WO2010064958A2

WO2010064958A2 - Protein kinase c inhibitors exhibiting an anti-inflammatory, anti-allergic and anti-asthma effect

Info

Publication number: WO2010064958A2
Application number: PCT/RU2009/000652
Authority: WO
Inventors: Александр Викторович ДЕМИН; Виталий Aфанасьевич МАРТЬЯНОВ; Александр Михайлович ШУСТЕР
Original assignee: Закрытое Акционерное Общество "Мастерклон"
Priority date: 2008-12-01
Filing date: 2009-11-27
Publication date: 2010-06-10
Also published as: WO2010064958A3

Abstract

The invention relates to the field of medicine, and specifically to a new use for derivatives of indole and the use of these in the manufacture of medicinal preparations for the treatment of conditions associated with protein kinase C dysfunction, including for the treatment of inflammatory, allergic and dermatological diseases, asthma, diabetes, ischemia, cardiovascular diseases and disorders caused by the inflammatory process, including hepatitis, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, and neurodegenerative diseases. The aim of the invention is to expand the arsenal of drugs for the treatment of conditions in which protein kinase C plays a part in the pathology and to prepare a drug having a wide spectrum of pharmacological action and high effectiveness with negligible toxicity. Pharmaceutical compositions for external and internal use are described.

Description

PROTEINKINASE C INHIBITORS WITH

ANTI-INFLAMMATORY, ANTI-ALLERGIC AND

ANTI-ASTHMATIC ACTION

The field of technology.

The invention relates to the use of known indole derivatives as protein kinase C inhibitors, as well as to the creation on their basis of pharmaceutical compositions having anti-inflammatory, anti-asthma, anti-allergic activity.

The prior art.

Protein kinase C (PKC) or Ser / Thr-specific protein kinase is the common name for a family of related enzymes that phosphorylate at serine and threonine residues. To activate protein kinases, calcium and phospholipids are necessary (J. Biol. Chem. 1977. 252,

7603-7609; J. Biol. Chem. 1977.252, 7610-7616). Additional stimulation of PKC occurs under the action of diacylglycerol, which plays the role of a chemical signal that activates PKC ip vivo (J. Biol. Chem. 1979. 254, 3692-3695; NATURE.

1984, VoI. 308. P. 693-698).

Protein kinase C is expressed in almost all mammalian cells and is one of the most important elements of the cellular signal transmission system, gene expression, and regulation of cell differentiation and growth. Currently, about twelve isozymes (isoenzymes) of PKC are known, which differ in enzymatic specificity, tissue distribution, and regulatory processes.

It is known that disturbances in the functioning of the network of signal transducing cell enzymes in which serine-threonine are involved protein kinases play a key role in the development of a significant number of diseases, including inflammatory diseases, diabetes, disorders of the central nervous system, cardiovascular diseases, various types of tumors, immune disorders, dermatological diseases. For example, in the work of Mulqueep MJ. with sotr. (Аgepts Аtiops. 1992 Sep; 37 (l-

2): 85-9) it was shown that PKC inhibitors block the negative regulation of CD3 in T-lymphocytes. Twotuu V. (Biochem Віорhus Res ResComp. 1990 Ser 28; 171 (3): 1087-92) demonstrated that PKC inhibitors have an effect on the oxidative emission of neutrophils. Therefore, compounds exhibiting the properties of PKC inhibitors can be used in the treatment of inflammation.

A consequence of inhibiting the oxidative release of neutrophils is the fact that PKC inhibitors are also applicable in the treatment of cardiovascular ischemia, as shown in an IP vivo study in dogs, by Sonoki H. (Kokuu Jun. 1989 Jun; 37 (6): 669-74).

In a Naga H. study (J Cerab Blood Metab. 1990 Seρ; 10 (5): 646-53) done on a rat model of ischemia, protein kinase C inhibitors prevent the neuronal damage observed in ischemic stroke. In several works (Вісхэм Віорхус Рес Commonmm. 1989 Jap

16; 158 (l): 105-9; Вісхэм Віорхус Res Resource 1990 Dec 31; 173 (3): 976-81.) It was shown that protein kinase C inhibitors block agonist-induced proliferation of smooth muscle cells, which allows them to be considered as potential therapeutic agents for the treatment of cardiovascular diseases.

A lot of work is devoted to the activity of PKC associated with proliferation, differentiation, apoptosis and cell growth, including the effect on tumor growth and use in cancer (for example, Semip Opt. 2006 Aug; 33 (4): 466-78; Int J Sapper. 1989 Mau 15; 43 (5): 851-6; Sapser Res. 1991 Ser 15; 51 (18): 4888-92, etc.).

Protein kinase C is also associated with various aspects of diabetes, in particular with diabetic retinopathy, nephropathy, and cardiovascular disease (Pharmacol Res. 2007 Jιm; 55 (6): 498-510).

Protein kinase C inhibitors have been shown to reduce the number of cytokines, inhibit the activation of peripheral T cells and the proliferation of keratinocytes, thereby exerting a positive effect on dermatological disorders such as contact dermatitis and psoriasis (J CHn Ipvest. 2008 Sep; 118 (9): 3151 -9; Argh Dermatol Res. 1992; 284 (3): 179-83 and Arch Dermatol Res. 1991; 283 (7): 456-60).

Contact dermatitis is an inflammation of the skin in acute or chronic form, which is either the result of irritation by any substance present in the external environment or the result of sensitization. Contact dermatitis is usually divided into primary irritable

(inflammatory) dermatitis and allergic contact dermatitis.

Clinical signs of irritable and allergic dermatitis are similar. In mild cases, the symptoms of dermatitis include itching, burning, or redness of the skin. In more acute cases, vesiculation and edema may occur, followed by wetting and crusting. The most acute cases may be accompanied by the appearance of bleeding vesicles and severe swelling.

