed in a sterile room.

Oil phase: Compound 25 was taken in Examples 1-2, heated at low temperature, dissolved in an appropriate amount of dimethyl sulfoxide, added oleic acid monoglycerides, heated to dissolve, and then added to phospholipids, heated at low temperature, mixed uniformly.

Water phase: glycerol, Poloxamer 188, adding water for injection, was heated with vigorous stirring, gradually added to the oil phase and stirring continued for 3 minutes. Evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-3

Compound 34 in examples 1-2 16 mg Human Albumin 20% 1 ml

Technology process: all of the following processes are performed in a sterile room.

Oil phase: compound 34 was taken in examples 1-2, heated and dissolved at a low temperature in a mixture of tetrahydrofuran and methylene chloride.

The aqueous phase: an aqueous solution of human albumin was poured into the oil phase, ultrasonic treatment was carried out for 1 minute, evaporated in a vacuum rotary evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-4.

Compound 122 in examples 1-2 10 mg Polyethylene glycol 400 100 mg Lecithin 50 mg Hydroxypropyl-β-cyclodextrin 200 mg

Technology process: all of the following processes are performed in a sterile room.

Oil phase: took compound 122 in examples 1-2, was dissolved in an alkaline solution of ethanol, adding lecithin and polyethylene glycol 400, was stirred until dissolved.

Water phase: hydroxypropyl-β-cyclodextrin was dissolved in a solution of 70% ethanol, poured into the oil phase, adding an appropriate amount of water for injection, stirred until uniform, evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-5.

Compound 110 in examples 1-2 10 mg Poloxamer 188 200 mg Lecithin 30 mg Hydroxypropyl-β-cyclodextrin 300 mg 0.9% sodium chloride solution 10 ml

Technology process: all of the following processes are performed in a sterile room.

They took compound 110 in examples 1-2, dissolved in an alkaline solution of ethanol, added poloxamer 188, heated, stirred to dissolve, cooled, added 0.9% sodium chloride solution mixed with an appropriate amount of ethanol, added lecithin, mixing evenly, added hydroxypropyl β-cyclodextrin, mixing uniformly, was sonicated for 1 minute, evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-6

Compound 30 in examples 1-2 10 mg Poloxamer 188 200 mg Soya lecithin 20 mg Hydroxypropyl-β-cyclodextrin 400 mg

Technology process: all of the following processes are performed in a sterile room.

Compound 30 was taken in Examples 1-2, dissolved in an alkaline ethanol solution, adding soya lecithin and Poloxamer 188 until dissolved, and then hydroxypropyl-β-cyclodextrin, water for injection was added and dissolved with a suitable amount of ethanol; evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-7

Compound 18 in examples 1-2 10 mg Polyethylene glycol monolaurate 200 mg Lecithin 20 mg Hydroxypropyl-β-cyclodextrin 200 mg

Technology process: all of the following processes are performed in a sterile room.

Oil phase: took compound 18 in examples 1-2, was dissolved in an alkaline solution of ethanol, adding lecithin and polyethylene glycol monolaurate, stirring until dissolved.

The aqueous phase: hydroxypropyl-β-cyclodextrin was added to a 70% ethanol solution, poured into the oil phase, altering; evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-8.

Compound 19 in examples 1-2 10 mg Polyoxyethylene (40) Castor Oil 100 mg Lecithin 50 mg Hydroxypropyl-β-cyclodextrin 200 mg

Technology process: all of the following processes are performed in a sterile room.

Oil phase: took compound 19 in examples 1-2, was dissolved in alkaline ethanol, adding lecithin and polyoxyethylene (40) castor oil, stirring until dissolved.

The aqueous phase: hydroxypropyl-β-cyclodextrin was dissolved in a 70% ethanol solution, poured into the oil phase by adding a suitable amount of water for injection, altered uniformly, evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-9

Compound 18 in examples 1-2 20 mg 20% human albumin 5 ml

Technology process: all of the following processes are performed in a sterile room.

