New drug preparation for inhibiting aggregation of proteins involved in diseases related to aggregation of proteins, and neurodegenerative diseases

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound presented by formula (E) , wherein X, Y and L are independently non-directionally specified in -C(R1)(R2)-, -C(R3)=, -N(R4)-, -N= and -O-; M and Z are independently non-directionally specified in ; ---- means an optional double bond; R1, R2, R3, R4 and R6 are independently specified in hydrogen; C1-4 alkyl; group -C1-4 alkylene-halogen; group -C1-4 alkylene-OH; Hal is specified in F, Cl, Br and I; RE1 and RE2 are attached to neighbouring carbon atoms, and RE1 and RE2 together non-directionally form the structure -T-(CRE7RE8)n-V-, wherein T is specified in CRE9RE10 and O or NH, and V is specified in CRE9RE10 and O or NH, as well as respective structures comprising a double bond; at least one of T or V represents O or N; RE7 and RE8 represent H or F; RE9 and RE10 represent H; n takes on the values of 1 to 2; RE3 represents C1-6 alkyl group; m takes on the values of 0 or 1; RE4 represents a halogen atom; p takes on the values of 0 or 1; as well as to pharmaceutical diagnostic compositions of the above compound.

EFFECT: preparing the new pharmaceutical compounds.

41 cl, 17 dwg, 2 tbl, 17 ex

 

The INVENTION

The present invention relates to a compound represented by the formula (E). The present invention relates also to the compound represented by formula (E), for use in the treatment or prevention of diseases associated with protein aggregation and/or neurodegenerative diseases. In addition, the present invention relates to pharmaceutical and diagnostic compositions comprising the compound according to the present invention, as well as to the set, including a connection according to the invention. The invention relates also to method of visualization accumulations of aggregated proteins. Also disclosed set for connection with a detectable label according to the present invention.

In the present invention are referred to some documents. The content of the documents cited in this invention (including any descriptions, instructions, manufacturers etc), entered into this invention by reference.

The LEVEL of TECHNOLOGY

M. Ono et al. (Bioorganic & Medicinal Chemistry 16 (2008) 6867-6872) describe some beta-amyloid probes on the basis of 3,5-diphenyl-1,2,4-oxadiazole.

In the US 2007/02760034 describes some of bis - and Tris-dihydroxyaryl compounds and their methylenedioxy analogs and pharmaceutically acceptable esters, which, allegedly, are suitable for treatment sinall is Opti.

In WO 2008/131148 describes specific diphenylchloroarsine derivatives and their use for binding and imaging amyloid plaques.

Heterocyclic compounds, which are applicable as NURR-1 activators described in WO 2004/072050.

Ethylene glycol or propylene glycol with a radioactive label is used as group-label in compounds that can be used for visualization of tissues, as described in WO 2007/002540.

In WO 98/17652 describes several derivatives of oxadiazole, which, as stated, are suitable for the treatment of neurodegenerative disorders and cerebral ischemia.

There are a large number of neurological and neurodegenerative diseases, many of which are currently not treatable. These diseases include painful conditions such as Parkinson's disease, Huntington's chorea, a disease Hallervorden-Spitze, Alzheimer's disease, senile dementia, disease Creutzfeldt-Jakob, arteriosclerotic dementia, peripheral occlusive cerebral obliterans, dementia with calves levy (DLB), multiple system atrophy (MSA), and many other painful conditions.

Prion diseases, which include diseases such as disease Creutzfeldt-Jakob (CJD), scrapie and bovine spongiform encephalitis (BSE), pathologically characterized by spongiforme money what neratzia brain. Prion diseases are caused by a particular pathogen, which consists mainly of incorrectly folded in the form of beta-sheets aggregated isoform PrPSc membrane beta-glikoproteid PrPC.

Prion diseases are of great interest to public health due to the emergence of BSE. Scientific evidence suggests that BSE was transmitted to humans, causing a new variant of the disease of Creutzfeldt-Jakob disease (nvCJD) (Willet al. 1996, Bruce et al. 1997). It is not known how many people are infected and how many more will be exposed to vCJD infection in the future. Currently available data do not exclude the possibility of the spread of the epidemic, affecting a large number of patients (Andrews et al., 2000). This increases the need to develop effective therapies in addition to the already enacted measures, warning of further spread of the disease. In addition, recent data suggests that it may occur secondary transmission of the disease through blood transfusion (LLewelynet al., 2004).

The main event in the pathogenesis of prion diseases is the conversion of the cellular prion protein PrPC in pathological isoform PrPSc, which is aggregated into large clusters of protein. Such formation of accumulations of PrPSc is the hallmark of the pathogenesis of prion diseases. The present water proof. istva testify, that PrPSc acts as a matrix of this transformation and how neurotoxic means causing neuronal dysfunction and cell death (Prusiner 1998, Giese and Kretzschmar 2001). Therefore, the most promising therapeutic approach to the treatment of prion diseases is the suppression of PrPSc amplification. Data obtained from studies in cell culture and in living organisms, suggests that when the inhibition of the formation of PrPSc may be a clearance of PrPSc (Mallucci 2003). Thus, this therapeutic strategy could also be effective at the end of incubational period and even after developing clinical signs of disease and is of great importance when the address used to combat prion disease of humans.

It has been shown that some compounds are effective in suppressing PrPSc amplificationin vitrosuch as, for example, derivatives of acridine, Congo Red, the porphyrin/phthalocyanine, Cu-60, peptides that destroy the beta layer, and varieties of PrP (Caugheyetal. 1998, Chabryet al. 1998, Demaimay etal. 2000, Horiuchietal. 2000, Perrier et al. 2000, Rudyket al. 2000, Sotoet al. 2000). To date, however, none of these compounds has not found application in the treatment of disease or as a starting compound to produce compounds with high therapeutic efficacy and pharmacological properties.

Connection, identify the new as potential therapeutic agents, were discovered, mostly by accident. There are several methods of testingin vitrosuitable for high-throughput screening of large libraries of compounds to identify potential antiprion medicines. In a recently published studies have proposed two different approaches to systematic screening: one based on yeast (Bach et al. 2003), and in the other are infected ScN2a cell culture (Kocisko et al. 2004, Kocisko et al. 2003). However, these approaches allowed for screening libraries, limited to 2500 and 2000 connections, respectively, and was tedious.

In addition to the use of low molecular weight compounds currently tested three possible approaches. In the first approach to suppress the formation of PrPSc used antibodies against PrP. This method was successfully used in the test cell culture and also in the test mice at intraperitoneal injections (Enariet al., 2001; Whiteet al., 2003). Another approach is the use of CpG oligonucleotides, which, as established, increasing the duration of the incubation period, scrapie-infected mice (Sethiet al., 2002). However, the mechanism of action of this method has not been explained yet. Finally, we study the suppression of expression of PrPC in neurons infected animals or people with the help of the siRNA. It was shown that this method leads to inhibition of the formation of PrPSc in cell cultures (Daudeet al., 2003). All three of these methods have the same drawback, namely the passage of molecules through the blood-brain barrier. Due to this drawback of these approaches is applicable only to prevent the established exposure in peripheral organs, but are not suitable for therapeutic treatment of diseases of the Central nervous system.

Another class of neurodegenerative diseases, the so-called synucleinopathies, characterized by intracellular accumulation of protein clusters, oligomers, protofibrils and fibrils containing mainly α-synuclein. It is believed that in cases of synucleinopathies pathological effects on nerve cells caused by the formation of oligomeric aggregates of α-synuclein and subsequent formation of membrane pores. Examples of synucleinopathies are Parkinson's disease (PD), dementia with calves levy (DLB) and multiple system atrophy (MSA). To date there is no reasonable therapeutic strategies to suppress aggregation of α-synuclein.

Thus, there is a need to identify new compounds that are suitable for the treatment of diseases associated with aggregation of proteins such as prion diseases and synucleinopathies.

So the WMD technical problem, it forms the basis of the present invention is to create compounds for the treatment of prion diseases, synucleinopathies and other diseases characterized by the aggregation of proteins, in particular Parkinson's disease. In addition, there is a need to create compounds that are suitable probes for visualization of clusters of aggregated proteins in the above-mentioned disorders.

DESCRIPTION of FIGURES

1: SAR-map made for DIVERSet 1 and 2, the screening was conducted using 2D-SIFT and testing in cell culture. The map shows clusters of structurally similar compounds (indicated by asterisks or squares), formed from 837 best compounds identified in primary screening in cell culture from the DIVERSet 1 and 2. The clusters, in turn, are arranged so that similar clusters are in close proximity to each other. Symbols representing clusters are the scale, form and colour according to their size and quantitative relationships between activities in SIFT-analysis and cellular structures, respectively, as indicated in the notes. Thus, large clusters containing a large ratio of SIFT-activities and activities in cell cultures, marked with large red stars. Selected five clusters, called the s DPP_1-DPP_5, and shows the connections-prototypes representing these clusters.

Figure 2:All connections, distributed in the clusters identified above (figure 1), presented in figure 2 (A-F) and their activities in various tests. They all belong to the chemical class of derivatives of 3,5-diphenylpyrazine (DPP) (structure DPP shown in figure 1). On fig.2F shows additional cluster DPP_6, which is not contained in the SAR map presented in figure 1, and includes only one connection is active against cell culture, with the N atom attached to the pyrazole nucleus cycle. On fig.2F additionally shows 4 connections from 33 DPP from the DIVERSet 1 and 2, as installed, is not active in cell culture and which, as determined by the DM program, not similar with six connections class DPP. Applicants invention identified these compounds by searching in the library structures DPP.

3: List of compounds that were synthesized according to the methods described in example 2, on the basis of the results of the initial screening and medical chemical considerations. These compounds were tested using different methods of analysis (i.e. SIFT, analyses in models of cell cultures of prion diseases, testsin vivoanimal or biochemical analysis of aggregation of α-synuclein).

4: The impact of treatment on the period and survived the of mice after intracerebral infection RML, scrape. Compounds were administered daily for 14 days starting from 80 days after infection (50 μl of 10 mm connections). In the diagram (A) shows that treatment with compound 10353_F11 increases the duration of survival intracerebrally infected mice (p<0,05). In the diagram (C) shows the average duration of survival after treatment with various compounds. Daily intraperitoneally injection compounds anle138b and sery149 significantly increases the duration of survival after infection RML, scrape (anle138b: p<0,01; sery149: p<0,05). Mean values of survival time is expressed in days ± standard deviation.

5: The impact of treatment on the levels of PrPSc in the spleen of mice administered intraperitoneally infected with the RML scrapie. (A) After inoculation of scrapie-prions mice were treated once daily with compound (25 μl 100 mm connection for intraperitoneal introduction and 50 mg/kg for oral administration). (C) Densitometric analysis of the levels of PrPSc in the spleen in the dot-blot analysis. Treatment with anle138b causes a steady decline in the levels of PrPSc in comparison with the control (p=0.001). (C) Immunohistological analyses of the spleens of scrapie-infected mice. After treatment with anle138b percentage spleens low PrPSc accumulations increases ( + ) and strong PrPSc accumulations are missing the ( +++ ). In (D) shows two sample painted PrPSc accumulations (arrows) in the spleens. The figure on the left shows highly colored PrPSc accumulation ( +++ ), while the right shows PrPSc accumulation with minor staining (quality +). Error bars in the flowcharts show the average value of PrPSc ± standard deviation.

6: Immune blotting and histological analysis of mouse brain. (A) Treatment of mice after intracerebral of inoculation with RML, scrape started after 80 days after infection. Compounds were injected at the indicated time points (25 μl 100 mm connection to I.P. Pavlova. introduction and 50 mg/kg for oral administration). (C) quantification of the levels of PrPSc in brain homogenates prion-inoculated mice at different time points. Treatment with anle138b completely blocks the accumulation of PrPSc in the brain. The amount of PrPSc 106 day is the number identified in day 80. Treatment with anle186b leads to reduced accumulation of PrPSc in the brain of mice. (C) the Change in relative levels of PrPSc after treatment with compounds compared to untreated control at day 80. (D) Histological examination of apoptotic cells (arrow in (E)) in H&E stained thin sections of the brain at the specified time points. The graph shows the average number of apoptotic glue is OK ± standard error. In (E) shows the painted area with apoptotic cell (arrow).

7: Increases the duration of survival with the introduction of daily compounds according to the present invention. Mice that were intracerebrally infected with the RML strain of scrapie show a longer survival period before reaching the end stage of scrapie infection.

Fig: Suppression of aggregation of α-synuclein compounds according to the present invention. (A) DPP-connection 351F11 able to inhibit the formation of multimeric complexes of α-synuclein dependent on dose. (B)(C) dose-Dependent inhibitory effect on the aggregation of α-synuclein identified for other DPP-related compounds.

Fig.9: Culture of cells infected with prions, treated with DPP-related compounds according to the present invention. DPP-related compounds showed high reduction of PrPSc in cell culture at low micromolar and even submicromolar concentrations.

Figure 10: The effect of daily treatment with connection anle138b to the accumulation of PrPScand prion pathology in mice infected with the RML scrapie. (A) Slices of the brain stained for PrPSc(top row: bark and hippocam, bottom row: the cerebellum), show that treatment with compound 138b by the accumulation of PrPSccompared to animals, processed DMSO. (C) Quantification of the content of PrPScin brain homogenates of prion-inoculated mice at the indicated time points shows that the accumulation of PrPScin anle138b-treated mice is significantly reduced even after the start of treatment to the last stage of the disease (120 days after infection). (C) Histological determination of the number of apoptotic cells in the cerebellum in H&E stained sections shows that the inhibition of PrPScaccumulation leads to inhibition of the death of neural cells. (D) a Control mouse treated with a mixture of DMSO+peanut butter without compounds show a progressive loss of mass. Treatment with anle138b, since 80 days after infection, prevents the mass loss at ~100 days. Treatment, starting with 120 days after infection, inhibits the mass loss at ~70 days. Error bars in b and C indicate standard error (n=4 mice). Captions to figv also applicable to figs and D.

11: Comparison of different methods of treatment. Treatment with anle138b at different times and on different circuits, as shown in this figure, significantly increases the duration of survival after the introduction of the RML scrapie (p<0,01). The average survival period is presented in days ± standard deviation.

Fig: A dose-dependent loads is by the introduction of anle138b in the levels of PrPSc in the brain. C57BL/6 mice were inoculable intracerebral (i.c.) 30 μl of 1% brain homogenate (RNL of scrape). Treatment was started at 80 days after infection with different amounts of anle138b (as shown in the figure), administered orally in a mixture with a buffer DMSO + peanut butter. At 120 days the animals were killed and determined the amount of PrPScin the brain in comparison with the animals slaughtered on day 80 after infection. Error bars show standard error (n=4 mice).

Fig: Quantitative determination of the content of PrPScthe Western blot turns brain tissue from uninfected mice treated 1 mg per day of the compounds of anle138b in a mixture with a buffer DMSO + peanut butter for 1 week. Each slat error shows the error for groups of four mice.

Fig: Pharmacokinetic analysis of anle138b. A single dose anle138b was administered to uninfected C57BL/6 mice, as shown. At various time points after administration of compounds number of compounds were identified in the brain and serum in 2 animals for each time point and experimental groups using LC-MS.

Fig: Inhibition of formation of α-synuclein clusters using different connections. Presents the structure of the compounds, the test results presented in table 2.

Fig: Quantitative determi the bookmark loss of neurons in MRTR-treated mice compared with MRTR-untreated mice using the tyrosine hydroxylase-(TH)-positive cells in the substantia nigra pars compacta (") 50 µm sections, immunoscreening antibody against TN. Every second slice " were analyzed using the software Stereo investigator (MicroBrightfield, Colchester, VT, USA). Immunoalkaline cells were counted using the fractional method using a 20× lens. Stereological counts were performed blindly by two independent researchers.

Fig: Effects anle138c on Abeta aggregation. Abeta aggregation was analyzed using dynamic light scattering. Monomeric and oligomeric Abeta in the absence (upper graph) and in the presence of anle138C (middle graph). The bottom graph shows the size distribution of amyloid fibrillar state Abeta40, quantitatively determined after centrifugation of the sample.

DETAILED description of the INVENTION

The invention is described using embodiments presented in the claims. You should imagine that the combination of all of the preferred embodiments of the invention given hereinafter and in the claims, should be considered as part of the scope of the present invention.

The present invention relates to a compound represented by the General formula (E)

X, Y and L in cycle D non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N=, -N+(R5)=, -O - and-S-;

p> M and Z non-directional independently selected from

- - - - indicates an optional double bond,

Of course, that X, Y, Z, L and M will be selected in accordance with the appropriate valence and stability of the compounds.

R1, R2, R3, R4, R5, R6and R7independently selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH; group-C1-4alkylen-C1-4alkoxy; -C(O)-C1-4of alkyl; and C6-10aryl, where aryl cycle can be optionally substituted C1-4by alkyl or halogen. C6-10aryl group specifically limited and may be selected, for example, from phenyl and naphthyl. The halogen atom can represent F, Cl, Br or I and usually represents F or Cl.

Preferably R1, R2, R3, R4, R5, R6and R7independently selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH; group-C1-4alkylen-C1-4alkoxy; and-C(O)-C1-4the alkyl. More preferably R1, R2, R3, R4, R5, R6and R7selected from hydrogen, C1-4of alkyl; and the group-C1-4alkylene-halogen.

The selection of the Deputy may depend on the intended use of the compounds of formulas is (E). In one preferred embodiment of the invention, at least one of R1, R2, R3, R4, R5, R6and R7(more preferably, at least one of R4, R5and R7) is a group-C1-4alkylene-halogen. This is particularly true if connection should be used as a probe to visualize the accumulation of aggregated proteins, because in this group, you can quickly and efficiently enter detectable label, such as a detectable isotope of a halogen. Examples of distinct isotopes of halogen include18F,125I123I131I77Br and76Br, in particular18F. of Course, you can use detectable isotope of a halogen as any other halogen atoms present in the compounds according to the present invention, such as halogen atoms attached to the phenyl cycle.

Alternative,11C can be used as detectable labels in the compounds according to the present invention.11C may be present, at least one of R1, R2, R3, R4, R5, R6and R7(more preferably, at least one of R4, R5and R7or in any other part of the connection according to the present invention.

In al the alternative preferred embodiment of the invention R 1, R2, R3, R4, R5, R6and R7independently selected from hydrogen and C1-4the alkyl, preferably hydrogen.

Cycle D is not specifically limited. Typical examples of cycle D include

Particularly preferred examples of cycle D are

In the above formulas, R8selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH; group-C1-4alkylen-C1-4alkoxy; -C(O)-C1-4of alkyl; and C6-10aryl, where aryl cycle can be optionally substituted C1-4by alkyl or halogen. Preferably R8selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH; group-C1-4alkylen-C1-4alkoxy; and-C(O)-C1-4the alkyl. More preferably, R8selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen. In one embodiment of the invention R8selected from hydrogen; and (C1-4of alkyl, more preferably represents hydrogen. In an alternative embodiment of the invention R8represents a group-C1-4alkylene-halogen. As indicated above, R8may be labeled with a detectable label, if necessary.

In the above, f is mulah R 9selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH; group-C1-4alkylen-C1-4alkoxy; -C(O)-C1-4of alkyl; and C6-10aryl, where aryl cycle can be optionally substituted C1-4by alkyl or halogen. Preferably R9selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH; group-C1-4alkylen-C1-4alkoxy and-C(O)-C1-4the alkyl. More preferably, R9selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen. In one embodiment of the invention R9selected from hydrogen; and (C1-4of alkyl, more preferably represents hydrogen. In an alternative embodiment of the invention R9represents a C1-4alkylene-halogen. As indicated above, R9can be marked distinguishable detectable label, if necessary.

In an additional embodiment of the invention R8and R9represent hydrogen. In yet another embodiment of the invention R8represents a group-C1-4alkylene-halogen and R9represents hydrogen.

Hal is selected from F, Cl, Br and I, and preferably represents F, Cl or Br, more preferably Cl or Br, more preferably Br.

R E1selected from a hydroxyl group, a C1-6alkoxy and-NRE5RE6.

RE2selected from hydrogen, halogen, a hydroxyl group, a C1-6alkoxy and-NRE5RE6preferably RE2selected from hydrogen, a hydroxyl group, a C1-6alkoxy and-NRE5RE6.

In an alternative embodiment of the invention RE1and RE2together and can array to form a structure-T-(CRE7RE8)n-V-, as well as the corresponding structures in which there is a double bond, if they are attached to adjacent carbon atoms. In this structure, T is selected from CRE9RE10, NH and O, V is selected from CRE9RE10, NH and O. Preferably at least one of T and V represents NH or O. Examples of such structures include-O-(CH2)n-O-, -O-(CF2)n-O-, -O-(CH2)n-CH2-, -NH-(CH2)n-NH-, -NH-(CF2)n-NH-, -NH-(CH2)n-CH2or the corresponding structure, which contains a double bond. For example, if n=1, then-N=CH-NH - represents a structure in which there is a double bond and which corresponds to the-NH-CH2-NH-. Preferably RE1and RE2together form the structure-O-(CH2)n-O-. It is understood that this group is also subject to hydrolysisin vivoto the guy who rossilini groups.

n takes values from 1 to 3; preferably n is 1 or 2, more preferably n is 1.

RE5and RE6independently selected from hydrogen and C1-6of alkyl; preferably RE5and RE6independently selected from hydrogen and C1-4the alkyl.

RE7and RE8independently represent H or F, preferably represent N.

RE9and RE10independently represent H or F, preferably represent N.

Position, in which RE1and RE2attached to the phenyl cycle may vary.

In one embodiment of the invention RE1and RE2independently represent a hydroxyl group or alkoxygroup and attached in the meta - or para-position relative to the carbon atom that binds the phenyl ring cycle D.

In another embodiment of the invention RE1and RE2represent the structure-T-(CRE7RE8)n-V or a corresponding structure in which the double bond is present, and is attached in the meta - and para-position relative to the carbon atom that binds the phenyl cycle with the cycle of D. above the preferred structure determination-T-(CRE7RE8)n-V is similarly applicable to this version of the implementation.

In the third embodiment, domestic the invention R E1represents-NRE5RE6and attached in the para-position relative to the carbon atom that binds the phenyl cycle with cycle D.

In addition to RE1and RE2on the phenyl cycle may not necessarily be present for more substituents RE3. RE3can be a1-6alkyl group or a C5-10aryl group (such as phenyl or naftalina group), preferably1-6alkyl group, more preferably1-4alkyl group. The number of substituents of m are not specifically limited and is usually in the range from 0 to 2, preferably from 0 to 1, usually 0.

May also contain additional substituents RE4. Usually they represent a halogen atom, a C1-6alkyl group or a C5-10aryl group (such as phenyl or naftalina group), preferably a halogen atom or With1-6alkyl group, more preferably1-6alkyl group, most preferably1-4alkyl group. The number of the substituents R are not specifically limited and is usually in the range from 0 to 2, preferably 0 or 1, usually 0.

In some embodiments of does not include the following compounds:

3(5)-(2-hydroxy-5-were)-5(3)-(4-chlorophenyl)pyrazole (DE 4126543: compound 26 in table 1)

ortho-hydroxyphenyl-5 dichloro-3'-4'-phenyl-3 methyl-2 pyrazole (FR 2.104.932: Example IV);

ortho-hydroxyphenyl-5 dichloro-3'-4'-phenyl-3-phenyl-2 pyrazole (FR 2.104.932: Example IV);

These compounds are described as compounds IA-44, IA-47, IA-81, IA-106 and IA-115 in WO 2004/080972.

