Anti-inflammatory composition

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented 3-(2',2'-dimethylpropanoylamino)-tetrahydropyridin-2-one , (S)-3-(2',2'-dimethylpropanoylamino)-tetrahydropyridin-2-one and pharmaceutical compositions prepared with using said compound or its isomer, as well as the use thereof for preparing a therapeutic agent for preventing or treating inflammatory conditions, and a related method of treating. What is shown is the advantage of the compounds as related to the other broad spectrum chemokine inhibitors (BSCI) on anti-inflammatory activity; it possesses the pharmacokinetic, toxicological properties and the pharmaceutical safety parameters: (S)-3-(2',2'-dimethylpropanoylamino)-tetra-hydropyridin-2-one is 5-25 times more active than (R)-isomer.

EFFECT: higher anti-inflammatory activity.

13 cl, 10 dwg, 9 tbl

 

The present invention describes 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-he and pharmaceutical compositions prepared using the specified connection, and the use of the compounds for the preparation of a medicinal product, aimed at the prevention or treatment of inflammatory conditions.

Inflammation is an important component of the physiological immune protection of an organism. However, there are many studies showing that irregular spatio-temporal regulation of the inflammatory response plays a role in a wide range of diseases, including those that explicitly involved leukocytes (such as autoimmune diseases, asthma or atherosclerosis), as well as in diseases that are not traditionally associated with the activities of leukocytes (such as osteoporosis or Alzheimer's disease).

Chemokines are a large family of signaling molecules homologous to interleukin-8, which is involved in the regulation of the movement of leukocytes both in physiological and pathological conditions. More than fifty ligands and twenty-receptors involved in signaling system involving chemokines that promote transmission of information sufficient to direct leukocytes through a complex immunonephelometric processes from the bone marrow to the periphery, and then back through the secondary lymphoid organs. However, the complexity of this system chemokines in the first time hindered the development of pharmacological approaches to the modulation of inflammatory responses through the blockade of receptors chemokines. Was difficult to determine which receptors chemokines must be Engibarov(s) in order to induce a favorable therapeutic effect in specific inflammatory disturbance.

It was recently described family of agents, which also block the transmission signal a wide range of chemokines (Reckless et al., 1999 // Biochem J. 340, 803-811). The first such agent is a peptide, called "peptide 3", for which it is shown that it inhibits leukocyte migration induced by five different chemokines, whereas migration, which occurs in response to other chemoattractant (such as fMLP or TGF-beta), does not change. Join this group, including the peptide and its analogues, such as NR58-3.14.3 (i.e. SEQ ID NO No. 1, c(DCys-DGl-DIle-DTrp-DLys-DGIn-DLys-DPro-DAsp-DLeu-DCys)-NH2), were called "Inhibitors of chemokines broad-spectrum" (Broad Spectrum Chemokine Inhibitors, BSCl). Grainger et al. (2003, Biochem. Pharm. 65, 1027-1034) further showed in a number of disease models in animals that BSCl have potentially beneficial anti-inflammatory activity. Interesting is the fact that at the same time, the second siege of many chemokines has no obvious connection with the development of acute or chronic toxicity, and this suggests that this approach may be a useful strategy for the development of new anti-inflammatory drugs, which will have the advantages of steroid drugs, but have less side effects.

However, peptides and derivatives of the peptides, such as NR58-3.14.3, may not be optimal in vivo. The synthesis of these substances is quite expensive, and they have relatively unfavorable pharmacokinetic and pharmacodynamic properties. For example, the connection NR58-3.14.3 has no bioavailability for oral use, and the elimination half-life from plasma after intravenous injection is less than 30 minutes.

Adopted two parallel strategies to identify new drugs that retain the anti-inflammatory properties of peptide 3 and NR58-3.14.3, but have improved characteristics for use as pharmaceuticals. First, it was developed a number of peptides, some of which are characterized by a longer half-life from plasma than NR58-3.14.3, and the synthesis of which is considerably cheaper. Secondly, the analysis was performed on the structure/activity of these peptides in order to determine pharmacophore and offer low-molecular compound nabatid is second nature, which retain the useful properties of the original peptide.

The second approach was received several series with different structures from different substances that retain anti-inflammatory properties of these peptides, including a 16-amino - and 16-aminoalkyl-derived alkaloid yohimbine, as well as a series of N-substituted 3-aminoglutethimide (see: Fox et al., (2002) / J. Med. Chem. 45, 360-370; WO 99/12968 and WO 00/42071). All these substances are inhibitors of chemokines wide range of activities that keep the selectivity compared to non-chemokines by chemoattractants, and it is shown that some of them inhibit acute inflammation in vivo.

The most powerful and selective action of these substances has a S-3-(undec-10-enoyl)-aminoglutaramic (NR58.4), which inhibits induced chemokines migration in vitro with ED50 equal to 5 nm. However, further research it was shown that could happen enzymatic cleavage aminoglutethimide ring in the serum. Therefore, in some cases (for example, if the inflammation that is subjected to treatment that is chronic, as in the case of autoimmune diseases) properties of these compounds may not be optimal, and a more stable substance with similar anti-inflammatory properties may be more efficient.

As the approach is to identify such stable analogues have investigated the stability in the serum of various derivatives of S-3-(undec-10-enoyl)-aminoglutethimide. One such derivative, 6-desoxo-analogue of S-3-(undec-10-enoyl)-tetrahydropyridine-2-it is completely stable in the serum of a person within a period of not less than 7 days at 37°C, but is characterized by a significantly lower activity compared to the parent molecule.

One of these families of stable inhibitors of chemokines broad-spectrum are 3-amino-caprolactam with semiclean monobactams ring (see, for example, WO 2005/053702 and WO 2006/016152). However further useful anti-inflammatory agents were also received from other 3-aminoethanol with different size rings (see, for example, WO 2006/134385). When making other modifications lactam ring, including the introduction of heteroatoms and design of systems with two lactam rings were also obtained compounds having the activity of inhibitors of chemokines wide spectrum of action (see, for example, WO 2006/018609 and WO 2006/085096).

To date, identification of broad classes of active substances having the activity of inhibitors of chemokines wide spectrum of action and, therefore, anti-inflammatory properties in vivo, was based on optimizing the activity of inhibitors of chemokines wide spectrum of action. For example, according to the results of previous observations have shown that double substitution at position 2 (on alpha or Central the th carbon atom in the acyl side-chain acyl-3-aminoethanol) leads to a significant increase in activity as BSCl as in vitro, and in vivo in models of acute inflammation, when disubstituted in position 2 acyl group is an open chain (see WO 2005/053702), one cycle (see WO 2006/134384) or polycyclic system (see WO 2006/016152).

However, the power of the desired pharmacological action is important but only one of the factors that determine whether a substance useful pharmaceutical preparation for human use. In particular, the pharmacokinetic parameters (or the distribution of the active substance in the body) are the most important in determining the usefulness of individual active substance. Pharmacokinetics, defined in its broadest sense, as the study of the action of the body on the drug substance (in contrast, pharmacodynamics studies the effects of medicinal substances on the body depends on a complex physiological processes in the body, including without limitation: absorption, stability in plasma, volume of distribution (and, especially, the speed of establishment of the equilibrium concentration in target tissues), metabolic transformation (including the metabolic processes in the liver, such as oxidation under the action of cytochrome P450 and metabolic reactions phase II, such as sulfation and glukuronirovanie, as well as the metabolic processes taking place outside the liver, the same is as modifications under the action of enzymes serum) and excretion (such as renal clearance and excretion of faeces). These processes are often jointly referred to as "ADME" properties of the active substance (ADME: Absorption, Distribution, Metabolism and Excretion - the absorption, distribution, metabolism and excretion").

Another important factor in determining the usefulness of the active substance as a drug when used for a person is his security. Many, if not all used substances have many effects on the human body, among which are favorable pharmacological action is usually only one of the effects. Other actions can cause damage (toxic effect) or inconvenience (side effects) for the patient. The study of properties of potential ingredients of pharmaceuticals called toxicology or pharmacology safety studies. Undesirable effects can generally be attributed to two types. The effects of Class are closely linked with the desired pharmacological effect and (to a greater or lesser extent) are the inevitable consequence of impacts on selected molecular target. For example, substances intended for preventing pathological inflammation, can, to some extent, to suppress the immune system and lead to an increased risk of developing infections. This is because damage to the fabric of SLE is due to inflammation and infection depend on the extent of the activity of the immune system. As a result, all molecules which are directed to the same pharmacological target will be to a greater or lesser extent, have similar effects class. On the contrary, the Specific effects associated with the special structure of the compound, and is caused by the interaction (usually unexpected) with a target other than the intended pharmacological targets. In principle, it is possible to find another molecule, which has the same intended pharmacological effect, but completely devoid of specific side effects. Some specific compounds are common (such as interaction with hERG potassium channels in the heart, which can lead to dangerous prolongation of QT interval during pacing, which leads to potentially fatal cardiac arrhythmia), while other specific activity can, apparently, be unique for each substance.

Importantly, despite decades of accumulated experience in the development of pharmaceutical agents, still there is no conventional way to predict how pharmacokinetic and Toxicological properties of the agent or its pharmacological safety. For this reason, studies using model systems in vitro (such as cell lines, hERG), then animal testing and, finally, conducting phase I clinical trials on humans are common worldwide regulatory requirement when developing a new pharmaceutical product.

Were described methods for predicting certain pharmacokinetic properties on the basis of studying the structure of molecules and obviously qualified specialists in the field of medical chemistry can properly exclude many connections, guided only by theoretical considerations. An example of such "rules of thumb" is proposed by Lipinski "rule of five"is based on the observation that the majority of approved pharmaceutical drugs meet certain criteria, which include molecular weight, number of links, around which it can rotate, and polarity. Similarly, it is well known that molecules with large hydrophobic groups are more likely to have undesirable effects on hERG potassium channels.

Such General requirements, even in case of simultaneous use, can be useful to exclude unsuitable molecules, but a large number of very inappropriate (for various reasons) molecules, however, cannot be eliminated. Currently, no one would likely choose the medicinal substance from CL the SSA active compounds only on the basis of theoretical considerations. Therefore, the selection of certain compounds from the class of substances, which are characterized by favorable ADME, pharmacokinetic and Toxicological properties, as well as the pharmacological properties of security requires extensive hands-on experimental work among the promising compounds that provide new information that cannot be predicted even by specialists in this field.

In this application, the authors of the present invention describe a new connection - 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-he (I)that has not previously been described:

This compound belongs to a wide class of inhibitors of chemokines broad-spectrum (BSCl), which have been described previously (see, for example, WO 2006/134385). However, the authors of the present invention in experimental comparison with other substances belonging to the same class of compounds, showed that while all the molecules belonging to the specified class, have BSCl activity, the compounds of formula (I) have significant advantages when used as a pharmaceutical for humans by combining their ADME, pharmacokinetic and Toxicological properties and parameters of pharmaceutical safety.

The carbon atom in position 3 of the lactam is th ring is asymmetric, and, therefore, the data connection in accordance with the present invention can exist in two different forms, which are "R" and "S" configurations. This invention covers both enantiomeric forms and all combinations of these forms, including racemic RS mixture. For simplicity, when in the structural formula does not reflect any specific configuration, it should be understood that presents these two different enantiomeric forms and mixtures thereof. Because of the structural differences between the enantiomers have no effect on the key ADME properties that define the advantages of the compounds according to the present invention (and, in addition, have only a minor effect on the activity of the compounds as BSCl), both enantiomeric forms and mixtures thereof are examples that offer significant advantages in relation to other representatives of this class of compounds.

Preferably the connection according to the present invention described by formula (I)is a compound of formula (I')

This compound (I')having the S-configuration of the stereocenter is 5-25 times more active as an inhibitor of chemokines broad-spectrum, R-enantiomer.

The present invention also provides pharmaceutical compositions in which the active substance contains a compound described by the General formula (I) or (I'), or a pharmaceutically acceptable salt of this compound and at least one pharmaceutically acceptable excipient and/or carrier.

Under a pharmaceutically acceptable salt see, in particular, the salts obtained by the accession of inorganic acids, such as hydrochloride, hydrobromide, codgitate, sulfates, phosphates, diphosphate and nitrates, or organic acids such as acetates, maleate, fumarate, tartarate, succinate, citrates, lactates, methansulfonate, R-toluensulfonate, palmoate and stearates. Also in the scope of the present invention, when they can be used are salts formed with bases such as sodium hydroxide or potassium hydroxide. Other examples of pharmaceutically acceptable salts are described in the paper: "Salt selection for basic drugs", Int. J. Pharm. (1986), 33: 201-217.

The pharmaceutical composition may be presented in solid dosage form, for example in the form of powders, granules, tablets, gelatin capsules, liposomes or suppositories. Suitable particulate fillers can be used, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, who ellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and wax. Other pharmaceutically acceptable excipients and/or fillers known to specialists in this field.

Data pharmaceutical compositions in accordance with this invention can also be presented in liquid form, for example in the form of solutions, emulsions, suspensions or syrups. Suitable liquid fillers can be water, organic solvents such as glycerol or glycols, and mixtures thereof with water, cooked in different proportions.

The invention also encompasses the use of compounds of formula (I) or (I') or pharmaceutically acceptable salts of the compounds for the preparation of medicines intended for the treatment of inflammatory conditions.

The present invention comprises compounds, compositions and their use, as defined herein, in the case where the specified connection is hydrated or solvated form.

The advantage of the present invention compared to prior art based on the fact that it was unexpectedly found that 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-he has better ADME properties compared with the total lactam classes BSCl, which have been described previously (see, for example, between narodnye applications listed above). Although it was known that such compounds possess pharmaceutically acceptable pharmacodynamic properties (which means that they possess strong anti-inflammatory effect in vivo as a result of their BSCl activity), and an assumption was made that one must have acceptable pharmacokinetic and therefore ADME-properties, the direct evaluation of ADME properties unexpectedly showed that 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it has significant advantages (see examples below).

In particular, although, as follows from the literature data (see, for example, Fox et al. J. Med. Chem. 2005 48:867-74), previous studies of stability in serum in vitro indicated that the lactam BSCl were significantly better than the previously described kidnie BSCl (see, for example, WO 99/12968), it is now clear that many (or possibly all) representatives of the class lactam BSCl are undesirable effect of metabolic reactions in vivo. The authors of the present invention synthesized and tested more than twelve BSCl belonging to aminoethanol class, and showed that all tested to date lactam BSCl, except 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it, undergo rapid metabolic transformation in the liver (hydroxylation in action and cytochrome P450, and/or reaction phase II metabolism).

At least partially due to reduced metabolism in vivo, the total clearance of 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it is significantly lower than the total clearance of other tested lactam BSCl. As a result, the impact of 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-she after administration of single oral doses more than 10 times higher. Thus, 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it is more suitable for use as a pharmaceutical product for use in humans than most (if not all) of the previously described lactam BSCl, especially if to achieve effective action requires continuous oral administration.