Oil phase: Compound 18 was taken in Examples 1-2, dissolved in a mixed solution of tetrahydrofuran and trichlorethylene by ultrasonic treatment.

The aqueous phase: human albumin was dissolved in an appropriate amount of a 0.1 mol / L hydrochloric acid solution, poured into the oil phase with vigorous stirring, and vigorous stirring was continued for 5 minutes until uniform. The mixture was homogenized (600 bar) and cooled with water. The operation was repeated six times. Evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 7-10

Compound 120 in examples 1-2 10 mg Medium chain triglyceride 600 mg Vitamin E Polyethylene Glycol Succinate 200 mg Lecithin 100 mg Poloxamer 188 200 mg Glycerol 225 mg

Technology process: all of the following processes are performed in a sterile room.

Oil phase: compound 120 was taken in examples 1-2, after dissolving with ethanol, vitamin E polyethylene glycol succinate was added, medium chain triglyceride and poloxamer 188 were heated to dissolve.

Water phase: 8 ml of a 2.25% glycerol solution was poured into the oil phase, stirred, adding a lecithin solution, subjected to ultrasonic treatment for 10 minutes, evaporated in a rotary vacuum evaporator at low temperature to remove organic solvents.

The resulting emulsion was sterilized, filtered, poured into a lyophilized sterile vial in a laminar state, frozen, dried, sealed, and closed with aluminum caps for sealing.

Example 8. Solubility Test

Compounds 129 and 91 are very similar in structure, the difference between them is only in the type of atoms in the 7-position (see formulas below), they were used as a solvent to check the changes in the solubility of 7-azaindirubin compared to indirubin.

Test method: at room temperature (20 ° C), respectively, compounds 129 and 91, 5 mg each were taken, added to 2 ml of the corresponding solvent, dissolved after stirring, the results are presented in table 9.

Table 9 Comparison of the solubility of compound 129 and control product 91 Solvent 129 91 deionized water slightly soluble Insoluble Methanol soluble slightly soluble Ethanol slightly soluble Soluble Acetone slightly soluble Easily soluble Chloroform slightly soluble Easily soluble Petroleum ether (boiling: 60 ~ 90 ° C) insoluble slightly soluble

According to the results presented in the table above, it can be seen that in the derivatives of 7-azaindirubin (129), the water solubility increased significantly, and the solubility in fat decreased. In general, the water solubility and fat solubility of indirubin is not good, but the treated solubility of indirubin increased fat solubility and water solubility decreased, compound 91 is exactly the same. This change in solubility reflects the need to study derivatives of 7-azaindirubin. Studies of the state of absorption of drugs in the body and the choice of dosage forms will bring benefits.

Mentioned in this invention, all references to the literature are cited in this application as manuals, as all articles were separately cited as references. In addition, it should be understood that after reading the foregoing content in the present invention, technical personnel in this field can make various kinds of changes or modifications, these equivalent forms also fall within the scope of the limitation of the claims appended to this application.

References

[1] Lundberg AS, Weinberg RA., Control of the cell cycle and apoptosis, Eur J Cancer, 1999, 35: 531-539.

[2] Sherr CJ. Canaer cell cycles, Science, 1996, 274: 1672-1677.

[3] Keyomarsi K., Pardee AB., Redundant cyclin overexpression and amplification in breast cancer cells, Proc Natl Acad Sci USA, 1993, 90: 1112-1116.

[4] Gray N, Detivand L, Meijer L et al., ATP-site directed inhibitors of cyclin-dependent kinases, Curr Med Chem, 1999, 6: 859-875.

[5] Malumbres M et al., Targeting cell cycle kinases for cancer therapy, Curr Med Chem, 2007: 14 (9): 969-85.

