Synthetic immune protection mimetics and use thereof

FIELD: chemistry.

SUBSTANCE: present invention relates to a preparation which inhibits microbial growth, which includes an arylamide compound as an active compound and kleptose or captisol.

EFFECT: methods of producing the preparation, use and method of treating microbial infections are disclosed.

21 cl, 5 dwg, 11 tbl, 16 ex

 

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention is directed, in particular, on arylamine compounds and methods for their preparation and use.

PRIOR art

Antimicrobial peptides (AMP) are the first line of defense against microbes for many species. AMP usually are small (12-80 amino acids) cationic amphiphile. There are two types of AMP, including ribosome and primocane synthesized peptides. It was identified more than 700 AMP, and they are usually α-helix (magainin and cecropin) or enriched disulfide β-folded layers (bactenecin and defensin). Although peptides are composed of many different sequences, their physiological properties highly similar. They take amphiphilic structure with positively charged groups, isolated on one side of the secondary structure and hydrophobic groups on the opposite surface. In mammals, the peptides are produced and secreted into the skin, mucosal surfaces and in neutrophils and act locally in response to infection. There are General physical and chemical properties, which are mainly responsible for the biological activity of these peptides.

Some antimicrobial activity of the proteins of the immune protection were associated with direct cytotoxic de the actions and modulation of the innate immune system. It is assumed that their direct antimicrobial activity include both membrane and remembrance effects. Antimicrobial peptides remain effective remedy bacterial infections during the time evolution, suggesting that their mechanism of action inhibits bacterial responses that lead to resistance to toxic substances. This assumption is supported by direct experimental data, showing that there is no significant resistance to the action of antimicrobial peptides is observed after multiple serial passages of bacteria in the presence of sublethal concentrations of peptides.

There is an urgent need to develop new antimicrobial agents that attack new targets, to elude products resistance that limit the applicability of many antibiotics. In addition, these new viewer should show their antimicrobial activity through mechanisms that make the resistance of bacteria is not effective. Produced a series of ones analogs, which have many advantages over peptides because of their small size, which increase the stability and improve the distribution in tissues, and have the opportunity to fine-tune their physical properties to optimize performance and security. It was found that the series arylamine compounds that mimic the structural properties of antimicrobial peptides, are effective antimicrobial activity and a broad index of selectivity against mammalian cells.

The INVENTION

The present invention provides compounds of Formula I

,

where: each A is independently-C=O, C=S or-CH2; each D is independently O or S; each R1represents independently hydrogen, C1-3alkyl, C1-3alkoxy, halo, or Gialos1-3alkyl; each R2represents independently hydrogen, C1-3alkyl, C1-3alkoxy, halo, or Gialos1-3alkyl; each R3represents independently hydrogen, C1-4alkyl, C1-4alkoxy, halo, or Gialos1-4alkyl; and each R4represents independently hydrogen, C1-3alkyl, C1-3alkoxy, halo, or Gialos1-3alkyl; or their pharmaceutically acceptable salt.

In some embodiments of the invention each A represents-C=O.

In some embodiments of the invention, each D represents O.

In some embodiments of the invention each R1represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, or Gialos 1-3alkyl. In some embodiments of the invention each R1represents independently hydrogen, methyl, methoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R1represents independently hydrogen, methyl or methoxy. In some embodiments of the invention each R1represents hydrogen.

In some embodiments of the invention each R2represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R2represents independently hydrogen, methyl, methoxy or halo. In some embodiments of the invention each R2represents hydrogen.

In some embodiments of the invention each R3represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R3represents independently methyl, methoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R3represents independently halo, or Gialos1-3alkyl. In some embodiments of the invention each R3represents independently Gialos alkyl. In some embodiments of the invention each R3represents trifluoromethyl.

In some embodiments of the invention each R4represents independently hydrogen, methyl, ethyl, methoxy, ethoxy or Gialos1-3alkyl. In some embodiments of the invention each R4represents independently hydrogen, methyl, methoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R4represents independently hydrogen, methyl, methoxy or halo. In some embodiments of the invention each R4represents hydrogen.