Peptides known from the prior art (such as NR58-3.14.3), are characterized by the following shortcomings: (a) the high cost and the need for solid-phase synthesis (at least for longer molecules), (b) rapid excretion through the kidneys, and (C) in General, significantly lower activity (more than 25 times less powerful effect in vitro and more than 10,000 times in vivo).

Known from the prior art, aminoglutethimide are cheaper, not excreted rapidly via the kidneys and have a stronger effect in vitro, BUT they are extremely unstable in serum (as d is a query result opening amidnogo ring under the action of enzymes; see, e.g., Fox et al. J. Med. Chem. 2005 48:867-74). Therefore, the action aminoglutethimide BSCl, such as S-3-(undec-10-anolamine) glutarimide, at least 250 times less powerful in vivo than the effect of 2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it, even when used in models of acute inflammation (such as endotoxicosis, induced by lipopolysaccharides with typical systemic production of TNF-α, where the influence of stability of connection and ADME properties less pronounced).

Other structurally related (but functionally very different) compounds, which are described in the literature, represent bacterial autoinducer, and, as a rule, the basis of their structure is a 6-membered homoserine, usually with 3-oxazoline side chain (see, for example, the publication Bycroft et al. US 5,969,158, which stated a wide range of such substances). Despite the fact that these publications disclose the General equation, which cover both lactams and lactones, only a small number of examples described compounds (if any such there are), having the properties of autoinduction bacteria have lactam end groups. It is known that all these compounds (especially those that have an end of the lactone ring and/or 3-oxoacyl group) relatively unstable, which limits the possibility of their application as a medical prep the preparations.

Advantages of 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it is described in this document, are that the synthesis of this compound is cheap (and described in this description of the method allows the direct synthesis even in quantities measured by weight), and that this connection is characterized by a high metabolic stability not only in an isolated serum in vitro (a property that applies to the entire class described earlier lactam BSCl), but also in vivo. Based on the foregoing, the compounds according to the present invention (as compared with the compounds, are widely studied to date), efficiency, strength and pharmaceutical properties, such as ADME, pharmacokinetic, Toxicological properties and parameters of pharmaceutical safety that are uniquely optimized for the treatment of inflammatory diseases in humans.

In accordance with the present invention inflammatory disorders that can be prevented or treated using the compounds described by formulas (I) or (I'), or pharmaceutically acceptable salts of these compounds, or pharmaceutical compositions containing these compounds as active ingredients include the following diseases:

- autoimmunization, for example, such as multiple sclerosis, rheumatoid arthritis, common lupus, irritable bowel syndrome, Crohn's disease;

- vascular disease, including cerebrovascular accident (stroke), coronary heart disease, myocardial infarction, unstable angina, atherosclerosis or inflammation of the blood vessels, such as the syndrome behceta, giant cell arteritis diagnostics, rheumatic rheumatica, Wegener's granulomatosis, syndrome Cerca-Strauss, purple's disease-Selana and Kawasaki disease;

- a viral infection or viral replication, for example infections caused by viruses or multiplication of viruses, including poxvirus, herpes virus (e.g., Herpesvirus samiri), cytomegalovirus (CMV), hepatitis or lentiviruses (including HIV);

- asthma and related disorders of respiratory system, such as allergic rhinitis and chronic nonspecific lung disease;

- osteoporosis (low density bone mineral substances);

- growth of the tumor;

- rejection of transplanted organs and/or delay in the functioning of the graft or organ, for example, in patients undergoing kidney transplantation;

- disorders characterized by increased levels of TNF-α;

- psoriasis;

- damage to the skin and other fibrotic disorders, including hypertrophic ru is s (keloid formation), the formation of adhesions after General or gynaecological surgery, lung fibrosis, liver fibrosis (including alcoholic liver disease) or fibrosis of the kidney, unclear etiology or developed as a consequence of the underlying disease, such as diabetes (diabetic nephropathy);

- disorders caused by intracellular parasites such as malaria and tuberculosis;

- neuropathic pain (such as postoperative phantom limb pains, post herpetic neuralgia, and others);

allergic reactions or

- Alzheimer's disease.

In accordance with the present invention to other inflammatory disorders include:

- amyotrophic lateral sclerosis;

a reaction caused by the presence of antigens; suppression of the immune response.

These clinical indications in the broadest sense refers to inflammatory disorders or disorders characterized by increased levels of TNF-α.

In order to avoid wrong interpretation, it should be noted that the primary target of action BSCl, including compounds described in this application is the immune system. Therefore, the claimed beneficial effect on diseases such as viral infection and/or viral replication and tumor growth (status, which in themselves are not a primary disease of the immune si themes) is a consequence of the modulation of the immune system against infection and/or replication of the virus or the growth and spread of tumors. Because BSCl, including compounds according to the present invention, in General, directly not affect virus replication or tumor growth, we should expect that they will not have any effect in an isolated system (for example, infected in vitro cell line or on the line of proliferating tumor cells)that lack a holistic and immune system. Therefore, the known data relating to the effect of any of the compounds in such isolated systems cannot provide information for the development of BSCl, affect the immune system.

In that case, if permitted by law, the present invention can also be used for treatment, improvement or prevention of the symptoms of inflammatory diseases (including adverse inflammatory reaction, which arose in response to any agent) by introducing a patient compounds, compositions or medicaments according to the present invention in an amount to provide an anti-inflammatory effect.

Introduction of medication in accordance with the present invention it is possible to perform local, oral, parenteral routes through ADAP is remisen injections and other methods.

Enter the dose estimated for the medicinal product in accordance with the present invention is from 0.1 mg to 10 g, depending on the composition of the drug and method of administration.

In accordance with the present invention compounds of General formula (I) or (I') can be synthesized using the process described below.

Obtaining compounds of General formula (I) or (I')

All compounds of General formula (I) or (I') can be easily obtained by using General methods known to experts in this field.

The compound (I) is a colorless crystalline substance, which can be derived from ornithine and 2,2-dimethylpropanamide. For the synthesis of compound (I') use pure enantiomer S-ornithine. It is possible to carry out the ring closure effects on ornithine or its methyl ester. This amino acid can be etherification in dry methyl alcohol in the in situ formation of hydrochloric acid using trimethylsilylpropyne. Alternatively, it is possible to carry out the ring closure of the isolated ester using triethylamine. The crude product can then be allievate after an exchange of solvent.

Acyl-lactam product (I) is characterized by a significant solubility in water, and consequently the conditions of acylation, ispolzuemye more hydrophobic related products (see, for example, WO 2006/134385), were unsatisfactory. The use of three equivalents of sodium carbonate as base resulted in significant precipitation of by-product sodium bicarbonate, despite the fact that used large amounts of water (>4 ml/mmol of ornithine). Using these concentrations, the extraction product dichloromethane is inefficient. Therefore, three equivalents of sodium carbonate was replaced with 2.5 equivalents of potassium hydroxide (which will neutralize 2.5 equivalents of triethylamine hydrochloride, obtained in stage ring closure). Using this Foundation, you can use significantly less water (less than 1 ml/mmol of ornithine) (final pH is from 8 to 9). When extraction of the aqueous layer with ethyl ether acetic acid (3×2 ml/mmol of ornithine) and repeated crystallization from ethyl acetate (0.5 ml/mmol, hot) and 40-60 petroleum ether (5 ml/mmol) primary product yield was 43% (of 4.25 g of 50 mmol of ornithine).

It should be noted that if, during the selection of the product of the reaction the pH of the aqueous layer is too low, then a layer of ethyl ester of acetic acid extracted a small amount of triethylamine hydrochloride. When washing this solution ethyl acetate water is the extraction of significant amounts of lactam product is (I) with an amine hydrochloride. This can be avoided by increasing the pH of the aqueous layer 12 (for example, the addition of approximately one equivalent of potassium hydroxide with respect to the acid chloride) to conduct extraction with ethyl ether, acetic acid, then with lactam product (I) is extracted only free base of triethylamine, which will be easier to remove by evaporation or in the process of re-crystallization.

Below are the next preferred way of synthesis:

S-ornithine monohydrochloride (50 mmol) are suspended in 100% methyl alcohol (100 ml) by the addition of trimethylsilyl chloride (75 mmol). The reaction mixture is heated in a flask under reflux for 24 hours. Then add triethylamine (150 mmol) and the resulting mixture is heated in a flask under reflux for 48 hours. Methyl alcohol is then removed under reduced pressure (optionally in the later stages add toluene to facilitate the removal of alcohol, the precipitate is dissolved in water (20 ml) with addition of potassium hydroxide (125 mmol).

The resulting mixture was cooled to 0°C., then slowly add 2,2-dimethylpropionic (50 mmol) and the reaction mixture stirred for 18 hours while warming to room temperature. Then add solid potassium hydroxide (50 mmol) and the donkey, as it dissolves, the reaction mixture is extracted with ethyl ether acetic acid (3×100 ml). The combined organic layers quickly dried over a mixture of K2CO3and Na2SO4under reduced pressure. The obtained solid residue is then subjected to re-crystallization with ethyl ether acetic acid (25 ml) / 40-60 petroleum ether (200-250 ml)to obtain the lactam (I') in the form of a crystalline solid (yield is more than 50%).

The identity and purity (>95%) of this product was then confirmed by way of the proton NMR (δ [n] (400 MHz, CDCl3) 6,63 (1H, br s, NH); 6,01 (1H, br s, NH); 4,20 (1H, dt, J 11, 5.5, CHNH); 3,40-and 3.31 (2H, m, CH2NH); 2,61 (1H, dq, J 13, 4.5, CH2); 1,97-of 1.88 (2H, m, CH2); 1,50 (1H, dddd, J 13, 12, 9.5, 7.5, CH2); to 1.22 (9H, s, 3×CH3).

DEFINITION

The term "approximately" refers to the period of around a given value. As used in the Appendix to this description, "about X" means the interval from the value of X minus 10% of the value of X to X plus 10% of the value of X, and preferably the interval from X minus 5% of the value of X to X plus 5% of value of X.

Using numeric values in this description are intended to clearly include in this volume describe all individual values in a given range and all combinations of upper and lower limit values in the gra who izah this interval. Accordingly, for example, range from 0.1 mg to 10 g, defined in relation to (inter alia) dose 3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-it is intended to apply, introduced in order to include all doses between 0.1 mg and 10 g, and smaller and smaller intervals, each defined by a combination of upper and lower limit values, regardless of the examples explicitly or not.

In this document, the term "includes/contains" should be understood as "comprising" and "consisting of". Therefore, if the invention relates to a pharmaceutical composition which includes/containing the active ingredient", this terminology is intended to describe how the compounds, which can be other active ingredients and compositions, which consist of only one active ingredient, as defined.

If not defined otherwise, all technical and scientific terms used in the compilation of this document have the same meaning as commonly understood by ordinary specialist in the field to which the invention relates. All publications, patents, all patents and other references mentioned in the text, are included in this description by reference (in those cases when it is permitted by law).

The following examples are provided to ensure that the s to illustrate the above procedure and does not in any way limit the scope of this invention.

FIGURES

Figure 1 (parts a-E) shows the graphs of the dependence of concentration on time for five of the studied compounds (I') to (V) after oral administration to rats with a single dose of 3 mg/kg in 1% solution of carboxymethyl cellulose. Three lines for each connection correspond to the three repetitions of the experiment (three experimental animals). On the Y-axis presents the concentration in units of ng/ml (0-3000); on the X-axis represents time in minutes (0-480).

Figure 2 shows the main metabolites identified using the method of liquid chromatography/tandem mass spectrometry (when scanning in full range) in the urine, which was collected after 24 hours, rats received oral introduction of a single dose of 3 mg/kg in 1% solution of carboxymethylcellulose each of the five investigated compounds (I') to (V). It is noteworthy that the main metabolite of the compound (IV) was identified, but its structure has not been determined by comparison with the patterns of fragmentation/rearrangement of the public databases by the metabolites. Although the concentration of individual metabolites were not determined, the order of their quantitative content in the urine in the left side of each row shows the types of metabolites that are present in the greatest quantity.

Figure 3 (parts A-F) presents graphs of the current from the time the new cell, expressing the hERG gene product when exposed to media or one of the five compounds (I') to (V), each in a separate experiment. In each experiment, additional cells were exposed connections, taken as positive control, which completely blocks the current conducted by the channel hERG. On the Y-axis presents the current; on the X-axis represents time in seconds. In terms of figure G shows hERG trace current (area under the curve of current versus time in the parts of figure a-F) by repeating the experience in the cells exposed to each compound or 0.1% DMSO as a carrier, or connection, is taken as the positive control, E-4031 (+ media). In part G of the figure on the Y-axis of the histogram presents the percentage inhibition trace hERG current relative to cells not exposed to.

Figure 4 shows a typical curve of binding of the compound (I'). In this experiment, in each reaction was added to 10 nm labeled compounds BN83250 (lactam BSCl, which binds to the same receptors that the compound (I')), together with various competing substances (100 μm - 1 PM). Total specific binding was defined as the number of displaced large excess (100 μm). Each column in the graph represents the average of three repeated definitions, whiskers represent the mill is artnow the error of the mean. On the Y-axis presents the radioactivity, who contacted each experiment in units of counts per minute (imp./min). The upper dotted line shows the total binding in the conditions of the experiments, while the lower dotted line shows the nonspecific binding; the distance between the dashed lines shows specific binding.

Figure 5 shows a profile cross-reactivity for each of the five compounds (I') to (V) (part a: (II): (III); (V); D (I') and E: (IV)) against a set of 75 different receptors, which constitute the set of CEREP (see text). These compounds were tested at a single concentration (10 μm), and described the inhibition of binding (Y-axis of each histogram) for a known ligand with each of the 75 receptors (-100%, thus, represents a 2-fold increase in binding of the specific ligand in the presence of the tested compound). All reactions were carried out in two repetitions, and the columns in the graph represent the average value (to simplify the graphs, the error between reps (not shown). When conducting this test, the binding was considered to be statistically and biologically significant only at 50% inhibition (or stimulation) or above (which corresponds to the average effective dose below 10 μm for the interaction of the compounds with a separate R is zeptogram). For five of these compounds were studied in this application, only in one case the interaction (the interaction of the compound (II) with the NK2 receptor, marked by the arrow in part a of the figure) can be potentially significant, even though this interaction was weak (the value of the average effective dose was 5-10 μm).