In view of the above, contact dermatitis is a serious problem for many people, and allergic dermatitis in addition, is often an occupational disease. The problem becomes especially acute in cases where professional allergic contact dermatitis can lead to the need for a job change and, consequently, loss of pay and other inconveniences for employees. Currently used treatment of dermatitis is complex and includes the use of antihistamines, desensitizing, diuretics and detoxifying agents.

The first stage of treatment should be to remove the damaging agent, however, in many cases, the irritant cannot be identified or removed. Therefore, in practice, in cases of mild to moderate severity, treatment is based on the use of topical glucocorticosteroids (GCS). Modern external corticosteroids (hydrocortisone, fluocinolone acetonide, flumethasone, etc.) have a strong anti-inflammatory, antipruritic and anti-allergic effect. However, corticosteroids have well-known side effects, in particular, they reduce immune defense, which can lead to infections. Discontinuation of treatment should be gradual and carried out under the supervision of a physician, as after discontinuation of treatment, the opposite effect may follow. GCS should not be used during the acute phase of wetness. In addition, corticosteroids can cause systemic effects, so their use for long-term treatment of dermatitis, especially in children, is not recommended.

Thus, the need for finding effective topical treatments for contact dermatitis that are safe and suitable for long-term use becomes apparent. It is known that protein kinase C, including protein kinase C alpha, is directly involved in the signaling cascades that occur in asthma. The amount of PKC is increased in the smooth muscles of the respiratory tract in patients with asthma. It is assumed that the activation of PKC leads to the proliferation of smooth muscle cells of the respiratory tract and, as a result, remodeling and tissue damage in asthma (Exrerimptal apd Molecular Medisipe, VoI. 39, JNeI, pp. 97-105; Сhiп Med J J, 2008, 121 (20): 2070-2076). Activation of PKC is also a necessary step in the inflammatory cascade of the leukotriene receptor (JPET, 2008, 325 (3), pp. 1024-1030), which is actively involved in asthma pathology. Bronchial asthma is a chronic inflammatory disease of the respiratory tract characterized by a variety of symptoms, such as airway obstruction, bronchial hyperreactivity and inflammation. The interaction of these symptoms determines the clinical manifestations and severity of asthma. Thus, the modern definition of bronchial asthma includes the main provisions that reflect the inflammatory nature of the disease.

Asthma occurs in response to various stimuli, such as antigens, pollutants, cold air, physical exposure. The treatment of asthma is to suppress bronchospasm and inflammation, and also to eliminate the effects of allergens or allergy mediators. The total number of patients with asthma worldwide is 300 million. And, despite the achievements of modern medicine, this number is growing, especially among children. In this regard, it is especially important to search for new drugs with pronounced anti-asthma activity, reduce inflammation and bronchospasm.

Currently, for the treatment of asthma, glucocorticosteroids, β ₂ -adrenomimetics, antagonists of leukotriene receptors, xanthines, and their combinations are used. As the disease develops, sensitivity to the drug used decreases, which makes it necessary to prescribe more and more potent drugs.

The action of glucocorticosteroids, the main group of drugs for the treatment of bronchial asthma, is based on the suppression of the production of anti-inflammatory cytokines, the reduction in the number of inflammatory cells, the suppression of their migration and activation. Glucocorticosteroids are used inhaled or orally. The use of glucocorticosteroids often leads to side effects; with inhalation use - such as hoarseness, candidiasis of the oral cavity and pharynx, skin manifestations, angioedema of the face and oropharynx; with systemic use - bradycardia, arterial hypotension, dizziness, headaches, convulsions, nausea, vomiting, the development of a steroid gastric ulcer, decreased immunity (for example, see the Vidal Handbook Medications in Russia: A Handbook, M .: AstraFarmService, 2002, pages B-69-73, as well as Wager Peer M. Neue Epptupek ip der Asthma teraris, Wie wirkep díe sípzelpep Leukotriep antagonisten // Schweiz.-Rupdsh. Med Prah., 1998, 87 (8), pp. 271-275). To reduce the risk of side effects, glucocorticosteroids are administered by inhalation. But this necessitates the use of an inhaler, which is inconvenient and requires skill in handling. Glucocorticosteroids for oral administration begin to be prescribed with the ineffectiveness of inhaled glucocorticosteroids. Moreover, bronchial asthma is characterized as steroid-dependent.

In addition to glucocorticosteroids, β2-adrenergic agonists are actively used to treat asthma, which relax the smooth muscles of the bronchi and, thereby, facilitate breathing. β2-adrenomimetics have their own set of side effects: palpitations, headache, abdominal pain, nausea, vomiting. And if monotherapy with β2-adrenomimetics is prescribed less and less, combinations of glucocorticosteroids and β2-adrenomimetics are the most popular drugs for the treatment of asthma and similar symptoms of chronic obstructive pulmonary disease. Such combinations can more effectively relieve the symptoms of the disease. But the use of such combinations is accompanied by frequent side effects characteristic of both groups of drugs. Thus, Glaso-Smith-Klein's Seretide, a third of the US and European markets, causes headaches in every tenth patient. Candidiasis of the oral cavity and pharynx, palpitations, muscle cramps are a little less common (Sétide Assuhaler. Raskagé Léflat: Ipformatioporte User).