In some embodiments of the invention each A is independently-C=O, C=S or-CH2; each D is independently O or S; each R1represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl; each R2represents independently hydrogen, methyl, methoxy, halo, or halomethyl; each R3represents independently C1-3alkyl, C1-3alkoxy, halo, or haloalkyl; and each R4represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl.

In some embodiments of the invention each A is an independently researched the mo-C=O or C=S; each D is independently O or S; each R1represents independently hydrogen, methyl, methoxy, halo, or halomethyl; each R2represents independently hydrogen, halo, or halomethyl; each R3represents independently methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl; and each R4represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen, halo, or halomethyl; each R2represents independently hydrogen or halo; each R3represents independently methyl, methoxy, halo, or halomethyl; and each R4represents independently hydrogen, methyl, methoxy, halo, or halomethyl.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently methyl, halo, or halomethyl; and each R4represents independently hydrogen, methyl, halo, or halomethyl.

In some embodiments, the implementation of izopet the of each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently halo, or halomethyl; and each R4represents independently hydrogen or halo.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently methyl, halo, or halomethyl; and each R4represents independently hydrogen, methyl, halo, or halomethyl.

In some embodiments of the invention the compound is a

or its pharmaceutically acceptable salt.

The present invention also provides pharmaceutical compositions comprising one or more compounds described above, or a salt of any of the compounds described above and a pharmaceutically acceptable carrier.

The present invention also provides preparations comprising one or more compounds described above, where the product comprises saline solution, water, a solution of cyclodextrin or a buffered solution at pH 3-9. In n which are variants of the invention, the drug is an orally is not absorbed by the composition. In some embodiments of the invention, the preparation includes excipient selected from purified water, propylene glycol, polyethylene glycol 400 (PEG 400), glycerol, DMA, ethanol, benzyl alcohol, citric acid/sodium citrate (pH 3), citric acid/sodium citrate (pH 5), Tris(hydroxymethyl)amino methane-HCl (pH 7.0), 0.9 percent saline solution and 1.2% saline solution, or any combination thereof. In some embodiments of the invention the composition includes excipient selected from propylene glycol, purified water and glycerin. In some embodiments of the invention, the preparation includes excipient selected from the 20% weight/volume of propylene glycol in saline, 30% weight/volume of propylene glycol in saline, 40% weight/volume of propylene glycol in saline, 50% weight/volume of propylene glycol in saline, 15% weight/volume of propylene glycol in purified water, 30% weight/volume of propylene glycol in purified water, 50% propylene glycol in purified water, 30% weight/volume of propylene glycol and 5 weight/volume of ethanol in purified water, 15% weight/volume of glycerol in purified water, 30% weight/volume of glycerol in purified water, 50% weight/volume of glycerol in purified water, 20% weight/volume Kleptos in purified water, 40% weight/volume Kleptos in purified water and 25% weight/volume of Captisol in cleansing the Noi water.

The present invention also provides methods of obtaining Compound A, including:

(a) the interaction of (R)-(-)-N-Boc-3-pyrrolidinone with a strong base to obtain a mixture; then the interaction of the mixture with 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene with obtaining compounds having the Formula II

b) interaction of the compounds of Formula II with an alcohol and a catalyst of transition metal in the presence of hydrogen to obtain the compounds of Formula III

(C) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine with obtaining the compounds of Formula IV

d) the interaction of the compounds of Formula IV with N-Boc-guanidine butyric acid to obtain the compounds of Formula V

e) removing the protective groups of the compounds of Formula V with Connection A. In some embodiments of the invention in a) the strong base is an NaH; and (b) the transition metal catalyst is a Pd/C, and alcohol is an ethanol.