In other embodiments of the invention these compounds are not excluded.

Preferred examples of the compounds represented by formula (E)include compounds represented by formula (A)

Determine X, Y, Z, M, L, cycle D, m, p and Hal above for formula (E), as applicable to the formula (A).

RA1and RA2each independently selected from hydrogen, halogen, hydroxyl group, With1-6alkoxygroup and-NRA5RA6provided that at least one of RA1and RA2represents a hydroxyl group, With1-6alkoxygroup or-NRA5RA6. Preferably, RA1and RA2independently selected from hydrogen, hydroxyl group, With1-6alkoxygroup and-NRA5RA6.

Alternative, RA1and RA2together array to form a structure-T-(CRE7RE8)n-V-. The definitions for RE1and RE2included in this structure, and in particular, the above definition of RE7, RE8, T, and V, likewise applicable to RA1and RA2forming such a structure.

In one embodiment of the invention RA1and RA2independently represent a hydroxyl or alkoxygroup.

In the second embodiment of the invention RA1and RA2represent the structure-T-(CRE7RE8)n-V or a corresponding structure, which contains a double bond. The above preferred definitions for patterns-T-(CRE7RE8)n-V is similarly applicable to this variant embodiment of the invention.

In the third embodiment of the invention RA1represents-NRA5RA6and RA2represents hydrogen.

In addition to RA1and RA2on the phenyl cycle may not necessarily be present for more substituents RA3. RA3can be aWith1-6alkyl group or a C5-10aryl group (such as phenyl or naftalina group), preferably1-6alkyl group, more preferably1-4alkyl group.

There may be additional substituents RA4. Usually they represent a halogen atom, a C1-6alkyl group or a C5-10aryl group (such as phenyl or naftalina group), preferably the volume of the halogen or 1-6alkyl group, more preferably1-6alkyl group, most preferably1-4alkyl group.

RA5and RA6independently selected from hydrogen and C1-6of alkyl; preferably RA5and RA6independently selected from hydrogen and C1-4the alkyl.

Preferred examples of the compounds represented by formula (E)include compounds represented by the formula (In)

Determine X, Y, Z, M, L, cycle D, m, p and Hal above for formula (E), as applicable to the formula (In).

RB1selected from a hydroxyl group, With1-6alkoxygroup and-NRB5RB6. Preferably, RB1represents a hydroxyl group or a C1-6alkoxygroup.

RB2selected from hydrogen, halogen, hydroxyl group, With1-6alkoxygroup and-NRA5RA6preferably, RB2selected from hydrogen, hydroxyl group, With1-6alkoxygroup and-NRA5RA6.

In one embodiment of the invention RB1represents a hydroxyl or1-6alkoxygroup and RB2represents hydrogen.

RB5and RB6independently selected from hydrogen and C1-6the alkyl, preferably RB5and RB6independently selected from hydrogen and C1-4the alkyl.

On fenil the th cycle in addition to R B1and RB2there may be additional substituents RB3, RB3can be a1-6alkyl group or a C5-10aryl group (such as phenyl or naftalina group), preferably1-6alkyl group, more preferably1-4alkyl group.

There may be additional substituents RB4. Usually they may represent a halogen atom, a C1-6alkyl group or a C5-10aryl group (such as phenyl or naftalina group), preferably a halogen atom or With1-6alkyl group, more preferably1-6alkyl group, most preferably1-4alkyl group.

Preferred compounds according to the present invention include

The definition given above for RE7,RE8and Hal, similarly applicable to these compounds.

R is selected from hydrogen; C1-4of alkyl; the group-C1-4alkylene-halogen; and (C6-10aryl (such as phenyl and naphthyl), where aryl cycle can be optionally substituted C1-4by alkyl or halogen. Preferably R is selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen. In one embodiment of the invention R is selected from hydrogen; and (C1-4of alkyl, bol is e preferably represents hydrogen. In an alternative embodiment of the invention R is a group-C1-4alkylene-halogen. As explained above, R may contain a detectable label, if necessary.

RA7represents H or C1-6alkyl, preferably H or C1-4alkyl.

RA8represents H or C1-6alkyl, preferably H or C1-4alkyl.

RA9represents H or C1-6alkyl, preferably H or C1-4alkyl.

RA10represents H or C1-6alkyl, preferably H or C1-4alkyl.

RB7represents H or C1-6alkyl, preferably H or C1-4alkyl.

The following compounds are particularly preferred because, as installed, are highly effective in the inhibition of protein aggregation or visualization of aggregated proteins:

where Hal represents Cl or Br, preferably Hal represents Br.

Most preferred are the following compounds

Compounds according to the present invention can also be presented in the form of prodrugs, esters, solvate, or salt.

Compounds according to the present invention form salts that are also in the scope of this invention Should be present, the link in the description for the connection according to the present invention includes a reference to salts thereof, unless otherwise stated. The term "salt(s)", when used herein, means acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when the compound contains basic and acid fragment can be formed zwitterions ("inner salts"), which are also included in the scope of meanings of the term "salt(s)"used in this description. Pharmaceutically acceptable (i.e. non-toxic, physiologically acceptable) salts are preferred, but can also be used and other salts, for example, in the stages of separation or purification in the production process. Salts of the compounds according to the present invention can be obtained, for example, the interaction of the compound with the amount of acid or base, such as an equivalent amount, in a medium such as the medium in which the salt precipitates or in an aqueous medium followed by lyophilization.

Compounds that contain a basic fragment, can form salts with various organic and inorganic acids. Examples of the acid additive salts include acetates (such as acetates formed with acetic or trigalogenmetany acid, for example, triperoxonane to what slotow), adipate, alginates, ascorbates, aspartate, benzoate, bansilalpet, bisulfate, borates, butyrate, citrates, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, econsultancy, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyethanesulfonic (for example, 2-hydroxyethanesulfonic), lactates, maleate, methanesulfonate, naphthalenesulfonate (for example, 2-naphthalenesulfonate), nicotinate, nitrates, oxalates, pectinate, persulfates, phenylpropionate (for example, 3-phenylpropionate), phosphate, picrate, pivalate, propionate, salicylates, succinate, sulfates (for example, salts formed with sulfuric acid), sulfonates (e.g., salts that are specified in the description), tartratami, thiocyanates, toluensulfonate, such as tozilaty, undecanoate etc.

Compounds that contain acid fragment can form salts with various organic and inorganic bases. Typical basic salts include ammonium salts, alkali metal salts, such as sodium, lithium and potassium salts, alkaline earth metals such as calcium and magnesium salts, salts with organic bases (for example, organic amines)such as benzathine, dicyclohexylamine, geranamine (formed with N,N-bis(Degi roubier)Ethylenediamine), N-methyl-D-glucamine, N-methyl-D-picamide, tert-butylamine, and salts with amino acids such as arginine, lysine and other Basic nitrogen-containing groups can be quaternidinum using such compounds as the lower alkylhalogenide (for example, methyl-, ethyl-, propyl - and butylchloride, -bromides and iodides), diallylsulfide (for example, dimethyl-, diethyl-, dibutil and dimycolate), halides with a long carbon chain (e.g., decyl-, lauryl-, myristyl and sterilgarda, -bromides and iodides), aralkylated (for example, benzyl and peptibody) etc.

Prodrugs and the solvate of the compounds according to the present invention is also included in the scope of the present invention. The term "prodrug", when used herein, means a compound, which after administration to a subject, undergoes chemical conversion by metabolic or chemical processes to obtain compounds according to the present invention or its salts and/or MES.

The solvate of the compounds according to the present invention include, for example, hydrates.

Esters of the compounds according to the present invention include complex1-6alkalemia, preferably complex1-4alkalemia esters.

Compounds according to the present invention may exist in their tautomeric form (for example, in the form amide is whether simple aminoether). It is implied that all such tautomeric forms are part of the present invention.

Assumes that all stereoisomers of the compounds according to the present invention (for example, connections that may exist due to the presence of asymmetric carbons on various substituents), including enantiomeric and diastereomeric forms included in the scope of the present invention. Individual stereoisomers of the compounds according to the invention can be, for example, essentially free from other isomers (for example, a pure optical isomer or substantially pure optical isomer, having a specific activity) or can be mixed, for example, as racemates or with all other, or otherwise selected stereoisomers. The chiral centers of the compounds according to the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations.

The racemic forms can be separated by physical methods such as fractional crystallization, separation or crystallization of diastereomeric derivatives or separation using a chiral column chromatography. The individual optical isomers can be obtained from the racemate by any suitable means, including, but without limitation, obtaining salt with an optically active acid followed by crystallization.

All spatial isomers of the compounds according to the present invention refers to either as a mixture or in pure or essentially pure form. The definition of the compounds according to the present invention includes a CIS (Z) and TRANS (E) alkylene isomers and CIS - and TRANS-isomers of hydrocarbon cycles or heterocycles.

In this description of the group and the substituents may be selected, subject to receipt of stable fragments and connections.

All the compounds of formula (E) can be provided in the form of a pharmaceutical or diagnostic compositions, which optionally include pharmaceutically acceptable carrier.

Applying system of biochemical analysis based on the method of "scanning intensely fluorescent targets (SIFT), in combination with cellular analysis in cell culture models of prion diseases by the applicants of the present inventionin vitrowas the screening of large libraries of synthetic compounds as inhibitors of the aggregation processes accompanying neurodegenerative diseases, and in particular, prion disease and binucleate, at the molecular level. Such inhibitors may be potentially new therapeutic drugs to treat these diseases.

This analytical system is sure to exceed all of the analysis is the cue system, used to identify new drugs for the inhibition of protein aggregation, degree of automation, speed measurement (75 seconds per sample), the quantities of chemical compounds (only 200 picomoles in the primary analysis), as well as the required number of sample (for example, only the equivalent of 0.2 mg brain from CJD-patient analysis). These relatively low needs sample time and cost effectively assess and analyze such a large number (i.e. 20000) compounds. In addition, the mapping of all data screening in a centralized database and automated analysis allows to effectively evaluate and analyze the relationship "structure-activity". The combination with the methods of screening in cell culture, which were included in the present invention, allows to identify compounds that are active in the biochemical analysis and testing at the cellular level. Thus, the identified compounds that are active not only in terms ofin vitrobut at the cellular level, which, for example, guarantees the right stability and reactivity identified compounds for further development in the applications ofin vivo.

Thus, the applicants of the present invention in this primary screenin the e was identified a series of compounds, the activity which was subsequently confirmed in a series of dilutions to identify compounds that are active in very low concentrations. Compounds identified as active in primary screening were subjected to a cluster analysis, which identified a group of five close clusters (from DPP_1 to DPP_5; figure 1), including highly active compounds derived class 3.5-diphenylpyrazine (DPP) (compare fragment DPP figure 1).

The authors of the present invention have introduced different substituents identified class of compounds to identify related compounds that are suitable as inhibitors of the aggregation processes accompanying neurodegenerative diseases, in particular prion disease, and synucleinopathies at the molecular level. When using such a medico-chemical approach was synthesized a number of additional compounds. These compounds together with the compounds selected in the primary screening were subjected to additional testing, including SIFT analysis, assays based on cell culture experiments on mice in terms ofin vivoand biochemical analyses focused on the aggregation of α-synuclein (see examples). Thus, it was verified the activity of these compounds asin vitroandin vivo. Detecting that the data connection can also effectively inhibit multimeric formation of α-synuclein, is the exact evidence that the identified compounds can function not only as antiprion connection, but also have therapeutic potential for the treatment of synucleinopathies, such as Parkinson's disease, DLB and MSA, directional effect on the disease mechanism at the molecular level. Moreover, the inhibitory activity of these compounds against aggregation of the prion protein and α-synucleinin vitrocan testify to their General protivoraketnoi activity against a broader spectrum of diseases associated with protein aggregation, where the protein is incorrectly formed in a predominantly beta-sheet conformation, forms the basis for the subsequent aggregation of proteins into amyloid fibrils. Thus, these compounds and compounds related to the compounds of the class of DPP derivatives have potential use as therapeutic agents for casatenovo treat the entire list (neurodegenerative) diseases associated with protein aggregation, including, but without limitation, Parkinson's disease, prinou disease, Alzheimer's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal marrow and the cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary't neuropathic amyloidosis, Finnish hereditary systemic amyloidosis.

The present invention relates also to the connection according to the present invention, as well as its prodrug, complex ether, MES or salt for use in the treatment or prevention of a disease associated with protein aggregation and/or neurodegenerative diseases. Additional variants of implementation of the present invention is the use of compounds according to the present invention for obtaining a pharmaceutical composition for the treatment or prevention of diseases associated with protein aggregation and/or neurodegenerative diseases, as well as a method of treatment or prevention of diseases associated with protein aggregation and/or neurodegenerative diseases, which include the introduction of a therapeutically effective amount of compound according to the present invention to a patient in need of such introduction.

The term "aggregation", according to the present invention, tositsa to the formation of oligomeric or multimeric complexes, usually one or more types of proteins, which may be accompanied by integration in the complex biomolecules, such as carbohydrates, nucleic acids and lipids.

The term "protein involved in a disease associated with protein aggregation and/or neurodegenerative disease", when used herein, refers to diseases that are characterized by the presence of aggregated proteins. Such aggregated proteins can form clusters in specific tissues, more preferably, in the nervous tissue or brain tissue. The degree of aggregation depends on the particular disease.

The present invention relates also to the use of compounds according to the present invention, as defined above, to obtain a pharmaceutical composition for the treatment or prevention of diseases associated with protein aggregation, and/or neurodegerative disease.

According to the present invention, the term "pharmaceutical composition" refers to compositions for administration to a patient, preferably a human. The pharmaceutical composition according to the present invention includes compounds defined above, and, optionally, additional molecules that can alter the characteristics of the compounds according to the invention, for example, stabilizing, modulating and/or activating their function is tion. The connection can be in solid, liquid or gaseous form and may be, inter alia, in powder form(s)tablet(s), solution(s) or aerosol(s). The pharmaceutical composition according to the present invention can, optionally and additionally, to include pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solution, water, emulsions such as emulsions oil-in-water, various types of wetting agents, sterile solutions, organic solvents, including DMSO, etc. Compositions comprising such carriers can be obtained by standard methods, well known in this technical field.

The pharmaceutical composition will be obtained and measured way that is consistent with traditional medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, method of introduction, the scheme of administration and other factors known to practitioners. Thus, the term "effective amount" of the pharmaceutical compositions according to the present invention corresponds to the above considerations. A qualified specialist in the art knows that an effective amount of the pharmaceutical compositions, BBO is imich individual, will, inter alia, depend on the nature of the input connections.

The pharmaceutical compositions according to the invention can be administered orally, rectally, parenterally, intracisternally, intrawaginalno, administered intraperitoneally, locally (powders, ointments, drops or transdermal patch), buccal or oral or nasal spray. The term "pharmaceutically acceptable carrier" means a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or auxiliary additives of any type. The term "parenteral", when used herein, refers to modes of administration, which include intravenous, intramuscular, intraperitoneally, intrasternally (sternum), subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition also appropriately introduced with the gradual release of the active substance. Suitable examples of compositions with the gradual release of the active substance include semi-permeable polymer matrices in the form of shaped particles, e.g. films, or microcapsules. Matrix with the gradual release of the active substance include polyactide (U.S. patent No. 3773919, EP 58481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly(hydroxyethylmethacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutiric acid (EP 133988). Pharmaceutical composition with the gradual release of the active substance also includes liposome captured the connection. Liposomes containing pharmaceutical composition to be obtained by methods known in the art: DE 3218121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; patent application Japan 83-118008; U.S. patent No. 4485045 and 4544545; and EP 102324. Typically, liposomes are a single layer of particles of small size (about 200-800 angstroms), in which the lipid content is more than about 30 mol%, moreover, the selected proportion is adjusted to achieve the optimal therapeutic effect.

Pharmaceutical composition for parenteral administration are usually obtained by mixing the compounds of the desired degree of purity in the form of a standard dose for injection (solution, suspension, or emulsion) with a pharmaceutically acceptable carrier, i.e. a carrier that is non-toxic to recipients at the dosages and concentrations and is compatible with other ingredients of the drug.

Usually medications get homogeneous thorough mixing of the components of the pharmaceutical composition with a liquid which carriers or finely powdered solid carriers, either one or the other. Then, if necessary, the resulting product is formed into the desired product. Preferably the carrier is a carrier for parenteral administration, more preferably a solution that is isotonic with the blood of the recipient.

Examples of these carriers diluents include water, saline solution, ringer's solution and dextrose. Applicable non-aqueous diluents, such as non-volatile oil and etiloleat, and liposomes. Media appropriately contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such additives are nontoxic to recipients at the dosages and concentrations and include buffer additives such as phosphates, citrates, succinate, acetic acid and other organic acids and their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, for example, polyalanine or tripeptides; proteins, such as albumin serum, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or text the ins; hepatoblastoma agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as Polysorbate, poloxamer or PEG.

Components of pharmaceutical compositions intended for therapeutic administration, must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 μm and outreach to consumers). Therapeutic components of the pharmaceutical compositions generally are placed into a container with sterile inlet, for example, a bag of IV solution or a bottle with a stopper that can be pierced by a needle for subcutaneous injection.

The components of the pharmaceutical compositions typically will be stored in containers with a single dose or multiple doses, for example, sealed ampoules or vials, as an aqueous solution or in lyophilized form for recovery. As an example, obtain a lyophilized vials of 10 ml fill in 5 ml of sterile-filtered 1% (wt./about.) aqueous solution and the resulting mixture lyophilizer. The solution for infusion is obtained by recovery of lyophilized(s) connection(s) using bacteriostatic water for injection.

The present izobreteyonija also to a method of treatment or prevention of disease, associated with protein aggregation and/or neurodegenerative disease, the method includes the administration to a patient in need of such introduction, a therapeutically effective amount of compound according to the present invention.

The term "therapeutically effective amount", when used herein, refers to a quantity sufficient to achieve the desired biological response. In the present invention, the desired biological response is the inhibition of protein aggregation.

The present invention relates also to a method of identifying compounds with increased efficiency of inhibiting aggregation of a protein involved in a disease associated with protein aggregation and/or neurodegenerative disease, the method includes the stages of (a) contacting labeled Monomeric protein and otherwise labeled unit of the specified protein in the presence of (1) and/or in the absence (2) potential inhibitor of aggregation, which is a derivative of the compounds defined above; (b) the determination of the number of nearby labels, showing the degree of binding Monomeric protein with the unit specified protein; and (C) comparing the result obtained in the presence and in the absence of a specified connection, where the reduction fo the data near the label in the presence of the specified connection is an indicator of the ability of the compound to inhibit the aggregation of the specified protein.

The term "Monomeric protein" refers to the molecular unit consisting of a single (poly)peptide chain with three-dimensional conformation specific to each protein, which preferably is soluble in aqueous solutions is usually to nanomolar, micromolded or millimolar concentrations and can be modified by covalent binding of one or more carbohydrates, carbohydrate derivatives, lipids, phosphate, sulfate, fatty acids and nucleotides with individual amino acids in the chain. Preferably, this modification is the phosphorylation, glycosylation, proteolytic processing, glycation, oxidation and nitration. The term "(poly)peptide", when used in this invention, refers to a group of molecules that includes the group of peptides consisting of up to 30 amino acids, as well as the group of polypeptides consisting of more than 30 amino acids. The term "protein"when used in this invention also relates to a (poly)peptides.

The term "aggregated protein" means ecovalence linked oligomers or multimer one or more types of monomer(s) protein(s) or polypeptide(s)", as defined above, which are characterized by a modified three-dimensional conformation of the complex units of the protein relative to the Monomeric single the C protein and usually low solubility of the complexes in aqueous solutions.

The term "compound for inhibiting the aggregation of a protein" refers to a compound that is capable of preventing the formation of protein clusters and/or which is able to disintegrate or destroy existing clusters of protein, where these compounds derived from the compounds according to the invention precolumn. Preferably, such compounds are developed computer simulation, where the term "computer simulation" means the use of means of virtual screening to search for compounds that bind to Monomeric or aggregated form of the protein, or both. Usually these methods are based on the three-dimensional structure of proteins, preferably proteins crystallized together with the substrate. More preferably, the substrate is replaced by a potential modulator or inhibitor.

The term "labeled ... protein" refers to a protein that is attached label. The specific label may be joined directly or indirectly. Indirect labelling, in particular, relates to labeled (poly)peptides, or more precisely to the labeled antibody. Attach the label can be carried out by a number of methods known to the expert of the art, and are described in standard textbooks (see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1998).

The term "different way mehanny the protein" means, what is aggregated and Monomeric isoforms of the protein attached to different labels. A typical example is the joining "FITC" to the aggregated protein and "Texas red" to the Monomeric protein. Since these labels can be detected by different wavelengths of light, you can determine the number and/or arrangement of the isoforms of the protein. More precisely, the use of different labels, it is possible to quantitatively determine the presence of closely spaced tags, i.e. tags that are found in close proximity to each other.

Defining a number of closely-spaced labels" can be carried out, for example, a single dimension (i.e. specific wavelength) number of single photons of at least two different wavelengths coming from the same small volume element, usually less than 1 femtolitr, sample within a very short period of time, usually less than 100 μs, with subsequent computerized comparison of the corresponding number of photons that can be graphically represented in a multidimensional histogram with one axis for the number of photons of a specific wavelength. Thus, in the case of two wavelengths, the number of photons in a specific period of time can be represented as single points in a two-dimensional histogram of fluorescence intensity.

The term "comparison of the result obtained is of CSOs in the presence and in the absence of the specified connection" means an assessment of the impact of connections on education and/or the number of clusters of proteins. In this description to reduce the number located in close proximity tags more than 10%, more preferably more than 25%, even more preferably more than 50% and most preferably more than 95%, in the presence of a compound that is a potential inhibitor of aggregation, is an indicator of the ability of compounds to inhibit protein aggregation. The term "absence of the specified connection" means that the inhibitor or potential inhibitor was not added in egregiously protein. In special cases it can be used to add a negative control, i.e. compounds which do not have the ability to influence protein aggregation. The term "absence of the specified connection" refers to such cases. Similarly, any of the compounds of the present invention that inhibit protein aggregation, can be used as a positive control in assays to identify new inhibitory compounds. It is obvious that the term "presence" also refers to the quality. For obvious reasons, the compounds mentioned in the present invention, have different effective concentrations. Preferably, the effective concentration is less than 100 microns, more preferably less than 10 μm, even more preferably less than 1 micron.

The method according to the present izaberete the Oia is particularly applicable for the identification of new compounds, capable with high efficiency to prevent aggregation of the protein. This allows the screening of large libraries of derivative compounds and allows high accuracy to identify compounds with inhibitory activity. In accordance with one aspect of the present invention the method is based on fluorescence correlation spectroscopy. In recent years, fluorescence correlation spectroscopy (FCS) has been recognized as a method that allows high-sensitivity analysis of aggregation of the protein at the molecular level in neurodegenerative diseases such as prion diseases (Bieschke and Schwille 1997, Bieschke et al. 2000, Giese et al. 2000, Post et al. 1998). In addition, FCS increasingly miniaturized and automated and becomes a method, adapted for high-throughput screening in the pharmaceutical industry (Koltermann et al. 1998). Fluorescence correlation spectroscopy in its confocal form analyzes the signal fluctuations caused by diffusion of single fluorescently labeled molecules through the open volume element defined by the beam of the excitation laser, focused through a high-aperture lens of the confocal microscope and displayed on counting single photon detector (Schwille et al. 1997). In the most preferred version done by the means of the invention the method according to the present invention based on this technology. This method is suitable for high-throughput screening based on inhibition of, for example, binding of PrPC into PrPSc accumulation or formation of oligomers or protofibrils or fibrils of α-synuclein.