Figure 6 (parts a-E) shows representative curves of the dose dependence of inhibition of chemokine-induced migration of leukocytes in vitro, for each of these five compounds (I') to (V) in the analysis of migration of cells between the holes in the ChemoTx system™. In each experiment induced the migration of cells TNR-1 with a maximum effective dose of the chemokine MCP-1 in the presence or absence of various doses (from 10 PM to 1 μm) of each compound. When conducting each experiment used appropriate media as a control. The percentage inhibition of cell migration induced by MCP-1 (defined as the number of cells migrated in the presence of MCP-1, minus the number of cells migrated in the absence of MCP-1 in the lower chamber) at each concentration of each test compound is shown as the average of the three repetitions of the experience, whiskers on the graph show the standard error of the mean. The average effective dose was assessed by whether anoy interpolation of the graphs. On the Y-axis of each graph shows the percentage of inhibition of cell migration induced by MCP-1; on the X-axis shows the concentration present test connection in nm (0.01 to 1000).

7 shows representative curves of the dose dependence of inhibition induced by lipopolysaccharides production of TNF-α in vivo in a model of sublethal endotoxemia in mice. In each experiment, groups of six mice received prior treatment with different doses of five compounds (part a: (II): (III); (V); D (I') and E: (IV)orally (circles) or subcutaneously (triangles). After 30-60 minutes, the animals were injected intraperitoneal lipopolysaccharide and after 3 hours of whole blood were prepared serum. The concentration of TNF-α in the blood was measured using the method enzyme immunoassay, and the degree of inhibition induced by lipopolysaccharide synthesis of TNF-α (defined as the concentration of TNF-α in mice exposed to lipopolysaccharides, minus the concentration of TNF-α in mice, which as a control were injected saline solution with phosphate-saline buffer containing no endotoxin) is shown on the Y-axis of each graph as the average of six animals, the whiskers show the standard error of the mean. The concentration of TNF-α in mice which were injected lipopolysaccharide, but who had not received treatment BSCl was about 5000 to 6000 PG/ml (compared with less than 10 PG/ml mice, not affected). The average effective dose was estimated by linear interpolation of the graphs. On the X-axis of each graph shows the dose of each compound that was administered to each mouse in group mg (1E-07 to 1).

On Fig shows the effect of compound (I') with pneumonia in a representative experiment, which was assessed by the number of cells in bronchoalveolar fluid in a model of asthma in rodents. Columns show the average number of cells shown on the Y-axis in units of 106cells for groups of five animals, the whiskers show the standard error of the mean; (p<0,01 against "sensitizing" using unpaired student test for one sample under the assumption of a symmetric distribution with the same variations). The horizontal line shows the average number of cells present in bronchoalveolar fluid from the lungs of rats not subjected to the specified impact. All rats in the remaining three groups were subjected to the same sensitizatio and mode of exposure, but at the same time or were exposed to only media ("sensitizirovannykh"), or the effect of compound (I') in an amount of 0.3 mg/kg of body weight or montelukast ('Singulair™') in an amount of 30 mg/kg body weight; in all cases, the introduction was carried out by daily oral route.

Figure 9 demonstrates the effect is soedineniya (I') on the polarization of T-helper cells in a representative experiment, which was evaluated by the method of flow cytometry product IFN-γ (marker cytokine for Th1) and IL-4 (marker cytokine Th2) CD4+ splenocytes in a model of asthma in rats. Bar in the graph represent the average value of the ratio of Th1/Th2 shown on the Y-axis for groups of 10 animals, the whiskers show the standard error of the mean; (p<0,05 versus untreated rats; † <0,05 against sensitizing rats and rats treated with the drug, in both cases for the evaluation of equal variances was used t-student test for one sample under the assumption of a symmetric distribution with the same variations). All rats (except group "BN-rats"who have not received ovalbumin) were subjected to the same sensitizatio and the mode of application of the drug, but gave only the media ("sensitizirovannykh + media)or media together with compound (I') in an amount of 0.3 mg/kg body weight or with montelukast ("Singulair™") in an amount of 30 mg/kg body weight; all options were administered daily orally through a feeding tube.

Figure 10 presents a plot of half-life from plasma (in minutes) of exposure for oral use for a number of different BSCl compounds in rats. In each case, an estimate of the time half-life was carried out using the standard chamber model concentrations in plasma through 0; 0,25; 0,5; 1; 2; 4; 6 and 8 hours after nutrive the aqueous injection of a single bolus dose of the compounds in quantities of 1 mg/kg dissolved in 1% solution of carboxymethyl cellulose, 0,9% DMSO in saline. The impact for oral use (AUC 0-t min·ng/ml) was determined after administration of single oral doses of the compound in an amount of 3 mg/kg dissolved in 1% solution of carboxymethyl cellulose, 0,9% DMSO in saline. R-enantiomer of compound (I), which is the inverse of the compound (I') and identified as "R-(I)marked with an empty circle.

EXAMPLES

In each of the following examples 1 to 6 were compared S-3-(2',2'-dimethylpropanolamine)-tetrahydropyridine-2-she (compound I') with a number of other lactam BSCl, which were selected to be representative of different subclasses. For example, 3-(adamantane-1-carbylamine)-caprolactam (II) was chosen as a typical representative of the subclass polycyclicaromatic BSCl (such as previously described in WO 2006/016152)

Similarly, 3-(1'-methylcyclohexylamine)-caprolactam (III) was chosen as a typical representative of the subclass monocyclic lactam BSCl (such as previously described WO 2006/134384)

The compound 3-(1',1'-dimethylethylenediamine)-caprolactam (IV) was chosen as a typical representative of a subclass of BSCl with simple (non-cyclic) alkyl side chains (such as previously described in WO 2005/053702), as well as connection is sulfonylamine linker (in contrast to the urea linker in the remaining selected connections)

The last selected compound 3-(3'-hydroxyadamantane-1 carbylamine)-caprolactam (V) was typical for BSCl substituted with acyl side chain (simple linear, branched, mono - or polycyclic structure)

It is important to note that all data BSCl connection from (II) to (V) were directly described previously, and all have a strong BSCl action in vitro (mean effective dose was less than 1 nm at the inhibition of migration TNR-1 cells induced MCP-1). All compounds are characterized by excellent stability in serum, in vitro and in perspective in theory are wonderful substances-candidates for the development of pharmaceutical drugs with anti-inflammatory properties, for use in humans in vivo.

All of the above compound (I', II, III, IV and V) were tested in the S-configuration lactam of the stereocenter.

Example 1 Pharmacokinetic parameters after administration of a single dose

Compounds were administered in a single dose (1 mg/kg body weight in 5% DMSO intravenously or in an amount of 3 mg/kg in 1% solution of carboxymethylcellulose oral) three adult rats (using different animals for each connection and each method of administration).

Then took blood samples at different points in the time (including immediately before dose) for up to 24 hours after a dose and evaluate the levels of various compounds using a validated analysis method liquid chromatography/tandem mass spectrometry. Briefly, 3-5 µl deproteinizing sample was applied to a column chromatography in reversed-phase Waters Atlantis (C18, 20×2.1 mm, grain size 3 μm), equilibrated with 0.1% solution of methanoic acid in a mixture of water:acetonitrile in the ratio 95:5. After 3.5 minutes of bound peroxidase material was subjected to gradient elution of 0.1% solution of methanoic acid in a mixture of water:acetonitrile in the ratio of 5:95, then step-by-step elution of 0.1% solution of methanoic acid in a mixture of water:acetonitrile in the ratio 95:5. The obtained eluent is then investigated by the method of tandem mass spectrometry using a mass spectrometer detector (Applied Biosystems API 4000/3200 QTrap with ion source Turbolonspray™operating mode of positively charged ions. The surface temperature was set To 650°C, retention time was 40 MS for each MRM transition when the monitor the following ions:

td align="center"> (I')
Analytemass Q1 (Amu)weight Q3, (u)
(II)291,14135,1
(III)253,1697,0
(V)307,15151,1
199,1557,2
(IV)249,15to 129.3
Internal standard213,1957,1

As an internal standard for each definition used related compound S-3-(2',2'-dimethylpropanolamine)-caprolactam, which was introduced into the sample before deproteinization. The value of the lower limit of quantitation (NPCA) for this method of analysis was 2.4 ng/ml for each compound, except (I'), for which the lower limit of quantitation amounted to 38.1 ng/ml.

After analysis of each selected sample by the method of liquid chromatography/tandem mass spectrometry modeling of pharmacokinetic parameters of the compounds was carried out using the software Kinetica is a well - known software package for solving such problems.

Results

Graphs of the individual concentrations over time for each rat, which was subjected to each connection in oral introduction, shown in figure 1. Obviously, data from five distinguished by the structure of lactam BSCl only the compound (IV), (V) and (I') are characterized by some l the Bo action for oral use and which of these compounds (I') is much better than others.

The parameters of a simple single chamber pharmacokinetic model are presented in Table 1. First, these parameters show that the effects of compound (I') is approximately 20 times stronger than the next connection with the best indicators to (V). The reason for this best exposure (which was defined as the area under the curve of the dependence of concentration on time in min·ng/ml) were also evident: the clearance of the compound (I'), which was defined as theoretical amount of blood, which is completely cleared of drug substances every minute (ml/min/kg, more than 10 times lower than that of other lactam BSCl.

Bioavailability (%)t1/2 (min)Clearance (ml/min/kg)The volume of distribution at steady state (l/kg)Exposure (min·ng/ml)
I')691962,60,7939000
II)<116840,8477
III)511550,62900
IV)4519320,641400
V)5924*311,457100

Table 1. Pharmacokinetic parameters differ in the structure of lactam BSCl. Bioavailability for oral use (bioavailability, %), the main half-life from plasma (t1/2, min), clearance (ml/min/kg), volume of distribution (volume of distribution at steady state (l/kg) and exposure (AUC 0→∞, min·ng/ml) with a simple single chamber pharmacokinetic model; for each connection, the average values obtained for the three rats. * 24 minutes - this is the main half-life of the compound (V)when the clearance was more than 95% of the administered dose; of secondary importance t1/2 was 110 minutes. In all cases, the value of Cmaxwas achieved within 30 minutes in accordance with optimal absorption.

The clearance of the compounds (II) and (III) approaches the speed pot is ka blood in rat liver, what convincing evidence in favor of the fact that both compounds undergo almost complete metabolic transformation during the first passage through the liver. Similarly, the clearance of both compounds (IV) and (V)exceeds the rate of blood flow in the kidneys in rats approximately 3-4 times, which again argues in favor of what is presumably the main part due to metabolic clearance is also carried out in the liver. In contrast, the clearance of the compound (I') is 2.6 ml/min/kg, which is less than half the speed of blood flow through the kidneys (usually tell about 7-9 ml/min/kg), suggesting a minimal effect of metabolic clearance. Since the compound (I') is extremely soluble in water, the value of the volume distribution corresponds to the equilibrium value of free allocation in the entire volume of water contained in the body (0.7 l/kg), it seems that the value of clearance below the rate of blood flow through the kidneys is a re-absorption of water in the distal renal tubules (more likely explanation than, for example, reduced impact on the kidneys because of the isolation of the compounds in lipophilic compartments).

In accordance with a significantly lower clearance of the compound (I') compared with the other compounds, the compound (I') is characterized by knowledge is sustained fashion over a prolonged half-life from plasma (more than 3 hours (I') and less than 30 minutes for the remaining four connections).

Not all BSCl are bioavailability for oral use, even though all five of the selected compounds possess bioactivity for oral use in the final stage of acute inflammation. Most likely, this reflects the rapid metabolic conversion of compounds (II) and (III) in the liver, which is effectively absorbed, but at the first passage through the liver into metabolites that retain some activity as BSCl (see example 2).

Based on the pharmacokinetic analysis for professionals in this field it is obvious that, despite similar chemical stability and stability in vitro in isolated serum for these five compounds, as well as the similarity of their theoretically predicted properties, the compound (I') is significantly superior to all other connections. In particular, the clearance of this compound is considerably lower, which probably reflects the lower susceptibility of this compound metabolic reactions in the liver, which leads to 10 times longer half-life of this compound from plasma and nearly 20 times better effect for oral use, than for the next best properties of the investigated compounds.

When conducting a separate experiment, the compound (I') was compared with the second best connection is (V) pharmacokinetic parameters in other species of animals (not rodents) - in dogs. A single dose (1 mg/kg in 5% DMSO intravenously or 3 mg/kg in 1% solution of carboxymethylcellulose oral) was administered one dog one connection in accordance with a simple cross-experiment plan, which was provided washout period between the two routes of administration duration of 1 week.

The results are presented in Table 2.

236
Pharmacokinetic parameters of intravenous (l·mg/kg)
ConnectionSpeciesThe half-life (minutes)Clearance (ml/min/kg)The volume of distribution at steady state (l/kg)
(V)Rat24330,8
Dog667,40,6
(I')Rat1962,60,7
Dog1,90,6

Table 2. Comparison of pharmacokinetic parameters of both compounds lactam BSCl with the best properties in rats and dogs. Pharmacokinetic parameters in dogs, in General, similar to the parameters in rats. Both types of compound (I'), characterized by a significantly lower clearance and longer half-life from plasma and, therefore, greater exposure (AUC, min·ng/ml). In each case, the main half-life (faster t1/2α) is responsible for eliminating more than 95% of the administered dose.

The results of these observations indicate that the predominant manifestation pharmacokinetic properties of the compound (I') is not species-specific and, therefore, likely that similar properties will be observed in humans.

Example 2 Determination of primary metabolites

Rats were kept in metabolic cages and exposed to a single oral dose (3 mg/kg in 1% solution of carboxymethyl cellulose) each connection within 24 hours of collected urine. Pooled urine sample was then subjected to a full analysis of the way mass spectrometry using the same conditions for liquid chromatography/mass spectrometry, as described above in the description of p is the iMER 1. Then conducted further analysis of the ion product by means of tandem mass spectrometry and evaluated the possible fragmentation/rearrangement of compounds according to publicly available database of metabolites.

For major metabolites predicted patterns were confirmed by the synthesis of authentic samples using well-known in the field of methods, which were then analysed by the method of liquid chromatography/tandem mass spectrometry in the same conditions as the urine samples.

Note that studies to determine the identity of the metabolites only provide a quantitative assessment of the relative amounts of the various metabolites present and would need a separate quantitative analyses using validated methods and appropriate internal standards to determine the number of each type of metabolites.

Results

Figure 2 presents the metabolites that have been identified for five of the analysed compounds in order of detected concentrations. It is important to note that the methodology used here is not necessarily exhaustive and additional (especially secondary) metabolites may also be present in quantities below the detection limit used for the above analysis is e ways. As a General rule, you can assume that will be detected metabolites that constitute 10% or more of the injected dose (although not necessarily structurally identified used here).

For compounds (II) and (III) the main route of metabolism is hydroxylation mediated by cytochrome P450, which is consistent with the values of the fast clearance at speeds approaching the speed of blood flow in the liver (see example 1 above). The main target, hydroxylation for both compounds is cycloalkyl terminal group, the second (slower) hydroxylation occurs in the lactam end groups. Note that the hydroxylation products of lactams appeared when determining the method of tandem mass spectrometry at -2 u (as opposed to +16 Amu), due to the instability of the 7-hydroxy in the source elektrorazpredelenie.