The present invention also provides additional methods of obtaining Compound A, including:

a) removing the protective groupstert-butyl ether (R)-3-hydroxypyrrolidine-1-carbon is th acid and the interaction of the compounds with 2-chloro-1,3-dinitro-5-triftorperasin with getting tert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid;

b) recoveringtert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid in the presence of alcohol, the transition metal catalyst and hydrogen with gettingtert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid;

c) a combination oftert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid with pyrimidine-4,6-dicarboxylic acid in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride with obtaining bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;

d) the interaction of bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid ({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanol acid in the presence of phosphorus oxychloride to obtain bis-{[3-(5-({[(tert-butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)pentanediamine)-2-((R)-1-(tert-butoxycarbonyl-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;

e) removing the protective groups of the bis-{[3-(5-({[(Tr is t -butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)pentanediamine)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid to obtain crude bis-{[3-(5-guanidino-pentanediamine)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid; and

f) purification of the crude bis-{[3-(5-guanidino-pentanediamine)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid using, for example, chromatography with reversed phase.

The present invention also provides additional methods of obtaining Compound A, including:

a) removing the protective groupstert-butyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid and further interaction of the compounds with 2-chloro-1,3-dinitro-5-triftorperasin with gettingtert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid;

b) recoveringtert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid in the presence of alcohol, the transition metal catalyst and hydrogen with gettingtert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid;

c) a combination ofthe pet -butyl ether (R)-3-(2,6-diamino-4-triptoreline)-pyrrolidin-1-carboxylic acid with pyrimidine-4,6-dicarboxylic acid in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride with obtaining bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;

d) the interaction of bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid with N-Cbz acid in the presence of tonila chloride;

e) recovering the obtained compound (d) in the presence of alcohol, the transition metal catalyst and hydrogen;

f) the interaction of the obtained compound (e) with di-Boc-pyrazole; and

g) removing the protective group of the obtained compound (f) with Connection A.

The present invention also provides methods of obtaining pharmaceutically acceptable salt of Compound A, including:

(a) the interaction of (R)-(-)-N-Boc-3-pyrrolidinone with a strong base to obtain a mixture; then the interaction of the mixture with 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene with obtaining compounds having the Formula II

b) interaction of the compounds of Formula II with an alcohol and a catalyst of transition metal in the presence of hydrogen to obtain the compounds of Formula III

c1) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine with obtaining the compounds of Formula IV

c2) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC1) and anhydrous pyridine to obtain the compounds of Formula IV

d) adding a compound of Formula IV with N-Cbz acid in a solution comprising anhydrous pyridine, dimethylaminopropylamine and any one of tonila chloride, POCl3, (EtO)2POCl or oxalyl chloride to obtain the compounds of Formula Va

e) hydrogenolysis of the Cbz group of compounds of Formula Va with obtaining the compounds of Formula VI

f) protection of the compounds of Formula VI to obtain the compounds of Formula VII

g) removing the protective groups of the compounds of Formula VII to obtain pharmaceutically acceptable salt of Compound A.

The present invention also provides methods of inhibiting growth of a microbe comprising contacting the microbe with any of the compounds described above, or its pharmaceutically acceptable salts.

The present invention also provides methods of treatment mlekopitayuschih is, suffering from a microbial infection, comprising the administration to a mammal in need of this, an antimicrobial effective amount of any of the compounds described above, or its pharmaceutically acceptable salts.

In some embodiments of the invention, the microbe or microbial infection are gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic, Mycobacterium or yeast. In some embodiments the invention, the gram-negative aerobe is an Escherichia coli, Citrobacter freundii, Citrobacter diverus, Citrobacter koseri, Enterobacter cloacae, Enterobacter faecalis, Klebsiella pneumoniae, Klebsiella oxytoca, Morganella morganii, Providencia stuartii, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter lwoffii, Haemophilus influenzae, Stenotrophomonas maltophilia, or Pseudomonas aeruginosa. In some embodiments of the invention are gram-positive aerobe is an Enterococcus faecalis, Enterococcus faecium, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus pneumoniae, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus colmii, Staphylococcus sciuri, Staphylococcus warneri, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus anginosus, Streptococcus mitis, or Streptococcus oralis. In some embodiments of the invention gone anaerobic gram-negative represents Bacteroides fragilis. In some embodiments of the invention are gram-positive gone anaerobic represents Clostridium difficile or Clostridium perfringns. In some embodiments of the invention Mycobacterium is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti or Mycobacterium microti. In some embodiments of the invention yeasts are Candida albicans or Candida krusei.