This test system to identify inhibitors of protein aggregation can be used to search for new therapeutic agents for the treatment of neurodegenerative diseases, which is associated with aggregation of specific proteins, such as Alzheimer's and Parkinson's disease. In addition, it should allow for screening potential therapeutic agents for the treatment of all diseases, the pathogenesis of which plays an important role in the formation of multimer regardless of the nature of its components.

In a preferred embodiment of the present invention these labels are fluorescent labels.

Preferably, the label is selected from the group comprising fluorochrome, such as isothiocyanate fluorescein (FITC), rhodamine, Texas Red, Alexa 488, Alexa 647, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, such as32P,35S3H; etc. Label also mo is et to represent the system, consisting of two parts, where, for example, a protein or (poly)peptide or compound according to the present invention anywhereman(o) with Biotin, Heptene etc. with high affinity to a binding partner, for example, adivino, specific antibodies, etc. where the binding partner anywhereman with a detectable label.

In another preferred embodiment of the present invention specified label attached to the antibody or antibody fragment that specifically associated with the specified protein.

The term "specific binding" for antibodies can be described, for example, on the basis of their cross-reactivity. Preferably, the term "antibody specifically associated with"refers to antibodies that do not bind (poly)peptides encoded by protein aggregation, with identity less than 98%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70% and less than 65% (according to calculations by methods known in the art). However, antibodies can also be described or specifically defined by their affinity for binding. Preferably, the affinity for binding includes a binding affinity to the binding, the dissociation constant or Kdis less than 5×10-6M, 10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10 -10M, 10-10M, 5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M, 5×10-15M and 10-15M.

The term "antibody" refers to polyclonal, monoclonal, chimeric, "humanized" antibodies, the antibody is a single chain antibody single chain Fv, an antibody, or fragments of antibodies that are similar to, inter alia, Fab fragments. Antibody fragments or derivatives thereof also include fragments F(ab')2, Fv or scFv (see, for example, Harlow and Lane (1988), (1999), loc. cit.). In the art there are known various techniques that can be used to generate such antibodies and/or fragments. Thus, derivatives (antibodies) can be obtained by using peptidomimetics. Further, techniques described for obtaining antibodies, single chain (see, inter alia, U.S. patent No. 4946778), can also be adapted to generate antibodies with single chain specific for the polypeptide(s) and the hybrid proteins of the present invention. In addition, for the expression of humanized antibodies specific for the polypeptides and hybrid proteins according to the present invention can be used in transgenic animals. Most preferably, the antibody according to this invention is a monoclonal antibody. To obtain monoclonal antibodies can be used any met the dick, which provides antibodies obtained using cultures of stable cell lines. Examples of such methods include a method of hybridoma (Köhler and Milstein Nature 256 (1975), 495-497), the method creamy, the method humanized b-cell hybridoma (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma to obtain monoclonal human antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Surface plasma resonance, which is used in the BIAcore system can be used to improve the efficiency of antibodies against bacterial virus that bind the epitope of the polypeptide according to the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The context of the present invention also provides that the term "antibody" includes genetically engineered construct antibodies, which can be expressed in cells, for example, the construct of antibodies, which can be transfection and/or transformed through, among others, the virus or plasmid vectors. Qualified it is known that in many cases, antibodies may be replaced by other specific binding compounds, such as peptides, eksponirovanie on the surface of a bacterial virus (visual image of the bacterial virus), or isolated (poly)peptides. The antibody or (poly)peptides can be unlabeled or mechen the mi of any stamp, described in this invention. Preferably, the antibodies may be derived from human, mouse, rat, goat or rabbit.

In a more preferred embodiment of the present invention, the specified antibody is able to distinguish between aggregated and Monomeric protein.

The term "capable of distinguishing" refers to an antibody that is specific for Monomeric or aggregated the isoforms of the protein. Preferably, the value of Kdthe specified antibodies in 5 times the value of Kdisoforms of the protein, more preferably 10 times. Therefore, this antibody is able to communicate with one isoform of the protein, while not intrinsically associated with a different isoform.

In another preferred embodiment of the present invention, the number of tags that are in close proximity, is determined using the method of "scanning intensively fluoresceine label (SIFT)" (Bieschke et al. 2000), FRET or confocal visualization of high resolution.

Preferably, the specified confocal visualization of high resolution is carried out using a laser scanning confocal microscope or a microscope, which uses the technology of spinning-disk.

In yet another preferred embodiment of the present invention mentioned mono is Ernie and egregiousness proteins selected from the group including prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, lysozym, fibrinogen, huntingtin, ataxin and other proteins with a poly-Q-sequence, and fragments or derivatives of these proteins. In a preferred embodiment of the invention monomer and egregiousness proteins selected from the group comprising amyloid protein precursor (APP) and alpha synuclein. In a more preferred embodiment of the invention monomer and egregiousness proteins are alpha synuclein.

Preferably, these proteins with a poly-Q-sequence represent proteins containing at least 36 consecutive glutamine residues. More preferably, these proteins c with poly-Q-a sequence selected from the group comprising huntingtin and ataxin.

Preferably, these fragments or derivatives selected from the group modified by phosphorylation, glycosylation, proteolytic processing, glycation, oxidation and nitration. (Poly)peptides described in this invention can contain one or more carbohydrates, proizvodi the x carbohydrates, lipids, phosphate, sulfate, fatty acids and nucleotides, are attached to the individual amino acids in the chain. Preferably, this modification is the phosphorylation, glycosylation, proteolytic processing, glycation, oxidation and nitration.

Protein can be a protein or spinal bespozvonochnykh animal. Preferably, the protein is a protein of a mammal or avian protein. More preferably, the protein of a mammal selected from a protein of a Primate, human, mouse, rat, cattle, pig or sheep. In special cases it may be preferable to use mixed isoforms, i.e, for example, aggregated forms of PrPSc derived from human, and Monomeric forms received from the mouse.

Proteins can be isolated from animal or tissue culture or can be obtained recombinante. The applicants of the present invention provides that the proteins can be chemically modified or processed by enzymes, such as protease or glycosidase, to facilitate the work with them in the analytical system.

In another preferred embodiment of the present invention Monomeric protein is a prion protein and aggregated protein is PrPSc (Prusiner, 1998).

In another more preferred variants of the e implementation of the present invention Monomeric protein is an alpha-synuclein and the specified aggregated protein selected from the group including oligomers, protofibrils or fibrils of alpha-synuclein.

The present invention relates also to a method of identifying compounds with improvedin vivoto be effective in the treatment of diseases associated with protein aggregation and/or neurodegenerative diseases, this method includes (a) the introduction of an investigational compound that is a derivative of the compound, as defined in this invention, in a cell culture or an animal with the protein isoform, which may be subject to aggregation, as defined in this invention; (b) determining the number obnaruzhivaemykh clusters; and (C) the identification of compounds that can reduce the accumulation or formation of clusters of the specified protein.

This method according to the present invention allows testing of candidate connectionsin vivoi.e. in cells within a living organism, or outside of a living organism. Candidate connections, testin vivorepresent, for example, compounds shown in the examples (see below). Testing candidate compoundsin vivogives important additional information, including information regarding toxicity, stability in the presence of complex chemical environment and the ability to reach the location where achieved the desired molecular action.

Preferably the compound is injected in different concentrations to determine the concentration at which it can be achieved the effect on protein aggregation, and to calculate the value of EU50, which is the (molar) concentration of a compound that provides 50% of the maximum possible response for this connection.

The present invention relates also to the use of compounds as defined above, for inhibiting aggregation of the protein in the animal body,in vitroorex vivo.

In a preferred embodiment of the invention the animal is not a person.

The present invention relates also to a pharmaceutical or diagnostic composition comprising the compound according to the invention and, optionally, a pharmaceutically acceptable carrier or excipients.

In accordance with the present invention, the term "diagnostic composition" refers to compositions for the diagnosis of individual patients on their possible response to pharmaceutically acceptable composition or treatment using the pharmaceutical compositions according to the present invention. The term "diagnostic composition" refers to compositions for determining the presence of aggregated proteins, which form the basis of the diseases mentioned above. Diagnostic composition with the according to the invention includes compounds above. The diagnostic composition may further include a suitable buffer additives, enzymes such as reverse transcriptase, thermostable polymerase, etc. Diagnostic compositions can rasfasovyvaetsya in the container or multiple containers.

In a more preferred embodiment of the invention the effectiveness of the compounds further enhanced by derivatization.

The term "derivatization" in accordance with the present invention relates to the production of chemically related compounds that contain a modification in at least one part of the molecule.

In a preferred embodiment of the present invention the specified connection is detected or marked detectable label. In accordance with the present invention it is clear that the connection is detected or marked detectable label, if his presence can be monitored by standard methods, such as NMR spectroscopy, optical detection, positron emission tomography (PET), electron microscopy, magnetic resonance testing (MRI), spectrometry, chromatography, ELISA analysis, detection of radioactive radiation, preferably scintillation counting or gamma counting, preferably PET.

When connect the deposits according to the present invention are intended for use as probes for the visualization of aggregated proteins, in particular, amyloid accumulation, they should be labeled. The specific nature of the label will depend on the method that should be used for visualization. Usually can be used radioactive labels that emit positrons (PET) and have a short half-life, such as18F111C,125I123I131I77Br and76Br, in particular,18F and11C. Due to the short half-life of labeled compounds according to the present invention must be obtained immediately prior to their application for testing. Therefore, the diagnostic composition according to the present invention may also be provided in the form of a kit including the precursor compounds according to the present invention that interact with obtaining the target compound. This set is particularly suitable if the connection according to the present invention contains at least one fragment representing the X, Y or L, which, in turn, represents-N(R4)-, where R4includes a detectable label.

In a preferred embodiment of the present invention compound, which is used for visualization, contains a fragment-N(R4)as X, Y or L, where R4represents a group-C1-4alkylen-Gal is a gene where the halogen atom is radioactive. In another preferred embodiment of the present invention compound, which is used for visualization, contains a fragment-N(R4)as X, Y or L, where R4represents-C1-4alkyl, containing at least one11With the isotope.

A qualified technician will be able to develop ways in which the detectable label can join the compounds according to the present invention. The following diagram may serve as illustrative examples.

2-[18F]Forecastability2applicable as a precursor to the introduction of18F (half-life 109,8 minutes) via toretrieve compounds, including oxygen-, gray -, and nitrogen-containing nucleophiles, or through reaction of methylation, mediated by different metals (R. Schirrmacher et al.,J.Label. Compd. Radiopharm. 2002, 45, 763-774). 2-[18F]forecastability can be synthesized by two-step synthesis by direct nucleophilic substitution of the ethylene glycol-1,2-ditosylate1with the addition of K[18F]/Kryptofix 2.2.2 to receive agent18F-toretrieve2. Nonradioactive reagent2can be used for the synthesis of non-radioactive compounds sery363A, sery363B and sery388B in accordance with the scheme A. the same conditions may apply is camping for the synthesis of radioactive analogues of these compounds.

Scheme And

Can also be used positron emitter11(Half-life 20,38 minutes), which can be entered through a [11C]methyliodide (J. Eriksson et al.,J. Label. Compd. Radiopharm. 2006, 49, 1177-1186) via nucleophilic substitution of the same type. Nonradioactive methyliodide was used for the synthesis of non-radioactive sery392A and sery392B in accordance with the scheme C. the same conditions can be applied for the synthesis of radioactive analogues of these compounds.

Circuit

Although it is preferable to attach a detectable fragment to the cycle D, since such compounds are particularly easy to synthesize, it is not a necessary condition. Equally it is possible to obtain compounds according to the present invention, in which the detectable fragment is in a different position in the molecule.

The present invention provides a method of visualization of the aggregated protein, which comprises the following stages:

(i) introduction the subject of detectable amounts of a composition comprising a detectable labeled compound according to the present invention;

(ii) provide a period of time sufficient to associate the connection with the aggregated protein;

(iii) detection of the connection associated with egregia the protein data.

In a preferred embodiment, the method of visualization of aggregated protein selected from the group comprising amyloid protein precursor (APP) and alpha synuclein. In a more preferred embodiment, the aggregated protein is an alpha-synuclein.

Composition comprising a detectable labeled compound may be administered to a subject by any route of administration described above, such as, for example, oral or parenteral administration. Labeled compound can be administered to a patient and, after the expiration of a period of time sufficient to ensure that the connection has become associated with the aggregated protein, the labeled compound is detected within the patient atraumatic way. Alternatively, the labeled compound can be administered to the patient after a period of time sufficient to ensure that the connection has become associated with the aggregated protein, then the patient tissue is removed and the labeled compound is detected in the tissue separately from the patient. The tissue sample may also be removed from the patient's body before the introduction of the labeled compound in the tissue sample. After a time sufficient for the compound was associated with aggregated proteins, the connection can be found.

Detection methods labeled with the organisations, associated with aggregated proteins, are well known in the art and include, but without limitation, magnetic resonance testing (MRI), positron emission tomography (PET) or computed tomography emission of a single positron (SPECT) for the detection of radioactively labelled compounds. The label, which is entered in the connection depends on the method of detection that should be applied. Thus, for example, if the detection method selected PET, the connection must contain a positron-emitting atom, such as11With or18F.

Visualization of aggregated proteins can also be carried out in such a way that can be determined and the amount of aggregated protein.

In a preferred embodiment of the invention, the disease associated with protein aggregation, characterized by the presence of aggregated forms at least one protein, fragment or derivative, where the protein is selected from the group comprising the prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, secrete lysozyme, fibrinogen, huntingtin, and other ataxin proteins with a poly-Q-placentas is lacking. In a preferred embodiment of the invention the protein is selected from the group comprising amyloid protein precursor (APP) and alpha synuclein. In a more preferred variant of the invention, the protein is an alpha-synuclein.

Preferably, these proteins with a poly-Q-sequence represent proteins containing at least 36 consecutive glutamine residues. More preferably, these proteins with a poly-Q-a sequence selected from the group comprising huntingtin and ataxin.

The specialist in the art it is known that these proteins can exist in various isoforms, including proteins, modified by phosphorylation, glycosylation, proteolytic processing, etc. the Term "at least one ..." refers to the fact, well-known specialist in this area, the disease may be associated with the presence of more than one protein in aggregate form. For example, in Alzheimer's disease can usually be found accumulations of fragments of the amyloid protein precursor (APP) and the accumulation of the Tau protein.

In this description, the term "neurodegenerative disease" includes diseases such as Parkinson's disease, prion disease, Alzheimer's disease, multiple system atrophy, diffuse disease Taurus Leo is, fronto-temporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy. In addition, the term "disease associated with protein aggregation" refers to diseases that occur primarily outside the nervous system, and includes such diseases as primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary't neuropathic amyloidosis and Finnish hereditary systemic amyloidosis.

In another preferred embodiment of the invention the disease is selected from the group including Parkinson's disease, prion disease, Alzheimer's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), Diab is t type II, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary't neuropathic amyloidosis and Finnish hereditary systemic amyloidosis. In a more preferred embodiment of the invention the disease is a disease of Parkinson.

In a preferred embodiment of the invention specified prion disease is selected from diseases of Creutzfeldt-Jakob disease, variant disease Creutzfeldt-Jacob, inherited prion diseases of humans, bovine spongiform encephalopathy (BSE), scrapie.

Finally, the present invention relates to a kit including a compound as defined in this invention, and, optionally, the antibody or antibody fragment that specifically bind with the specified connection; and/or Monomeric or aggregated protein as defined in the present invention; and/or Monomeric or aggregated protein as defined in the present invention, it is not necessarily associated in a complex with the specified connection, and instructions for use in one or more containers.

GENERAL experimental METHODS

Compounds according to the present invention can be obtained using any of the suitable methods of organic synthesis. Many of these techniques are described in detail in L.F. Tietze, Th. Eicher "Reaktionen und Synthesen", 2. Edition (Georg Thieme Verlag, Stuttart, NY, 1991), by T.W. Greene, P.G.M. Wuts "Protective Groups in Organic Synthesis", Third Edition, (John Wiley & Sons, NY, 1999), and in J. March "Advanced Organic Chemistry", Third Edition, (John Wiley & Sons, NY, 1985).

A number of typical examples of methods for producing compounds according to the invention are presented below. These methods are intended only to illustrate the essence of these methods get and are not intended to limit the scope of suitable methods.

Usually the reaction conditions, such as temperature, duration of reaction, solvents, processing methods, etc. are standard in the art for the particular reaction, which should be carried out. These references along with their contents include a detailed description of such conditions. Usually the temperature will be in the range from -80°C to 150°C, the solvents are aprotic and proton and the duration of the reaction will be in the interval from 10 seconds to 10 days. Treatment usually involves clearing any unreacted reagents with subsequent distribution between the system water/organic layer (extraction) and the separation layer containing the product.

Standard methods of synthesis, such as the use of anhydrous reaction conditions (e.g., inert gas), are traditional for this area of technology and will be used when possible.

Modification of each is below Oh schemes leads to various analogs of specific examples of the compounds received below. The explanation below describes appropriate methods of organic synthesis, applicable to such modifications.

In each of the following typical examples of schemes can be helpful to separate the reaction products and/or to separate them from the original substances. Target products of each phase are separated and/or purified (hereinafter - allocated) to the desired degree of homogeneity using methods conventional to the art. Typically, these cues include multiphase extraction, crystallization from a solvent or mixture of solvents, distillation, sublimation or chromatography. Chromatography can include any number of methods, including, for example, exclusion chromatography or ion exchange chromatography, chromatography high, medium or low-pressure chromatography of small samples or preparative thin-layer chromatography, and methods thin-layer chromatography of small samples and flash chromatography.

Another class of methods for isolating includes processing the mixture with a reagent selected to bind or to give the target product, unreacted educt or by-product, etc. other properties, allowing it to allocate. Such reagents include adsorbents, such as activated carbon, molecular sieves, ion exchange media and other Alternatively, the reagents can be an acid in the case of the base material, the base case of acid material, binding reagents, such as antibodies, binding proteins, selective hepatoblastoma substances such as simple crown ethers, liquid/lipid ion extraction, etc.

The selection of appropriate methods of selection depends on the nature of the materials involved, such as boiling point and molecular weight during the distillation and sublimation, the presence or absence of polar functional groups in chromatography, stability of materials in acidic or basic environments with multi-stage extraction, etc. Specialist this area will be able to apply methods most appropriate to achieve the desired allocation.

In particular, the compounds according to the present invention can be obtained in a manner analogous to the methods described, for example, in M. Ono et al. (Bioorganic & Medicinal Chemistry 16 (2008) 6867-6872), WO 2008/131148, WO 2004/080972, WO 2004/072050, WO 98/17652. Alternative ways are also discussed in Examples according to the present invention.

The following examples are intended to illustrate the invention. However, they should not be considered as examples, limiting the scope of the present invention.

EXAMPLES

Example 1: Identification of a new class of compounds is of deposits for inhibiting protein aggregation

To identify inhibitors of the spread of prion diseases are testing two subgroups commercial compound libraries DIVERSet (ChemBridge Corp., San Diego, CA, USA), each containing 10,000 compounds, named applicants invention DIVERSet 1 and 2, the testing is performed using antiprion analysis 2D SIFT (Bertsch et al. 2005) and models of prion disease in cell culture. In both tests the primary inhibitors identify test compounds at a single concentration with subsequent review of the activity in a series of dilutions. Additionally, the identified primary inhibitors of cell culture experience using a different cell line.

2D SIFT screening

To assess the inhibitory effects of drugs on the accumulation of PrPC and PrPSC in high-performance and vysokomarzhinalnoj screening test applicants using the method of "scanning intensely fluorescent targets (SIFT), which is used to install the inverted dual color confocal microscope with single detectors protons for the two colors of fluorescent light. Samples get in 96 - or 384-well titration microplate with sliding glass bottoms. Analytical mixture consists of recombinant mouse PrP (rPrP), monoclonal antibodies (mAb) L42, which does not recognize the mouse is th PrP, but recognize human PrP and PrPSc accumulations derived from CJD human brain. Molecules rPrP and mAb outline of green and red fluorophores, respectively. The binding of certain molecules rPrP and mAb with PrPSc accumulations will lead to the formation of three complexes, exhibiting a large number of red and green attached fluorophores. Such clusters can be identified and analyzed using the SIFT method, because at the same time is called high intensity in both fluorescent channels. The distribution of the intensities of red and green fluorescence can be measured using two-dimensional histograms of fluorescence intensity. In any case, when the analytical system is an inhibitor of Association rPrP and PrPSc, the intensity of the three complexes should be reduced. The color distribution of the clusters in the 2D histogram will then move in the direction of the "red" sectors of this histogram.

Primary SIFT-screening 20000 connections

The above analytical framework is applied to two libraries, each library contains 10,000 different compounds with properties similar to drugs (ChemBridge; “DIVERSet1” and “DIVERSet2”) in 96-well title microplate for primary screening, including negative controls any more the positive compounds and positive controls, containing 17 μm DOSPA, as well as controls without CJD sticks and connections (used to verify the absence of aggregation in mixtures of antibodies and rPrP).

Samples containing DOSPA, show reduced SIFT signal in those sectors that control signals aggregation, predominantly labeled green rPrP. This indicates that these controls less rPrP associated with DJD prion sticks. Because prion sticks marked with red marks antibodies, their fluorescence is still SIFT generates a signal in the "red" sectors. Most of the compounds does not affect the distribution of SIFT signal. But some of the DIVERSet compounds reduced the number of clusters found in "green" sectors. SIFT the curves are shifted in the direction of DOSPA controls. Thus, the compounds can be considered as the primary candidate connections in potential antiprion medicines. From time to time, some technical problems lead to artifacts that make strange result obtained on all title microplate. In addition, only about 7% of the measurements, which was not recorded changes in the distribution, should be treated as "emissions" and not suited for automated SIFT analysis due mainly internal fluorescence feel soy is ineni. This rather low percentage of connections not be processed SIFT analysis, is unexpected and highlights the versatility and reliability of this analysis. Identification of problematic dimensions and connections facilitated vysokomarzhinalnoj the nature of the data obtained in this SIFT analysis. For each sample simultaneously written several fluorescence parameters, such as, for example, the average value of the fluorescence intensity of each color channel. They are displayed together with the total value of events of high intensity in each sector of the histogram of the color distribution. Therefore, samples with high internal fluorescence can easily be excluded from the analysis.

Primary candidates obtained in the SIFT analysis, and verification of their activity through a series of dilutions

For the connection, which should be classified as a primary candidate connections, applicants invention analyze the amount of columns in "green" sectors from 1 to 5 to determine the value of approximately 50% inhibition of the effect of DOSPA compared to untreated control as the minimum desired effect. Based on the results of SIFT-screening, DIVERSet connections assign scalar values SIFT activity (tp1_sift), characterizing their inhibitory effect on prion aggregation, campisano in the publication Bertsch et al. 2005. Activity values less than zero indicate the absence of inhibitory effect, while values greater than zero indicate inhibitory action. In this case, values close to unity indicate a significant inhibitory action, equal inhibitory effect of the positive control on the microplate.