The action of the following group of drugs, antagonists of leukotriene receptors, is based on blocking the signal of leukotriene receptors, which leads to dilatation of the bronchi and a decrease in inflammation. These drugs are used as an adjunct to basic treatment, for example, with glucocorticosteroids, to relieve residual symptoms of asthma. Or, more often, for the treatment of asthma in children, since the use of glucocorticosteroids in this case is undesirable due to the negative effect on vision and bone growth (The Asthma, COPD & Allergis Ripitis Markut Out 2011, Buseps Ipsights, 2006.). Among the side effects of leukotriene receptor antagonists are: abdominal pain, nausea, headache, cough, drug-induced hepatitis (for example, Vidal Handbook Medications in Russia: Handbook, M .: AstraFarmService, 2002, p. B-17). Xanthines are also used to reduce bronchospasm. But their use is very limited due to a long list of side effects: dizziness, headache, insomnia, agitation, anxiety, irritability, tremors, palpitations, tachycardia, arrhythmias, decreased blood pressure, cardialgia, increased frequency of angina attacks, gastralgia, nausea, vomiting , heartburn, exacerbation of peptic ulcer, diarrhea, etc. (for example, Vidal Handbook of Medicinal Products in Russia: Handbook, M .: AstraFarmService, 2002, pp. 3-17, 3-290).

Given all of the above, the search for new drugs for the treatment of asthma remains very relevant, since currently used drugs often do not cover all the clinical manifestations of asthma and have a significant number of side effects.

Currently, in addition to the already known classes of compounds, such as glucocorticosteroids, β2-adrenergic antagonists, antagonists of leukotriene receptors and xanthines, a fairly large number of compounds have been proposed that have activity as anti-asthma agents. For example, derivatives of substituted 3-amino-III-indole-2-carboxylic acid (WO 03091214, WO 03091215), pyrazoloantrone derivatives (WO 0112609), heterocyclic compounds (WO 020724548), etc. However, since many of the listed compounds exhibiting anti-asthma activity have not yet been clinically tested and, therefore, can only potentially be drugs, we chose glucocorticosteroid fluticasone (MHH), which has proven anti-inflammatory and anti-asthma activity, as a close analogue.

Fluticasone (trade name Flixotide (Glaxo), is used to treat asthma and chronic obstructive pulmonary disease, and belongs to the latest generation of glucocorticosteroids. It is administered by inhalation to reduce the number of side effects inherent in this class of drugs. Inhalation therapy is inconvenient in itself and requires training patients for accurate dosing of the drug, but even it does not completely avoid the effects of fluticasone. According to published clinical trial results (http://us.gsk.com/products/assets/us_flovent.pdf), the use of fluticasone led to a number of side effects, the most common being oral irritation (up to 22%), upper respiratory tract infections (up to 21% ) and headache (up to 14%).

Until now, it was not known that the compounds of formula I exhibit anti-inflammatory, in particular, anti-asthmatic activity, which was first shown by the authors of this invention.

One of the objectives of the present invention is the search for compounds that suppress the activity of the transcription factor NF-kV, which allows, on the basis of the laws known from the prior art, to offer them as drugs for the treatment of diseases directly related to the onset of the inflammatory process by enhancing the transcription of inflammatory protein genes during help NF-kV. Similar conditions occur not only in asthma, but also in chronic obstructive pulmonary disease, hepatitis, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neurodegenerative diseases, contact dermatitis. Therefore, based on the presence of a common mechanism for the development of inflammation, we suggested that the compounds proposed in this invention may become a potential drug with the ability to reduce inflammation in these diseases.

Thus, the compounds according to the proposed invention are applicable in the treatment of conditions in which protein kinase C in one way or another affects the pathological process. Conditions for which this effect is confirmed by the studies are: inflammatory diseases, diabetes mellitus, asthma, ischemia, cardiovascular diseases, dermatological diseases.

The crucial role of PKC in physiology is caused by the growing interest in the preparation of compounds that are active as inhibitors of PKC. Such compounds, exhibiting a selective effect against PKC or its isozymes, can become effective therapeutic agents. However, along with effectiveness, an equally important condition for a potential therapeutic agent is low toxicity.

A known inhibitor of PKC is staurosporin, a microbial indolocarbazole (Biochem. Biorhus. Res. Comms. 135: 397-402 (1986); Biochem. Pharmacol. 40: 343-350 (1990)). However, its use is limited due to the unacceptable toxicity inherent in this compound. Staurosporin became a kind of starting point in the synthesis of protein kinase inhibitors, and most of the further work was aimed at creating a close structural analogue of staurosporin, which has less toxic properties. Examples of such analogues are, in particular: sotrastaurin - international application WO-00238561, cephalon - international application WO-09721677, PKC inhibitors described in international application WO-09741127, PKC inhibitors described in Eurasian patent 000598, RF patent 2191175. Unfortunately, most of the potential drugs, which are PKC inhibitors, do not even pass the second phase of clinical trials (Nat Rev Rev Sapser. 2007 Jul; 7 (7): 554-62). Therefore, despite numerous studies, the creation of effective and low-toxic PKC inhibitors remains an urgent need.

Therefore, the main objective of the present invention is to expand the arsenal of drugs useful in the treatment of conditions in which protein kinase C plays a role in pathology, in particular, associated with inflammatory diseases, dermatological diseases, asthma, diabetes, ischemia, cardiovascular diseases.

This invention relates to protein kinase C inhibitors having a structure fundamentally different from that of staurosporin analogues. The compounds of this invention are, quite unexpectedly, effective inhibitors of PKC, as well as PKC alpha isozyme. The compounds of the present invention can therefore be used to treat a variety of diseases, including anti-inflammatory, anti-allergic, and anti-asthmatic diseases.

SUMMARY OF THE INVENTION The technical result of the present invention is the use of indole derivatives having the properties of protein kinase C inhibitors to create a medicament for treating conditions in which protein kinase C plays a role in pathology, in particular associated with inflammatory diseases, ischemia, asthma, cardiovascular diseases dermatological diseases, which will expand the arsenal of drugs for the treatment of these diseases.