Product dihydroxypropane of compound (II) is present in a quantity sufficient for detection in urine, while for compound (III) did not find any product dihydroxypropane. In both cases, it seems that also formed additional minor products in quantities below the detection limit of the used method (e.g., 3,5-dihydroxy - 3,5,7-trihydroxyphenyl-derivative is connected to the I (II), and glukuronirovanie derivatives as hydroxy-(II), and hydroxy-(III), especially since it was discovered glukuronirovanie derivative of compound (V)).

For compound (V) glukuronirovanie derived was the main metabolite, although in rats, only a minor fraction of glucuronate was eliminated in the urine, most of it was derived from faeces (in person, on the contrary, this glukuronirovanie will be derived primarily excreted in the urine). May also be formed of other metabolites of phase II (such as 3'-O-sulfate), but only in quantities (at least in the urine), too low to be detektirovanie with the use described in this application methods. For compounds (II) and (III) also identified a small number of product gidrauxilirovannogo on lactam end groups (mainly as the ionic product - 2 u).

The main metabolite of the compound (IV) could not be identified, although the loss of the main compound (see Table 3) clearly corresponded to the formation of unidentified metabolite (less than 10% of the administered dose of the compound (IV) was found unchanged). Provided that the compound (IV) was the only one that contains sulfonamidnuyu communication, probably, but at the moment it is not proved that happened metabolic cleavage (or other modification) of this innovation is due. We also observed a small amount of product hydroxylation lactam end groups.

Unlike all other compounds no significant metabolites of the compound (I') was not detected in the urine, which corresponds to the appearance of more of the administered dose in the urine unchanged (see Table 3), and velocity values clearance are the same or lower value of the velocity of renal blood flow (see example 1 above). The lack of formation of metabolites is a major and unexpected advantage of the compound (I') compared to other tested here compounds for drug development for use in humans and is, at least partially, explain the preferential pharmacokinetic properties, described in example 1 above.

In order to provide a quantitative assessment of the degree of metabolic transformations of each of the investigated compounds, the number of compounds which have undergone changes, measured using the same validated method of analysis by liquid chromatography/mass spectrometry, described in example 1, with the use of S-3-(2',2'-dimethylpropanolamine)-caprolactam as an internal standard. The results are presented in Table 3. In addition, it was determined the concentration of the studied compounds in the distinctions of the different target tissues.

but = not determined; NPCO = 2.4 ng/g

ConnectionHeartEasyKidneyLiverMuscleThe brainUrine (ng/ml)
(II)butbutbutbutbutbutbut
(III)butbutbutbutbutbutbut
(V)butbutbutbutbutbut16300
(I')5,3of 21.95,13,63,11,6 24567
(IV)butbutbutbutbutbut1010

Table 3. The distribution of compounds in various tissues within 24 hours after administration of a single dose in rats. Only compound (I'), (V) and (IV) could be detected in the urine after 24 hours after administration of a single oral dose (3 mg/kg in 1% solution of carboxymethyl cellulose). Among them, the compound (I') has undergone significantly less metabolic transformations (more than 60% of the injected dose was found in urine). Moreover, only the compound (I') can be detected in other tissues 24 hours after administration of a single oral dose. The obtained data are likely to reflect the benefits of distribution and greater exposure to the compound (I') in comparison with other investigated here the connection.

Significantly lower level of metabolism of the compound (I') in comparison with other compounds shows that an unexpected way connection (I') is much superior and a wide range of lactam BSCl previously described for the development of medicines for use in humans. This reduced level of metabolism (and therefore improved ADME their is TBA), apparently, is the explanation for the improved pharmacokinetic properties above in the description of example 1. Moreover, because BSCl are designed to develop anti-inflammatory drugs directed against unwanted presence of cells in different tissues, unexpected discovery that the compound (I') is found in all tissues of the body under test 24 hours after administration of a single dose, while all other investigated lactam BSCl was not present, a clear indication of particular usefulness of this new connection.

Example 3. Pharmacological studies security

These five compounds were subjected to the standard Ames test in order to assess their potential genotoxicity. Three auxotrophic for histidine strain S.typhinurium (TA, ÒÀ98 and TA100) were treated with each of the five studied compounds in five concentrations (up to 5 mg/ml) in the presence and in the absence of microsomal metabolism of rat S9. Then determine the number of revertant colonies by sowing on minimal medium containing trace amounts of histidine.

The results obtained (table 4) show that none of the five of these compounds does not increase significantly the formation of revertant colonies (with or without the metabolic activate is) for all tested strains.

At= rare bacterial
concentration of 5 mg/ml:lawn
= low reference value
* this experiment
= significant decrease number
revertant colonies
ConnectionTA100TA100+S9TATO+S9ÒÀ98ÒÀ98+S9
(II)0,59*0,730,50*0,930,58*0,60*
(III)0,790,81 0,690,880,680,55
(V)0,780,880,931,090,960,91
(I')0,970,950,820,932,00**1,23
(IV)0,890,900,850,880,820,90
the positive control6,897,185,972,659,6819,05

Table 4. The formation revertant colonies when conducting the Ames test. None of the tested compounds did not lead to a significant increase in the formation revertant colonies at any of the concentrations used (data recip is installed with only the highest dose). Note that the compound (II) resulted in suppression of growth of the bacterial lawn at a concentration of 5 mg/ml

When conducting a separate experiment, all five of the investigated compounds were tested for interaction with protein ion channel hERG. The use of compounds which interact with hERG, is risky, as they may cause prolongation of QT interval and potentially fatal cardiac arrhythmia. Compounds that inhibit trace the hERG current by more than 50% at a concentration of 10 μm, in General, are considered high-risk for the development of drugs for use in humans.

Cells NEC subjected stable transfection for expression of hERG protein, washed with liquid containing the compounds at a concentration of 10 µm (0.1% DMSO). Then write the values of hERG trace current from the three cells by the method of fixation of the membrane potential after depolarization to +20 mV for 5 sec. the Force of interaction with hERG any compounds that have been shown to be significantly modulated at a concentration of 10 μm, was then determined using the curve dose-response relationships, built on 4 points.

The results (Figure 3) showed that none of the five of the studied compounds at a concentration of 10 μm did not engage in significant interaction with protein hERG channel.

The authors did C the conclusion is, from the point of view of pharmacology security all five of the studied compounds, including (I')are equally suitable for development of drugs intended for use in humans. In particular, significantly predominant ADME and pharmacokinetic properties of the compound (I')above in the description of Example 1 and Example 2, does not correspond to the worst data on pharmacological security.

Example 4. General pharmacology

Evaluating the overall pharmacological parameters investigated these five compounds was carried out as in the interaction with the specific receptor-targeted, and in response to a wide range of other receptors, many of which are similar in structure to the receptor target. Binding with a specific receptor target was assessed by competitive binding of [3H]-BN83250 (BN83250 is S-3-(2',2'-dimethyldodecylamine)-caprolactam; Fox et al. J Med Chem. 200; 48(3): 867-74; a substance which binds to the same receptor-a target that lactam BSCl described in this document). Binding to receptors that are not target were evaluated by competitive binding with different radioactively labeled ligands that are specific to other receptors, is well known to specialists in this field.

For specific binding to cell lines melomanie the s human cells resuspendable in the buffer for binding (20 mm HEPES, 150 mm NaCl; pH 7,4; 106 cells per reaction) at 4°C in the presence of 10 nm [3H]-BN83250 (prepared from 1 μm stock solution in 100% ethanol; 30 CI/mmol) and different agents for competitive binding (1% DMSO, the maximum concentration of media). The reaction was carried out for 2 hours on ice, then filtered through GF/C filters pre-saturated 0.5% polyethylenimine. Unbound material was removed 5x washing with 5 ml ice-cold buffer for washing (20 mm HEPES, 150 mm NaCl; pH 7,4) with a weak vacuum. It was previously shown that the use of these terms is achieved equilibrium binding, at least 80% of binding is specific (able to compete with 10 μm unlabeled BN83250).

Competitive specific binding of [3H]-BN83250 and compounds (I'), (II) and (V) were then determined at various concentrations from 1 PM to 10 μm. Compound (III) and (IV) not investigated in the conduct of these experiments. Typical competitive binding curve for the compound (I') shown in Figure 4.

Then to the competitive binding curves for various compounds were applied nonlinear modeling in order to compare their properties as agents that bind to the receptor target (defined in the site specific interaction with the connection BN83250). The parameters of the model are presented in Tables is 5.

ConnectionThe functional value of the average
effective dose against MCP-1 (PM)
Toandreceptor target (PM)The hill coefficient
(II)808200-0,5
(III)80Not testedNot tested
(V)12010000-0,5
(I')5050of-1.0
(IV)800Not testedNot tested

Table 5. Nonlinear modeling of curves of competitive binding.

It is important to note that for the compound (I') as opposed lactam BSCl (II) and (V) was shown to be an ideal and predictable binding to the receptor target. In particular, a specific value of the binding affinity to the receptor was similar to the functional value of the average is ffektivnoe dose analysis for the inhibition of migration. Similarly, the value of the hill coefficient was approximately -1,0 (theoretically expected value for a simple model of non-cooperative competitive binding), while the study of other lactam BSCl were identified significantly less significant coefficients hill. The reason for the differences from the ideal of the receptor binding target for compounds (II) and (V) is unknown, but this difference properties underlies the unexpected superiority of the compound (I').

Binding to receptors non-target were evaluated using similar protocols using specific radioactively labeled ligands for each receptor, which is well known to specialists in this field. Each connection is explored in a competitive specific binding of each ligand only at one concentration (10 μm). If inhibition of binding was between 20% and 80%, determined the value of Kandfor this interaction. If the inhibition was less than 20%, believed that such a connection does not have the ability to competitively bind to the receptor. If the inhibition was greater than 80%, believed thatandfor this interaction is less than 1 μm. Detailed description used in this study receptors, radioactively labeled ligands and cell types n is located at

The results (Figure 5) indicate that all tested lactam BSCl connection free from the main cross-reactively based on the study of this set of 75 receptors (there were no interactions, characterized Byand<1 μm). Was found only a weak (but statistically significant) cross-reaction of the compound (II) and NK2 receptor). Based on this, the compound (I') was recognized indirectly more specific interaction with the receptor-target than the compound (II), but all tested lactam BSCl compounds were suitable for development of pharmaceutical preparations intended for use in humans on the basis that they did not possess the ability to communicate with false targets, as shown in this high-throughput analytical screening.

Example 5. Inhibition of chemokines broad-spectrum in vitro

Biological activity of compounds described in this invention can be demonstrated using any of a wide range of methods of functional studies of the migration of leukocytes in vitro, including but not limited to) the camera Boyden and related experiments on migration between the holes, experiments on migration under a layer of agarose and the use of cameras for direct visualization, such as Luggage Dunn.

For example, to show the inhibition of the migration of leukocytes in response to chemokines (but not other chemoattractant), used the system for analysis of migration between wells, consisting of 96 microwell ChemoTx™produced Neuroprobe (Gaithersburg, MD, USA). In General, this analysis uses two cameras separated by a porous membrane. The chemoattractant is placed in the lower compartment, and the cells placed in the upper compartment. After incubation for a certain period of time at 37°C. the cells move in the direction of chemoattractant and the number of cells in the lower compartment becomes proportional to the activity of the investigated chemoattractant (relative to the results of a series of control experiments).

This method of analysis can be used with a wide range of populations of leukocytes. For example, can be used fresh leukocytes from human peripheral blood. Otherwise, can be prepared some types of cells, including polymorphonuclear cells, or lymphocytes, or monocytes using methods well known to specialists in this field, such as centrifugation in density gradient or separation of the magnetic beads. You can also use an immortalized cell line to the e have been widely used as models of leukocytes in human peripheral blood, including (but not limited to, cells TNR-1 as a model of monocytes or Jurkat cells as a model of naive T cells.

Although many conditions are acceptable when conducting experiments to demonstrate the suppression of the migration of leukocytes induced by chemokines (see, for example, recommendations Frow et al. Med Res Rev. 2004; 24(3): 276-98 on the conditions required for the interpretation of results obtained in experiments on the study of cell migration in vitro), in this application describes the conditions used in this specific case.

Materials

System for studying cell migration between the holes produced Neuroprobe, Gaithersburg, MD, USA. Used in experiments tablets: tablets ChemoTx™ (Neuroprobe 101-8) and the transparent plate 30 ál (Neuroprobe MR).

Balanced salt solution Gace (SSR) were purchased from Sigma (Sigma G-9779). BSA containing no fatty acids were purchased from Sigma (Sigma A-8806). MTT, for example, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma (Sigma M-5655). RPMI-1640, containing no phenol red, were purchased from Sigma (Sigma R-8755).

Cell line TNR-1 (European collection of cell cultures) were used as a population of cells.

Protocol test

The following procedure was used for testing is soedineniya according to the present invention, to determine its ability to specifically block leukocyte migration induced by chemokines:

First, to prepare a cell suspension, which was then placed in the upper compartment. Cells TNR-1 was besieged by centrifugation (770 g; 4 min) and washed with balanced salt solution Gace containing 1 mg/ml BSA (SSR+BSA). This washing was then repeated and the cells re-besieged before resuspending them in a small volume SSR+BSA for counting, for example, using standard counting chamber.

Volume SSR + BSA was then selected depending on the numbers of cells so that the final cell density was of 4.45×106cells in 1 ml SSR + BSA. In each 25 μl of the solution will contain 100,000 cells TNR-1, which will be placed in the upper chamber of the tablet.

In order to test a separate connection for its ability to inhibit leukocyte migration induced by chemokines, it is necessary to prepare two series of cells. Cell suspension TNR-1 with a density of 4.45×106cells/ml were placed into two containers. In one container add the studied inhibitor to the desired final concentration in a suitable medium (for example, at a concentration of 1 μm in 1% DMSO). Into the second tank add an equal volume of SSR + BSA plus the nose of the tel as follows (for example, no more than 1% DMSO) as a control.

Then prepare a solution of chemoattractant, which is then placed in the lower compartment. For example, MCP-1 diluted in SSR + BSA to a final concentration of 25 ng/ml This number is divided between the two tanks as a cell suspension. In one tank type test the connection to the same final concentration, while in another vessel, add an equal volume of SSR + BSA plus suitable medium (for example, not more than 1% DMSO). Otherwise, you can use other chemokines (SDF-1α concentration of 7.5 ng/ml; RANTES concentrations of 50 ng/ml; IL-8 at a concentration of 10 ng/ml using neutrophils as a population of target cells). In each case it is important to determine (in a separate experiment) the concentration of each chemokine, which leads to maximum stimulation of migration of the selected population of cells membranes. This maximum concentration should then be used in experiments to test the inhibitory activity of this compound, which is the subject of this invention. Because chemokines usually induce leukocyte migration, which describes kolokoloobraznuyu curve dose-response relationships, the use of sub - or supra-maximal concentration of chemokine may lead to artifactual results (for example, the connection, the cat is itself an inhibitor chemokines, may cause paradoxical stimulation of migration, if the experiment was incorrectly selected supramaximal concentration chemoattractant. Additional evidence of the importance of this factor in the planning of experiments on the migration of leukocytes in vitro were provided in the work Frow and employees (Med Res Rev. 2004; 24(3): 276-98). In addition, chemoattractant non-chemokines may also be used to demonstrate the selectivity of the biological activity of the studied compounds, which is the subject of this invention, in respect of chemokines (for example, Sa at a concentration of 25 ng/ml using neutrophils as a population of target cells or TGF-β1 at a concentration of 10 ng/ml using TNR-1 as the population of target cells).