The dependence of the inhibitory action on the dose of each of the compounds identified in the SIFT analysis, in relation to binding PrPC-PrPSc check using a range of dilutions of the six points (0.1 to 100 μm). Curves "dose-effect" of these compounds confirm the dependence of the inhibitory activity concentration. Compared to the effect of 17 μm DOSPA, fifty percent of the maximum inhibition of the binding of rPrP with prion chopsticks observed in the values of EC50in the interval from 0.3 to 60 microns.

Primary candidate connections in the screening cell culture and verification of the activity through a series of dilutions

In addition to the SIFT analysis, DIVERSet library is subjected to screening in models of cell cultures of prion diseases. In these analyses antiprion activity DIVERSet connections first experience at a concentration of 20 μm. The results of the initial screening in cell culture (SMB cells at 20 μm) encode a binary variable (tp_3 reduktion), where the value is 0,1" encodes "active" sample, and the value "0,0" encodes "inactive". The activity of compounds identified in the primary screening test to identify compounds that are active even at very low concentrations. Verification of primary cell culture is carried out in a series dilution at four concentrations (1 μm, 4 μm, 10 μm, 40 μm) for DIVERSet 1 and only at two concentrations (0.2 μm, 2 μm) for DIVERSet 2. The results (tp3_reduktion_mue) marked "1,0" for active at 1 μm (DS1) or 0.2 μm (DS2) and "0,5" to active when 2 μm (DS2). The connection is inactive at concentrations of more than 20 μm, refer to "0,0" (DS1 + 2). Furthermore, the activity of the primary candidates identified in the SMB cell culture test at single concentrations in ScN2a cells (tp3_scn2a), where again "0,0" and "1,0" means inactive and active compounds, respectively.

Diphenylpyrazine (DPP) and related compounds as new compounds with antiprion activity

The creation of the SAR maps

DIVERSet compounds characterized as "active" in the primary screening in cell culture, subjected to cluster analysis using the software package Benchware HTS DataMiner (DM; Tripos, Inc., St. Louis, MO, USA) to obtain the SAR-map presented in figure 1. Since a large number of compounds included in the two libraries (20000), is too large as the original t is his for cluster analysis using DataMiner, analysis of the first limited primary candidates (837 compounds) screening in cell culture DIVERSet 1 and 2. Thus, the clusters determined only for active compounds. In this case, the DM program is grouped into clusters of structurally similar active compounds and, therefore, suitable to identify potential relevant new lead structures. In the second stage, certain thus the classification applied to other compounds of the library, which is inactive in cell culture. In this case, the DM program adds to glenrowan clusters (inactive) connections, if applicable measurement indicates high structural similarity.

The results of cluster analysis graphically represent using DateMiner in the form SAR-map, on which clusters of compounds S are indicated by symbols. DM has a character such that they are in close proximity to each other, if the clusters are structurally similar. The size, shape and color of the symbols is assigned on the basis of the cluster-specific properties that can be selected by the user.

So, determines that the symbol size is proportional to the cluster size /S/, i.e. the number of connections that are contained in it. The shape of the symbols determined in accordance with the formula

of those compounds in the respective cluster's primary activity(C), as defined SIFT screening, above the selected threshold amin=0,25. Thus, clusters, for which PSIFTabove 50%, denoted by asterisks, while the rest, mainly inactive clusters, denoted by the square. Similarly, the color of characters is encoded in accordance with formulas clusters

relevant primary inhibitors identified in the test in cell culture, where the characters clusters with more than 50% of the active substances are red, and the remaining clusters are gray. Figure 1 shows the SAR-map obtained by the above-described cluster analysis. Large red asterisk represent clusters with a high proportion of the compounds showed a positive effect in the SIFT-analysis and the analysis in cell culture. These clusters represent potentially leading patterns. Using DataMiner clusters of interest, subject to further analysis and identify the group consisting of the five closest clusters (DPP_1-DPP_5), which are shown in figure 1 in bold. All connections highlighted in these clusters are presented in figure 2 together with their activities, as identified in various analyses. The fact that these clusters are located is received in close proximity to each other, means that they contain compounds that are structurally similar, ie they all belong to the chemical class of derivatives of 3,5-diphenylpyrazine (DPP) (compare fragment DPP presented in figure 1).

Example 2. Synthesis of new drugs for inhibition of aggregation from the point of view of the medico-chemical aspects

On the basis of the opening of the new leadership structure, as described above, the selective substitution of different substituents are synthesized, the number of additional connections as shown below.

Scheme 1: Synthesis of isoxazoles

(E)-1-(3,4-acid)-3-(3-forfinal)-2-propen-1-he(1)[Nam et al., 2004]

A solution of 3,4-dimethoxyacetophenone (1.8 g, 10 mmol), 3-forventelige (1.24 g, 10 mmol), NaOH (50 mg, 1.25 mmol) and Ba(OH)2·8H2O (100 mg, 0.32 mmol) in methanol (10 ml) was stirred at room temperature for 24 hours. The reaction mixture is cooled to +4°C, the precipitate is collected by filtration, recrystallized from methanol and dried, obtaining1(1.65 g, 58%) as yellow powder.

2,3-dibromo-1-(3,4-acid)-3-(3-forfinal)propane-1-he(2)[Harris et al., 1977]

To a solution of1(715 mg, 2.5 mmol) in chloroform (11 ml) was added dropwise a solution of bromine (400 mg, 2.5 mmol) in chloroform (4 ml) at 0°C. the Mixture is stirred for 2 hours at 0°C, then rasb the keys petroleum ether (20 ml), the resulting mixture is frozen (-24°C) and maintained at this temperature for 10 hours, the precipitate is collected by filtration, washed with n-hexane (10 ml) and dried, obtaining2(780 mg, 70%) as a white powder.

3-(3,4-acid)-5-(3-forfinal)isoxazol(3)[Harris et al., 1977]

Solution2(450 mg, 1 mmol) in ethanol (6 ml) is treated with hydroxylamine hydrochloride (306 mg, 4.4 mmol) and then a solution of NaOH (460 mg, 11.5 mmol) in water (1.5 ml). The mixture is refluxed for 2 hours, cooled and treated with water (3 ml). The mixture is cooled (4°C), left overnight at the same temperature, the product is collected by filtration, washed with water (5 ml) and dried, obtaining3(180 mg, 60%) as a white powder.

3-(3,4-Dihydroxyphenyl)-5-(3-forfinal)isoxazol (4)[Vanelle et al., 2000]

Solution3(100 mg, 0.33 mmol) in dichloromethane (5 ml) cooled to -78°C, treated with tribromide boron (0.16 ml, 1.7 mmol), stirred at -78°C for 3 hours and then overnight at room temperature. The mixture is cooled to -78°C and quenched with methanol (5 ml). The mixture is stirred for 3 hours at room temperature, then the solvents removed under reduced pressure, the residue is subjected to four times the co-evaporation with methanol (10 ml). The resulting residue is refluxed in 5 ml of chloroform, after cooling, the product is collected by filtration and dried, getting4(60 mg, 67%) as a white powder.

Scheme 2: Synthesis of pyrazoles

1,3-Bis(3,4-acid)propane-1,3-dione (5)[Anselme, 1967]

60% suspension of sodium hydride in mineral oil (0.4 g, 10 mmol) is washed twice with petroleum ether (20 ml), add anhydrous DMSO (10 ml). The mixture is stirred for 30 minutes at room temperature in an argon atmosphere, add THF (5 ml), the flask was cooled to 15°C and add ethyl-3,4-dimethoxybenzoate (2.1 g, 10 mmol). The temperature of the mixture allow to drop to 10°C and to this mixture a solution of 3,4-dimethoxyacetophenone (1.08 g, 6 mmol) in DMSO (4 ml) at such a rate that the temperature of the mixture did not rise above 15°C. After completion of the reaction, the reaction mixture is stirred for 72 hours at room temperature and then slowly poured into crushed ice (250 g), containing 85% phosphoric acid (1 ml). The precipitate is collected by filtration, washed with water (50 ml) and dried, obtaining5(2.1 g, 99%) as yellow powder.

3,5-Bis(3,4-acid)pyrazole (6)[Hauser et al., 1957]

Solution5(1.0 g, 2.9 mmol) and hydrazine hydrate is added (218 mg, 4.4 mmol) in ethanol (15 ml) is refluxed under stirring for 3 hours. Transparent yellow solution evaporated under reduced pressure, water is added, the precipitate Sobir the Ute filtering, washed with water and dried, obtaining6(960 mg, 97%) as yellow powder.

The hydrobromide 3,5-bis(3,4-dihydroxyphenyl)pyrazole (7)[Vanelle et al., 2000]

Solution6(120 mg, 0.35 mmol) in dichloromethane (5 ml) cooled to -78°C, treated with tribromide boron (0,34 ml, 3.5 mmol), stirred at -78°C for 3 hours and then overnight at room temperature. The mixture is cooled to -78°C and quenched with methanol (5 ml). The mixture is stirred for 3 hours at room temperature, then the solvents removed under reduced pressure, the residue is subjected to four times the co-evaporation with methanol (10 ml). The resulting residue is refluxed in 5 ml of chloroform, after cooling, the product is collected by filtration and dried, obtaining7(108 mg, 85%) as yellow powder.

1-(1,3-Benzodioxol-5-yl)-3-(3-bromophenyl)propane-1,3-dione(21)[Anselme, 1967]

1. Method

A dry three-neck flask with a volume of 500 ml provide a magnetic stir bar coated with Teflon (Teflon®), a rubber membrane, thermometer and reflux condenser is attached to T-shaped tube connected to a source of pure nitrogen. The remaining point of connection between the T-shaped tube is connected with a bubbling fluidised bed device so that the flow rate of nitrogen could be regulated in p is acesse reaction.

The device features so that the bulb could be periodically cooled in a water bath. After purging the flask with nitrogen in a flask maintain a constant atmosphere of nitrogen for the reaction. In the flask is charged with a 60% dispersion of sodium hydride (5 g, 0.125 mol) in mineral oil (note 1). Mineral oil out of hydride using petroleum ether 40/60 (3×40 ml) (note 2). The upper petroleum ether layer is removed using a hypodermic syringe with Luer lock and needle stainless stage, is inserted through the rubber membrane. Residual sodium hydride is mixed with 80 ml of dimethyl sulfoxide (note 3) and the rubber membrane replace drip funnel, a compensating pressure. A solution of 16.4 g (0.1 mol) of 1-(1,3-benzodioxol-5-yl)ethanone (note 4) and to 26.9 g (0.125 mol) of methyl-3-bromobenzoate (note 5) in 60 ml of dimethyl sulfoxide was placed in an addition funnel. The funnel is closed, begin stirring and the contents of the flask cooled in a water bath to 15°C. To the contents of the flask slowly add 1-(1,3-benzodioxol-5-yl)Etalon and methyl-3-bromobenzoate so that hydrogen gas was controlled speed and temperature did not exceed 20°C for 60 minutes (note 6). When you are finished adding bath removed and the reaction mixture was stirred at room temperature (23°C) for 15 hours is. The remaining homogeneous reaction mixture is reddish-brown color slowly poured into a mixture of 500 ml of ice and water containing 5 ml of 85% orthophosphoric acid (note 7) under stirring. The mixture is stirred for 1 hour, after which the product is removed by filtration (note 8), washed with water using a vacuum filter (2×100 ml) and dried in vacuum to constant mass at 40°C for 6 hours, getting 34.4 g of crude (note 9) product. Recrystallization from 200 ml of 99.9% ethanol and 200 ml of ethyl acetate (note 10) and drying in vacuo to constant weight at 40°C for 6 hours to yield 28.5 g (82% yield) of pure (note 11) 1-(1,3-benzodioxol-5-yl)-3-(3-bromophenyl)propane-1,3-dione, TPL 136-137°C, the filtrate is concentrated under reduced pressure to a volume of approximately 30 ml, solid crystals, which precipitate is collected by filtration, washed using vacuum filter (2×10 ml) and dried in vacuum to constant mass at 40°C for 6 hours, getting extra yield crude product. The crude product is twice recrystallized from 20 ml of 99.9% ethanol and 20 ml of ethyl acetate and dried in vacuum to constant weight 40°C for 6 hours, with the added 1.8 g (5% yield) of pure 1-(1,3-benzodioxol-5-yl)-3-(3-bromophenyl)propane-1,3-dione (note 12). The total yield of 30.3 g (87%).

2. Note

1. And is using sodium hydride in the form of 57-63% oil dispersion, available under serial number 13431 from Alfa Aesar GmbH & Co KG, Karlsruhe.

2. Use petroleum ether GR for analysis (interval boiling point 40-60°C), available at ordinal 101775 from Merck KGaA, Darmstadt.

3. Used without additional purification dimethyl sulfoxide GR for analysis, ordinal 102952 from Merck KGaA, Darmstadt.

4. Using 1-(1,3-benzodioxol-5-yl)Etalon, 98%, sequence number A13597, available from Alfa Aesar GmbH & Co KG, Karlsruhe.

5. Use of methyl-3-bromobenzoate, 98%+, serial number A16174, available from Alfa Aesar GmbH & Co KG, Karlsruhe.

6. In the process of adding observe the formation of foam. The use of mechanical stirrers and antifoaming agent, such as polyethylenglykolmonobutylether, it may be necessary to increase the output.

7. Use phosphoric acid, 85 wt. -%/mass. aqueous solution, GR for analysis, ordinal 100573 from Merck KGaA, Darmstadt.

8. The pH of the reaction mixture is 7. Upon acidification to pH=2 by addition of 15 ml of phosphoric acid can be obtained 1.3 g of 3-bromobenzoyl acid.

9. The purity of the product, according to HPLC, equal to 96.3%.

10. Use absolute ethanol 99.9% of GR for analysis, ordinal 100983, and the ethyl acetate GR for analysis, ordinal 109623 from Merck KGaA, Darmstadt.

11. The purity of the product, according to HPLC, equal to 99.3 percent. Analytical HPLC is performed using HPLC system Waters photodiode detective is rum Waters 996 Photodiode Array Detector. All divisions include using a mobile phase of 0.1%.about. triperoxonane acid (TFUC) in water (solvent A) and 0.1% vol./about. TFUK in acetonitrile (solvent B) using columns with reversed phase (RP) Eurospher RP 18, 100 Å, 5 μm, 250×4.6 mm at a speed of expiration of 1 ml/min Compound dissolved in acetonitrile GR for HPLC at a concentration of 1 mg/ml Peaks with values of retention time (RT) of 20.9 and 10.3 min correspond to the enol and keto forms, respectively, ANLE 138A. Re-injection of separately collected peaks 20,9 or 10.3 min again gives the same two peaks with identical values of the retention time (RT).

12. The purity of the product, according to HPLC, equal to 98.4%.

3-(1,3-Benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole (22)[Hauser et al., 1957]

1. Method

A mixture of 28.4 g (81,8 mmol) of 1-(1,3-benzodioxol-5-yl)-3-(3-bromophenyl)propane-1,3-dione (note 1) and 200 ml of n-butyl alcohol (note 2) is placed in a round bottom flask with a volume of 500 ml, equipped with a magnetic stirrer coated with Teflon (Teflon®), a reflux condenser and a heating jacket with electric heating. Begin mixing and heating, add 6 ml (6.2 g, 123,4 mmol) of hydrazine monohydrate (note 3) dissolution of solid substances and the reaction mixture is refluxed with stirring for 4 hours. The reaction mixture is cooled to 20°C, videris the Ute for 1 hour at 0°C, precipitated precipitated product is collected by filtration and vacuum pump. Rinsing with water (100 ml) and drying in vacuo to constant weight at 40°C for 36 hours results of 26.8 g (95% yield) of 3-(1,3-benzodioxol-5-yl-5-(3-bromophenyl)-1H-pyrazole (note 4) TPL 195-197°C.

2. Notes

1. 1-(1,3-Benzodioxol-5-yl)-3-(3-bromophenyl)propane-1,3-dione receive in accordance with the method of obtaining ANLE 138A.

2. Using n-butyl alcohol (99.4% of GR "Baker analyzed"), ordinal 8017, available from J.T. Baker B. V., Deventer, Holland.

3. Use hydrazinoacetate GR purum, serial number 53850, available from Sigma-Aldrich Chemie GmbH, Taufkirchen.

4. The purity of the product, according to HPLC, equal to 99.3 percent. Analytical HPLC is performed using HPLC system Waters photodiode detector Waters 996 Photodiode Array Detector. All divisions include using a mobile phase of 0.1%.about. triperoxonane acid (TFUC) in water (solvent A) and 0.1% vol./about. TFUK in acetonitrile (solvent B) using columns with reversed phase (RP) Eurospher RP 18, 100 Å, 5 μm, 250×4.6 mm at a speed of expiration of 1 ml/min Compound dissolved in acetonitrile GR for HPLC at a concentration of 1 mg/ml

Scheme 3: Synthesis of imidazoles

4-(3,4-Acid)-2-phenylimidazole (8)[Li et al., 2000]

A mixture of the hydrochloride of benzamidine (313 mg, 2 mmol) and sodium bicarbonate (672 mg, 8 mmol) in THF (6 ml) and water is (1.5 ml) is refluxed. To the mixture for 30 minutes, add a solution of α-bromo-3,4-dimethoxyacetophenone (518 mg, 2 mmol) in THF (1.5 ml) by boiling under reflux. After complete addition, the reaction mixture is refluxed for 2 hours and the THF is evaporated under reduced pressure. To the mixture are added ethyl acetate (20 ml), the organic layer is separated, washed with saturated salt solution (5 ml), dried over sodium sulfate and the mixture is evaporated under reduced pressure. The resulting crude product was then purified column chromatography on silica gel (chloroform/methanol 100:1), obtaining the8(470 mg, 84%) as a solid product.

The hydrobromide of 4-(3,4-dihydroxyphenyl)-2-phenylimidazole (9)[Vanelle et al., 2000]

Solution8(190 mg, of 0.68 mmol) in dichloromethane (5 ml) cooled to -78°C, treated with tribromide boron (0,32 ml, 3.4 mmol), stirred at -78°C for 3 hours and then overnight at room temperature. The mixture is cooled to -78°C and quenched with methanol (5 ml). The mixture is stirred for 3 hours at room temperature, the solvent is evaporated under reduced pressure, the residue is subjected to four times the co-evaporation with methanol (10 ml). The resulting residue is refluxed in 5 ml of chloroform, after cooling, the product is collected by filtration and dried, obtaining9(192 mg, 85%) as a powder.

Scheme 4: Synthesis of Pyrrhus is fishing

(E)-1-Phenyl-3-(3,4-acid)-2-propen-1-he (10)

Connection10receive in accordance with the method of obtaining1. Output 90%.

3-(3,4-acid)-4-nitro-1-phenylbutane-1-he (11)[Hall et al., 2005]

Solution10(774 mg, 2.9 mmol) in MeOH (30 ml) is treated with diethylamine (of 1.55 ml, 15 mmol) and nitromethane (0,81 ml, 15 mmol) and the resulting mixture is refluxed for 24 hours. The solution is cooled, partitioned between dichloromethane (60 ml) and water (50 ml) and acidified with 1M hydrochloric acid. The organic layer is separated, the aqueous layer was extracted with dichloromethane (20 ml). The combined organic layers washed with water (50 ml) and salt solution (50 ml) and dried over sodium sulfate. The solvent is removed under reduced pressure and the resulting oil purified column chromatography on silica gel (n-hexane/ethyl acetate 3:2), receiving11(780 mg, 82%) as a solid.

4-(3,4-acid)-1-phenylpyrrole(12)[Hall et al., 2005]

Solution11(400 mg, 1,22 mmol) in methanol (13 ml) and THF (26 ml) at room temperature with stirring, treated with potassium hydroxide (343 mg, 6.1 mmol). After 1 hour the reaction mixture is added dropwise to a solution of sulfuric acid (2,44 ml) in methanol (13 ml) at 0°C and the resulting mixture is stirred for 1 hour at room temperature. To the mixture add the water (20 ml) and ice (20 ml), the mixture is neutralized 1M aqueous solution of sodium hydroxide and extracted with dichloromethane (2×50 ml). The combined organic fractions washed with salt solution (25 ml), dried over sodium sulfate and evaporated under reduced pressure. The oil obtained is treated with acetic acid (8 ml) and ammonium chloride (470 mg), the solution is heated to 100°C and maintained at this temperature for 1 hour. The reaction mixture is cooled, add ice (50 ml) and the mixture is neutralized 1M aqueous solution of sodium hydroxide. The solution is extracted with dichloromethane (2×50 ml). The combined organic fractions washed with salt solution (25 ml), dried over sodium sulfate and evaporated under reduced pressure. The crude product is purified column chromatography on silica gel (n-hexane/ethyl acetate 3:1) and then recrystallized from a mixture of n-hexane/ethyl acetate (2:1), obtaining the12(150 mg, 44%) as a solid.

4-(3,4-Dihydroxyphenyl)-1-phenylpyrrole (13)[Vanelle et al., 2000]

Solution12(80 mg, 0.29 mmol) in dichloromethane (5 ml) cooled to -78°C, treated with tribromide boron (of 0.13 ml, 1.4 mmol), stirred at -78°C for 3 hours and then overnight at room temperature. The mixture is cooled to -78°C and quenched with methanol (5 ml). The mixture is stirred for 3 hours at room temperature, the solvent is evaporated under reduced pressure, the remainder of the basement is Laut four times co-evaporated with methanol (10 ml). The resulting crude product was then purified column chromatography on silica gel (chloroform/methanol 95:5) and then recrystallized from chloroform and a few drops of acetonitrile, getting13(36 mg, 50%) as a solid.

Scheme 5: Synthesis of pyrazolines

3-(3,4-Acid)-5-(3-forfinal)-4,5-dihydro-1H-pyrazole (14)

Suspension1(57 mg, 0.2 mmol) and hydrazine hydrate is added (0.5 ml, 10 mmol) in water (0,14 ml) is heated to 100°C and maintained at this temperature for 1.5 hours under stirring. The reaction mixture is cooled, water is added (0.2 ml), the precipitate is collected by filtration, washed with water and dried, obtaining14(37 mg, 62%) as a white solid.

Scheme 6: Synthesis of N-Ac-pyrazolines

(E)-1-(3,4-methylenedioxyphenyl)-3-phenyl-2-propen-1-he (15)

Connection15receive in accordance with the method of obtaining1. Yield 64%.

1-Acetyl-3-(3,4-methylenedioxyphenyl)-5-phenyl-4,5-dihydropyrazol (16)[Chimenti et al., 2004]

Solution15(504 mg, 2 mmol) and hydrazine hydrate is added (250 mg, 5 mmol) in acetic acid (12 ml) is heated to 120°C and maintained at this temperature for 24 hours under stirring. The reaction mixture is cooled, add chilled water (40 ml), the precipitate is collected by filtration, paracrystalline is live from ethanol and dried, getting16(458 mg, 74%) as a white solid.