[6] Rudolph J., Inhibiting transient protein-protein interactions: lessons from the Cdc25 protein tyrosine phosphatases, Nat Rev Cancer, 2007, 7 (3): 202-11.

[7] Huwe A., Mazitschek R., Giannis A., Small molecules as inhibitors of cyclin-dependent kinases, Angew Chem Int Ed, 2003, 42: 2100-2138.

[8] Ji Xiujuan, Wu Kemei, Huang Liang, etc., Indigo Naturalis, see: edited by the Institute of Medicine at the Chinese Academy of Medical Sciences. Modern Studies of Chinese Herbal Medicine (Volume 1), First Edition, Beijing: Joint Publishing House of Beijing Medical University and Sehe Chinese Medical University, 1995: 227-257.

[9] Nam S., Buettner R, Turkson J. et al., Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in human cancer cells, PNAS, 2005, 102 (17): 5998-6003.

[10] Roy KK., Sausville EA. Early development of cyclin dependent kinase modulators, Curr Pharm Design, 2001, 7 (16): 1669-1687.

[11] Doklady, Akad Nauk SSSR, 1958, 118: 302-305.

[12] Christoph MS, Pascale H., John M. et al., Synthesis and evaluation of comparative of 10H-indolo [3,2-b] -quinoline as G-quadruplex stabilizing ligands and potential inhibitors of the enzyme telomerase, Org . Biomol. Chem., 2004, 2: 981-988.

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Claims (6)

1. Derivatives of azaindole-indole and their pharmaceutically acceptable salts according to the formula

where Y is an azaindole group of the formula (Y1), (Y2)

Z is an indole group of the formula (Z1) or (Z2)

"=" means a double bond located at the 3-position of the azaindole group Y and between the 2'- and 3'-positions of the indole-Z group;
in the above formulas, Y1, Y2, Z1 and Z2, R ¹ and R ¹ independently mean H or the following groups which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ -C ₆ alkyl, aryl, aralkyl, acyl, aroyl, glucosyl or biosil protected by acyl, glucosyl or biosil; wherein the substituents described are selected from the group: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino;
R ² , R ³ , R ⁴ , R ^{2 '} , R ^3' , R ^{4 '} , R ^5' independently mean H, halogen, hydroxyl, sulfhydryl or the following groups, which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ -C ₄ alkyl, nitro, amino, amido, amide, C ₁ -C ₄ alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonic group, sulfamoyl, isocyanate or alkyl isocyanate, wherein the substituents described are selected from the group: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino;
R is oxygen, sulfur, selenium, or an NR ⁶ or NOR ⁶ group in which R ⁶ is H, or the following groups, which may be unsubstituted or substituted with 1 to 3 substituents: C ₁ -C ₆ straight-chain alkyl or branched bond , aryl, aralkyl, C ₃ -C ₆ alicyclic group, acyl, aroyl, sulfonyl or phosphoryl; wherein the substituents described are selected from the group: halogen, hydroxyl, C ₁ -C ₃ alkyl, nitro or amino. 2. The compounds or their pharmaceutically acceptable salts according to claim 1, as they are represented in formulas (I), (II), (III) or (IV), in which formula (I) is a derivative of 5-azaindirubin, formula (II ) are derivatives of 5-azaisoindigo, formula (III) are derivatives of 7-azaindirubin, and formula (IV) are derivatives of 7-azaisoindigo