Note that the volume of liquid, which is added in the composition of the solution under test connection, you need to consider at the nal concentration of MCP-1 in the solution for the lower compartment final concentration of cells in the upper compartment.

When solutions chemoattractant to the bottom of the cells and fluids of the cells to the upper compartments cooked, you should collect the camera for migration. Put 29 μl of a solution of the desired chemoattractant in the lower chamber capacity. Experiments should be conducted not less than three repetitions of drakedog conditions. When all the lower chamber is full, the camera is placed a porous membrane in accordance with manufacturer's instructions. Finally, in each upper chamber is placed in 25 μl of a suitable solution of cells. Put the plastic cover on top of all fixtures in order to avoid evaporation.

Prepared camera incubated at 37°C, 5% CO2within 2 hours. Cell suspension in SSR + BSA also incubated under identical conditions in vitro, these cells will be used to plot a standard curve to determine the number of cells that migrated to the lower chamber under specified conditions.

At the end of the incubation liquid cell suspension carefully removed from the top of the camera, add in the upper chamber 20 ál of ice-cold 20 mm EDTA in phosphate-buffered saline and incubated fixture at 4°C for 15 minutes. This procedure leads to the fact that all cells attached to the bottom of the membrane, fall into the lower chamber.

After this incubation the filter, gently wash solution SSR + BSA to remove EDTA, then the filter is removed.

The number of cells that migrated to the lower chamber under specified conditions, can be determined by applying a number of ways, including direct counting, tagging fluorescent or radioactive the mi markers or by using dyes for in vivo staining of cells. Usually for intravital staining of cells using MTT. 3 µl of stock solution of MTT was added to each well, the tablet then incubated at 37°C for 1-2 hours; during this time the dehydrogenase enzymes contained within cells that convert soluble MTT to insoluble blue product formazan, which can be quantified by a spectrophotometric method.

In parallel, carry out the construction of the standard curve by 8 points. Cells are added to the plate in 25 μl with 3 µl of stock solution of MTT, since the number of cells added to each upper chamber (100000), and reduce the number of cells, performing a 2-fold serial dilution solution SSR + BSA. Tablet for standard curve incubated near the tablet to study cell migration.

After this incubation, the liquid is carefully removed from the bottom of the camera, trying not to muddle the precipitated precipitate the reaction product, which is formazan. After a short drying air in each of the bottom camera add 20 ál of DMSO to dissolve the dye is blue and measure the optical density at 595 nm using a reader for 96-well plate. Then assess the number of cells in each lower chamber by interpolating the obtained values of optical is some density for each well using the standard curve.

Migration, which is stimulated with chemoattractant, is determined by subtracting the average number of cells that reached the lower compartment in the holes where not added any chemoattractant, the average number of cells that reached the lower compartment, attended by the chemoattractant.

The effect of the compounds is determined by comparing the migration caused by the action of chemoattractant, which happened in the presence or absence of various concentrations of test compounds. Usually the suppression of migration expressed as a percentage of total migration, caused by the action of chemoattractant, which was ingibirovany in the presence of this compound. For most compounds the plotting dose-response relationships is performed by determining the suppression of the migration induced by the action of chemoattractant, which occurs when different concentrations of compounds (usually the concentration range from 1 nm to 1 μm or above in the case of low activity of compounds). The inhibitory activity of each compound is then expressed as the concentration required to reduce migration, caused by the action of chemoattractant 50% (concentration corresponding to the mean effective dose). Usually, the migration of cells TNR-1, caused by the action of MCP-1, used is as standardized test system to compare the biological activity of a wide range of compounds (see, for example, Reckless & Grainger Biochem J. 1999 Jun 15;340 (Pt 3): 803-11; Reckless et al. Immunology. 2001 Jun; 103(2): 244-54; Fox et al. J Med Chem. 2002 Jan 17; 45(2): 360-70; Fox et al. J Med Chem. 2005 Feb 10; 48(3): 867-74; the International applications supra). Compounds which inhibit leukocyte migration induced by more than one chemokine, but not chemoattractants non-chemokines (such as TGF-β or Sa), defined as inhibitors of chemokines broad-spectrum Broad-spectrum Chemokine Inhibitors, BSCl; see, e.g., Grainger & Reckless Biochem Pharmacol. 2003 Apr 1; 65(7): 1027-34; Grainger et al. Mini Rev Med Chem. 2005 Sep; 5(9): 825-32).

Results

Figure 6 shows a typical curve dose-response relationships for the compound (I'), which inhibits the migration of TNR-1 cells induced MCP-1, together with comparable dose curve according to other selected lactam BSCl for which it is known that they are characterized by particularly strong (i.e. less than 1 nm) effect. The activity of compound (I') in action against various chemokines and chemoattractants that do not belong to the chemokines expressed in values of the mean effective doses are shown in Table 6 and compared with other previously described lactam BSCl.

From the analysis of these data it is clear that compound (I') can be classified as BSCl (because it strongly and actively inhibits leukocyte migration induced by a number of chemokines, but has no effect on migration is aconito, induced chemoattractant, which are not chemokines, in this case, Sa anaphylatoxins). Moreover, it is clear that compound (I') is at least the same potent and active, as BSCl in vitro, as selected lactam BSCl, which have been described previously (for example, the compound (II) in WO 2006/016152 or the compound (IV) in WO 2005/053702). All investigated here lactam BSCl are much more active than any of selectany BSCl that have been described to date (including imides, such as NR58.4, yohimbine, lysergamide and peptide 3 and related structure compounds such as NR58-3.14.3). Moreover, compound (I') is more potent as BSCl in vitro (at least to inhibit migration induced by MCP-1)than any other connection, open or previously described. Although this action as BSCl is quantitatively superior in comparison with previously known prototypes BSCl (albeit to a small extent), this is not a property, which mainly distinguishes the compound (I) as having unexpected advantages compared to previously known prototypes inhibitors of chemokines wide spectrum of action. On the contrary, it shows that these unexpected and greatly predominant ADME and pharmacokinetic properties of the compound (I) compared to Shiro who they range previously described BSCl were achieved without any reduction in strength or activity of this compound as inhibitors of chemokines broad-spectrum in vitro.

ConnectionMCP-1SDF-1αRANTESIL-8, neutrophilsSa neutrophils
(II)80100100600>1000000
(III)80nononono
testedtestedtestedtested
(V)120200250500>1000,000
(I')505080600>1000000
(IV)800no nonono
testedtestedtestedtested

Table 6. The action selected lactam BSCl on leukocyte migration in vitro. In each case, the indicated dose of the compound (in PM), which is required to suppress the migration of leukocytes in response to the maximum dose approved chemoattractant 50% (median effective dose). Unless otherwise stated, the data included in the report, obtained for the cell lines for TNR-1. It was shown that none of the tested compounds even at the highest concentration (1 μm) did not suppress the migration of neutrophils induced Sa.

Example 6. Anti-inflammatory activity in vivo

We used the model of sublethal endotoxemia induced by lipopolysaccharide in order to demonstrate generalized anti-inflammatory properties previously described BSCl in vivo (see, e.g., Fox et al. J Med Chem. 2002; 45(2): 360-70; Fox et al. J Med Chem. 2005; 48(3): 867-74). When conducting this study, mice were subjected to non-specific Pro-inflammatory effects of bacterial endotoxin (lipopolysaccharide), and article is stump the development of systemic inflammatory response (reaction quantitatively assessed by the concentration of proinflammatory cytokine TNF-α in serum, which normally is not present in the blood, but its level is rising rapidly in response to a wide range of proinflammatory stimuli). The applicants have chosen this model, although it is not close to the terms of any inflammatory diseases in humans, since it is known that the concentration of TNF-α is important in many diseases (including rheumatoid arthritis, autoimmune disease, Crohn's disease, atherosclerosis, asthma and many others). Therefore, the active substances that suppress the synthesis of TNF-α, already used in the clinic (e.g., Enbrel™ and other antibodies against TNF-α) to treat a wide range of such diseases. Thus, in this model was shown activity that inhibits TNF-α, which is very effective for predicting the anti-inflammatory action, clinically useful in a wide range of diseases.

Mice (each group contained six animals) were subjected to preliminary exposure to different doses of each compound, which was administered subcutaneously 30 minutes prior to the injection of lipopolysaccharides or forcibly orally 60 minutes before the injection of LPS. These mice were then injected intraperitoneally 750 µg of bacterial lipopolysaccharides and were killed after 3 hours. Sera were prepared from whole blood and determined the concentration of TNF-α method immunofermentnogo the analysis (R& D Systems). In each experiment a group of six mice, which was used as a negative control, did not receive any lipopolysaccharides, the second group of mice received only LPS (without any inhibitor). The concentration of TNF-α in the serum of these animals that received LPS without prior treatment with medicines, taken as 100% (typically, this concentration was approximately 6000 PG/ml compared to concentrations less than 10 PG/ml in animals from group negative control). Previously, applicants have shown that the synthetic corticosteroid dexamethasone (a well known anti-inflammatory drug, which is used in a wide range of inflammatory diseases) in this model inhibits the synthesis of TNF-α, induced by the action of the lipopolysaccharides, at least 90%, whereas thalidomide (another well-known inhibitor of the production of TNF-α, which operates more on the level of intracellular synthesis of TNF-α than as an inhibitor of attracting leukocytes, as described in this application BSCl) inhibits the synthesis of TNF-α, induced by the action of lipopolysaccharides, approximately 60%.

The action of compound (I') when used in different doses, as well as other selected lactam BSCl shown in Fig.7. In accordance with what itaniemi this compound strongly inhibited the synthesis of TNF-α, the induced effect of lipopolysaccharides, subcutaneous (circles) or oral (triangles) application. When using this connection in quantities of more than 1 μg/mouse, the concentration of TNF-α-induced activity of lipopolysaccharides, is usually decreased by more than 90%, which was comparable with the effect of corticosteroid dexamethasone.

Other tested lactam BSCl also inhibited the synthesis of TNF-α, induced by the action of lipopolysaccharide, a dose-dependent manner (7), although the activity of the compound (I') in vivo was higher than any other tested compounds (and indeed higher than the activity of the other previously described elsewhere lactam BSCl, which were tested in this analysis). This quantitative (albeit small) increase in activity is not the main reason that the applicants hereby claim that the compound (I) has the unexpected advantage compared to previously known prototypes of BSCl. On the contrary, it shows that unexpected and greatly predominant ADME and pharmacokinetic properties of the compound (I) in comparison with a wide range of early described BSCl were obtained without loss of activity or power of action as an anti-inflammatory agent in vivo. In addition, these data clearly show the usefulness of the compound (I when used as an anti-inflammatory agent in vivo models of inflammation, which indicates the usefulness in a wide range of inflammatory diseases, which are accompanied by increased levels of synthesis of TNF-α as a component of the pathogenic mechanism.

It is important to note that Verhoture inflammation that is observed in this model, especially insensitive to ADME and pharmacokinetic properties of the tested anti-inflammatory agents. Because stimulation by lipopolysaccharides carried out only after 30 minutes after administration of the drug, even agents with very short times the retention in plasma (such as compounds (II) and (III)remained in the plasma in sufficient concentrations to exert strong anti-inflammatory effect. Thus, during this test was not emphasized the superiority of the claimed compounds over previously known prototypes that nevertheless shows the usefulness of this connection.

The utility of the claimed compounds was shown during the studies on animal models of asthma person (Sirota response, inflammatory response, which was observed in response to administration of lipopolysaccharides may not be typical for any human disease, although it is clear that it is a useful model for studying acute inflammation in General). When is provedenii research data rodents (usually rats) received ovalbumin in accordance with the plan of the experiment:

Adult rats Brown Norway (body weight 200-300 g; the number of animals = 10 per group) were subjected to sensitization by a single intraperitoneal injection of 0.1 mg of ovalbumin in the 0-th day. Then each rat was injected endotracheal 1% solution of ovalbumin (weight to volume) on day 8 and 2% solution of ovalbumin (weight to volume) in the days of the 15th, 18th and 21st. These animals were then killed 3 hours after the last injection on day 21. Note that in the experiments used ovalbumin (Sigma; the highest degree of purification), which is optionally purified from endotoxin on EndoTrap columns™ Red (acquired Cambrex; used in accordance with the manufacturer's instructions), and it was shown with the use of LAL-system for quantitative determination of endotoxins (QCL-1000™; Cambrex; the test was carried out according to the manufacturer's instructions; 1 mg of standard endotoxin contains ~900000 EE/mg), the concentration of endotoxin was less than 5 EE/mg protein. The obtained data show that the response to inflammation in the lungs rather is the result of an allergic response to ovalbumin than unintentional stimulation by lipopolysaccharides, which develops even with the use of commercial preparations of ovalbumin with a very high degree of purification; thus, it was confirmed that when using this mod is whether it is better reproduced molecular pathology, underlying the development of asthma in humans.

One group of mice (which was used as the primary control) did not receive ovalbumin, but all the rest was subjected to an identical impact. Mice from the second group (positive control) received ovalbumin, but had not received the medication. Mice of the third group were treated identically but received a daily dose of compound (I') in an amount of 0.3 mg/kg orally force in the days from the 8th to the 21st one hour prior to any subsequent administration of ovalbumin prepared on the same day. The compound (I') was introduced in the form of a sterile solution free from endotoxins phosphate-saline buffer solution. Mouse from the fourth group received montelukast (the active ingredient of the commercial drug used in the treatment of asthma, produced Singulair™) in an amount of 30 mg/kg orally for the treatment identical to the use of compound (I').

After driving animals evaluated the overall participation of leukocytes in the lungs by analysis of bronchoalveolar washings from the introduction through the tracheal cannula 4 episodes in 3 ml of sterile phosphate-saline buffer solution. For each animal obtained bronchoalveolar washings were combined and determined the total number of cells (using counting chambers). In addition,were used to define types present leukocytes using flow cytometer in accordance with the methods, well-known specialists in this field.

Also was collected from each mouse spleen and placed in a solution of nutrient medium RPMI + 10% serum (FCS) and antibiotics. Then the spleen punching through a nylon mesh with a small (100 μm) holes in sterile containers for filtration, which were placed in sterile Petri dishes to obtain suspensions consisting of single cells. The obtained cell suspension was then centrifuged (328 g; 5 min) and were washed in culture medium RPMI + 10% serum + antibiotics before applying conducted re-suspension in fresh medium and determined the number of cells in the counting chamber.