1-Acetyl-3-(3,4-dihydroxyphenyl)-5-phenyl-4,5-dihydropyrazol(17)[Vanelle et al., 2000]

Solution17(70 mg, 0.23 mmol) in dichloromethane (3 ml) cooled to -78°C, treated with tribromide boron (0,11 ml of 1.16 mmol), stirred at -78°C for 3 hours and then overnight at room temperature. The mixture is cooled to -78°C and quenched with methanol (5 ml). The mixture is stirred for 3 hours at room temperature, the solvent is evaporated under reduced pressure, the residue is subjected to four times the co-evaporation with methanol (10 ml). The resulting crude product was then purified column chromatography on silica gel (n-hexane/ethyl acetate 1:1), obtaining the17(25 mg, 37%) as a white solid.

Scheme 7: Synthesis of 1,2,4-oxadiazoles

3,4-Dimethoxybenzamide (18)[Chalquest, 2001]

A solution of 3,4-dimethoxybenzonitrile (4.0 g, 24.5 mmol), hydroxylamine hydrochloride (2.0 g, 28.8 mmol), N,N-diisopropylethylamine (5.0 ml, 29.2 mmol) in ethanol (70 ml) was stirred at room temperature for 48 hours. The ethanol is evaporated under reduced pressure, add chilled water (60 ml), the precipitate is collected by filtration and dried, obtaining18(3.4 g, 71%) as a white powder.

3,5-Bis(3,4-acid)-1,2,4-oxadiazol (19)[Korbonits, 1982]

To Rast is Ouro 18(700 mg, of 3.57 mmol) and ethyl-3,4-dimethoxybenzoate (834 mg, of 3.97 mmol) in ethanol (12 ml) is added tert-piperonyl potassium (425 mg, with 3.79 mmol) and the resulting mixture is refluxed for 12 hours. The mixture is cooled, the precipitate is collected by filtration, washed with warm ethanol and dried, obtaining19(540 mg, 44%) as a white powder.

The hydrobromide 3,5-bis(3,4-dihydroxyphenyl)-1,2,4-oxadiazole(20)[Vanelle et al., 2000]

Solution19(220 mg, 0.64 mmol) in dichloromethane (6 ml) cooled to -78°C, treated with tribromide boron (0,59 ml, 6.1 mmol), stirred at -78°C for 3 hours and then overnight at room temperature. The mixture is cooled to -78°C and quenched with methanol (5 ml). The mixture is stirred for 3 hours at room temperature, the solvent is evaporated under reduced pressure, the residue is subjected to four times the co-evaporation with methanol (10 ml). The resulting residue is refluxed in 5 ml of chloroform, after cooling the mixture the product is collected by filtration and dried, obtaining20(190 mg, 81%) as a powder.

The examples above are examples of the synthesis of the target compounds and their derivatives. Other connections shown in figure 3, are synthesized in accordance with these examples. These chemically synthesized compounds together with the selected primary connections the d uke is Inga, subjected to additional tests, including SIFT analysis, the analyses in cell culture,in vivoexperiments on mice, as well as biochemical tests for aggregation of α-synuclein (see below). The list of tested compounds are presented in figure 3.

Example 3: Used material and methods

Compound libraries

Library, subjected to screening, contain 10,000 connections each and named applicants invention DIVERSet1 and DIVERSet2, because they include only part of a larger DIVERSet library (ChemBridge Corp., San Diego, CA). DIVERSet is a collection of rationally selected a variety of small molecules with properties similar to drugs. Connections come in the form of a solution in dimethyl sulfoxide (DMSO) and 96-well titration microplate. The database containing the molecular structure and some physicochemical data for each of the connections available on http://www..

The preparation of recombinant mouse PrP 23-231

Recombinant PrP receive and purified essentially as described in the publication Liemann et al. (1998) with the difference that the cells of the bacterial expression BL21DE3 RIL E. coli (Novagen) transformed with the plasmid pET17b-MmPrP23-231WT31 mouse PrP23-231. In addition, bacteria are grown to an optical density of 0.5 before producing the protein, which induce the addition of 1 mm IPTG, and cells and harvested after two hours. Bacteria are lysed by addition of 0.5% Triton X-100 to lessnau buffer and incubation for 30 minutes at 37°C instead of applying plunger (French press). Next, filter replace gel phase chromatography on affinity to chelate Nickel. The final stage cation exchange chromatography, after refolding is also missing.

In particular, PrP, pre-purified by ion exchange chromatography, subjected to oxidation as described, the oxidation finish by adding 0.1 mm EDTA and bringing the pH to about 6. After adding 0.1 mm NiCl2up to 50 mg PrP applied to 2 ml of chelate forming sepharose (Pharmacia), pre-charged with NiCl2in accordance with the manufacturers instructions, and pre-equilibrated with buffer A (8 M urea, 10 mm MOPS pH 7.0). The binding of PrP with Ni-chelate matrix can be performed during at least 3 hours at room temperature, continuously turning over the mixture. The matrix is transferred into a chromatographic column (poly-prep, BioRad) and washed with a stream passing through the column. The column is washed twice with 5 ml of buffer B (8M urea, 10 mm MOPS pH 7.0 500 mm NaCl) and then sequentially elute 6 times with 5 ml of buffer D (7,2M urea, 10 mm MOPS pH 7.0, 150 mm NaCl, 50 mm imidazole). The fractions containing purified PrP, unite, concentrate in the centrifuge (centriprep device) and finally diluted 1:50 for refolding in 0 mm MES (pH of 6.0).

The introduction of fluorescent labels in antibodies and recombinant PrP

In monoclonal antibody L42 (r-biopharm, Darmstadt, Germany) enter the label Alexa Fluor 647 (Alexa-647; Invitrogen, Eugene) in accordance with the manufacturer's instructions. In recombinant PrP 23,231 mouse injected label Alexa Fluor 488 (Alexa-488; Invitrogen, Eugene) in 20 mm phosphate potassium buffer (pH 6, 0.1% of Nonidet P40), 40 mm bicarbonate buffer sodium bicarbonate (pH 8.3). Unbound fluorophores separated by gelfiltration on PD10 columns (GE Healthcare, Freiburg, Germany), equilibrated to 20 mm phosphatecalcium buffer (pH 6, 0.1% of Nonidet P40). Quality control reaction injection label and the ratio of introduction of the label is carried out using fluorescence correlation spectroscopy (FCS) on the sensor reading Reader Insight (Evotec Technologies, Humburg, Germany). The ratio of introduction of the label is approximately 1.3 fluorophore molecule rPrP.

Analysis of the Association of PrPC-PrPSC

PrPSc derived from brain CJD patients in accordance with the publication (Safar et al. (1998)), aliquots of the final pellets, resuspendable in 1×PBS+0.1% of sarcosine solution, diluted five-fold in buffer A (20 mm phosphatability buffer at pH 6.0, 0.1% of Nonidet P40) and treated with ultrasound device for treatment with ultrasound in a water bath for 60 seconds. The treated mixture is centrifuged at 1000 rpm for 1 min and the resulting supernatant was diluted 100 times with buffer for analysis.

The mixture of labeled mouse rPrP and labeled L42 monoclonal antibodies prepared in 20 mm fosfornocalzievogo buffer (pH 6, 0.1% of Nonidet P40) so that the labeled molecules are approximately equal in excess of 2-6 mm. In the analytical volume of 20 ál mix volume 8 μl of a mixture of rPrP/antibody, 2 μl of compound and 10 μl of the diluted drug PrPSc. Samples are loaded into 96-well microplates with sliding glass bottoms (Evotec-Technologies, Humburg, Germany) and carried out measurements for Reader Insight.

Determining the number of single particles and analysis

The FIDA measurement is carried out at excitation energies of 200 µw for the 488 nm laser and 300 µw for the 633 nm laser. Scanning parameters: path length scanning 100 μm, the frequency of the scanning beam 50 Hz, the movement of the coordinate table 2000 μm. The measurement time of 10 seconds. Fluorescence from the two fluorophores read separately detectors of single photons and the number of detected photons during the time intervals of constant duration (Bina) summarize, using the duration Bina 40 microseconds. Determine the number of read red and green fluorescent photons and analyze it in a two-dimensional histogram of the intensity distribution as described above (Bieschke et al., 2000).

Data distribution of intensities evaluated using the software module 2D SIFT Evotec-Technologies, Hamburg, Germany) by summing the bins high intensity sectors. Values off for Banovich intensities for each series of measurements regulate manually in accordance with the control measurements.

Example 4: therapeutic application of new potential antiprion compounds in scrapie-infected mice after 80 days after infection

To prove the effectiveness of these new potential antiprion compounds in suppressing infectious spongiform encephalopathies (TSE) or prion diseases are conducting an experiment on animals, in which compounds with antiprion activity in accordance with the SIFT analysis and/or analysis on the culture of scrape cells, used for the treatment of mouse infected with the RML strain of scrapie, at a late stage of the incubation period. Applicants choose intraperitoneally the use of compounds with an interval of 14 days after 80 days after infection, because usually this is the time when animals infected prion strain, are initial subclinical symptoms. This should correspond to the earliest time when a person infected with TSE and showed the first symptoms of the disease, should get a real therapeutic treatment. The choice of such hard conditions for therapeutic treatment, the applicants do for assessment of the functionality of the test compounds as therapeutic agents in the real world. Still, as a rule, a therapeutic agent for the treatment of TSE were to test only on preventive action after exposure in experiments on animals, where they are applied at approximately the same time as the infection with prions. In real life, only in very rare cases, perform this therapeutic regime for patients infected with TSE. For most patients infected with TSE, the time of infection or the beginning of the incubation time for hereditary and sporadic cases (which constitute the vast majority) is unknown and cannot be identified. Therefore, the majority of patients infected with TSE, can receive treatment only after the first symptoms of TSE.

Experimental methods:

Females C57BI6 mice aged 6-7 weeks inoculant RML, scrape intracerebral injection of 30 μl of 1% sterile brain homogenate in phosphate buffer solution (PBS) from mouse, terminally ill RML, scrape-strain. After 80 days after infection mice treated with selected new potential antiprion compounds or diluent (water sulphoxide, DMSO). For the treatment of select five potential antiprion compounds in accordance with their antiprion activity in model cell culture, which were designated as 10353F11 in accordance with the Leith-position plate-position) in the Diverset library of chemical compounds (Chembridge Corp., San Diego, USA) and anle138b, anle143b, sery106 and sery149. Treatment of these compounds is carried out in the next 14 days intraperitoneally by injection of 50 µl of the day in connection 10353F11 when diluted with 25 µl of other compounds in the solvent (DMSO). Connection 10353F11 used at a concentration of 10 mm, and the connection anle138b, anle143b, sery106 and sery149 injected at a concentration of 100 mm injection during the entire period. Animals are examined daily for signs of illness trained attendants for animals after 80 days after infection. Are slaughtered when they reach end-stage disease, which is characterized by certain clinical symptoms (ataxia, tremor, difficulty out of the prone position on his back in the normal position and the stiffness of the tail) and agonizing. Usually the disease that has passed through the final stage, will lead to the death of the animal within one or two days. From slaughtered animals one hemisphere and half of the spleen frozen fresh at -80°C for Western blotting, and the second hemisphere and the second part of the spleen, as well as all the internal organs fixed in 4% solution of formaldehyde (immune) histology.

Results

As shown in figure 4, treatment of mouse, which was intracerebrally infected with the RML strain of scrapie, at the last stage of the incubation period (80 days ri.) the village is edstam daily intraperitoneal the introduction of selected potential antiprion drugs leads to an increase in the length of the incubation period until while not reached the last stage of scrapie infection for 10353F11 (figa) and anle138b and sery149 (pigv). The average survival period defined for compounds of anle138b and sery149 when tested in groups of seven and eight animals each, increased by 14.9 and 11.5 days, respectively, compared with a group of twelve animals, which take only the diluent 100% DMSO. For connections sery106 and anle143b increase the duration of survival in the group of eight treated animals compared with the same control group following statistically significant values. When treating connection 10353F11 the survival period specified for groups of eight animals each, increased by 11 days compared with the control group. The observed increase in the duration of survival in the experiments corresponds approximately to the duration of treatment. This may mean that the treatment data of drugs stops the progression of the disease, while injected drugs. In this case, the treatment data of drugs could lead to an increase in the duration of the lives of TSE-infected patients and to stabilize their health, protecting it from further deterioration by stopping the disease.

Example 5.Therapeutic applications is the potential antiprion connections in scrapie-infected mice after intraperitoneal infection

For evidence on the effectiveness of this new antiprion compounds in suppressing infectious spongiform encephalopathies (TSE) or prion diseases is carried out an additional experiment on animals. In this experiment, mice infect intraperitoneal RML, scrape and treat the connection anle138b, which has proven antiprion activity in the experiment on animals in the last stage of the development of prion disease (example 4). Applicants select a combination of intraperitoneal and oral administration of the compound and begin treatment immediately after infection.

Methods experiment:

Scrap infection and treatment of mice

Females C57BI6 mice aged 6-7 weeks inoculant RML, scrape intraperitoneally inoculation of 100 ál of 1% sterile brain homogenate in phosphate buffer solution (PBS) from mouse, terminally ill RML, scrape-strain.Treatment of these mice with connection anle138b or diluent, which is used as an aqueous dimethylsulfoxide (DMSO), beginning immediately after infection. Treatment of this compound is carried out in the next 14 days intraperitoneally injection of 25 μl per day connection, diluted with diluent (DMSO) and then orally administered for 4 and then 5 days 50 μl of compounds per day in a mixture of vegetable oil/DMSO c using the probe for feeding (figa). Connection anle138b used at a concentration of 100 mm at intraperitoneally use and dose of 50 mg/kg oralintroduction. Animals slaughtered at 35 days after infection, when PrPSc can clearly be detected in the spleen administered intraperitoneally infected mice. One part of the spleen slaughtered animals frozen fresh at -80°C for Western blotting, while the second part of the spleen and all the internal organs fixed in 4% formaldehyde solution for immunohistology.

PET blotting

The formalin-fixed brain tissue is cut into blocks with a thickness of 2 mm, disinfected in concentrated formic acid for 1 hour, postfixed in 4% phosphate-buffered formalin solution for 48 hours in accordance with the method described in the publication Brown et al. (1990) and pour in the wax. Sections (5-7 μm) are cut on a microtome, placed in a water bath (55°C), collected on a pre-wetted nitrocellulose membrane with a pore size of 0.45 μm (Bio-Rad, Richmond, CA) and dried for at least 30 minutes at 55°C. the Nitrocellulose membrane deparaffinized xylene. Xylene is replaced by isopropanol and then Paladino registryroot. At the last stage of rehydration in distilled water add Tween 20 at a final concentration of 0.1%. The membrane is dried and stored at room temperature for the of months without loss of quality subsequent PrPSC staining.

After pre-wetting TBST (10 mmol/l Tris-HCl, pH 7.8; 100 mmol/l NaCl; 0.05% of Tween 20) conduct biological process using 250 μg/ml proteinase K (Boehringer) in phosphate-potassium buffer (10 mmol/l Tris-HCl, pH 7.8; 100 mmol/l NaCl; 0.1% Of Brij 35) for 8 hours at 55°C. At this stage of the membrane-attached proteins immobilized on the membrane. After three times washing TBST proteins on the membrane are denatured 3 mol/l guanidinoacetate in 10 mmol Tris-HCl (pH 7.8) for 10 minutes. Guanidine washed three times with TBST. Immunological analysis is performed after pre-incubation in blocking solution (0.2% casein in TBST) for 30 minutes. As a primary antibody using a polyclonal antibody rabbit against recombinant mouse PrP, as indicated by the CDC1, at 1:500 dilution in the antibody-diluent (Ventana). The mixture is incubated for at least 1 hour. After washing three times with TBST, the incubation is conducted for at least 1 hour alkalinemanganese-linked antibody rabbit anti mouse antibodies (Dako, Hamburg) at a dilution of 1:500. After five washes in TBST for 10 minutes, the pH value of the membrane is brought to an alkaline value by incubating twice for 5 minutes in NTM (100 mmol/l Tris-HCl, pH of 9.5; 100 mmol/l NaCl; 50 mmol/l MgCl2). Visualization of antibody response carried out by reaction of formazan with COI is whether the NBT/BCIP. The blots appreciate using preprofile Olympus magnifier.

Results

Fabric spleen extracted after the death of the mice treated with the medication, and mice treated with diluent, immunohistochemically paint for detecting the presence of PrPSc accumulations and examined for the levels of splenic PrPSc using Western blot turns. As shown in figv, the levels of PrPSc in the spleen of animals treated, is significantly reduced compared to mice treated with diluent. The study of the splenic tissue from infected mice shows that accumulation of PrPSc reduced after treatment with compound anle138b. The percentage of spleens with a low content of PrPSc accumulations increases, and the content of solid PrPSc accumulation is reduced (figs). On fig.5D presents examples of two PET blots PrPSc accumulations. The results clearly show antiprion the effectiveness of this compound in the peripheral tissues in the selection of the plan of the experiment and schema therapy.

Example 6: therapeutic application of new antiprion compounds for the treatment of scrapie-infected mice after 80 days after infection

To confirm the results obtained in example 4 that the increase in survival time corresponds to the duration of treatment is Oia animal experiment on animals in which connections are introduced in higher dosages and for longer periods of time, treatment of mice infected with the RML strain of scrapie, at a late stage of the incubation period. Applicants choose the combination intraperitoneal and oral administration of the compounds after 80 days after infection, because usually this period of time corresponds to the time of occurrence of the first sub-clinical symptoms in animals infected prion strain.

Methods experiment:

Females C57BI6 mice aged 6-7 weeks inoculant RML, scrape intracerebral injection of 30 μl of 1% sterile brain homogenate in phosphate buffer solution (PBS) from mouse, deadly sick RML, scrape-strain. After 80 days after infection of these mice are treated with new antiprion compounds or diluent DMSO. Use two potential antiprion connection: anle138b and anle186b. Treatment of these compounds is carried out in the next 14 days intraperitoneally injection of 25 μl per day compounds diluted in DMSO, and the subsequent two oral doses for 5 days at 50 μl per day connection in a mixture of vegetable oil/DMSO by probe feeding figa). 3 control mice 2 mice treated with compound 138b, impose additional connection oral, WA what I 109 day by day 136, providing pellets of food peanut butter mixed with basic solutions of DMSO/connection. These compounds are used at a concentration of 100 mm for intraperitoneal injection in a dose of 50 mg/kg for oral administration. From each group at the indicated time points scored four animals (figa). Eight animals from each group trained personnel checks daily for signs of disease, ranging from 80 days after infection. Are slaughtered when they reach end-stage disease, which is characterized by certain clinical symptoms (ataxia, tremor, difficulty out of the prone position on his back in the normal position and the stiffness of the tail) and agonizing. Usually the disease that has passed through the final stage, will lead to the death of the animal within one or two days. From slaughtered animals one hemisphere and half of the spleen are freezing fresh at -80°C for Western blotting, and the second hemisphere and the second part of the spleen, as well as all the internal organs fixed in 4% solution of formaldehyde for histology.

Results

The levels of PrPSc in brain homogenates and the death of apoptotic cells after infection

Brain homogenates of mice receiving the treatment, and the mice receiving diluent, analyze what and levels of PrPSc by immunoblot analysis. As shown in figv, the levels of PrPSc in the brain of all animals tested at specified points in time, from a group receiving treatment with connection anle138b, can be maintained at the levels of PrPSc in the body untreated mice 80 day, while the levels of PrPSc in the control group increases. The results obtained for the group of animals treated with the compound anle186b is between the control group and groups receiving anle138b. The increase in PrPSc could be delayed (pigv). On figs shows the change in relative levels of PrPSc after treatment with compounds compared to untreated control 80 day. The levels of PrPSc in the brain of animals slowly decreases after treatment with compound anle138b. Histological analysis of H&E stained sections of brain tissue of mice treated treatment compounds anle138b and anle186b within a specified time points, showed a decrease of pathological changes. The number of apoptotic cells in the layer of cells of the grains of the cerebellum of infected mice from groups who took the treatment, is reduced compared with the control group (fig.6D). These results indicate that both compounds can cross the blood brain barrier. Therefore, therapeutic treatment can prevent further deposition of PrPSc and the development of the disease in the brain of the LM is now in the process of treatment. These results indicate that treatment with compound anle138b suspends the progress of the disease, while injected drugs. These results also show that it is possible to influence the development of the disease with therapeutic treatment using this connection, influencing the formation of PrPSc. In this case, the treatment should lead to an increase in life expectancy TSE-infected individuals and stabilization or, perhaps, improve their health, protecting it from further deterioration of health by stopping the development of the disease.

The increase in the length of incubation period

As shown in Fig.7, the treatment of mice that were intracerebrally infected with the RML strain of scrapie, at the last stage of the incubation period (80 day ri.) by daily injection of compounds leads to an increase in the length of the survival period up until not reached the final stage of scrapie infection (7). In addition, longer periods of survival of two mice who received additional treatment, starting from 109 days on day 136, introduction connections anle138b in a mixture with peanut butter showed that i) the survival correlates with the duration of treatment, (ii) the compound is effective by oral administration and (iii) the connection preodolev the t blood-brain barrier.

Example 7: Suppression of aggregation of α-synuclein in terms ofinvitro

Synucleinopathies represent (neurodegenerative) diseases characterized by intracellular accumulation of clusters and fibrils, consisting mainly of the protein α-synuclein (see review: Goedert, 2001). The most well-known neurodegenerative synucleinopathies are Parkinson's disease (PD), dementia with calves levy (DLB), multiple system atrophy (MSA).

It was shown that the aggregation of α-synucleinin vitrotakes place in the presence of compounds such as organic solvents that mimic the dielectric conditions that occur under natural conditions in the immediate vicinity of biological membranes (Munishkina et al., 2003). Were developed tests of aggregation in terms ofin vitrothat provide a model system to study the main aspects of the incorrect addition and aggregation of α-synuclein and formation of toxic species clusters during the course of the disease (Kostka et al., 2008).

To test selected compounds for their ability to inhibit the aggregation of α-synuclein applicants invention, usein vitrowhere used, an organic solvent, dimethylsulfoxide (DMSO) at low concentrations (<3%) and, in some experiments, trivalent is elezo to induce aggregation of α-synuclein in vitro. Multimeric formation control using installation fluorescence correlation of single particles by cross-correlation analysis and SIFT analysis applied to mixtures of monomers α-synuclein labeled green Alexa488 - or red Alexa647 the fluorophores, respectively. Such α-synuclein mixture of aggregate in parallel to the samples, where the connection type in the response.

Methods experiment:

The introduction of fluorescent labels in α-synuclein

The introduction of labels in α-synuclein carried out using fluorescent dyes that can interact with the amino group of Alexa Fluor-488-O-Succinimidyl ester) or Alexa Fluor-647-O-Succinimidyl ester (Molecular Probes, USA), respectively. After completion of the reaction, unbound dye molecules separate exclusion chromatography of reaction mixtures pass through two series-connected PD10 column (Amersham Bioscience, Germany) according to the manufacturer's instructions. The effectiveness of the introduction of the label and remove unbound dye determined by direct measurement of light scattering (FSC) with appropriate dilutions of the fractions containing the labeled monomers of α-synuclein.