The specified result is achieved by using indole derivatives to create a pharmaceutical composition, the active principle of which are the compounds of general formula (I): R

where: R ₁ represents a substituent selected from optionally substituted alkyl, hydroxyalkyl or cycloalkyl; optionally substituted heterocyclyl or optionally substituted aryl, with lower alkyl, halogen, hydroxyalkyl, hydroxy group, amino group or several of these substituents simultaneously being used as substituents.

R ₂ - H, -CH ₂ N (R'R ") ₂ , where R 'and R" independently represent an amino substituent selected from hydrogen or optionally substituted alkyl, R ₃ is an alkoxy group, or OH,

R ₄ - represents a substituent selected from hydrogen, - halogen, cyano group, NO ₂ group, trifluoromethyl, optionally substituted aryl or optionally substituted heterocyclyl;

A - S ₅ SO ₅ SO ₂ B - (CH ₂ ) _k —Rs _> where R ₅ is optionally substituted aryl, optionally substituted alkene, optionally substituted alkyl or hydroxyalkyl, optionally substituted heterocyclyl; n is 0-l; k = 0-2 ₅ or hydrates and pharmaceutically acceptable salts of a compound of formula I ₅ having C ₅ protein kinase inhibitor activity as well as a pharmaceutically acceptable carrier and / or excipient.

This pharmaceutical composition can be used for the preparation of drugs for both internal and topical application, for example, in the form of tablets, granules, capsules, solutions, solutions for injection, suspensions, ointments, gels.

Mentioned compounds corresponding to formula I, including their synthesis and physicochemical properties, are described in the literature (Journal of Pharmaceutical Chemistry, NaI, 1995, pp. 51-53, Pharmaceutical Chemistry Journal; N ° 2; 1995, p. 5-8, Journal of Pharmaceutical Chemistry, N ° 5, 1988, pp. 555-569; Biorg. Med. Chem., Na 14, 2006, pp. 911-917), as well as in PCTYRU 2007/000246. All final chemical compounds of the present invention are obtained by the methods described in the literature, or can be obtained by similar methods, while these methods are known to ordinary specialists in the field of organic chemistry.

Previous studies have shown that the compounds described by formula I can be useful in the treatment of viral diseases such as influenza A and B (RF patent 2008004), SARS (RF patent Ns2256451), rotavirus infection (RF patent 2266742), diabetes (patent RF N ° 2361581).

However, to date, it was not known that the compounds of formula I are active as protein kinase C inhibitors, have anti-inflammatory, anti-allergic and anti-asthmatic effects, which was first shown by the authors of the present invention.

The ability of the compounds of formula I to inhibit protein kinase C allows us to offer them new uses as components of drug compounds for treating diseases or conditions for which the effectiveness of the use of protein kinase C inhibitors has been confirmed.

In vitro experiments, we showed that compounds corresponding to formula I effectively inhibit protein kinase C, as well as its isozyme protein kinase C alpha (examples 1-3). The possibility of using the mentioned compounds for the treatment of diseases or conditions in which protein kinase C in one way or another affects the pathological process, we have confirmed on models of allergic and contact dermatitis, asthma and inflammation (examples 4-9). In addition, in our previous study (RF patent N ° 2361581), we confirmed the effectiveness of the compounds mentioned in this application on a model of diabetes mellitus. This knowledge, together with the inhibitory effect we discovered for the first time with respect to protein kinase C, allows us to take a fresh look at the mechanism of action of the studied compounds, based on which new applications of known indole derivatives can be proposed.

Most preferably, the following compounds are used as the active component of pharmaceutical compositions (chemical formulas and brief notations are given in Table 1 for ease of use in the further text of the description and claims):

Table 1

Formulas, names and designations of the compounds according to the present invention.

MC-I lO l-methyl-2-phenylcyl-methyl-3-carbetoxy-4-dimethyl l aminometh l-5 - oxy-6-bromindole

The proposed compounds inhibit PKC with an IC _{5O value} below 50 μm. In addition, the compounds of this invention selectively inhibit alpha isozyme of protein kinase C and have an IC ₅₀ value relative to this isozyme below 10 μm.

In accordance with this invention, pharmaceutical compositions containing, as an active substance, a compound of general formula I or a racemate, or an optical isomer thereof, or a pharmaceutically acceptable salt and / or hydrate thereof, are used for the manufacture of medicaments for the treatment of diseases or conditions associated with inflammatory diseases, ischemia, asthma, cardiovascular diseases, dermatological diseases.

Another aspect of the present invention is a method for treating diseases or conditions associated with inflammatory diseases, ischemia, asthma, cardiovascular diseases, dermatological diseases, which consists in administering to an animal or human or topical administration of drugs containing, as an active substance, a compound of formula I or its racemate, or its optical isomer, or its pharmaceutically acceptable salt and / or hydrate. Thus, drugs comprising the compound of formula I as an active ingredient can be administered orally, parenterally (e.g., intravenously, subcutaneously, intraperitoneally) or applied topically. A pharmaceutical formulation containing a compound of formula I ₅ may further include one or more pharmaceutically acceptable carriers, diluents, or excipients. These pharmaceutical formulations are prepared by known methods. Finished compositions for internal administration may be in the form of tablets, powders, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, injectable solutions.

The compositions of this invention can be formulated so as to provide quick or sustained release of the active ingredient after administration to the patient. The compositions are preferably prepared in unit dosage form, each dose containing from ~ 50 to -1000 mg, preferably 100 to 500 mg of the active ingredient.

In addition to the above formulations for internal use, the compounds of this invention can be administered topically. Formulations for topical administration are ointments, creams and gels.