The total number of splenocytes 4×106(except red blood cells) were cultured overnight at 37°C, 5% CO2in a nutrient medium RPMI + 10% serum + antibiotics in the presence of 2 U/ml (10 ng/ml murine IL-2 in 4 wells of 96-hole tablet (volume of 100 µl per well / 1×106cells/well) from each mouse. Approximately 24 hours 4 wells were divided into two groups of 2 cells: two wells were not treated in the other two wells were added to 500 ng/ml ionomycin and 50 ng/ml PMA and kept at 37°C for 4 hours. In the last two hours of incubation in one hole from each group were added to 10 μg/ml of brefeldin A (stock solution 1 mg/ml in ethanol). Brefeldin And block the plans of the transport proteins in the Golgi apparatus and, thus, contributes to the accumulation of proteins in the endoplasmic reticulum.

The wells, which were not added brefeldin And incubated further for 48 hours at 37°C. After incubation, the cell suspension was centrifuged (328 g; 5 minutes) and the resulting supernatant was analyzed by application of the method enzyme immunoassay for the presence of murine IL-4 (Th2 marker) cells and murine interferon-γ (IFN-γ; marker Th1) cells (R&D Systems; the analysis was carried out in accordance with the manufacturer's instructions).

The wells, which added brefeldin And stained for intracellular IL-4 and IFN-γ immediately after 4-hour incubation as follows: held staining CD4-FITC-antibodies (eBioscience Rat lgG2b, Cat. Code. 11-0041) for 30 minutes on ice, then washed with phosphate-saline buffer solution, Dulbecco and fixed with 2% solution of paraformaldehyde (final concentration) in phosphate-saline buffer solution, Dulbecco for 20 minutes. After fixation, cells were treated with 1% BSA/0.5% saponin in phosphate-saline buffer solution, Dulbecco (Sigma S7900) for 10 minutes at room temperature so that the cells become permeable. Then, cells from each well were distributed in three separate test tubes for holding fluorescent sorting of cells and added:

- IFN-g-PE (eBioscience Rat IgG1, Cat. Code. 12-7311-82, 100 µg)

or

IL - 4-PE (eBioscience Rat IgG1, Cat. Code. 12-7041-82, 100 µg)

or

- Isotype control (a mixture of Rat IgG2b-FITC, eBioscience Cat. Code 11-4031 and Rat IgG1-PE, eBioscience Cat. Code 12-4301)

and incubated for 30 minutes at room temperature. Then cells were washed twice with a solution of BSA/saponin in phosphate-buffered saline and then with a solution of BSA in phosphate-buffered saline without saponin to the membrane pores were closed) and resuspendable in phosphate-saline buffer solution, Dulbecco for analysis by the method of flow cytometry.

Cell-specific painted on CD4 FITC (what defines them as T-helper cells)were analyzed for the presence of specific staining for IL-4 or IFN-γ for RE. The ratio of CD4+ cells positively stained for IFN-γ to the number of CD4+ cells stained positive for IL-4, and then included in the report as the ratio of Th1/Th2. Rat line Brown Norway, not subjected to, was characterized by the ratio of Th1/Th2 in the spleen is approximately equal to 2.7 (this means that the spleen is approximately 2.7 times more CD4+ cells produce INF-γ than IL-4). After sensitization and reprocessed by ovalbumin this ratio decreased to <1,5, reflecting a significant Th2-polarization, which is accompanied by changes in asthma both in rats and in humans (the lower the ratio of Th1/Th2 indicates the relative Th2 on the arizala, while the increase in the ratio of Th1/Th2 indicates the relative Th1-polarization).

Daily application of compound (I') and used as positive control, montelukast significantly reduced the number of leukocytes in bronchoalveolar aspirates (reduced by 70% when using the compound (I'); p<0,01; unpaired student test; Fig). The data obtained clearly show that the tested compound that is the subject of this invention possess useful anti-inflammatory effects in models of asthma person that follows from the ability of this compound to inhibit leukocyte migration induced by chemokines. The magnitude of the resulting effect is at least comparable to the action of commercial products intended for the treatment of asthma in humans (such as Singular™), and the primary pharmacokinetic parameters and the biodistribution of the compound (I') define a significantly greater effect of compound (I') compared with montelukast (in order to cause such a reduction in the number of leukocytes in bronchoalveolar lavage fluid, which occurs when the introduction of only 0.3 mg/kg of compound (I'), required dose montelukast 30 mg/kg).

Daily application of compound (I'), in the absence of the introduction used as a positive control, montelukast, much the way changed in the opposite direction Th2-polarization (Fig.9), which is considered the main driving force in the pathogenesis of asthma as used in conducting the research model of pneumonia, which is caused by injection of ovalbumin and asthma in humans. In the treatment using the compound (I'), even with the introduction of low doses, such as 0.3 mg/kg oral, completely stops the Th2 polarization caused by chronic exposure to allergens such as ovalbumin, so that the ratio of Th1/Th2 animals that received the compound (I'), practically does not differ from rats, Brown Norway, which was not exposed to the allergen.

Interestingly, other chronic inflammatory diseases such as atherosclerosis, are associated with Th1-polarization (as opposed to Th2 polarization). Diseases of both types of violation of the balance of cytokines produced by T-helper cells, described as the main pathogenetic factor of development of chronic inflammatory component of the disease. In models of disease associated with Th1-polarization (such as atherosclerosis), applicants previously observed a significant shift towards Th2 in the treatment with the use of BSCl, such as compound (I')described in this document. Mice with homozygous deletion of the gene encoding apoE (apoE-/-mice), developing multiple foci of lipid damage inside blood vessels, on the same at normal power, and such animals characterized by the ratio of Th1/Th2 is approximately equal to 8 (compared to 3.2 in use as control wild-type mice lines C57BI6). However, subsequent treatment with the use of BSCl for 3 months (aged 12 to 24 weeks - the period of life during which development occurs most lipid lesions lesions of blood vessels) normalizes the ratio of Th1/Th2 (and in this model at very high doses, even causes Th2-polarization). These data and data obtained from the study of models of inflammation of the lungs, which is caused by injection of ovalbumin, together show that the use of BSCl can normalize or to restore the balance of the synthesis of cytokines by T-helper cells, regardless of the defect underlies the pathology: Th2-polarization (as in asthma) or Th1-polarization (as in atherosclerosis). According to the applicant, currently, inhibitors of chemokines wide spectrum of action are the only agents that are characterized by such "restoring the balance" effect on the population of T-helper cells. These mechanistic view in the future can confirm (together with data on the efficiency, which were obtained by studying the numerous models of various diseases in animals with inflammatory component), our statement about h what about the BSCl, and in particular the compound (I), stated in this document (as a result of unexpectedly superior pharmacokinetic parameters and bearsdley this connection), are useful as drugs for the treatment of an unusually wide range of conditions associated with an inflammatory component.

Example 7. The comparison substance (I') and close structure analogs

After identification of the compound (I) as having unexpectedly superior pharmaceutical properties, especially with respect to its pharmacokinetic parameters compared with other acylaminoalkyl BSCl, then the applicants conducted a direct comparison of the connection with the closest structural analogues, which were described earlier.

The original set of compounds that were compared in examples 1-6 was so that they are structurally more different, in the framework of the previously described acylaminoacyl BSCl. On the basis of the structure of the compound (I), other compounds having a 6-membered lactam end groups, were included in the study, although there is no particular reason to assume that these compounds generally have the advantage (or, conversely, less efficient) compared to compounds containing 7-membered lactam ring (see WO 2006/134385 for a wide compare inogo analysis acylaminoalkyl analogues with different ring sizes in relation to their effectiveness as BSCl in vitro). Similarly, in the set of investigated compounds included only example of connection with sulfonamidnuyu communication (IV) as different from other molecules with amide bond.

In respect of end groups (tailgroups) in the list of the studied compounds were included two simple alkyl groups (both pivol) and three cyclic alkyl groups (methylcyclohexyl group and two groups adamantane, one of which was replaced). All five compounds had a well-established confirmed double substitution in position 2'2 tetrahedral angular relationship to the Central carbon atom (in position 2 relative to the amide carbonyl cardonia, 4 carbon atoms, associated with the Central carbon atom in the tetrahedral structure). It was previously shown (WO 2006/016152)that the presence of this double substitution at position 2'2' at the Central carbon atom gives BSCl more efficient compared to linear alkyl terminal groups or other entities that do not have four carbon atoms, linked to the Central carbon atom it is in the tetrahedral structure.

The result of these end groups in the original set compounds were selected with this double substitution in position 2',2', in order to ensure proper efficiency in vitro, n is also a requirement for a similar acceptable physical properties of the resulting molecules. For example, expected that the presence adamantanol, tsiklogeksilnogo group and short chain branched in position 2'2 alkyl groups will lead to the fact that the compounds will be in crystalline (or semicrystalline) solid form and to have acceptable solubility in water. In contrast, other possible end groups, such as dimethyldodecylamine group in position 2',2' (which otherwise corresponds to the desired configuration of carbon atoms to the Central carbon atom in position 2), were excluded from the list on the grounds that in the solid state are waxy, amorphous substances, not having a solubility in physiological buffers. These properties are the reason why these compounds are less suitable for pharmaceutical development even to determine their ADME and Toxicological properties, because of the obvious difficulties in handling these compounds, for example, when writing the formula of the drug or pelletizing, and the presence of such features increases the likelihood of undesirable ADME parameters such as accumulation in adipose tissue in constant use or insufficient bioavailability for oral use.

Thus, these five compounds studied in examples 1-6, had approximately equal the s chances to be suitable for pharmaceutical development. To determine ADME properties was not possible to predict which of these compounds is the best.

When the research described in examples 1-6 was completed, however, it became clear that compound (I) is significantly and unexpectedly superior to other studied compounds. Since the compound (I) was the only compound in this set, which had a 6-membered ring, it is possible that the pre-emptive properties were due to the presence of the specified lactam ring. In order to confirm or refute this, the authors of the present invention conducted a review of examples of all previously described acylaminoalkyl compounds with activity of inhibitors of chemokines broad-spectrum, and chose the two compounds most similar in structure to compound (I). From all previously known compounds with BSCl activity, these two compounds were characterized by the most similar structure to the compound (I).

Selected compounds were represented as 3-(2',2'-dimethyldodecylamine)-tetrahydropyridine-2-he (example 3 of WO 2006/134385; here the connection VI) and 3-(adamantane-1-carbylamine)-tetrahydropyridine-2-he (example 1 of WO 2006/134385; here the connection VII):

Danyushevsky, as the compound (I)containing 6-membered lactam ring, amide linker, a double substitution in position 2',2' to 4 carbon atoms, linked to the Central carbon atom in the 2-nd position relative to the amide carbonyl group, which form an important tetrahedral structure.

Conducted a pharmacokinetic analysis of the compounds (VI) and (VII) in rats using exactly the same protocols that were used previously in the study of compounds (I)to(V) in Example 1. It should be noted that when conducting these experiments used the S-enantiomers of both compounds (VI) and (VII), as expected, that they will be more active enantiomers, as the others described to date acylaminoalkyl BSCl.

Results

Unfortunately, the compound (VI) represented difficultly soluble waxy solid. It was hard to get the drug of the compound (VI)that does not contain solvent, but over time, this drug was obtained by substantially drying for many weeks. However, the resulting sample was completely insoluble in 5% DMSO in saline, which was used as a carrier for intravenous administration in the study of other compounds. Therefore, in the study of compound (VI) was necessary to use different media for Thu who would prepare the solutions, suitable for intravenous administration. The compound (VI) was dissolved in 50% PEG 400/20% solutol/30% solution of sodium chloride for intravenous injection. All connections, including (VI), suspended (or dissolved) in a 1% solution of carboxymethyl cellulose in saline solution for oral dosing. Although a comparison of values of bioavailability for oral use, obtained in experiments using a variety of media is generally not recommended (because the media can have a significant impact on the absorption of the compounds in the gut), however, it is unlikely that the choice of medium significantly influenced the values of elimination half-life after intravenous administration of the compound (VI) when compared with other compounds, such as (I).

The parameters of a simple single chamber pharmacokinetic model are shown in Table 7. Data for compound (I') from Table 1 above for comparison. It is obvious that both compounds (VI) and (VII) are significantly less effective than compound (I'): clearance is more than 10 times higher, and the action for oral use in 25-50 times lower than when using the compound (I')

Bioavailability (%)t1/2(minutes)Clearance (ml/min/kg) The volume of distribution at steady state (l/kg)Exposure (min·ng/ml)Bioavailability (%)
(VI)1646of 40.31,9511000
(VII)352031,10,732500
(I')691962,60,7939000

Table 7. Pharmacokinetic parameters selected acylaminoalkyl BSCl with six rings. Determined the bioavailability for oral use (bioavailability, %), the main half-life from plasma (t1/2, min) after intravenous clearance (ml/min/kg), volume of distribution (volume of distribution at steady state (l/kg) and exposure for oral use (AUC 0-t, min·ng/ml) from a simple onecompartment pharmacokinetic model is La each connection, presents the average data obtained for three rats. For compound (VII) Cmaxwas achieved within 15 minutes in accordance with the best absorption, as the compound (I'). The hydrophobic compound (VI) was absorbed more slowly, however, the value of Tmaxamounted to between 60 and 120 minutes.

Indeed, a comparison of the data of Table 7 and Table 1 it is obvious that the potency of the compounds (VI) and (VII) not as good even as compounds (IV) and (V). Despite the fact that these compounds contain a 6-membered lactam ring, as well as the compound (I), compounds (VI) and (VII) have a significantly less pronounced ADME properties and no better than a wide range of other acylaminoalkyl BSCl compounds that have been described previously.

In accordance with higher values clearance for the compounds (VI) and (VII) compared to compound (I') these compounds are characterized by a significantly shorter period of time half-life from plasma than the compound (I') (less than 1 hour, compared with more than 3 hours).

On the basis of the results obtained in this pharmacokinetic analysis, specialists in this field it is obvious that, despite similar numbers of chemical stability, as well as similar to theoretically predicted properties and similarity patterns, however, the compound (I) has a significant advantage compared with the compounds (VI) and (VII), as has 25-50 times the best action for oral use, than any of these analogues 6-membered lactam ring. The applicants concluded that unexpectedly preferential ADME properties of compounds (I') can not be explained neither by the presence of 6-membered lactam rings (because other compounds having a 6-membered lactam ring, such as (VI) and (VII)do not have the same favorable ADME characteristics), nor by the presence of amide linker (as other compounds having an amide linker, including the compound (II), (III) and (V)do not have the same favorable ADME characteristics)or the presence of end groups of pepole (as the compound (IV) has this group and does not have the same outstanding ADME characteristics as the compound (I')). On the contrary, unexpected and greatly predominant ADME characteristics of the compound (I') depend on the particular combination of its structural features, which cannot be predicted.