Targeting fluorescence correlation spectroscopy of single particles

In a volume of 20 μl in the wells specifically what about tiralongo microplate with sliding glass bottom for measurement by fluorescence correlation spectroscopy (Evotec-Technologies, Germany) are the aggregation of α-synuclein in buffer containing 50 mm Tris at pH 7.0 and the mixture of monomers α-synuclein labeled with Alexa488 - or Alexa647-fluorophores, respectively, at a final concentration of approximately 5-10 nm of each type α-synuclein. The measurements were carried out on the sensor Insight (Evotec Technologies, Germany) using a 40× lens microscope 1,2 NA (Olympus, Japan) c FIDA optical configuration, the diameter of the perforated hole 70 μm and the excitation energy of 200 µw for the 488 nm laser and the excitation energy of 300 µw for the 633 nm laser. The measurement time is 10 seconds, during which the focus of the laser is moved along the through hole of the scanning beam path length scanning 100 microns, with a scanning frequency of 50 Hz, the frequency of the scanning beam 50 Hz, the movement of the coordinate table 2000 µm). This is equivalent to scanning speed of approximately 10 mm/s the result is a two-dimensional histogram of the distribution of intensities that are analyzed using 2-D SIFT software (Evotec OAI, Germany).

Results: Suppression of aggregation of α-synuclein connections

Aggregation of α-synuclein caused by DMSO and DMSO/Fe3+manifests itself in the formation of multimeric complexes of α-synuclein that contain both green and red labeled units of α-synuclein in large quantities. Thus, the control implement the function without adding inhibitory compounds indicates the presence of a large number of complexes, which emit a large number of photons. Adding a DPP-compounds 351F11 an experienced solution can significantly inhibit the formation of multimeric complexes of α-synuclein dependent dose-dependent manner, as shown in figa. Therefore, the connection 351F11 able to effectively inhibit the formation of multimeric complexes of α-synuclein at low micromolar concentrations in thisin vitromodels of pathological aggregation of the protein found in enucleated. This is a clear indication that the connection 351F11 can function not only as antiprion connection, but also has the potential therapeutic drugs for the treatment of synucleinopathies, such as Parkinson's disease, DLB and MSA, which affects the pathological mechanism at the molecular level. The inhibitory effect dependent on the dose of the aggregation of α-synuclein can also be detected in other investigational DPP-derivatives (Fig-In, With). Therefore, these compounds represent a new group of compounds with proven ability to inhibit the aggregation of α-synucleinin vitrothat will allow you to develop a causal therapy against Parkinson's disease and other synucleinopathies.

In addition, the inhibitory activity of these compounds against aggregationin vitro/i> the prion protein and α-synuclein can testify to their General protivoraketnoi activity against a broader spectrum of diseases associated with protein aggregation, in which the incorrect addition of proteins predominantly β-sheet conformation is allows their aggregation into amyloid fibrils. Therefore, the data connection and other members of the class of DPP-derivatives have potential use as therapeutic agents for the causal treatment of the full list (neurodegenerative) diseases associated with aggregation of proteins, including Alzheimer's disease, prion disease, Parkinson's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2 microglobulinuria amyloidosis, hereditary't neuropathic amyloidosis and Finnish hereditary systemic amyloidosis.

Example 8: Inhibition of PrPSc in cell culture

u> Methods experiment:

Prion-infected cell culture process newly synthesized compounds as described above for the primary screening. Compounds added to the concentrations indicated in Fig.9. The structure of the compounds shown below, and figure 3.

5-(3,5-dibromophenyl)-3-(3,4,5-trihydroxyphenyl)pyrazole (anle145d)

5-(3-bromophenyl)-3-(3,4-acid)pyrazole (sery255b)

Results:

A very high proportion of representatives of the class of DPP-derived manifests effective reduction of PrPSc in cell culture at low micromolar and even when submicromolar concentrations. This indicates that these compounds represent a group derived from antiprion activity.

Example 9: Inhibitory effect of different DPP-derivatives in the brain and spleen

Compounds are tested for their inhibitory activity against the accumulation of PrPScin vivoin accordance with the following three methods:

(a) C57BL/6 mice inoculant intracerebral (i.c.) 30 μl of 1% brain homogenate (RML, scrape). Treatment begins after 80 days after infection by oral administration of 1 mg of compound per day mixed with DMSO + coconut oil. The levels of PrPScin the brain determined by immunoblot analysis on 120 day after infected with the project;

(b) C57BL/6 mice inoculant intracerebral (i.c.) 30 μl of 1% brain homogenate (RML, scrape). Treatment begins after 80 days after infection intraperitoneally injection 0.84 mg of the compound (in DMSO) daily for 14 days followed by treatment with 2×5 days (with a break between this treatment 2 days) 1 mg of the compound (in DMSO + peanut butter), injected oral tube for feeding. The levels of PrPScdetermine the 106th day after infection;

(C) C57BL/6 mice inoculant administered intraperitoneally (R) 100 μl of 1% brain homogenate (RML, scrape). The levels of PrPScin the spleen was determined on a 35 day after infection after 34 days of treatment with 1 mg per day of compound in DMSO + peanut butter buffer.

The relative inhibition of the accumulation of PrPSccompared with the group treated with DMSO, shown in table 1 (mean value of inhibition in animals receiving DMSO, defined as 0% inhibition, the mean value for control animals at the beginning of the treatment period is defined as 100% inhibition).

The experimental results show that given the experimental conditions (i) connection of anle138b and chemically similar compounds provide a highly efficient inhibition of amplification of prions in vivo; ii) there is a dependence of the activity on structure, which shows that for this application in these experimental conditions, the connection anle138b provides a relative optimum activity.

Example 10: The possibility of passing the blood-brain barrier and the observed interaction with pathological accumulations of proteins

C57BL/6 mice inoculant intracerebral (i.c.) 30 μl of 1% brain homogenate (RML, scrape). Treatment begins after 80 days after infection by oral administration of 1 mg of compound (sery383) or 3 mg of compound (sery363a) in a mixture of buffer DMSO + peanut butter. The levels of PrPScin the brain determine the 120-day Western blot turns.

Compared with control groups receiving DMSO, introduction sery363a reduce the accumulation of PrPSc35%introduction sery383 reduce the accumulation of PrPSc30%.

Sery363a synthesize and test data in the experiments because it is a modification of anle138b and is well suited for the introduction of isotopic labels, which are required for use as a tracer in PET imaging.

Sery383 tested in this analysis because it was determined that this compound and its structural analogues containing an-NH2 group and the halogen atom, the two who are highly effective for inhibiting the aggregation of α-synuclein (see example 16 Inhibition of the formation of clusters of α-synuclein various connections").

The results of these experiments show that both compounds are able to pass the blood-brain barrier and interact with pathological accumulations of proteins, and suggests that these compounds have properties that can be used in their application as therapeutic compounds, as well as diagnostic connection.

Example 11: The effect of daily treatment with connection anle138b to the accumulation of PrPScand prion pathology in mice infected with the RML scrapie

C57BL/6 mice inoculant intracerebral (i.c.) 30 μl of 1% brain homogenate (RML, scrape). Treatment begins after 80 or 120 days after infection by oral administration of 5 mg of the compounds per day mixed with buffer DMSO + peanut oil (figure 10).

Sections of the brain stained for detection of PrPSc(figa), show that treatment with compound anle138b reduces the accumulation of PrPSccompared to animals receiving DMSO. Quantification of levels in brain homogenate of prion-inoculated mice at different points in time shows that the accumulation of PrPScin organisms mice treated with compound anle138b, is significantly reduced, even when the treatment starts the Ute at a late stage of the disease (120 days after insulinopenia, figv). Histological quantification of apoptotic cells in a thin slice H&E stained brain tissue, indicates that the inhibition of the accumulation of PrPScleads to inhibition of the death of neural cells (figs). Control mice treated with buffer DMSO + peanut butter without compounds show a progressive loss of body weight (fig.10D). Treatment of compound anle138b, starting from day 80 after insulinopenia, prevents further loss of mass within approximately 100 days. Treatment with 120 days after insulinopenia inhibits the loss of mass within ~70 days.

The obtained experimental data show that treatment with compound inhibits the accumulation of PrPScthe death of neural cells and the development of clinical signs of disease even in the case when the treatment is started at a late stage of the disease after symptoms are obvious signs of illness.

Example 12: Comparison of different methods of treatment

C57BL/6 mice inoculant intracerebral (i.c.) 30 μl of 1% brain homogenate (RML, scrape). Treatment of compound anle138b in different times and according to different schedules (as indicated in the captions to 11) significantly prolongs the survival time after the introduction of the RML scrapie (p<0,01). The average duration of periods of survival are presented in days ± standard on klonnie.

As shown in figure 11, presents the results of the experiments show that (i) treating the compound is also effective for oral administration, (ii) the treatment is also effective when it begins at a late clinical stage of the disease, and iii) more prolonged treatment leads to a longer survival period.

Example 13: Dose-dependent effect of compounds anle138b on the levels of PrPScin the brain

C57BL/6 mice inoculant intracerebral (i.c.) 30 μl of 1% brain homogenate (RML, scrape). Treatment begins after 80 days after infection by oral administration of different amounts of compounds anle138b (as shown in Fig) in a mixture of buffer DMSO + peanut butter. On day 120 after infection of animals slaughtered and determine the number of PrPScin the brain in comparison with the animals slaughtered at 80 days after infection.

The data presented in Fig show that the connection anle138b reduces PrPScaccumulation in the brain is dependent on dose.

Example 14: Quantification of PrPCby Western blot turns brain tissue from uninfected mice, taking 1 mg per day of the compounds of anle138b in a mixture with a buffer DMSO + peanut butter in 1 week

As shown in Fig, there was no reduction of the levels of PrPCin mice, the principles of the host connection anle138b compared to control mice.

These experimental results show that therapeutic effect in scrapie-infected mice is not due to reduced expression of PrPCbut is the result of inhibition of the formation of pathologically aggregated varieties of proteins.

Example 15: Pharmacokinetic analysis of compound anle138b

A single dose of the compounds of anle138b enter uninfected C57BL/6 mice, as shown in Fig. At different time points after injection to determine the number of connections in the brain and serum for 2 animals at each time point and for each experimental group using LC-MS.

The results of this experiment show that there is a good bioavailability when administered orally and good penetration into the brain. Connection anle138c detected in blood of mice, so it is assumed that it is a metabolite of compound 138b.

Example 16: Inhibition of the formation of clusters of α-synuclein various connections

Aggregation of α-synuclein induce DMSO and 10 μm FeCl3and analyze confocal spectroscopy of single molecules, as described in the publication Kostka et al. (J Biol Chem (2008) 283: 10992-11003). The effect of various compounds added at a concentration of 10 μm, the effect of education intermediate II oligomers examined in comparison with control (no connection is possible), as shown in table 2. Patterns corresponding compounds represented at Fig.

Table 2
Inhibition of the formation of clusters of α-synuclein different compounds
ConnectionThe intermediate product IIConnectionThe intermediate product IIConnectionThe intermediate product II
anle138c<5%sery85<25%sery145<50%

sery117<5%sery335b<25%sery345<50%
sery384<5%sery275b<25%sery256b<50%
sery383<5%sery140/td> <25%sery161<75%
sery109<5%anle138b<25%sery315b<75%
sery320c<5%sery363b<25%sery316b<75%
sery319<25%sery292b<75%
sery329<75%

The results of this experiment show that these compounds inhibit the formation of toxic accumulations of α-synuclein and that these structurally similar compounds have potential use for the treatment of diseases associated with protein aggregation, and, in particular, for the treatment of diseases which can be observed aggregation of α-synuclein.

Example 16: The action of compoundsin vivoin models of Parkinson's disease in mice

Ek the pilot data confirm, in experimental models of Parkinson's disease using mitochondrial toxins such as MPTP and rotenone, in appropriate concentrations, can be observed the formation of aggregated α-synuclein that contributes to the death of neural cells. Mice injected with MPTP (daily, 30 mg/kg body weight) intraperitoneally injection to induce the degeneration of dopaminergic neurons in the substantia nigra. Animals (3-10 for the experimental group) treated with various compounds or diluent (daily 250 mg/kg body weight, oral administration (enteral nutrition connection of 12.5 ál of DMSO mixed with 487,5 µl of olive oil) 0-12 days). Quantitative determination of loss of neurons compared to mice not treated with MPTP (control, defined as 0% cell death), and mice treated with MPTP, which treat only the diluent (DMSO, defined as 100% cell death), performed on day 12. For the quantitative determination of tyrosine hydroxylase (TH)-positive cells (substantia nigra parts compacta - Agent) section thickness of 50 μm paint using immune-label anti-TN-antibody. Every second section inside the " analyze using software Stereo investigator (MicroBrightfield, Colchester, VT, USA). Cells stained using immune labels, and count with the optical fractionation column 20× object is om. Stereological calculations performed blindly by two independent researchers.

Experimental data are presented on Fig indicate that the test compound reduces cell death in models of Parkinson's disease in terms ofin vivo.

Example 17: The impact of connection anle138c on the aggregation of Abeta

Abeta40 at a concentration of 50 µm, incubated for 30 hours under the following conditions: 50 mm phosphate, 50 mm sodium chloride, 0.01% of sodium azide, pH 7.4, 37°C; stirred by magnetic stirring with addition or without addition of 50 μm compound anle138c. To the control sample. add DMSO at the same concentration as in the sample. The concentration of DMSO equal to 2% (vol./vol.), the concentration of compounds anle138c with the initial solution is 3 mm. The peptide solution was centrifuged at 16000 g for 15 minutes before DLS experiments.

While the largest peak in the monomer Abeta40 corresponds to a hydrodynamic radius of about 1.5 nm, the peak of the oligomer is at approximately 30 nm (top panel), the aggregate state of Abeta40 in the presence anle138C (middle panel) showed oligomer peak near 20 nm in addition to the peak of the monomer. The bottom panel shows the distribution of particle sizes for amyloid fibrillar state Abeta40, measured after centrifugation of the sample. The aggregation of ABeta analyze dynamic the static light scattering. DLS measurements performed twice at 25°C in the apparatus DynaPro Titan (Wyatt Technology Corp., CA) with laser 827,08 nm. The falloff angle is 90°. DLS measurement consists of twenty measurements with a duration of 10 seconds. The refractive index (RI) of the solution set on a lot in 1,333 at 589 nm and 20°C, and RI at length of a wave studies are in accordance with the equation Cauchy (Cauchy) with coefficient 3119 nm2. Viscosity equal 1,019 CPS (at 20°C, and the temperature-dependent changes estimated using a water model. The distribution of particle sizes determined by the method of limited regularization.

The obtained experimental data are presented on Fig show that the connection anle138c inhibits the formation of large Abeta40 oligomers, the connection anle138c and compounds of similar chemical structure can also influence the aggregation of Abeta and can be used for therapeutic and diagnostic purposes for diseases such as Alzheimer's disease, which neuropathologically characterized by the accumulation of aggregated Abeta.

Alternative compounds according to the present invention are summarized in the following paragraphs. These compounds can be used in a manner analogous to the method of applying the above compounds according to the invention.

1. The compound represented by formula (I)

where

X, Y and L non-directional independently selected from-C(R11)(R12)-, -C(R13)=, -N(R14)-, -N=, -N+(R17)=, -O - and-S-;

M and Z non-directional independently selected from

----- represents an optional double bond;

R1-R15 or R17, or R18 is independently selected from hydrogen, halogen, cyano, hydroxyl group, nitro, amino, azido, sulfonyl, thio, phosphonyl, carboxyl group, carbonamide, alkyl, alkenyl, quinil, alkoxy, acyl, acyloxy, acylamino, carbocyclic groups, carbonyloxy, carbosilane, carbosilane, aryl, arylalkyl, arylalkyl, arylalkyl, aryloxy, Allakaket, heterocyclic group, heterocyclic, geterotsiklicheskie, heteroaryl group, heteroaromatic, heteroallyl, heteroallyl, heteroaromatic, or two adjacent groups may be connected with the formation of the bridge group containing from 1 to 6 carbon atoms, where one or two carbon atoms may be replaced by-O-, -S - or-N(R')-, where R' is selected from H and C1-4of alkyl; each of which is optionally substituted; and its prodrug, ester, MES or salt;

provided that the compound is not one of the following compounds (a), (b) or (c)

2. The compound according to claim 1, where the cycle is A non-directional is selected from the following structures:

3. The compound according to claim 1 or 2, where R7 represents halogen, cyano, hydroxyl group, nitro, azido, alkoxy, thio, alkylthio, amino, halogenoalkane, alkyl or halogenated.

4. The compound according to any one of claims 1 to 3, where R2 and R3, each independently, selected from hydroxyl group and C1-6alkoxy; or R2 and R3 together form the structure-O-(CH2)n-O-, where n takes values from 1 to 3, preferably n is 1.

5. The compound represented by formula (I)

where

X, Y and L non-directional independently selected from-C(R11)(R12)-, -C(R13)=, -N(R14)-, -N=, -N+(R17)=, -O - and-S-;

M and Z non-directional independently selected from

----- represents a possible double bond;

R1-R15 or R17, or R18 is independently selected from hydrogen, halogen, cyano, hydroxyl group, nitro, amino, azido, sulfonyl, tigroup, phosphonyl, carboxyl group, carbonamide, alkyl, alkenyl, quinil, alkoxy, acyl, acyloxy, acylamino, carbocyclic groups, carbonyloxy, carbosilane, carbosilane, aryl, arylalkyl, arylalkyl, arylalkyl, aryloxy, Allakaket, heterocyclic group, heterocyclic, geterotsiklicheskie, heteroaryl group, heteroaromatic, heteroallyl, heteroallyl, heteroaromatic, or two adjacent groups can connect with education is the bridge Finance group, containing from 1 to 6 carbon atoms, where one or two carbon atoms may be replaced by groups-O-, -S - or-N(R')-, where R' is selected from H and C1-4of alkyl; each of which is optionally substituted;

and its prodrug, ester, MES or salt;

for use in the treatment or prevention of a disease associated with protein aggregation and/or neurodegenerative diseases.

6. The use of compounds represented by formula (I)as defined in claim 5, to obtain a pharmaceutical composition for the treatment or prevention of diseases associated with protein aggregation, or neurodegenerative diseases.

7. A method of treating or preventing diseases associated with protein aggregation and/or neurodegenerative diseases, including the introduction of a therapeutically effective amount of the compound represented by formula (I)as defined in claim 5, the patient who needs it.

8. The method of identifying compounds for inhibiting the aggregation of proteins involved in a disease associated with protein aggregation, or neurodegenerative disease, comprising the following stages:

contacts labeled Monomeric protein with a concentration otherwise specified labeled protein (1) in the presence and/or (2) in the absence of a possible candidate inhibitor of aggregation, which represent a compound according to claim 5;

the determination of the number of labels that are localized in close proximity to each other, the specified number indicates the degree of binding of Monomeric protein in the accumulation of a specified protein; and

the comparison result obtained in the presence of the specified connection, with the result obtained in the absence of a specified connection,

where the reduction in the number of labels that are localized in close proximity to each other in the presence of the specified connection, indicates the ability of the compound to inhibit the aggregation of the specified protein.

9. The method of claim 8, where these labels are fluorescent labels.

10. The method according to claim 8 or 9, where these labels are attached to the antibody or antibody fragment that specifically associated with the specified protein.

11. The method according to claim 10, where the aforementioned antibody or fragment antibodies can distinguish between aggregated and Monomeric protein.

12. The method according to any of PP-11, where the number of labels that are located in close proximity to each other, determined using the method of "scanning intensely fluorescent targets (SIFT)method of fluorescence resonance energy transfer (FRET) or a method of confocal visualization of high resolution.

13. The method according to any of PP-12, where these Monomeric and aggregated proteins selected from the group comprising the prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, secrete lysozyme, fibrinogen, huntingtin, ataxin and other proteins with a poly-Q-sequence, and fragments or derivatives of these proteins.

14. The method according to item 13, where these Monomeric protein is a prion protein and the specified aggregated protein is PrPsc.

15. The method according to item 13, where the specified Monomeric protein is an alpha-synuclein and the specified aggregated protein selected from the group comprising oligomers, protofibrils or fibrils of alpha-synuclein.

16. Method of screening compoundsin vivoeffectiveness for treatment or prevention of diseases associated with protein aggregation, or neurodegenerative disease, the method includes the following stages:

(a) introducing the compound candidate as defined in claim 5, in cell culture or animal that is not human, capable of containing the aggregation of the isoforms of the protein as defined in any of PP-15;

(b) determination of the quantity amenable to visualization clusters;

(C) identification and selection of compounds that could reduce congestion or education is the use of aggregations of a specified protein or able to increase the survival cell culture or animal, which is not a person.

17. The use of compounds as defined in claim 5, for inhibiting protein aggregationin vitroin the animal body orex vivo.

18. Pharmaceutical or diagnostic composition comprising a compound as defined in claim 5, and, optionally, a pharmaceutically acceptable carrier.

19. The compound according to any one of claims 1 or 5, the use according to claim 6, the method according to any of claims 7 to 16, use 17 or the composition according to p, where the specified connection selected from the group including

where each R16 is independently selected from H and C1-4of alkyl; or two adjacent groups R16 can be connected with the formation of the bridging group containing from 1 to 3 carbon atoms;

and its prodrug, ester, MES or salt.

20. The method according to any of PP or 19, the application on 17 or 19, the diagnostic composition according p or 19, where the specified connection observed detectable label.

21. The method according to any of p, 19, 20 or use according to any one of p, 19, 20, where two or more of these compounds are applied simultaneously.

22. The compound according to any one of pp.5 or 19, where the disease associated with aggregation, characterized by the presence of aggregated forms at least one protein or its fragment, or derivative, of debelak selected from the group including prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, secrete lysozyme, fibrinogen, huntingtin, ataxin and other proteins with a poly-Q-sequence.

23. The compound according to any one of pp.5, 19 or 22, where the disease is selected from the group comprising Alzheimer's disease, prion disease, Parkinson's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary't neuropathic amyloidosis and Finnish hereditary systemic amyloidosis.

24. Connection item 23 where the specified prion disease is selected from diseases of Creutzfeldt-Jakob disease, variant disease Creutzfeldt-Jakob, genetic prion diseases of humans, bovine spongiform is encefalopatia (BSE) and scrapie.

25. The kit including a compound as defined in any of pp.5 and 18-24 and, optionally, the antibody or antibody fragment that specifically bind with the specified connection; and/or Monomeric or aggregated protein as defined in PP-15; and/or Monomeric or aggregated protein as defined in PP-15. not necessarily associated in a complex with the specified connection; and instructions for use in one or more containers.

The term "halogen", when used herein, refers to a halogen atom selected from fluorine, chlorine, bromine and iodine, preferably, the bromine.

The term "carboxy", when used herein, refers to the group-COOH.

The terms "alkyl" and "ALK" refers to alonovoa (hydrocarbon) radical with a straight or branched chain, containing from 1 to 12 carbon atoms, preferably from 1 to 6 atoms, more preferably from 1 to 4 carbon atoms. Typical examples of such groups include, but without limitation, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl.

The term "alkenyl" refers to a hydrocarbon radical straight or branched chain, containing from 2 to 12 carbon atoms, preferably from 2 to 6 carbon atoms and at least one double carbon-carbon bond. Typical examples that the groups include ethynyl or allyl.

The term "quinil" refers to a hydrocarbon radical straight or branched chain, containing from 2 to 12 carbon atoms, preferably from 2 to 6 carbon atoms, and at least one atom of carbon to carbon triple bond. Typical examples of such groups include ethinyl.

The term "alkoxy" refers to an alkyl group as defined above attached through an oxygen bridge (-O-).

The term "acyl group" in accordance with this invention refers to a functional group in which the alkyl, aryl, heterocyclyl or heteroaryl attached to a carbonyl group. Examples of acyl groups are formyl group; (C1-6acylcarnitine group such as acetyl group, propylaniline group, Butyrina group and pivellina group; C2-6alkenylamine group, such as ethanola group, propanola group and bottomline group; arolina group, such as benzoline group and the like, preferably acetyl group.