The usual amount of topical preparation to be applied to the affected tissue will depend on the size of the affected tissue and the concentration of the compound in the preparation. As a rule, the finished form is applied to the affected tissue in an amount delivering from -0.05 to -100 mg of the compound per cm of the affected tissue. Preferably, the applied amount of the compound should be in the range from -1 to -10 mg / cm ² .

The dosage can be adjusted depending on the therapeutic efficacy and bioavailability of the active ingredients in the body, their metabolic rate and excretion from the body, as well as on the age, gender, presence of concomitant diseases, as well as the stage of the patient's underlying disease. The therapeutic dose administered should be determined by the physician depending on the condition to be treated, the choice of the compound administered and the chosen route of administration, therefore, the above dose ranges do not imply a limitation on the scope of the invention. The invention is illustrated by the following examples.

Example 1. Inhibition of protein kinase C (PKC).

Compounds were evaluated for their ability to inhibit protein kinase C according to known methods (Biotechnology, 1990, N ° 8, 732 - 735; Biochem J., 1993, 294: 335 - 337). To do this in reaction buffer (20mM Tris-HCl, ρH = 7,4; 10 mM MgCl _2, O.lmM CaCl _2; 8.7 mg / ml phosphatidyl serine, 5.9 mg / ml diacylglycerol) was added PKC test compound ( 10 μM) and histone NS (370 mg / ml) and incubated for 15 min at 25 ^° C. Then, [g-P] ATP (10 μM) was added and incubated for 15 min at 25 ^° C. The reaction mixture was applied to filters, washed with phosphoric acid (75 mM), transferred to scintillation vials and the inclusion of ³² P (pulse / min) was determined on a scintillation counter. For each reaction mixture, the percent inhibition was calculated. Compounds were analyzed in duplicate. The results are summarized in table N ° 2

Table N ° 2 Data on the inhibition of protein kinase C

Example 2. Determination of IC _5O for PCB.

For compounds that showed the strongest inhibitory properties, the standard parameter IC ₅₀ , the concentration of the substance at which 50% inhibition of the enzyme was achieved, was determined. The inhibition reaction was carried out under conditions similar to those in the JNbI example. For analysis, concentrations in the range from 0.03 to 100 μM were used. The obtained IC _5O values _are summarized in table 3.

Table 3

Example 3. Inhibition of protein kinase C alpha (PKCα).

In addition to the study of PKC, which is a mixture of isoenzymes, the ability of several compounds of this class to inhibit the activity of Ca protein kinase was analyzed by a known method (Biochem Bihorhus Res Comm., 1986, 135: 397-402). For this, PKCα, the test compound and histone (250 mg / ml) were added to the reaction buffer (20 mM HEPES; 8 mM MgCl ₂ , 0.08 mM CaCl ₂ ; 100 μg / ml diacylglycerol) and incubated for 15 min at 25 ⁰ C. Then [g- ³² P] ATP (10 μM) was added to the mixture and incubated for 10 min at 25 ⁰ C. The reaction mixture was applied to filters, washed with phosphoric acid (75 mM), transferred to scintillation vials, and inclusion ^{32 was} determined P (pulse / min) on a scintillation counter. The following data were obtained: Table 4

The above examples prove that the compounds of the present invention inhibit the activity of protein kinase C ₅ and protein kinase

Ca. Given the laws known from the prior art, it can be expected that such compounds will find use in the treatment of diseases associated with protein kinase C. To confirm this statement, we studied the behavior of the studied compounds on known models of inflammation, asthma, allergies and dermatitis.

Example 4. Inhibition of the activity of transcription factor NF-kV.

The transcription factor NF-kB plays a central role in the regulation of the activity of genes involved in the development of all inflammation processes, mainly due to increased transcription of inflammatory protein genes. Accordingly, suppressing the activity of NF-kV can reduce inflammation. This principle underlies the action of most modern inhibitors of the inflammatory process (IL-IO, glucocorticosteroids, non-steroidal anti-inflammatory drugs), which suppress the activity of NF-kV (for example, Molesular ipterveptiops, 2002, v.2, pp22-35; Theoretical medicine, 1 ( 4) 2007).

The experiment was carried out on Jurkat kBZ cells transformed with the β-galactosidase gene under the control of an enhancer that binds to NF-kB, according to the well-known method (Меsuremept оf ligapd-aspertiviсtеpеllесеpеllесепепепепепепепепепепепепепепепепепепепепепепепепепепепепепепепепепепепліпепріпеперпепепепепеперпепепепепепліпепрі 3972-3976). Compound MC-101 added to the culture medium in concentrations from 0.001 to 10 μM. NF-kV was activated by incubation with A23187 and PMA for 4 hours. The resulting IC _5O (concentration of compound MC-101 necessary for inhibiting 50% of the receptor) is 3.54 μM. This example confirms that the compound MC-101 has the ability to inhibit the NF-kV receptor (unknown from published sources), and thus the ability to block the inflammation reaction.

This ability allows the use of its compounds as medicines for various diseases associated with the development of inflammation, in particular asthma, chronic obstructive pulmonary disease, hepatitis, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neurodegenerative diseases, and dermatitis.

The anti-inflammatory effect of MC-101 was further confirmed in a mouse model.

Example 5. Anti-inflammatory effect on the model of inflammatory contact dermatitis.

Inflammatory dermatitis is the most common form of contact dermatitis. For example, the so-called unconditioned irritants can cause such dermatitis: friction, pressure, radiation and temperature effects, acids and alkalis, substances of some plants (nettle, cowberry, etc.). In other words, it is usually caused by irritating agents that cause dermatitis in all people with sufficient exposure.