where R, R ¹ , R ² , R ³ , R ⁴ , R ^{1 '} , R ^2' , R ^{3 '} , R ^4' and R ^{5 '} mean, as described above. 3. The compounds or their pharmaceutically acceptable salts according to claim 1, in which R ¹ and R ^{1 '} independently mean H, C ₁ -C ₆ alkyl, aryl, aralkyl, acyl, aroyl, glycosyl, protected by alkyl or glucosyl;
R ² , R ³ , R ⁴ , R ^{2 '} , R ^3' , R ^{4 '} and R ^5' independently mean H, halogen, hydroxyl, sulfhydryl, C ₁ -C ₄ alkyl, amino, amido, amide, C ₁ - C ₄ alkoxy, methylthio, phenyl, phenoxy, aryl, aralkyl, trifluoromethyl, acyl, aroyl, sulfonic group or isocyanate;
the aforementioned glycosyls are arabinose, xylose, ribose, mannose or glucose;
R is oxygen, sulfur, selenium, or an NR ⁶ or NOR ⁶ group in which R ⁶ is H, a C ₁ -C ₆ straight chain or branched bond, aryl, aralkyl, a C ₃ -C ₆ alicyclic group, acyl , aroyl, sulfonyl or phosphoryl. 4. The compounds or their pharmaceutically acceptable salts according to claim 1, in which these compounds are selected from the following: 5-azaindirubin derivatives (I) (table 1), 5-azaisoindigo derivatives (II) (table 2), 7-azaindirubin derivatives ( III) (table 3) and derivatives of 7-azaisoindigo (IV) (table 4)

where R ² -R ⁴ , R ^{1 '} , R ^2' , R ^{4 '} and R ^5' mean H, the remaining radicals are presented in table 1:
The structure of compounds of 5-azaindirubin (I)
Table 1
No. R ¹ R ³ R one 2 3 four one N N O 2 CH ₃ N O 3 CH ₃ IT O four CH ₃ OCH ₃ O 5 CH ₃ SCH ₃ O 6 C ₂ H ₅ N O 7 i-C ₃ H ₇ N O 8 n-C ₄ H ₉ N O 9 CH ₂ -Ph N O 10 CH ₃ Br O eleven CH ₃ Cl O 12 C ₂ H ₅ Br O 13 C ₂ H ₅ Cl O fourteen i-C ₃ H ₇ Br O fifteen i-C ₄ H ₉ Cl O 16 n-C ₄ H ₉ Br O 17 n-C ₄ H ₉ Cl O eighteen CH ₂ -Ph Br O 19 CH ₂ -Ph Cl O twenty CH ₂ -Ph F O 21 Triacetyl ribosyl N O 22 Phco OCH ₃ S 23 CH ₃ CH ₂ CO OCH ₃ S 24 CH ₃ Br N-OH 25 CH ₃ Cl N-OH 26 CH ₃ Ph N-OH 27 CH ₃ SCH ₃ N-OH 28 C ₂ H ₅ Br N-OH 29th C ₂ H ₅ Cl N-OH thirty i-C ₃ H ₇ Br N-OH 31 i-C ₃ H ₇ Cl N-OH 32 n-C ₄ H ₉ Br N-OH 33 n-C ₄ H ₉ Cl N-OH 34 CH ₂ -Ph Br N-OH 35 CH ₂ -Ph Cl N-OH 36 CH ₂ -Ph F N-OH 37 CH ₃ N N-OH 38 C ₂ H ₅ N N-OH 39 i-C ₃ H ₇ N N-OH 40 n-C ₄ H ₉ N N-OH 41 CH ₂ -Ph N N-OH 42 ribosyl N N-OH 43 Glycosyl N N-OH 44 CH ₃ N N-OCH ₃ 45 CH ₃ Br N-OCH ₃ 46 CH ₃ Cl N-OCH ₃ 47 C ₂ H ₅ N N-OCH ₃ 48 C ₂ H ₅ Br N-OCH ₃ 49 C ₂ H ₅ Cl N-OCH ₃ fifty i-C ₃ H ₇ N N-OCH ₃ 51 i-C ₃ H ₇ Br N-OCH ₃ 52 i-C ₃ H ₇ Cl N-OCH ₃ 53 n-C ₄ H ₉ N N-OCH ₃ 54 n-C ₄ H ₉ Br N-OCH ₃ 55 n-C ₄ H ₉ Cl N-OCH ₃ 56 CH ₂ -Ph N N-OCH ₃ 57 CH ₂ -Ph F N-OCH ₃ 58 CH ₂ -Ph Br N-OCH ₃ 59 CH ₂ -Ph Cl N-OCH ₃