The term "acyloxy", when used herein, refers to an acyl group, which is associated with-O-. Similarly, the term "acylamino" refers to an acyl group, which is associated with the group-N(R")-, where R ' represents H or C1-6alkyl.

The term "carbocyclic group" refers to a fully saturated ilicakoy hydrocarbon group, containing from 1 to 4 cycles, preferably 1 cycle, and from 3 to 8 carbon atoms in the cycle. Typical examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

The term "carbonyloxy" refers to carbocyclic group as defined above attached through an oxygen bridge (-O-).

The term "carbocyclic" refers to an alkyl group, a substituted carbocyclic group, where the carbocyclic group and represent alkyl groups defined above.

The term "carbosilanes" refers to alkenylphenol group, a substituted carbocyclic group, where the carbocyclic group and alkenyl represent groups defined above.

The term "aryl" refers to cyclic aromatic hydrocarbon groups that contain from 6 to 20, preferably from 6 to 10 carbon atoms in the main chain of the molecule and include from 1 to 3 aromatic cycle, in particular monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl. In the case when the group contains two or more aromatic cycles (bicyclic and so on), aromatic cycles aryl group can be connected at a single point (e.g., biphenyl), or may be condensed (for example, naphthyl, phenanthrene etc).

The term "arylalkyl" refers to an alkyl group, W is displaced aryl group, where the aryl and alkyl groups are defined above.

The term "arylalkyl" refers to alkenylphenol group, substituted aryl group, where the aryl and alkenyl represent groups defined above.

The term "arylalkyl" refers to alkenylphenol group, substituted aryl group, where the aryl and quinil represent groups defined above.

The term "aryloxy" refers to an aryl group as defined above attached through an oxygen bridge (-O-), for example, fenoxaprop, antioxycaps, biphenyloxy and the like, preferably fenoxaprop.

The term "Allakaket" refers to alkoxygroup, substituted aryl group, where the aryl and alkoxy groups are defined above.

The term "heterocyclic group" refers to fully saturated or partially or fully unsaturated cyclic groups (for example, 3 to 7-membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16-membered tricyclic systems)that contain at least one heteroatom in the cycle containing at least one carbon atom. Each cycle heterocyclic group containing a heteroatom can contain 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen and/or sulphur, where the heteroatoms nitrogen and sulfur may be optionally oxidized, and Goethe is otomy nitrogen may be optionally quaternidinum. (The term "heteroaryl" refers to a heteroaryl group containing quartersawing the nitrogen atom and, therefore, a positive charge). Heterocyclic group may join the rest of the molecule via any heteroatom or carbon atom of the cycle or cyclic system. Typical examples of the monocyclic heterocyclic groups include ethylene oxide, azetidine, pyrrolidine, pyrrolyl, pyrazolyl, oxetanyl, pyrazolines, imidazoles, imidazolines, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, diazolidinyl, isothiazolin, isothiazolinones, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinil, 2-oxopiperidine, 2-oxopiperidine, 2-oxopyrrolidin, 2-oxoazetidin, azepine, hexahydroazepin, 4-piperidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, morpholinyl, thiomorpholine, themorphological, themorphological, 1,3-dioxolane, tetrahydro-1,1-DIOXOLANYL etc. are Typical examples of bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzoxadiazole, benzothiazyl, hinokitiol, chinoline, tetrahydroisoquinoline, ethenolysis, benzimidazolyl, benzopyranyl, indolizinyl, benzofuran, Benzi razani, chromanol, coumarinyl, benzopyranyl, cinnoline, honokalani, indazoles, pyrrolopyridine, properidine (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl or furo[2,3-b]pyridinyl), dihydroergotoxine, dehydrodiconiferyl, dihydroisoquinolyl, dihydroindole, dihydroquinoline, dihydroquinazolines (such as 3,4-dihydro-4-oxothiazolidine), trainersair, tetrahydroquinoline etc. are Typical examples of tricyclic heterocyclic groups include carbazolyl, bunzendahl, phenanthrolines, dibenzofurans, acridines, phenanthridines, xantener etc.

The term "heterocyclic" refers to a heterocyclic group as defined above attached through an oxygen bridge (-O-).

The term "heteroseksualci" refers to an alkyl group, a substituted heterocyclic group, where the heterocyclic group and the alkyl groups are defined above.

The term "heteroaryl", when used herein, refers to a 5-6-membered aromatic cycle, which may contain as heteroatoms oxygen atom, sulfur and/or nitrogen and which may be condensed other aromatic cycle. Examples of heteroaryl groups, but without limitation, are benzofuranyl, furyl, thienyl, benzothiazyl, thiazolyl, imidazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, benzoate who was Salil, triazolyl, tetrazolyl, isoxazolyl, isothiazolin, pyrrolyl, pyranyl, tetrahydropyranyl, pyrazolyl, pyridyl, chinoline, ethenolysis, purinol, carbazolyl, benzoxazolyl, benzimidazolyl, indolyl, isoindolyl, pyrazinyl, diazines, pyrazin, triazinetrione, tetrazines, tetrazolyl, benzothiophene, benzopyranyl and benzimidazolyl.

The term "heteroaromatic" refers to a heteroaryl group as defined above attached through an oxygen bridge (-O-).

The terms "heteroaromatic", "heteroaromatic and heteroarylboronic" refer to groups in which the alkyl, Alchemilla or Alchemilla group is a substituted heteroaryl group, where heteroaryl, alkyl, alkenyl and quinil represent groups defined above.

The term "heteroaromatic" refers to alkoxygroup, substituted heteroaryl group, where the heteroaryl and alkoxygroup represent groups defined above.

The term "substituted", when used herein, refers to a group substituted by one or more substituents, preferably from 1 to 4 substituents, at any available point of attachment. Typical examples of substituents include, but without limitation, one or more of the following groups: alkyl, alkoxy, halogen, hydroxyl group, carboxyl group (i.e.- IS HE), alkoxycarbonyl, alkylcarboxylic, amino group (i.e.- NH2), Tolna or the nitro-group.

In a preferred alternative embodiment, R1 is selected from the group comprising hydrogen and alkyl, more preferably hydrogen.

In a preferred alternative embodiment, R2 is selected from the group comprising hydroxyl and alkoxygroup.

In a preferred alternative embodiment, R3 is selected from the group comprising a hydroxyl group and alkoxygroup.

In an additional preferred alternative embodiment, R2 and R3 are linked and together form the structure-O(CH2)nO-, where n takes values from 1 to 3, preferably n is 1.

In a preferred alternative embodiment, R4 is selected from the group comprising hydrogen, hydroxyl group, and alkoxygroup, more preferably hydrogen.

In a preferred alternative embodiment, R5 is selected from the group comprising hydrogen and alkyl, more preferably hydrogen.

In a preferred alternative embodiment, R6 is selected from the group comprising hydrogen and alkyl, more preferably hydrogen.

In a preferred alternative embodiment, R7 is selected from the group comprising hydrogen, halogen, cyano, nitro, hydroxyl group and alkoxygroup, more preferably R7 represents hydrogen, halogen, hydroxyl group or alkoxy is the SCP, even more preferably R7 is a halogen.

In a preferred alternative embodiment, R8 is selected from the group comprising hydrogen, hydroxyl group, and alkoxygroup.

In a preferred alternative embodiment, R9 is selected from the group comprising hydrogen, halogen, hydroxyl group, and alkoxygroup.

In a preferred alternative embodiment, R10 is selected from the group comprising hydrogen and alkyl, more preferably hydrogen.

In a preferred alternative embodiment, R11 is selected from the group comprising hydrogen and alkyl.

In a preferred alternative embodiment, R12 is selected from the group comprising hydrogen and alkyl.

In a preferred alternative embodiment, R13 is selected from the group comprising hydrogen and alkyl.

In a preferred alternative embodiment, R14 is selected from the group comprising hydrogen and alkyl.

In a preferred alternative embodiment, R15 is selected from the group comprising hydrogen and alkyl.

In another preferred embodiment, compounds according to the invention R7 represents halogen, cyano, hydroxyl group, or nitro, azido, alkoxy, thio, alkylthio, amino, halogenoalkane, alkyl or halogenated.

In a preferred alternative embodiment, R7 represents halogen, cyano, hydroxyl group, or n the tro, more preferably halogen.

In an additional preferred alternative embodiment, R2 and R3, each independently, selected from hydroxyl group and1-6alkoxy; or R2 and R3 together form the structure-O-(CH2)n-O-, where n takes values from 1 to 3, preferably n is 1.

In a preferred alternative embodiment, the compound is selected from the group including

where each R16 is independently selected from H and C1-4of alkyl; or two adjacent groups R16 can be connected with the formation of the bridging group containing from 1 to 3 carbon atoms; and its prodrug, ester, solvate, or salt.

In a more preferred alternative embodiment, the compound is selected from the group including

3-(4-hydroxyphenyl)-5-(3,4-dihydroxyphenyl)isoxazol

3,5-bis(3,4-acid)pyrazole

5-(3-bromophenyl)-3-(3,4-methylenedioxyphenyl)pyrazole

5-(3-forfinal)-3-(3,4-methylenedioxyphenyl)pyrazole

3-(3,4-dihydroxyphenyl)-5-(3-forfinal)pyrazole

the hydrobromide of 2,4-bis(3,4-dihydroxyphenyl)imidazole is

3-(3,4-acid)-5-(3,4,5-trimethoxyphenyl)pyrazole

the hydrobromide 5-(3,5-dibromophenyl)-3-(3,3,5-trihydroxyphenyl)pyrazole

5-(3-bromophenyl)-3-(2-hydroxyphenyl)pyrazole (10353_F11)

Connection of an alternative embodiment of the invention may contain a detectable label.

The list of references

1. The compound represented by formula (E)

where
X, Y and L non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N= and-O-;
M and Z independently is not directionally selected from;
---- indicates an optional double bond;
R1, R2, R3, R4and R6independently selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH;
Hal is selected from F, Cl, Br and I;
RE1and RE2attached to adjacent carbon atoms, and RE1and RE2together undirected form a structure-T-(CRE7RE8)n-V-, where T is selected from CRE9RE10and O or NH, and V is selected from CRE9RE10and O or NH, and the corresponding structure, which is a double bond, moreover, at least one of T or V represents O or N;
RE7and RE8represent H or F;
RE9and RE10represent H;
n takes values from 1 to 2;
RE3represents a C1-6alkyl group;
m takes on the values 0 or 1;
RE4represents a halogen atom;
p takes on the values 0 or 1;
and its ester, MES or salt;
provided that the specified list excluded the following connections:

2. The compound according to claim 1, where the compound represented by formula (E)is a compound represented by formula (A)

where
m, n, p, T, V, X, Y, L, M, Z, RE7, RE8and Hal take the values defined in claim 1;
RA1and RA2together undirected form a structure-T-(CRE7RE8)n-V-;
RA3represents a C1-6alkyl group;
RA4represents a halogen atom;
and its ester, MES or salt.

3. The compound according to claim 1, where the cycle D directly selected from the following structures:



where
R8and R9selected from hydrogen, -C1-4alkylene-halogen, -C1-4alkylene-OH.

4. Connect the tion according to claim 3, where cycle D directionally selected from the following structures:

where
R8and R9take the values defined in item 3.

5. The compound according to claim 1, where the compound is selected from the group including

where
R is selected from hydrogen, -C1-4alkylene-halogen, -C1-4alkylene-OH; and
Hal is selected from F, Cl, Br and I; and
RE7and RE8represent H or F,
and its ester, MES or salt.

6. The compound according to claim 1, where the compound is selected from the group including

or

where Hal represents Cl or Br;
and its ester, MES or salt.

7. The compound according to claim 1, selected from




,
and its ester, MES or salt.

8. The compound represented by formula (E)

where
RE1selected from-NRE5RE6;
RE2selected from hydrogen;
RE5and RE6independently selected from hydrogen and C1-6of alkyl;
X, Y and L non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N=;
M and Z non-directional independently selected from;
---- Osnach is no optional double bond;
R1, R2, R3, R4and R6independently selected from hydrogen;
Hal is selected from F, Cl, Br and I;
RE3represents a C1-6alkyl group;
m takes on the values 0;
RE4represents a halogen atom;
p takes on the values 0;
and ester, MES or salt compounds represented by the formula (E);
provided that the specified list excluded the following connections:
3(5)-(2-hydroxy-5-were)-5(3)-(4-chlorophenyl)pyrazole;
ortho-hydroxyphenyl 5-dichloro-3',4' phenyl-3 methyl-2-pyrazole;
ortho-hydroxyphenyl-5 dichloro-3',4' phenyl-3 phenyl-2 pyrazole;



9. The connection of claim 8, where the cycle D directionally selected from the following structures:

where
R8and R9represent hydrogen.

10. The connection according to claim 9, where the compound is a


11. The compound of claim 8 where the compound is a

or

where
R represents H;
Hal is selected from F, Cl, Br and I;
RA9represents H or C1-4alkyl;
RA10the stand is made by a H or C 1-4alkyl;
and its ester, MES or salt.

12. The compound of claim 8 where the compound is a

or

where
Hal is selected from Cl or Br;
and its ester, MES or salt.

13. The compound represented by formula (E)

where
RE1and RE2independently selected from hydroxyl group and C1-6alkoxy and attached in the meta and para with respect to the carbon atom that binds the phenyl ring cycle D;
X, Y and L non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N= and-O-;
M and Z non-directional independently selected from;
---- indicates an optional double bond;
R1, R2, R3, R4and R6independently selected from hydrogen or-C(O)-C1-4alkyl;
Hal is selected from F, Cl, Br and I;
RE3represents a C1-6alkyl group, methoxy group, or OH;
m takes on the values 0 or 1;
RE4represents a halogen atom;
p takes on the values 0 or 1;
and its ester, MES or salt.

14. The connection indicated in paragraph 13 where the compound represented by formula (E)is a compound represented by formula (A)

where
m, p, X, Y , M, Z, R1, R2, R3, R4and R6and Hal take the values defined in item 13;
where in the formula (A):
RA1and RA2each independently selected from a hydroxyl group and C1-6alkoxy;
RA3represents a C1-6alkyl group;
m=0, 1
RA4represents a halogen atom;
p=0, 1,
and its ester, MES or salt.

15. The connection of item 13 or 14, where the cycle D directionally selected from the following structures:


where
R8and R9represent hydrogen or-C(O)-C1-4alkyl.

16. The connection indicated in paragraph 15, where the cycle D directionally selected from the following structures:

where
R8and R9represent hydrogen or-C(O)-C1-4alkyl.

17. The connection indicated in paragraph 13, where the connection is a

where
R represents H;
Hal is selected from F, Cl, Br and I;
RA7represents H or C1-4alkyl;
RA8represents H or C1-4alkyl;
and its ester, MES or salt.

18. The connection indicated in paragraph 13 where the compound is selected from the group consisting of


where
Hal represents Cl or Br;
and its ester, MES or salt.

19. Link is, containing a detectable label, represented by formula (E)

where
X, Y and L non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N= and-O-;
M and Z non-directional independently selected from;
---- indicates an optional double bond;
R1, R2, R3, R4and R6independently selected from hydrogen, C1-4of alkyl; the group-C1-4alkylene-halogen; group-C1-4alkylene-OH;
Hal is selected from F, Cl, Br and I;
RE1and RE2attached to adjacent carbon atoms, and RE1and RE2together undirected form a structure-T-(CRE7RE8)n-V-, where T is selected from CRE9RE10and O or NH, and V is selected from CRE9RE10and O or NH, and the corresponding structures in which there is a double bond, and at least one of T or V represents O or N;
RE7and RE8represent H or F;
RE9and RE10represent H;
n takes values from 1 to 2;
RE3represents a C1-6alkyl group;
m takes on the values 0;
RE4represents a halogen atom;
p takes on the values 0 or 1;
and its ester, MES or salt.

20. The compound containing detectable label, represented by formula (E)

where
RE1selected from-NRE5RE6;
RE2selected from hydrogen;
RE5and RE6independently selected from hydrogen and C1-6of alkyl;
X, Y and L non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N=;
M and Z non-directional independently selected from;
---- indicates an optional double bond;
R1, R2, R3, R4and R6independently selected from hydrogen;
Hal is selected from F, Cl, Br and I;
RE3represents a C1-6alkyl group;
m takes on the values 0;
RE4represents a halogen atom;
p takes on the values 0;
and its ester, MES or Sol connection.

21. The compound containing detectable label, represented by formula (E)

where
RE1and RE2independently selected from hydroxyl group and C1-6alkoxy and attached in the meta and para with respect to the carbon atom that binds the phenyl ring cycle D;
X, Y and L non-directional independently selected from-C(R1)(R2)-, -C(R3)=, -N(R4)-, -N= and-O-;
M and Z non-directional independently selected from;
---- indicates an optional double bond;
R1, R2, R3, R4and R6independently selected from hydrogen or-C(O)-C1-4alkyl;
Hal is selected from F, Cl, Br and I;
RE3represents a C1-6alkyl group, methoxy group, or OH;
m takes on the values 0 or 1;
RE4represents a halogen atom;
p takes on the values 0 or 1;
and its ester, MES or salt.

22. Pharmaceutical composition suitable for treating or preventing diseases associated with protein aggregation and/or neurodegenerative diseases, comprising the compound according to any one of claims 1 to 18, as well as its ester, MES or salt, where disclaim specified in claims 1 to 8, does not apply, and a pharmaceutically acceptable carrier.

23. A diagnostic composition suitable for rendering deposits of aggregated protein, comprising the compound according to any one of claims 1 to 18, as well as its ester, MES or salt, where disclaim specified in claims 1 to 8, does not apply, and a pharmaceutically acceptable carrier.

24. The use of compounds according to any one of claims 1 to 18, as well as its complex ether, MES or salt, where disclaim specified in claims 1 to 8, is not applied in the treatment or prevention of diseases associated with protein aggregation and/or neurodegenerative diseases.

25. The application of paragraph 24, where the disease associated with the aggregation of proteins, characterized by the presence of aggregated forms at least one protein or its fragment, or it is derived, where the protein is selected from the group comprising the prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, secrete lysozyme, fibrinogen, huntingtin, ataxin and other proteins with a poly-Q sequence.

26. The application of paragraph 24, where the disease is selected from the group including Parkinson's disease, prion disease, Alzheimer's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary is not neuropathic amyloidosis and Finnish hereditary systemic amyloidosis.

27. The application of paragraph 24, where the prion disease is selected from diseases of Creutzfeldt-Jakob disease, type of disease Creutzfeldt-Jacob, inherited prion diseases of humans, bovine spongiform EN is evalita (BSE) syndrome (mad cow disease), scrapie.

28. The use of compounds according to any one of claims 1 to 18, as well as its complex ether, MES or salt, where disclaim specified in claims 1 to 8, does not apply to pharmaceutical compositions for the treatment or prevention of diseases associated with protein aggregation and/or neurodegenerative diseases.

29. Use p, where the disease associated with the aggregation of proteins, characterized by the presence of aggregated forms at least one protein or its fragment, or derivative, where the protein is selected from the group comprising the prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, secrete lysozyme, fibrinogen, huntingtin, and ataxin other proteins with a poly-Q sequence.

30. Use p, where the disease is selected from the group including Parkinson's disease, prion disease, Alzheimer's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary is th amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary't neuropathic amyloidosis and Finnish hereditary systemic amyloidosis.

31. Use p, where the prion disease is selected from diseases of Creutzfeldt-Jakob disease, type of disease Creutzfeldt-Jacob, inherited prion diseases of humans, bovine spongiform encephalitis (BSE) syndrome (mad cow disease), scrapie.

32. A method of treating or preventing diseases associated with protein aggregation and/or neurodegenerative diseases, this method includes the introduction of a therapeutically effective amount of a compound according to any one of claims 1 to 18, where disclaim specified in claims 1 to 8, not applicable, or its ether complex, MES or salt to a patient in need of such introduction.

33. The method according to p, where the disease associated with the aggregation of proteins, characterized by the presence of aggregated forms at least one protein or its fragment or derivative, where the protein is selected from the group comprising the prion protein, amyloid protein precursor (APP), alpha-synuclein, superoxide dismutase, Tau-protein, immunoglobulin, amyloid A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, secrete lysozyme, fibrinogen, huntingtin, ataxin and other proteins with a poly-Q sequence.

34. The method according to p, where the disease is selected from the group including Parkinson's disease, prion disease, Alzheimer's disease, multiple system atrophy, diffuse disease Taurus Levi, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, ataxia, associated with diseases of the spinal cord and cerebellum, and other poly-Q diseases, hereditary cerebral amyloid angiopathy, hereditary amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-localized amyloidosis, beta-2-microglobulinuria amyloidosis, hereditary is not neuropathic amyloidosis and Finnish hereditary systemic amyloidosis.

35. The method according to p, where the prion disease is selected from diseases of Creutzfeldt-Jakob disease, type of disease Creutzfeldt-Jacob, inherited prion diseases of humans, bovine spongiform encephalitis (BSE) syndrome (mad cow disease), scrapie.

36. The use of compounds according to any one of claims 1 to 18, where disclaim specified in claims 1 to 8, not applicable, or its ether complex, MES or salt for inhibiting protein aggregation in vitro, in organisms is e animal or ex vivo.

37. The kit including a compound according to any one of claims 1 to 18 or its ester, MES or salt, where disclaim specified in claims 1 to 8, does not apply, and optionally, the antibody or antibody fragment that specifically bind with the specified connection; and/or Monomeric or aggregated protein defined in A.25; and/or Monomeric or aggregated protein defined in A.25, not necessarily in the complex connection with the specified connection; and instructions for use, in one or more containers.

38. The visualization of clusters of aggregated protein, comprising the following stages:
(i) introduction the subject of detectable amounts of a composition comprising a compound with a detectable label defined in any one of claims 1 to 18, where disclaim specified in claims 1 to 8, not applicable, or its ether complex, MES or salt;
(ii) provide a period of time sufficient for binding of the compounds with the aggregated protein; and
(iii) detection of the connection associated with the aggregated protein.

39. The method according to § 38, where the detectable label is selected from the18F,11C,125I123I131I77Br and76Br, in particular18F and11C.

40. Set for connection with a detectable label according to any one of claims 1 to 18, where the kit includes at least two compounds is preceded by the Tonika, who in the reaction to form the compound according to any one of claims 1 to 18, where disclaim specified in claims 1 to 8, not applicable, or its ether complex, MES or salt, where at least one of X, Y, and L represents-N(R4)and R4includes a detectable label.

41. Set p, where the detectable label is selected from the18F,11C,125I123I131I77Br and76Br, in particular18F and11C.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to a novel derivative of N-acylanthranilic acid, represented by the following general formula 1, or to its pharmaceutically acceptable salt, in which R1, R2, R3, X1, X2, X3, X4 and A are determined in the invention formula.