Example 8. Attempt rational design acylaminoalkyl BSCl. with D properties of the compound (I'). During the investigated ADME properties of compounds (I') the applicants have analyzed the pharmacokinetic properties of a number of different acylaminoalkyl compounds with activity as BSCl in vitro at concentrations nepriemyshami nanomole. The applicant who ate expected to understand what factors determine ADME properties of molecules among this structural class of compounds. Applicants, therefore, tried to learn from experience in order to choose the second acylaminoacyl connection with BSCl activity of the compounds on their structure related to classes of substances, which were previously described with ADME properties, as far as possible close to the properties of the compound (I').

Relatively rapid clearance of the compound (IV) in comparison with the clearance of the compound (I') (see Table 1 above) suggests that the presence of amide linker can be correlated with favorable ADME properties. Applicants, therefore, have limited selection of compounds with the amide linkers.

The compound (II) was characterized by very good absorption, but has undergone rapid transformation in the compound (V) in vivo, presumably under the influence of isoenzyme (isoenzymes) of cytochrome P450 in the liver. The compound (V), apparently, was not subjected to further oxidation (probably due to their electron-withdrawing substituent on the Adamantine ring), but, nevertheless, was eliminated from the body relatively quickly. The absence of a detectable primary metabolites in the urine is a strong argument in favor of the fact that the rapid clearance was mediated by formation of a conjugate in phase II, such as glycerin is d, which rats excreted in faeces. Conjugates of phase II has already been formed on the hydroxyl groups, such as 3-hydroxyadamantane in the compound (V). In the result, the applicants suggested that the derivative of compound (II), which has a stable electron Deputy (in order to prevent hydroxylation Adamantine ring under the action of cytochrome P450 or other hydroxyls or oxidase liver), which was unable to form conjugates phase II, such as halosubstituted can have optimal ADME properties, which is sought by the authors of the present invention.

The result was synthesized and tested compound (VIII)below. Applicants selected chlorine-substituted analog (instead of the fluorine-substituted analogue, for example) because it was well-known compound (see example 8 in WO 2006/016152), which was already available path synthesis. Moreover, the introduction of aliphatic C-F bonds is challenging from the point of view of synthesis and may increase the cost of Europroduct because of the cost of the active pharmaceutical component on the order of magnitude or more

As before (see Example 7), it was decided to prepare and evaluate the S-enantiomer of this compound, since it is known that, in General, S-enantiomers of acylamine amnah compounds are characterized by higher BSCl activity in vitro.

The obvious anxiety that was associated with this molecule, was the stability of the C-Cl bonds. Therefore, applicants investigated the stability of the compound (VIII) in vitro in physiological saline solution, and in the preparation of microsomes of rat liver. Were provided encouraging data, as less than 2% of the compound (VIII) was subjected to hydrolysis (determined by the method of liquid chromatography/mass spectrometry dual ion detection of the hydrolysis product of the compound (V) and the starting compound (VIII)) even after 24 hours at 37°C. Similarly, during the incubation of compound (VIII) in a concentration of 1 µm in conjunction with the preparation of microsomes of rats for 1 hour at 37°C. it was shown that less than 10% of the compound was subjected to degradation compared with the control study, which was conducted in the absence of NADP·H". In the result, the applicants concluded that the compound (VIII) is similar stability in vitro and own ground clearance compared to the compound (I').

Thus, on the basis of theoretical considerations and application of the experience gained in the study of compound (I'), as well as General knowledge for predicting ADME properties, and application of generally accepted methods of screening in vitro, such as stability studies in vitro and own clearance in the preparations of microsomes PE the Yeni, it is expected that the compound (VIII) will have ADME properties comparable to the properties of the compound (I').

Accordingly, the compound (VIII) was subjected to pharmacokinetic analysis in rats using protocols that were used in the study of compounds (I')to(VII) in examples 1 and 7.

Results

The parameters of a simple single chamber pharmacokinetic model are presented in Table 8. Data for compound (I') from Table 1 above for comparison. It is obvious that the compound (VIII) is significantly less effective than compound (I'): the rate of clearance of more than 20 times higher, and found no bioavailability for oral use.

Bioavailability (%)t1/2 (min)Clearance (ml/min/kg)The volume of distribution at steady state (l/kg)Exposure (min·ng/ml)
(VIII)<1%1562,80,7<100
(I')691962,6 0,7939000

Table 8. Pharmacokinetic parameters for the selected lactam BSCl. Bioavailability for oral use (bioavailability, %), the main half-life from plasma (t1/2, min) after intravenous clearance (ml/min/kg), volume of distribution (volume of distribution at steady state (l/kg) and exposure for oral use (AUC 0-t, min·ng/ml) from a simple single chamber pharmacokinetic model for each connection shows the average values obtained for the three rats. For compound (VIII) the concentration of the analyte was below the limit of quantitation (~2 ng/ml) at all time points after oral administration.

In conclusion, the compound (VIII), which applicants are selected on the basis of theoretical considerations and the results of tests carried out in vitro, with an optimal ADME properties, however, were among the least suitable acylaminoalkyl compounds, at least among those for which you have determined the pharmacokinetic parameters for the development of pharmaceutically active substances. This connection is characterized by rapid clearance, what is the cause of the short half-life from plasma, was also shown the absence of any kind of bioavailability for oral use.

The degree of excellent is the navigation of the compound (I') above the other connections shown in Figure 10. The figure shows the graph for the two pharmacokinetic parameters (elimination half-life from plasma and effect for oral use) for each BSCl compounds were analyzed pharmacokinetic parameters in rats. The compound (I') lies far from the cluster representing the overall poor ADME properties of compounds in this structural space (and is not atypical for many generally unrelated classes of compounds are candidates for drug development)that indicates that the compound (I') an unexpected and unusual way has advantages.

Example 9. Comparison of S - and R-enantiomers of the substance (I)

It was shown that the compound (I') has an unexpected and significantly superior ADME properties (see example 1). This compound has an asymmetric carbon atom (at the junction of the ring with the amide linker) and can therefore exist in the form of two stereoisomers: S-enantiomer, designated as compound (I'), and the opposite R-enantiomer labeled compounds R(I).

As with all other described to date acylaminoalkyl BSCI S-enantiomer of compound (I) is more effective when applied as a BSCI in vitro than the corresponding R-enantiomer. For this reason, in many experiments have studied the compound (I'), instead of isolated R-enantiomer or mixture of enantiomers. However, unlike that in this case ADME properties associated with the primary pharmacological activity. Indeed, for many compounds ADME properties of pairs of enantiomers are very similar, because (in many cases) the physical properties of these compounds are particularly dominant in determining the distribution in vivo, and the physical properties of the enantiomeric pairs are identical.

Therefore, applicants examined whether R-enantiomer special preferential ADME properties of compounds (I'). Since the usefulness of any particular compound depends on the strength of its primary pharmacological action (action) and the distribution of the molecules of this compound in vivo (because, in order for the connection to be effective, the molecule of the active substance must be present in the targeted area-target areas target) in sufficient concentrations to be of pharmacological action), so the connection which is characterized by a lower activity, but more predominant ADME properties, may be more effective (or to have more action in vivo)than a connection that is taken as the comparison drug, which has more force action, but less desirable ADME properties.

Thus, applicants have synthesized from the new R-enantiomer of compound (I) (exactly as described here for the isolated S-enantiomer, but the substitution of the original material S-ornithine was carried out using the commercial preparation of R-ornithine high stereochemical purity) and determined the pharmacokinetic properties of the given active compound on rats using the same methods of analysis described above (see Examples 1, 7 and 8).

Results

The parameters of a simple single chamber pharmacokinetic model are shown in Table 9. Data for compound (I') from Table 1 are shown for comparison. It is obvious that the compound R-(I) has, to a large extent, unexpectedly excellent ADME properties, shows the compound (I'). As in the case of (I'), compound R-(I) is also characterized by a significant bioavailability for oral use and are affected after administration of a single oral dose of 3 mg/kg is more that is more than 5 times higher than the dose the next best compounds studied to date (see substance (V) in example 1; table 1).

td align="center"> Exposure (min·ng/ml)
Bioavailability (%)t1/2(minutes)Clearance (ml/min/kg)The volume of distribution at steady state (l/kg)
R(I)84818,60,9295000
(I')691962,60,7939000

Table 9. Pharmacokinetic parameters of isolated R - and S-enantiomers of compound (I). Bioavailability for oral use (bioavailability, %), the main half-life from plasma (t1/2, min) after intravenous dose clearance (ml/min/kg), volume of distribution (volume of distribution at steady state (l/kg) and exposure for oral use (AUC 0-t, min·ng/ml) from a simple single chamber pharmacokinetic model for each connection, presents average values measured in three rats. In both cases, the maximum concentration in plasma is usually observed after 30 minutes, which corresponds to good absorption.

Although the compound R-(I) shares largely unusually predominant ADME properties of the compounds (I'), nevertheless, there are some statistically significant differences. The most pronounced of these differences is the increased plasma clearance (~3 times higher in compound R-(I) compared to (I')), which leads to the fact that the half-life from plasma after intravenous injection of 2 times shorter, and impacts after the application of a single oral dose of 3 times lower.

The clearance of the compound R-(I) is close to the speed of glomerular filtration in rats (typically report the amount of 9 ml/min/kg), while the clearance of the compound (I') is significantly lower. One possible explanation of the obtained data is that the compound (I') is subjected to active reabsorption, probably in the distal tubules of the nephron, and this re-absorption is selective with respect to the S-enantiomer compared to the R-enantiomer. If, as is likely, the cause of sudden and vysokopreosviaschennij ADME properties of compounds (I') is partly reuptake in the nephron through the conveyor, is able to distinguish between the S-enantiomer, and R-enantiomer, then this explanation emphasizes the unpredictability of ADME properties and further demonstrates that the discovery is described in this document, is not obvious.

However, R-(I) is the predominant compound in accordance with this invention (as shown on the indoor range figure 10, which illustrates that it is closer in ADME properties to the compound (I'), than to a General class acylaminoalkyl compounds). In conclusion, therefore, it is shown that both compounds R(I) and (I') and consequently a more General connection (I) are characterized by unexpected and significant preferential ADME properties compared with other acylaminoalkyl BSCl, which were previously described. Moreover, compound (I') slightly, but significantly, in General, superior to the compound (I), and thus, is preferable.

1. The use of the compounds of formula (I) or pharmaceutically acceptable salts of the compounds for the preparation of medicines intended for the treatment of inflammatory States:

2. The use of the compounds of formula (I') or pharmaceutically acceptable salts of the compounds for the preparation of medicines intended for the treatment of inflammatory States:

3. Pharmaceutically acceptable composition for treating an inflammatory condition, which contains as active substance a compound of the formula (I) or pharmaceutically acceptable salt of the compounds and at least one pharmaceutically acceptable excipient and/or carrier:

4. Pharmaceutically acceptable composition for treating an inflammatory condition, which contains as active substance a compound of the formula (I') or a pharmaceutically acceptable salt of the compounds and at least one pharmaceutically acceptable excipient and/or carrier:

5. The compound of General formula (I):

6. The compound of General formula (I'):

7. The use of the compounds of formula (I) or (I') according to any one of claims 1 or 2, according to which an inflammatory condition selected from the group including: autoimmune diseases, disorders of the blood vessels, infection or viral replication, asthma, osteoporosis (low density bone mineral substances), tumor growth, rejection of transplanted organ and/or delaying the functioning of the graft or organ, a disorder characterized by excessive levels of TNF-α, psoriasis, skin damage, disorders caused by intracellular parasites, neuropathic pain, allergies, Alzheimer's disease, the response induced by antigen, suppression of the immune response, rheumatoid arthritis, multiple sclerosis, lateral Amyot-trophic sclerosis, fibrosis, and formation of adhesions.

8. The use of the compounds of formula (I) or (I') according to claim 7, according to which the criminal code of the related inflammatory condition is an asthma, allergic rhinitis or chronic nonspecific lung disease.

9. The use of the compounds of formula (I) or (I') according to claim 7, according to which the specified inflammatory condition is the formation of adhesions after gynecological or General surgery.

10. The use of the compounds of formula (I) or (I') according to claim 7, according to which the specified inflammatory condition is a renal disease, including diabetic nephropathy.

11. A method of treating, reducing the severity of the condition or prevent the development of symptoms of inflammatory conditions (including adverse inflammatory reaction to any agent), according to which the patient is administered a compound, composition or the medicinal agent according to any one of claims 1 to 10 in number, have anti-inflammatory action.

12. Pharmaceutically acceptable composition according to claim 3 or 4 for use in treating, reducing the severity or prevention of the symptoms of inflammatory conditions.

13. Pharmaceutically acceptable composition according to item 12, wherein the specified condition selected from the conditions listed in any of claims 7 to 10.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula I or use thereof to prepare a medicine for treating depression, anxiety or both: or pharmaceutically acceptable salts thereof, where m is 0-3; n is 0-2; Ar is: optionally substituted indolyl; optionally substituted indazolyl; azaindolyl; 2,3-dihydro-indolyl; 1,3-dihydro-indol-2-one-yl; optionally substituted benzothiophenyl; benzothiazolyl; benzisothiazolyl; optionally substituted quinolinyl; 1,2,3,4-tetrahydroquinolinyl; quinolin-2-one-yl; optionally substituted naphthalenyl; optionally substituted pyridinyl; optionally substituted thiophenyl or optionally substituted phenyl; R1 is: C1-6alkyl; hetero-C1-6alkyl; halo-C1-6alkyl; halo-C2-6alkenyl; C3-7cycloalkyl; C3-7cycloalkyl-C1-6alkyl; C1-6alkyl-C3-6cycloalkyl-C1-6alkyl; C1-6alkoxy; C1-6alkylsulphonyl; phenyl; tetrahydropyranyl-C1-6alkyl; phenyl-C1-3alkyl, where the phenyl part is optionally substituted; heteroaryl-C1-3alkyl; R2 is: hydrogen or C1-6alkyl; and each Ra and Rb is independently: hydrogen; C1-6alkyl; C1-6alkoxy; halo; hydroxy or oxo; or Ra and Rb together form C1-2alkylene; under the condition that, when m is 1, n is 2, and Ar is an optionally substituted phenyl, then R1 is not methyl or ethyl, and where optionally substituted denotes 1-3 substitutes selected from alkyl, cycloalkyl, alkoxy, halo, haloalkyl, haloalkoxy, cyano, amino, acylamino, monoalkylamino, dialkylamino, hydroxyalkyl, alkoxyalkyl, pyrazolyl, -(CH2)q-S(O)rRf; -(CH2)q-C(=O)-NRgRh; -(CH2)q-N(Rf)-C(=O)-Ri or -(CH2)q-C(=O)-Ri; where q is 0, r is 0 or 2, each Rf, Rg and Rh is independently hydrogen or alkyl, and each Ri is independently alkyl, and where "heteroaryl" denotes a monocyclic radical having 5-6 ring atoms, including 1-2 ring heteroatoms selected from N or S, wherein the rest of the ring atoms are C atoms, "heteroalkyl" denotes an alkyl radical, including a branched C4-C7-alkyl, where one hydrogen atom is substituted by substitutes selected from a group consisting of -ORa, -NRbH, based on the assumption that the bonding of heteroalkyl radical occurs through a carbon atom, where Ra is hydrogen or C1-6alkyl, Rb is C1-6alkyl. Pharmaceutical compositions based on said compound are also disclosed.