where R ² -R ⁴ , R ^{2 '} , R ^4' and R ^{5 '} mean H, the remaining radicals are presented in table 2:
The structure of compounds 5-azaisoindigo (II)
table 2
No. R ¹ R ^{1 '} R ^{3 '} one 2 3 four 60 CH ₃ N N 61 CH ₃ N Br 62 CH ₃ N Cl 63 CH ₃ N F 64 CH ₃ CH ₃ IT 65 CH ₃ CH ₂ CO N OCH ₃ 66 Phco N OCH ₃ 67 Triacetyl ribosyl N N 68 C ₂ H ₅ N N 69 C ₂ H ₅ C ₂ H ₅ Br 70 C ₂ H ₅ N Cl 71 C ₂ H ₅ N F 72 C ₂ H ₅ C ₂ H ₅ IT 73 i-C ₃ H ₇ N N 74 i-C ₃ H ₇ N Br 75 i-C ₃ H ₇ N Cl 76 i-C ₃ H ₇ N F 77 i-C ₃ H ₇ i-C ₃ H ₇ IT 78 n-C ₄ H ₇ N N 79 n-C ₄ H ₇ N Br 80 n-C ₄ H ₇ N Cl 81 n-C ₄ H ₇ n-C ₄ H ₉ it 82 n-C ₄ H ₇ N F 83 n-C ₄ H ₇ n-C ₄ H ₉ OCH ₃ 84 CH ₂ -Ph N N 85 CH ₂ -Ph N Br 86 CH ₂ -Ph CH ₂ -Ph Cl 87 CH ₂ -Ph N F 88 CH ₂ -Ph CH ₂ -Ph IT 89 ribosyl N N

where R ² , R ⁴ , R ^{1 '} , R ^2' , R ^{4 '} and R ^5' mean H, the remaining radicals are presented in table 3:
The structure of compounds 7-azaindirubin (III)
Table 3
No. R ¹ R ³ R ^{3 '} R one 2 3 four 5 92 H N N O 93 CH ₃ N N O 94 CH ₃ CH ₃ OH O 95 CH ₃ CH ₃ OCH ₃ O 96 CH ₃ CH ₃ SCH ₃ O 97 C ₂ H ₅ N N O 98 i-C ₃ H ₇ N N O 99 n-C ₄ H ₉ N N O one hundred CH ₂ -Ph N N O 101 CH ₃ N Br O 102 CH ₃ N Cl O 103 C ₂ H ₅ N Br O 104 C ₂ H ₅ N Cl O 105 i-C ₃ H ₇ N Br O 106 i-C ₃ H ₇ N Cl O 107 n-C ₄ H ₉ N Br O 108 n-C ₄ H ₉ N Cl O 109 CH ₂ -Ph N Br O 110 CH ₂ -Ph N Cl O 111 CH ₂ -Ph NHCH ₃ F O 112 Triacetyl ribosyl CH ₃ N O 113 Phco CH ₃ OCH ₃ S 114 CH ₃ CH ₂ CO F OCH ₃ S 115 CH ₃ N Br N-OH 116 CH ₃ N Cl N-OH 117 CH ₃ CF ₃ Ph N-OH 118 CH ₃ CF ₃ SCH ₃ N-OH 119 C ₂ H ₅ N Br N-OH 120 C ₂ H ₅ N Cl N-OH 121 i-C ₃ H ₇ N Br N-OH 122 i-C ₃ H ₇ N Cl N-OH 123 n-C ₄ H ₉ N Br N-OH 124 n-C ₄ H ₉ N Cl N-OH 125 CH ₂ -Ph N Br N-OH 126 CH ₂ -Ph N Cl N-OH 127 CH ₂ -Ph N F N-OH 128 CH ₃ N N N-OH 129 C ₂ H ₅ N N N-OH 130 i-C ₃ H ₇ N N N-OH 131 n-C ₄ H ₉ N N N-OH 132 CH ₂ -Ph N N N-OH 133 ribosyl CH ₃ N N-OH 134 glucosyl CH ₃ N N-OH 135 CH ₃ N N N-OCH ₃ 136 CH ₃ N Br N-OCH ₃ 137 CH ₃ N Cl N-OCH ₃ 138 C ₂ H ₅ N N N-OCH ₃ 139 C ₂ H ₅ N Br N-OCH ₃ 140 C ₂ H ₅ N Cl N-OCH ₃ 141 i-C ₃ H ₇ N N N-OCH ₃ 142 i-C ₃ H ₇ N Br N-OCH ₃ 143 i-C ₃ H ₇ N Cl N-OCH ₃ 144 n-C ₄ H ₉ N N N-OCH ₃ 145 n-C ₄ H ₉ N Br N-OCH ₃ 146 n-C ₄ H ₉ N Cl N-OCH ₃ 147 CH ₂ -Ph N N N-OCH ₃ 148 CH ₂ -Ph N F N-OCH ₃ 149 CH ₂ -Ph N Br N-OCH ₃ 150 CH ₂ -Ph N Cl N-OCH ₃