EFFECT: invention relates to an inhibitor of collagen production, a medication for treating diseases, associated with the excessive production of collagen, containing N-acylanthranilic acid derivative Formula 1.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a new compound of formula [I] or to its pharmaceutically acceptable salt, wherein A represents optionally substituted alkyl, wherein the substitute represents identical or different 1-3 groups specified in aryl optionally substituted by 1-3 groups specified in alkyl, halogen, alkoxy and alkanoyl; cycloalkyl optionally substituted by 1-3 groups specified in alkyl and halogen; hydroxy; alkoxy; halogen; an amino group and oxo; an optionally substituted carbocyclic group specified in a mono- and bicyclic group, wherein an aromatic ring and cycloalkyl are condensed; optionally substituted aryl, an optionally substituted completely saturated 5- or 6-merous monocyclic heterocyclic group each of which contains 1 heteroatom specified in nitrogen and oxygen, wherein the substitute of optionally substituted aryl, the optionally substituted carbocyclic group and the optionally substituted heterocyclic group for A represents identical or different 1-3 groups specified in alkyl, optionally substituted hydroxy, alkoxy, cycloalkyl or halogen; cycloalkyl optionally substituted by alkyl or alkoxy; alkoxy optionally substituted by halogen; halogen; hydroxy; oxo; heterocycle; alkyl sulphonyl; and mono- or dialkylcarbamoyl, optionally substituted amino, wherein the substitute represents identical or different 1 or 2 alkyl or aryl, or optionally substituted carbamoyl, wherein the substitute represents identical or different 1 or 2 alkyls optionally substituted by aryl, X represents optionally substituted methylene or -O-, wherein the substitute of optionally substituted methylene for X represents alkoxy or hydroxy, Q represents N or C-R4, L1 represents a single bond, methylene, -CH=CH-, -O-, -CO-, -NR11-, -NR11CO-, -CONR11- or -CH2NR11-, L2 represents a single bond, -CR6R7- or a bivalent 5- or 6-merous completely saturated monocyclic heterocyclic group each of which contains 1 heteroatom specified in nitrogen and oxygen, R1 and R2 are identical or different, and each represents hydrogen, alkyl or halogen, R3 and R4 are identical or different, and each represents hydrogen, alkyl, alkoxy, cyano or halogen, R1 and R3 are optionally bond thereby forming 5- or 6-merous cycloalkane, or a 5- or 6-merous aliphatic heterocycle containing oxygen atom, R5 represents a carboxyl group, an alkoxycarbonyl group or a bioisosteric group of the carboxyl group, R6 and R7 are identical or different, and each represents hydrogen or alkyl, or R6 and R7 are bond thereby forming cycloalkane, R8 represents hydroxy, alkanoylamino or alkyl sulphonylamino, R9 and R10 represent hydrogen or halogen, and R11 represents hydrogen or alkyl. Besides, the invention refers to specific compounds of formula [I], a drug based on the compound of formula [I], using the compound of formula [I], a method of treating based on using the compound of formula [I], and an intermediate compound of formula [II].

EFFECT: there are prepared new compounds possessing the agonist activity on thyroid hormone β receptor.

18 cl, 36 tbl, 344 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to novel compound of formula (1) or its pharmaceutically acceptable salt, possessing SNS inhibiting properties. In general formula R1 represents (1) hydrogen atom, (2) halogen atom, (3) C1-6alkyl group or (4) C1-6halogenalkyl group (whereR1 can be present in any substitutable position of benzene or pyridine ring); L represents (1) simple bond, (2) -O- or (3) -CH2O- (where L can be present in position 5 or 6 of condensed cycle); R2 represents (1) C6-10aryl group (C6-10aryl group is optionally condensed with C3-6cycloalkane), optionally substituted with substituent(s), X represents carbon atom or nitrogen atom. Other values of radicals are given in the invention formula.

EFFECT: obtaining compounds which can be used to prepare medication for treatment or prevention of such diseases as neuropathic pain, nociceptive pain, dysuria, disseminated sclerosis, etc.

19 cl, 47 tbl, 237 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of general formula I , where R1 is a hydrogen atom, a lower alkyl, CD3, -(CH2)n-CHO, -(CH2)n-O-lower alkyl, -(CH2)n-OH, -(CH2)n-cycloalkyl or is a heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with O); R2 is a hydrogen atom, a halogen atom, hydroxy, lower alkyl, di-lower alkyl, -OCH2-O-lower alkyl or lower alkoxy; or the piperidine ring along with R2 forms a spiro-ring selected from 4-aza-spiro[2,5]oct-6-yl; Ar is an aryl or heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N), optionally having one, two or three substitutes selected from a halogen atom, lower alkyl, lower alkyl having as substitutes, a halogen atom, a lower alkoxy having as substitutes, a halogen atom, cycloalkyl, lower alkoxy, S-lower alkyl, heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with N), or optionally having as substitutes, phenyl, optionally having R' as substitutes, and R' is a halogen atom, CF3, lower alkyl, lower alkoxy or a lower alkoxy having as substitutes, a halogen atom, or is a heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N and S); R is a lower alkyl, heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with O), aryl or heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N), Where the aryl and heteroaryl optionally have as substitutes, one or two R'; n equals 0, 1, 2 or 3; or to a pharmaceutically acceptable acid addition salt, a racemic mixture or a corresponding enantiomer and/or optical isomer of said compound. The invention also relates to pharmaceutical compositions based on a glycine reuptake inhibitor of a compound of formula I.

EFFECT: obtaining novel compounds and a pharmaceutical composition based thereon, which can be used in medicine to treat neurological and psychoneurological disorders.

22 cl, 1 tbl, 128 ex

FIELD: chemistry.

SUBSTANCE: invention relates to 5-membered heterocyclic compounds of general formula (I), their prodrugs or pharmaceutically acceptable salts, which possess xanthine oxidase inhibiting activity. In formula (I) T represents nitro, cyano or trifluoromethyl; J represents phenyl or heteroaryl ring, where heteroaryl represents 6-membered aromatic heterocyclic group, which has one heteroatom, selected from nitrogen, or 5-membered aromatic heterocyclic group, which has one heteroatom, selected from oxygen; Q represents carboxy, lower alkoxycarbonyl, carbomoyl or 5-tetrasolyl; X1 and X2 independently represent CR2 or N, on condition that both of X1 and X2 do not simultaneously represent N and, when two R2 are present, these R2 are not obligatorily similar or different from each other; R2 represents hydrogen atom or lower alkyl; Y represents hydrogen atom, hydroxy, amino, halogen atom, perfluoro(lower alkyl), lower alkyl, lower alkoxy, optionally substituted with lower alkoxy; nitro, (lower alkyl)carbonylamino or (lower alkyl) sulfonylamino; R1 represents perfluoro(lower alkyl), -AA, -A-D-L-M or -A-D-E-G-L-M (values AA, A, D, E, G, L, M are given in i.1 of the invention formula).

EFFECT: invention relates to xanthine oxidase inhibitor and pharmaceutical composition, which contain formula (I) compound.

27 cl, 94 tbl, 553 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel radiolabelled compounds of formula I in which R1 represents isopropoxy or 2,2,2-trifluoro-1-methyl-ethoxy; and R2 represents radiolabelled CH3 group, where radionuclide represents 3H or 11C. Invention also relates to pharmaceutical composition for diagnostic visualisation of GlyT1 transporter (glycine transporter type 1).

EFFECT: obtained are novel radiolabelled compounds, which can be applied in medicine as radioactive indicator in PET (positron emission tomography) for labeling and diagnostic molecular visualisation of functionality of glycine transporter type 1.

13 cl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to N-[2,4-dioxo-6-(tetrahydrofuran-2-yl)-7-trifluoromethyl-1,4-dihydro-2H-quinazolin-3-yl]methanesulphonamide and N-[6-(1-isopropoxyethyl)-2,4-dioxo-7-trifluoromethyl-1,4-dihydro-2H- quinazolin-3-yl] methanesulphonamide, having antagonistic activity on the AMPA receptor. The invention also relates to a pharmaceutical composition.

EFFECT: use of said compounds to produce drugs for treating AMPA mediated conditions and primarily for treating epilepsy or schizophrenia.

6 cl, 81 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining [1S-[1α,2α,3β(1S*,2R*),5β]]-3-[7-[2-(3,4-difluorophenyl)-cyclopropylamino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidine-3-yl]-5-(2-hydroxyethoxy)-cyclopentane-1,2-diol of formula (I) .

EFFECT: improving yield of the compound of the formula and its high quality when recrystallisation is not available.

5 cl, 1 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula I

or a pharmaceutically acceptable salt thereof, where R1 is H or R1 and R2 together with a nitrogen group can form where A, B, C and D are independently selected from a group consisting of CR1a and N; where at least one of A, B, C and D is CR1a; where R1a is selected from a group consisting of H, -ORi, -SRii, -S(O)Riii, -C(O)NRvRvi and CF3, where Ri is selected from a group consisting of methyl, ethyl, propyl, hydroxyethyl, hydroxypropyl, 2-oxo-2-phenylethyl, butyl, acetonitrile and benzyl; Rii, Riii and Riv denote methyl; Rv and Rvi are independently selected from a group consisting of H, methyl, ethyl, hydroxyethyl, hydroxypropyl, diethyalminoethyl, phenyl, pyridinyl, methoxyethyl, hydroxyethoxyethyl, benzyl, phenylethyl, 2-hydroxy-1-hydroxymethyl-2-phenylethyl and carbomoylethyl, or Rv and RVi together form morpholine or ethyl ester of piperazine; R2 is selected from a group consisting of phenyl, naphthyl, pyrazolyl and C1-C8alkylene phenyl; R3 is C1-C8alkylene; R4 is selected from a group consisting of H, C1-C8alkyl and -C=NH(NH2). The invention also relates to compounds of formulae I-A

I-B I-C

I-D I-E

values of radicals of which are given in the claim; a method of treating said pathological conditions, a pharmaceutical composition based on said compounds, a method of identifying a Trp-p8 agonist and specific compounds.

EFFECT: obtaining compounds which are useful as Trp-p8 modulators.

25 cl, 19 dwg, 8 tbl, 17 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel pyranyl aryl methylbenzoquinazolinone compounds of formula (I), which are positive allosteric modulators of the M1 receptor and which can be used to treat diseases associated with the M1 receptor, such as Alzheimer's disease, schizophrenia, pain disorders or sleep disturbance. In formula (I) X-Y are selected from a group comprising (1) -O-CRARB-, (2) -CRARB-O-, (3) -CRARB-SRC-, (4) -CRARB-NRC- and (5) -NRC-CRARB-, where each RA and RB is a hydrogen atom, and RC is selected from a group comprising (a) hydrogen, (b) -C(=O)-C1-6alkyl, (c) -C1-6alkyl, (d) -C(=O)-CH2-C6H5, (e) -S(=O)2-C1-6 alkyl, R1 is a hydroxy group, R2 is selected from a group comprising (1) -phenyl, (2) - heteroaryl, where the phenyl or heteroaryl group R2 is optionally substituted; the rest of the values of the radicals are given in the claim.

EFFECT: obtaining novel pyranyl aryl methylbenzoquinazolinone compounds.

28 cl, 12 tbl, 37 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to compounds, represented by formula (I) , where X1 and X2 independently represent CH or N; ring U represents benzene ring, pyrazole ring, 1,2,4-oxadiazole ring, 1,2,4-thiadiazole ring, isothiazole ring, oxazole ring, pyridine ring, thiazole ring or thiophene ring, m represents integer number, which has values from 0 to 1; n represents integer number, which has values from 0 to 3; R1 represents hydroxygroup or C1-6 alkyl; R2 represents any of (1)-(3): (1) halogen atom; (2) hydroxygroup; (3) C1-6 alkyl or C1-6 alkoxy, each of which can independently contain any group, selected from group of substituents α; group of substituents α includes fluorine atom and hydroxygroup, or its pharmaceutically acceptable salt. Invention also relates to pharmaceutical composition, possessing inhibiting activity with respect to xanthenes oxidase, including formula (I) compound or its pharmaceutically acceptable salt as active ingredient.

EFFECT: derivative, which contains condensed ring structure, intended as means for prevention and treatment of disease, associated with abnormal level of uric acid in serum.

15 cl, 11 tbl, 126 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of obtaining derivatives of ethyl 1,2,4-oxadiazole-5-carboxylates of general formula VII from respective amidoximes of general formula VI by interaction with diethyl ether of oxalic acid: Method includes mixing amidoxime of general formula VI with 3-fold excess of diethyl ether of oxalic acid with the following exposure at temperature 120°C for 3-4 hours. Separation of target ethyl 1,2,4-oxadiazole-5-carboxylate VII consists in cooling reaction mass to room temperature, filtering formed suspension and washing with dichloromethane.

EFFECT: method of obtaining derivatives of ethyl 1,2,4-oxadiazole-5-carboxylates with increased output of product without application of sodium ethoxide is elaborated Obtained derivatives of ethyl 1,2,4-oxadiazole-5-carboxylates can be applied in chemical industry as antibacterial medications or half-products in synthesis of antithrombotic, antiplatelet and antiparasitic medications.

1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of 1,2,4-oxadiazoles of formula: where R1 is a substituted or unsubstituted alkyl or -(CH2CH2O)nR6; R6 is hydrogen, substituted or unsubstituted alkyl, X in each case is independently F, C, Br or I, n equals an integer from 1 to 7, and m equals an integer from 1 to 5, which are used to produce 3-[5-(2-fluorophenyl-[1,2,4]oxadiazol-3-yl]benzoic acid.

EFFECT: method for synthesis of 1,2,4-oxadiazoles.

20 cl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pyridine derivatives of formula (I) wherein A, R1, R2, R3, R4, R5, R6 and R7 are presented in the description, preparing and using them as pharmaceutically active compounds possessing SP1/EDG1 receptor agonist activity.

EFFECT: using the declared compounds or pharmaceutically acceptable salts thereof for preparing a pharmaceutical composition for preventing or treating the diseases or disorders associated with the activated immune system.

13 cl, 76 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to crystalline forms of 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid (formula I), pharmaceutical compositions and dosage forms containing these crystalline forms, as well as to methods for preparing such crystalline forms and methods for using them for treating, preventing a disease or a disorder associated with premature terminating codon. There are prepared new crystalline forms of 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid which are non-absorbent and which can find application in medicine for treating or preventing such diseases or disorders as type III mucopolysaccharidosis, hemophilia A, hemophilia B, neurofibromatosis 1, neurofibromatosis 2, Parkinson's disease, cystic fibrosis, macular degeneration, cephalooculocutaneous telangiectasis, retinitis pigmentosa, tuberous sclerosis, Duchenne muscular dystrophy and Marfan's syndrome, cancer.

EFFECT: higher effectiveness of using the compounds and a method of treating.

46 cl, 11 dwg, 9 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention offers compounds presented by general formula (I): or their pharmaceutically acceptable salts wherein R1, R2, R3 and R4 are presented in the description and exhibit substantial COMT inhobotory activity. Besides, the present invention described pharmaceutical compositions inhibiting catechol-O-transferase activity which contain the compound or its pharmaceutically acceptable salt as an active ingredient, and a pharmaceutically acceptable carrier.

EFFECT: there are declared pharmaceutical combinations for treatment or prevention of Parkinson's disease which contain (1) the pharmaceutical composition containing the compound under any cl 1-8 or its pharmaceutically acceptable salt and the pharmaceutically acceptable carrier, and (2) at least one compound specified in L-dope or carbidole.

10 cl, 9 ex, 17 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing optionally substituted 4-(benzimidazo-2-yl methylamino)benzamidine of formula (I) in which R1 denotes a methyl group, R2 denotes a R21NR22 group, where R21 denotes an ethyl group which is substituted with an ethoxycarbonyl group, and R22 denotes a pyridin-2-yl group, R3 denotes an n-hexyloxycarbonyl group, where at step (a) phenyldiamine of formula (II) where R1 and R2 assume values given for formula (I), which reacts with 2-[4-(1,2,4-oxadiazol-5-on-3-yl)phenylamino]acetic acid, to obtain a product of formula (III) where R1 and R2 assume values given for formula (I), which is hydrogenated at temperature from 30 to 60°C at hydrogen pressure from 1 to 10 bar, over palladium on activated charcoal (Pd/C) in a mixture of tetrahydrofuran and water, and then, without any preliminary extraction of the hydrogenation product, the obtained compound of formula (I), in which R3 denotes hydrogen, in the presence of potassium carbonate reacts with a compound of formula (IV) R3-X (IV), where R3 assumes values given for formula (I), and X denotes a suitable splitting group.

EFFECT: simple method of producing optionally substituted 4-(benzimidazo-2-yl methylamino)benzamidine.

3 cl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention refers to new phenyl derivatives with the formula (I) where # symbol indicates two carbon atoms of the phenyl ring bearing R1, R2 and R3; to each of the latter components the A group can be linked; and where A represents or where asterisks indicate the link through which the formula (I) is linked to the phenyl ring bearing R1, R2 and R3; R1 represents hyndrogen or C1-3-alkyl; R2 represents C2-5-alkyl or C1-4-alkoxy group; R3 represents hydrogen, and in case when the A group is linked in the para-position in relation to the phenyl ring of the formula (I) bearing R1, R2 and R3, R3 can additionally represent the methyl group; R4 represents hydrogen; R5 represents C1-3-alkyl; R6 represents a hydroxy group, di-(hydroxy-C1-4-alkyl)-C1-4-alkoxy group, 2,3-dihydroxypropoxy group, -OCH2-CH(OH)-CH2-NR61R62 or -OCH2-CH(OH)-CH2-NHCOR64; R61 represents hydrogen; R62 represents hydrogen; R64 represents hydroxymethyl; and R7 represents C1-3-alkyl; and to its salt. The invention also refers to the pharmaceutical composition that is agonistic in relation to S1P1/EDG1 receptor on the basis of the mentioned compounds.

EFFECT: new compounds and the pharmaceutical composition based on them that may find their application in medicine as immunomodulating agents.

18 cl, 2 tbl, 28 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula (I): where R1 and R2 represent hydrogen and a group which is hydrolysed in a physiological environment, optionally substituted lower alkanoyl or aroyl; X represents a methylene group; Y represents oxygen atom; n represents the number 0, 1, 2 or 3 and m represents the number 0 or 1; R3 represents a group of pyridine N-oxide according to formula A, B or C which is attached as shown by an unmarked linking: where R4, R5, R6 and R7 independently represent aryl, heterocycle, hydrogen, C1-C6-alkyl, C1-C6-alkylthio, C6-C12-aryloxy or C6-C12-arylthio group, C1-C6-alkylsulphonyl or C6-C12-arylsulphonyl, halogen, C1-C6-haloalkyl, trifluoromethyl, or heteroaryl group; or where two or more residues R4, R5, R6 and R7 taken together represent an aromatic ring, and where P represents a central part, preferentially chosen from regioisomers 1,3,4-oxadiazol-2,5-diyl, 1,2,4-oxadiazol-3,5-diyl, 4-methyl-4H-1,2,4-triazol-3,5-diyl, 1,3,5-triazine-2,4-diyl, 1,2,4-triazine-3,5-diyl, 2H-tetrazol-2,5-diyl, 1,2,3-thiadiazol-4,5-diyl, 1-alkyl-3-(alkoxycarbonyl)-1R-pyrrol-2,5-diyl, where alkyl is presented by methyl, thiazol-2,4-diyl, 1H-pyrazol-1,5-diyl, pyrimidine-2,4-diyl, oxazol-2,4-diyl, carbonyl, 1H-imidazol-1,5-diyl, isoxazol-3,5-diyl, furan-2,4-diyl, benzole-1,3-diyl and (Z)-1-cyanoethene-1,2-diyl, and where the regioisomers of the central part include both regioisomers produced by exchanging the nitrocatechol fragment and the -(X)n-(Y)m-R3 fragment. Also, the invention refers to a method for making a compound of formula I, as well as to a method for treating an individual suffering central and peripheral nervous system disorders, to a pharmaceutical composition based on the compounds of formula I, and also to their application for preparing the drug and as COMT inhibitor.

EFFECT: there are produced and described new compounds which show a potentially effective pharmaceutical properties in treating a number of central and peripheral nervous system disorders.

25 cl, 64 ex, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to oxadiazolidinone compounds presented by following formula (I), or to their pharmaceutically acceptable salts, (symbols in the presented formula represent the following values, R1: -H, R0: lower alkyl, Rz: the same or different from each other, and each represents -H or lower alkyl, L: *-CH2-O- or *-CH2-NH-, where the symbol * in L represents binding with the ring A and a substitution position in the group L in the ring B represents the 4-position, the ring A: benzole, the ring B: benzole or pyridine, R2; the same or different respectively, and each represents -halogen or -R0, n: 0 or 1, R3: phenyl which can be substituted by a group selected from the group G3, The group G3: halogen, -R0, halogen-lower alkyl, -ORz, -CON(Rz)2, -CON(Rz)-heteroring group, -O-S(O)2-R0, -O-lower alkylene-ORz, -O-lower alkylene-O-COR2, -O-lower alkylene-N(RZ)2, -O-lower alkylene-N(Rz)CO-Rz, -O-lower alkylene-CO2Rz, -O-lower alkylene-CON(Rz)2, -O-lower alkylene-CON(Rz)-(lower alkyl substituted by the group-ORz), -O-lower alkylene-SR0, -O-lower alkylene-cycloalkyl, -O-lower alkylene-CON(Rz)-cycloalkyl, -O-lower alkylene-heteroring group and -O-lower alkylene-CON(Rz)-heteroring group, where lower alkylene in the group G3 can be substituted by halogen or -ORz, and cycloalkyl and the heteroring group in the group G3 can be substituted by the group selected by the group G1, The group G1: halogen, cyano, -R0, -ORz, -N(RZ)2, -S-R0, -SO2-R0, -SO2N(Rz)2, -CO-R2, -CON(Rz)2, -CON(Rz)-lower alkylene-OR2, -N(Rz)CO-Rz, oxo, -(lower alkylene which can be substituted by the group -ORz)-aryl, heteroring group and lower alkylene-heteroring group where aryl and the heteroring group in the group G1 can be substituted by the group selected from the following group G2, the group G2: halogen, cyano where the heteroring group means a group containing a ring selected from i) a monocyclic 5-7-members, saturated or unsaturated heteroring containing 1 to 3 heteroatoms selected from O, S and N, ii) a bicyclic heteroring in which the heterorings selected in i) mentioned above are ring-condensed where the condensed rings can be the same or different, and iii) the bicyclic heteroring in which the heteroring selected in i) mentioned above is condensed with a benzoic ring or 5-7-members cycloalkane, R4: -H. The invention refers to a pharmaceutical composition, to application of the compounds under cl.1, as well as to a method for preventing and/or treating diabetes.

EFFECT: making new biologically active compounds representing GPR40 agonist, an agent stimulating insulin secretion and/or an agent for preventing and/or treating diabetes.

9 cl, 27 ex, 138 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a fluorine-containing organosulphur compound of formula (I) and a composition based on said compound, which can be used in arthropod pest control: where m equals 0; n equals 0, 1 or 2; A is pyrazolyl, isoxazolyl, imidazolyl, oxazolyl or thiazolyl, optionally substituted with a group E1; R1 is C1-4 alkyl, a cyano group, a halogen atom or a hydrogen atom; R2 is C1-4 alkyl, a halogen atom or a hydrogen atom; Q is C1-5 haloalkyl containing at least one fluorine atom or a fluorine atom; group E1 is selected from halogen, tert-butyl, trifluromethyl, pentafluoroethyl, ethynyl, propargyl, propargyloxy, cyano, trifluoromethyl, trifluromethane thionyl and trifluoromethane sulphonyl.

EFFECT: efficient agent for arthropod pest control.

5 cl, 24 ex

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