The anti-inflammatory effect was evaluated in a mouse model of inflammatory contact dermatitis caused by phorbol ester. In the experiment, IRC mice weighing 22 ± 2 g were used. Arbidol in the amount of 3 mg / ear was applied as an ointment to the right ear of the mouse 30 minutes before and then 15 minutes after the application of phorbol ester (5 μg in 20 μl of ethanol). Arbidol before application was dissolved in a mixture of acetone: ethanol = l: l. Dexamethasone in the amount of OD mg / ear was used as a positive control. Placebo (acetone: ethanol = l: l), 20 μl / yxo, was used as a negative control. The control regimen was similar to Arbidol.

The results of suppression of inflammation were evaluated after 6 hours after application. The magnitude of the edema was determined by subtracting the thickness of the left ear (without applying an inflammatory agent) from the thickness of the right ear. The percentage of inhibition was calculated by the formula:

Inhibition = 100% * (T -T _ppav _lev) / T _lev; wherein T and T _lev _ppav corresponding to the thickness of the left and right ears in mm. In this model, the percentage of inhibition in excess of 30% is considered as confirmation of the anti-inflammatory effect. The data obtained are presented in Table 5.

Table 5

Confirmation that the analogs of MC-101 will also influence the inflammation processes that underlie a number of diseases, we also received on several mouse models. Example 6. Anti-asthma action

The studied compounds were tested on a model of asthma caused by sensitization by ovalbumin. Mice (BALB / s males, 5-6 weeks old, 10 animals in the group) were sensitized by intraperitoneal administration of an ovalbumin emulsion in aluminum hydroxide (0.2 ml / mouse) on days 1 and 14.

The emulsion was prepared as follows: 20 μl of 5% ovalbumin was dissolved in PBS buffer (pH = 7.4) to a volume of 2.5 ml. 5 ml Alum (a mixture of 40 mg / ml aluminum hydroxide and 40 mg / ml magnesium hydroxide) was added to the resulting solution. Thoroughly mixed and brought to a volume of 10 ml by adding PBS buffer (pH = 7.4).

On days 21, 23, and 25, mice were inhaled with 5% ovalbumin for 25 minutes in a special room.

The studied compounds and the comparison drug dexamethasone were administered to mice orally for six days (from 21 to 26 days) 1 hour before ovalbumin inhalation. On day 26, 1 hour after drug administration, an asthma attack was stimulated by inhalation of methacholine.

Dexamethasone is selected only as a traditional positive control for experiments of this kind. Being the most potent glucocorticosteroid, dexamethasone is rarely used for asthma therapy, as it has a lot of side effects.

To assess the effect of drugs, plethysmography was used. When plethysmography on special equipment, the pressure on the inhale and on the exhale, the temporary parameters of respiration are measured. The dimensionless complex index Rehp is derived from them.

The results are presented in tables 6 and 7.

JSGsb table

By reducing the Rehp index relative to placebo, the effectiveness of the drug is judged (table N ° 6). The effectiveness of therapy through the Rehp index was calculated by the formula:

PeЪxι (pzhzebo) - Rehiιζ preparation)

Bf f = * 100%

Pekp (matsebό) - P ehp (without sensitization)

Table Na7

The data in the table confirm that all the studied compounds had a positive effect on respiration parameters.

Example 7. Anti-inflammatory effect on an asthma model

Animals used in the experiment of Example 6 were sampled with bronchoalveolar fluid to determine inflammatory cytokines. The level of inflammatory cytokines in this experiment is the main indicator associated with the presence or suppression of inflammation. Bronchoalveolar fluid (BJ) was collected in animals 1 hour after taking the drugs on day 26. To obtain BZ, mice were euthanized with a propofol solution with a concentration of Humg / ml (50 μl per mouse). A catheter was inserted into the trachea, through which 0.25 ml of PBS with pH = 7.4 was twice introduced. One aliquot of the selected fluid (0.25 ml) was used to count the cells, the second (0.25 ml) was centrifuged, and the supernatant was selected to determine the number of cytokines (IL-5 and IL-13).

Anti-inflammatory effect was evaluated in 6 groups of animals. Data on the content of cytokines are shown in table 8.

Table 8

As can be seen from this table, the studied compounds significantly reduce the number of inflammatory cytokines, compared with placebo, thereby demonstrating a decrease in inflammation.

Example 8. Antiallergic effect on asthma models. Symptoms of bronchial asthma or rhinitis are often associated with ongoing contact with an allergen that causes allergic inflammation. The reaction of the human body to various allergens leads to increased production of antibodies (IgE immunoglobulins). The inflammatory process in this case is directly related to the cascade of reactions developing during the interaction of IgE antibodies and an allergen. Thus, an increased level of specific IgE antibodies in the blood indicates the presence of an allergic disease. To determine the level of IgE, blood samples of mice receiving test drugs were taken. Sampling was carried out an hour after the administration of the test compounds on the 26th day of the experiment, the details of which are set out in Example 6. Blood samples were centrifuged, after which the serum was taken to measure ovalbumin-specific IgE antibodies using a special test kit for the determination of IgE (OVA-resistance IgE ELISA Kit). The data obtained are collected in table 9. Table 9

From table 9 it is seen that almost all the studied compounds significantly reduced the level of antibodies in the blood compared with placebo, which indicates the potential use of these drugs as anti-allergic drugs, which, together with the presence of anti-asthma activity, opens up great prospects for the complex treatment of inflammatory diseases, associated with damage to the respiratory tract.

Example 9. Antiallergic effect on the model of allergic contact dermatitis

Allergic contact dermatitis, in contrast to irritant, does not develop immediately after contact with an irritant, and not during the first contact. In order for an allergic reaction (sensitization) to form, it takes from several hours to several weeks or even months from the first contact. This type of dermatitis can be caused by many substances that induce the reaction of individuals in physical contact. More specifically, this reaction causes a state of hypersensitivity in sensitive individuals.