EFFECT: obtaining novel compounds which can be used in medicine to treat depression, anxiety or both.

14 cl, 1 tbl, 28 ex

The invention relates to the derivatives of acanaloniidae formula (I)

or their pharmaceutically acceptable acid additive salts, where R1means a hydrogen atom or hydroxyl; R2means a hydrogen atom or methyl, X is-O - or-CH2-

The invention relates to new piperidine derivative of formula (I)

< / BR>
where X represents the group< / BR>
Ar represents phenyl, one-deputizing or disubstituted by a halogen atom or (C1-C3)alkyl, R1represents a chlorine atom, a bromine atom, (C1-C3)alkyl or trifluoromethyl, R2represents a group-CR3R4СОNR5R6, R3and R4represent identical radicals selected from methyl, ethyl, n-propyl or n-butyl, or R3and R4together with the carbon atom form a (C3-C6)cycloalkyl, R5and R6represent hydrogen, (C1-C3)alkyl or together with the nitrogen atom form a 4-morpholinyl, or their salts, solvate or hydrate

The invention relates to new derivatives of arylalkylamines, as well as containing their farbkomposition, which can find application in pathological conditions involving the system of neurokinin

The invention relates to new biologically active chemical compounds, in particular to cyclic amino compounds of the formula I

BANwhere In - perederina, piperidinyl or pyrrolidinyl group, each of which may be substituted by a lower alkyl group, lower alkylcarboxylic group, carbobenzoxy, afterburner (lower) accelgroup, phenylketone (lower) alkyl group, phenylcarbamoyl (lower) alkyl group or phenyl (lower) alkyl group, each of which may be substituted by a halogen atom or a lower alkoxygroup; p is 1 or 2; And -- is a bond, or two-, or trivalent aliphatic C1-6hydrocarbon residue which may be substituted by a lower alkyl group, oxo, hydroximino or hydroxy-group;means either simple or double bond, provided that when a represents a bond, thenmeans of a simple bond; R2and R3independent means ATO condition, both R2and R3are not hydrogen atoms, or R2and R3together with the adjacent nitrogen atom form piperidino, hexamethyleneimino, morpholino, pyrolidine, pieperazinove or 1-imidazolidinyl group, each of which may be substituted by a lower alkyl group, a phenyl (lower) alkyl group, a lower alkylcarboxylic group or diphenyl (lower) alkyl group or a physiologically acceptable salt additive acid

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to combined antipyretic drug in form of rectal suppositories. Drug contains paracetamol, dimedrol and papaverine hydrochloride as active components, and lipophilic suppository base Suppocire (Suppocire NA-15) as auxiliary substance, with the following component ratio in g per 1 suppository with 2.0 g weight: paracetamol 0.3-0.5; dimedrol 0.03-0.05; papaverine hydrochloride 0.03-0.05; Suppocire (Suppocire NA-15) - the remaining part (to 2.0 g).

EFFECT: increased efficiency of paracetamol suppositories as antipyretic drugs, with absence of narcotic and psychostimulating substances in their composition.

4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new compounds of general formula I [X]n-Y-ZR1R2, wherein the radicals are specified in the description, effective as heparan sulphate-binding protein inhibitors. The invention also refers to a pharmaceutical or veterinary composition having heparan sulphate-binding protein inhibitory activity for preventing or treating a disorder in a mammal, and to the use of these compounds and compositions for antiangiogenic, antimetastatic, anti-inflammatory, antimicrobial, anticoagulant and/or antithrombotic therapy in a mammal.

EFFECT: preparing the new compounds of general formula I [X]n-Y-ZR1R2, wherein the radicals are specified in the description, effective as the heparan sulphate binding protein inhibitors.

10 cl, 31 ex, 11 tbl, 40 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to an aminopropylidene derivative presented by formula wherein R1 and R2, which may be identical or different, represent hydrogen or a substitute specified in the following (a)-(c), provided the case of both representing hydrogen is excluded: (a) carbonyl substituted with hydroxy, alkoxy or hydroxy alkylamino, (b) carbonylalkyl substituted by hydroxy or alkoxy, and (c) acrylic acid including its alkyl ester, R3 and R4, which may be identical or different, represent hydrogen, alkyl which may be substituted by phenyl or cycloalkyl, or R3 and R4, which together form a heterocyclic ring with a nitrogen atom bound thereto, represent pyrrolidino, piperidino, which may be substituted by oxo or piperidino, piperazinyl substituted by alkyl or penyl, morpholino or thiomorpholino; A means oxo or is absento, B represents canbon or oxygen; one of X and Y represents carbon, while the other one represents sulphur, a part represented by a dash line represents a single bond or a double bond, and a wavy line represents a cys-form and/or a transform. Also, the invention refers to a pharmaceutical composition exhibiting histamine receptor antagonist activity on the basis of said compounds.

EFFECT: there are produced new compounds and pharmaceutical compositions thereof, which can be used in medicine for treating asthma, allergic rhinitis, pollen allergy, hives and atopic dermatitis.

10 cl, 12 tbl, 58 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, and concerns a pharmaceutical composition for treating skin diseases. The composition contains a combination of methylprednisolone aceponate and glycoceramides and a glycoceramide complex containing cholesterol in the amount of 1-3%, and phospholipids in the amount of 25-34%, and excipients. A method for preparing the composition consists in the fact that methylprednisolone aceponate is introduced into an emulsion base containing the glycoceramide complex in the form of a solution to form a coagulation structure.

EFFECT: new pharmaceutical composition is characterised by a wide spectrum of pharmacological properties, stability, uniform distribution of the active ingredients.

9 cl, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a compound of formula (I) presented below wherein the radicals and symbols have the values presented in the patent claim, and/or to its racemate, enantiomer, diastereomers or its pharmaceutically acceptable salts and/or esters. The invention also refers to a method for preparing it, using it in preparing a drug preparation and to drug preparations containing the compound of formula (I).

EFFECT: compound of formula has analgesic action and may be used as an active compound for pain management.

20 cl, 3 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound having formula (I): wherein each of the groups R1, R2, R3 are independently H or C1-4 alkyl group or C2-4 acyl group; each of the groups R4 and R5 are independently H or the group of formula -SO3R6, wherein R6 is H or the C1-4 alkyl group or the C2-4 acyl group; provided at least one of the groups R4 and R5 is a group having formula -SO3R6, or a pharmaceutically acceptable salt thereof. The invention also refers to a method for preparing the specified compounds of formula (I) and a pharmaceutical composition possessing activity in the inhibition of IL-1 anti-inflammatory cytokines based on these compounds.

EFFECT: there are produced new compounds and pharmaceutical composition on their basis which can find application in medicine for treating an inflammatory or autoimmune condition.

17 cl, 4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and immunology. Claimed is medication for treatment and prevention of TNF-dependent disorder, which contains molecule of TNF-binding nanobody, lyoprotector, surface-active substance and buffer, method of such medication obtaining, method of lyophilised preparation reduction and method of analysis of medication-manufacturing process, as well as method and set for treatment or prevention of TNF-dependent disorder.

EFFECT: invention ensures obtaining stable pharmaceutical preparations of TNF-binding nanobodies and can be applied in therapy of TNF-dependent diseases.

30 cl, 12 ex, 3 tbl, 32 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula I , wherein R2 means methyl, Y means carbon or nitrogen, and R1, R3 and R4 have the value specified in the patent claim. Also, the invention refers to a pharmaceutical composition for the use as a pharmaceutical drug having activity of a phosphatidylinositol-3-kinase inhibitor, to the use of the compounds of formula I for preparing the pharmaceutical drug for treating a disease mediated by phosphatidylinositol 3-kinase and to a method for preparing the compounds of formula I .

EFFECT: preparing the compounds of formula I possessing activity of the phosphatidylinositol-3-kinase inhibitor.

10 cl, 5 tbl, 51 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a method of intraocular pressure reduction and to a method of pain control involving the administration of a therapeutic compound representing , or its tautomer or stereoisomer forms wherein X represents NH; n is equal to 2 or 3; Ra, Rb, Rc and Rd represent stable functional groups independently consisting of: 0 to 4 carbon atoms, 1 to 9 hydrogen atoms; and Re represents H or C1-4alkyl. Furthermore, the invention refers to a compound represented by formula , or to its tautomer or stereoisomer form.

EFFECT: new compound is prepared; besides, the known compounds to be applied in pain and glaucoma control are studied.

8 cl, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new compounds of formula (I) or to its stereoisomers, or to a pharmaceutically acceptable salt, wherein Ra represents H or (C1-C6)alkyl; Rb is specified in an optionally substituted group consisting of -(CH2)n-aryl, -CH(CH3)-aryl, -(CH2)n-arylaryl, -(CH2)n-arylheteroaryl, -(CH2)n-(C3-C8) cycloalkyl, -(CH2)n-heteroaryl, -(CH2)n-heterocyclyl and -(C3-C8) cycloalkylaryl; or Ra and Rb taken together with a nitrogen atom form 2,3-dihydro-1H-isoindolyl, decahydroisoquinolinyl, optionally substituted piperidinyl or optionally substituted pyrrolidinyl; Y is specified in an an optionally substituted group consisting of 5,6,7,8-tetrahydro[1,6]naphthyridinyl, -NH-(CH2)n-heterocyclyl, wherein NH is attached to carbonyl, and -heterocyclylaryl, wherein heterocyclyl is attached to carbonyl; and n is equal to 0, 1 or 2; wherein each heterocyclyl represents an independent non-aromatic ring system containing 3 to 12 ring atoms, and at least one ring atom specified in a group consisting of nitrogen, oxygen and sulphur; wherein each heteroaryl represents an independent non-aromatic ring system containing 3 to 12 ring atoms and at least one ring atom specified in a group consisting of nitrogen, oxygen and sulphur; and wherein the optional substitutes are independently specified in a group consisting of C1-C6-alkyl, C1-C6-alkoxy, halogen, CN, CF3, OCF3, NH2, NH(CH3), N(CH3)2, hydroxy, cyclohexyl, phenyl, pyrrolidinyl, -C(O)-piperidinyl, -N(H)-C(O)-C1-C6-alkyl and N(H)-S(O)2-C1-C6-alkyl. The invention also describes a pharmaceutical composition having chemokine receptor antagonist activity and a method of treating such diseases, such as rheumatoid arthritis, psoriasis, lupus, etc.

EFFECT: there are prepared and described new chemical compounds that can be used as chemokine receptor antagonists and, as such, may be used in treating certain pathological conditions and diseases, particularly inflammatory pathological conditions and diseases and proliferative disorders and conditions, eg rheumatoid arthritis, osteoarthritis, multiple sclerosis and asthma.

23 cl, 59 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to an aminopropylidene derivative presented by formula wherein R1 and R2, which may be identical or different, represent hydrogen or a substitute specified in the following (a)-(c), provided the case of both representing hydrogen is excluded: (a) carbonyl substituted with hydroxy, alkoxy or hydroxy alkylamino, (b) carbonylalkyl substituted by hydroxy or alkoxy, and (c) acrylic acid including its alkyl ester, R3 and R4, which may be identical or different, represent hydrogen, alkyl which may be substituted by phenyl or cycloalkyl, or R3 and R4, which together form a heterocyclic ring with a nitrogen atom bound thereto, represent pyrrolidino, piperidino, which may be substituted by oxo or piperidino, piperazinyl substituted by alkyl or penyl, morpholino or thiomorpholino; A means oxo or is absento, B represents canbon or oxygen; one of X and Y represents carbon, while the other one represents sulphur, a part represented by a dash line represents a single bond or a double bond, and a wavy line represents a cys-form and/or a transform. Also, the invention refers to a pharmaceutical composition exhibiting histamine receptor antagonist activity on the basis of said compounds.

EFFECT: there are produced new compounds and pharmaceutical compositions thereof, which can be used in medicine for treating asthma, allergic rhinitis, pollen allergy, hives and atopic dermatitis.

10 cl, 12 tbl, 58 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of medicine, namely, to antidiabetic composition. Method of obtaining antidiabetic pharmaceutical composition includes preparation of trituracio mixture of active substance repaglinide, taken in therapeutically efficient quantity, with complex-forming substance, solubiliser and colloid silicon dioxide, further addition of filling agent, disintegrant and lubricant, and tabletting by method of direct pressing.

EFFECT: pharmaceutical composition in form of tablet, obtained by claimed method, is characterised by high degree of active substance release, satisfactory strength and has storage term longer than 2 years.

10 cl, 1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to pediatric neurology, and can be used for rehabilitation of neurological disorders in children in case of neuroinfections. For this purpose, at the background of adequate complex and pathogenetic therapy, parenteral introduction of actovegin in acute period of disease additionally from the first days of disease cytoflavin is introduced intravenously by drop infusion in dose 0.6 ml/kg or 10 ml per day for 3-5 days, elcar perorally in dose 70-100 mg/kg of weight per day for 3-4 weeks. In the period of early reconvalescence pantogam is additionally introduced perorally in dose 50-70 mg/kg of weight per day for 4 weeks. In case if multifocal affection of brain substance is present, gliatilin is introduced intravenously by drop infusion in dose 1 ml per 5 kg of body weight per day in combination with intramuscular introduction of ipidacrine in dose 5-15 mg per day for 7-10 days, after that gliatilin perorally in dose 50 mg/kg of weight per day together with ipidacrine inside in dose 1 mg/kg per day for 4 weeks.

EFFECT: method makes it possible to improve disease outcome due to reduction of frequency of residual neurological deficiency formation with reduction of term of hospital treatment.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel medication for treatment of purulent-inflammatory processes of skin and mucous membranes of different etiology. Medication is made in form of gel and contains the following ingredients with the following ratio of components in g per 100 g: biocomplex of metronidazole with zinc 0.5-2.0, biocomplex of furacilinum with copper 0.1-0.2, trimecaine 3.0-5.0, vitamins (vitamin A, vitamin E, vitamin PP, vitamin C or their mixture) 0.1-1.0, dimexide 10.0, hydrogel of methylsiliconic acid 1.0-5.0, hydrophilic base 3.0-7.0, water 82.3-69.8.

EFFECT: extension of assortment of wound-healing medications.

2 tbl, 1 ex

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