where R ² , R ⁴ , R ² , R ⁴ and R ⁵ mean H, the remaining radicals are presented in table 4:
Structures of 7-azaindigo (IV) compounds
Table 4
No. R ¹ R ^{1 '} R ³ R ^{3 '} one 2 3 four 5 151 CH ₃ N N N 152 CH ₃ N N Br 153 CH ₃ N N Cl 154 CH ₃ N N F 155 CH ₃ CH ₃ CH ₃ IT 156 CH ₃ CH ₂ CO N F OCH ₃ 157 Phco N CH ₃ OCH ₃ 158 Triacetyl ribosyl N CH ₃ N 159 C ₂ H ₅ N N N 160 C ₂ H ₅ C ₂ H ₅ N Br 161 C ₂ H ₅ N N Cl 162 C ₂ H ₅ N N F 163 C ₂ H ₅ C ₂ H ₅ CF ₃ IT 164 i-C ₃ H ₇ N N N 165 i-C ₃ H ₇ N N Br 166 i-C ₃ H ₇ N N Cl 167 i-C ₃ H ₇ N OCH ₃ F 168 i-C ₃ H ₇ i-C ₃ H ₇ N IT 169 n-C ₄ H ₉ N N N 170 n-C ₄ H ₉ N N Br 171 n-C ₄ H ₉ N N Cl 172 n-C ₄ H ₉ n-C ₄ H ₉ SCH ₃ it 173 n-C ₄ H ₉ N N F 174 n-C ₄ H ₉ n-C ₄ H ₉ OCH ₃ OCH ₃ 175 CH ₂ -Ph N N N 176 CH ₂ -Ph N N Br 177 CH ₂ -Ph CH ₂ -Ph SCH ₃ Cl 178 CH ₂ -Ph N OCH ₃ F 179 CH ₂ -Ph CH ₂ -Ph N IT 180 ribosyl N CH ₃ N 5. The compounds or their pharmaceutically acceptable salts according to claim 1, wherein said pharmaceutically acceptable salts include salts formed with inorganic acids or organic acids, while inorganic acids include the following: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid and sulfuric acid ; organic acids include the following: formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1, 5), Asian acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, butyl carboxylic acid, diethylacetic acid, malonic acid , succinic acid, fumaric acid, pimelic acid, hexanediol acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid and, nicotinic acid, isonicotinic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid and amino acids. 6. The pharmaceutical composition inhibiting cyclin-dependent kinase containing the compounds or their pharmaceutically acceptable salts according to claim 1 and pharmaceutically acceptable carriers.

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