The antiallergic effect was evaluated in a mouse model of allergic contact dermatitis caused by oxazolone. In the experiment, BALB / c mice weighing 23 ± 2 g were used. Seven days before the start of the experiment, the mice were sensitized by introducing 100 μl of a 1.5% solution of oxazolone in acetone into the abdominal cavity.

Arbidol in an amount of 1 mg / ear was applied as an ointment to the right ear of the mouse 30 minutes before and then 15 minutes after application of oxazolone (100 μl of a 1.5% solution of oxazolone in acetone). Indomethacin in an amount of 1 mg / ear was used as a positive control. Placebo (acetosetanol ^ l:!), 20 μl / yxo was used as a negative control.

The results of suppression of inflammation were evaluated 24 hours after application. The magnitude of the edema was determined by subtracting the thickness of the left ear (without applying an inflammatory agent) from the thickness of the right ear. The percentage of inhibition was calculated about the formula:

Inhibition = 100% * (T -T _ppav _lev) / T _lev; where T _a _T e and T _ppav corresponding to the thickness of the left and right ears in mm. In this model, a percentage of inhibition in excess of 30% is considered as confirmation of the antiallergic effect. The data obtained are presented in Table 10.

Table 10

The data obtained indicate that Arbidol applied to the site of damage has a good anti-allergic and anti-inflammatory effect, characterized by a decrease in the severity of clinical signs of contact allergic and contact inflammatory dermatitis.

Example 10. Preparation of a pharmaceutical composition for topical application.

5 g of l-methyl-2-phenylthiomethyl-3-carbetoxy-4-dimethylaminomethyl-5-hydroxy-6-bromindole monohydrate are thoroughly ground and dissolved in 10 g of dimethyl sulfoxide. The resulting solution was combined with stirring with 50 g of petroleum jelly. 15 g of lanolin is mixed with 20 g of water and, after obtaining a homogeneous mass, combined with a vaseline mixture. Get 5% ointment.

Example 11. The solution for local use.

100 g of a 0.25% solution contains, g: Arbidol 0.25 dimexide 5.0 polyethylene oxide - 400 20.0 purified water to 100 g.

Example 12. Liniment

100 g of 20% liniment contains, g: Arbidol 20.0

1,2-propylene glycol 60.0 purified water to 100 g. Example 13 Gel.

100 g of a 1.0% gel contains, g: Arbidol 1.0 Dimexide 5.0 gel polyethylene oxide 1500 to 100 g.

Example 14. Obtaining a pharmaceutical composition for the preparation of tablet mass.

A mixture is prepared consisting of 0.5 kg of compound MC-101; 2.26 kg of potato starch; 5.5 g of polyvinylpyrrolidone; 11.5 g of methyl cellulose; 7 g of calcium stearate; 0.10 g of stearic acid. The mass obtained after mixing is used for tabletting.

Example 15. Obtaining a pharmaceutical composition for the preparation of capsules containing 100 mg of active substance.

Thoroughly mix 100 g of compound MC-107; 28 g of potato starch; 59 g of microcrystalline cellulose; 2 g of silicon dioxide; 9 g of collidone 25; 2 g of calcium stearate. The prepared powder mixture is packaged in 200 mg in a suitable size gelatin capsule.

Example 16. The preparation of a pharmaceutical composition for injection. To 100 ml of the prepared solution of 0.9% sodium chloride, 1 g of compound MC-109 was added with stirring. The resulting solution was filtered. The finished product is ampouled and used for intramuscular and intravenous injection.

Example 17. Preparation of an aerosol. A chilled propellant is added to perfluorodecanoic acid (2 mg) in a glass aerosol ampoule, for example, trichlorormonofluoromethane, dichlorotetrafluoroethane, dichlorodifluoromethane, etc.) in an amount of 36 g. Then, finely ground powder of compound MC-I lO (100 mg) is added and the metering valve is screwed. The procedure is performed in a dry box.

Claims

CLAIM

1. The use of substituted indole of the general formula (I):

R

Where:

R ₁ represents substituted or unsubstituted alkyl, hydroxyalkyl, cycloalkyl, heterocyclyl or aryl, with lower alkyl, halogen, hydroxyalkyl, hydroxy, amino, or several of these substituents being used simultaneously as substituents.

R ₂ is H, —CH ₂ N (R′R ″) ₂ , where R ′ and R ″ independently of one another are a substituent selected from hydrogen or optionally substituted alkyl,

R ₃ is an alkoxy group, or OH,

R ₄ - represents a substituent selected from hydrogen, halogen, cyano, NO ₂ groups, trifluoromethyl, optionally substituted aryl or optionally substituted heterocyclyl;

A - S, SO ₅ SO ₂

B - (CH ₂ ) _k —R ₅ , ^g D ^e R-5 — optionally substituted aryl, optionally substituted alkene, optionally substituted alkyl or hydroxyalkyl, optionally substituted heterocyclyl; n is 0-l; k = 0-2, or its hydrates and / or pharmaceutically acceptable salts, for the preparation of a medicament having the properties of a protein kinase C inhibitor, anti-inflammatory, anti-asthma and anti-allergic activity.

5 2. The use according to claim 1, characterized in that l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxo-6-bromo-monohydrochloride hydrochloride is used as a substituted indole with anti-inflammatory and anti-asthmatic activity quantity.

YU

3. The use according to claim 1, characterized in that as a substituted indole for the preparation of a medicinal product intended for the treatment of a disease selected from the group: inflammatory disease, dermatological disease, cardiovascular disease, ischemia, asthma, l-methyl-2- is used phenylthiomethyl-3-carbetoxy-4-

15 dimethylaminomethyl-5-hydroxy-6-bromindole or its pharmaceutically acceptable salts and / or hydrates.

4. The use according to claim 1, characterized in that substituted indole is used to prepare a drug that inhibits the activity of the transcription factor NF-kB.

20 5. The use according to claim 4, characterized in that substituted indole is used for the preparation of a medicament for treating diseases based on the inflammatory process, including asthma, hepatitis, rheumatoid arthritis, inflammatory bowel disease, glomerulonephritis, neurodegenerative diseases.

25 6. The use according to any one of claims 1 to 5, characterized in that the drug is in the form of tablets, capsules, injections, aerosol or composition for topical use.

7. The use of a pharmaceutical composition containing, as an active agent, a substituted indole of general formula I

Where:

R ₂ is H, —CH ₂ N (R′R ″) ₂ , where R ¹ and R ″ independently of one another are a substituent selected from hydrogen or optionally substituted alkyl,

R3 is an alkoxy group, or OH,

R ₄ - represents a substituent selected from hydrogen, halogen, cyano group, NO group, trifluoromethyl, optionally substituted aryl or optionally substituted heterocyclyl;

A - S, SO ₅ SO ₂

B - (CH ₂ ) _I cR _5J where R ₅ is optionally substituted aryl, optionally substituted alkene, optionally substituted alkyl or hydroxyalkyl, optionally substituted heterocyclyl; n is 0-l; k = 0-2, or a pharmaceutically acceptable salt and / or hydrate thereof, in an effective amount and a pharmaceutically acceptable carrier for the treatment of inflammatory diseases or asthma.

8. The pharmaceutical composition according to claim 7, characterized in that l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl ~ 5-hydroxy-6-bromindole hydrochloride is used as the active agent.

9. The pharmaceutical composition according to paragraph 7 or 8, characterized in that it is in the form of a tablet, capsule, injection, or aerosol.

10. The pharmaceutical composition according to paragraph 7 or 8, characterized in that it is made in the form of a dosage form that provides effective delivery of the active component, including for enteral, parenteral or transdermal administration.

11. The use according to claim 1, characterized in that for the manufacture of a medicament intended for the treatment of inflammatory and allergic skin diseases, l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxo-6-bromindole hydrochloride is used monohydrate or its pharmaceutically acceptable salts and / or hydrates.

12. The use according to claim 11, characterized in that the inflammatory skin disease is dermatitis.

13. A topical pharmaceutical composition for the treatment of inflammatory skin diseases, containing, as the active ingredient, l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromindole or a pharmaceutically acceptable salt and / or hydrate thereof in an effective amount and pharmaceutically acceptable carrier.

14. The topical pharmaceutical composition according to claim 13, characterized in that it is intended for the treatment of contact dermatitis.

15. The topical pharmaceutical composition according to p. 13, characterized in that it contains the following quantitative ratio of components (mass%): l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxo-6-bromindol or its pharmaceutically acceptable salt and / or hydrate 0.2-30.0% pharmaceutically acceptable carrier

16. The use according to claim 1, characterized in that as a substituted indole for the preparation of a medicament having anti-inflammatory and anti-asthma activity, a compound selected from the group: l-methyl-2-β-naphthylthiomethyl-3-carbetoxy-4- is used dimethylaminomethyl-5-oxi-6-bromo indole hydrochloride; l-methyl-2-phenylcylmethyl-3-carbetoxy-4-dimethylaminomethyl-5-oxo

6-bromoindole; l-methyl-2-phenylcyclo-methyl-3-carboxyloxy-4-dimethylaminomethyl-5-hydroxy-6-bromindole; l-methyl-2-phenylcylmethyl-3-carbetoxy-5-acetoxy-6-bromindole; l-methyl-2-phenylcycloxymethyl-3-carbethoxy-5-oxo-6-bromindole.

17. The use according to claim 1, characterized in that substituted indole is used for the preparation of a medicament intended for the treatment of a disease selected from the group: inflammatory disease, dermatological disease, cardiovascular disease, ischemia, asthma.

18. The use according to claim 1, characterized in that the substituted indole l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxi-6-bromoindole or its pharmaceutically acceptable salts and / or hydrates are used to prepare a medicine possessing anti-inflammatory activity.

19. The use according to claim 1, characterized in that the substituted indole l-methyl-2-phenylthiomethyl-3-carbetoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole or its pharmaceutically acceptable salts and / or hydrates are used for preparation of a drug with anti-asthma activity.

20. The use according to claim 1, characterized in that the substituted indole l-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxo-6-bromoindole or its pharmaceutically acceptable salts and / or hydrates are used for the preparation of a medicament possessing antiallergic activity.

21. A method of obtaining a medicinal product for the treatment of inflammatory, allergic diseases, diseases caused by protein kinase C dysfunction and asthma by mixing substituted indole with the general formula (I):

R

where: R ₁ represents substituted or unsubstituted alkyl, hydroxyalkyl, cycloalkyl, heterocyclyl or aryl, with lower alkyl, halogen, hydroxyalkyl, hydroxy-group, amino group or several of the listed substituents simultaneously being used as substituents.

R ₃ is an alkoxy group, or OH, R ₄ - represents a substituent selected from hydrogen, halogen, cyano group, NO group, trifluoromethyl, optionally substituted aryl or optionally substituted heterocyclyl; A - S ₅ SO, SO ₂ B - (CH ₂ ) _Ic -R ₅ , where R ₅ is optionally substituted aryl, optionally substituted alkene, optionally substituted alkyl or hydroxyalkyl, optionally substituted heterocyclyl; n is 0-l; k = 0-2, or its hydrates and / or pharmaceutically acceptable salts with a pharmaceutically effective carrier and / or excipient and / or the formation of a dosage form suitable for administration to a patient.