The present invention also provides any of the compounds described above, for the treatment of microbial infection.

The present invention also provides any of the compounds described above, or their pharmaceutically acceptable salts for use in the manufacture of a medicine for the treatment of microbial infections.

The present invention also provides the use of any of the compounds described above, or their pharmaceutically acceptable salts for inhibiting growth of a microbe.

The present invention also provides the use of any of the compounds described above, or their pharmaceutically acceptable salts for the treatment of microbial infection of a mammal.

BRIEF DESCRIPTION of DRAWINGS

In figure 1A and figure 1B presents the results of the study time-eradication of Compound A againstS. aureusATCC27660 (figure 1B is an expanded view of Figure 1A).

The figure 2 shows the Association of the passage of S. aureus with norfloxacin with significant increase in MIC values [minimum inhibitory concentration] at passage 3 ( dual cultivation) for MSSA and MRSA.

The figure 3 shows the effectiveness of Compound A againstS. aureusin the mouse model of loads on the femur.

The figure 4 shows the effectiveness of Compound A compared with vancomycin againstS. aureusin a rat model of loads on the femur.

The figure 5 shows the effectiveness of Compound A againstS. aureuson a murine model of sepsis.

DESCRIPTION of embodiments of the INVENTION

As used herein, the term "about" means ±5% of described values. For example, about 100 means from 95 to 105.

As used in this description. the terms "C1-3alkyl, C1-4alkyl and(CH2)1-7" means saturated, monovalent unbranched or branched hydrocarbon chain containing from 1 to 3 carbons, from 1 to 4 carbons and from 1 to 7 carbons, respectively. Examples of alkyl groups include, but are not limited to, (C1-C7)alkyl groups such as methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2-methyl-1-pentyl, 2,2-dimethyl-1-propyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl and heptyl. The alkyl group can be unsubstituted is Oh or substituted by one or two acceptable alternates.

As used in this description, the terms "C1-3alkoxy" and "C1-4alkoxy" means-O-alkyl with alkyl defined as above. The alkoxy group can be unsubstituted or substituted by one or two acceptable alternates. Alkyl chain alkoxygroup contains from 1 to 3 or 1 to 4 carbon atoms in length.

As used herein, the term "halo" means a halogen, such as fluorine, chlorine, bromine or iodine.

As used in this description, the terms "Gialos1-3alkyl" and "Gialos1-4alkyl" means an alkyl group as defined above where one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or (10) of the hydrogens are substituted by halo, as defined above.

As used herein, "isolated" means that the compounds of Formula I are distinguished from other components of either (a) a natural source, such as a cell, such as bacterial culture, or (b) a synthetic organic chemical reaction mixture, such as those used in the conventional methods, the compounds of Formula I are cleared.

As used herein, the term "mammal" means a rodent (i.e., mouse, rat or Guinea pig, monkey, cat, dog, cow, horse, pig, or human. In some embodiments the invention, the mammal is man.

As used is in the description the term "microbe" means bacteria, fungi, protozoa or virus.

As used herein, the phrase "pharmaceutically acceptable salt(s)" includes, but is not limited to, salts of acidic or basic groups.

As used herein, the term "purified" means that when you isolate the isolate contains at least 90%, at least 95%, at least 98% or at least 99% of the compounds of Formula I by weight of the isolate.

As used herein, the phrase "appropriate office" means a group that does not negate synthetic or pharmaceutical value of compounds of the Formula I or of intermediates used to produce it. Examples of suitable substituents include, but are not limited to: (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)quinil, (C1-C4)alkoxy, -CN, -OH, oxo, halo, -NO2, -CO2H, -NH2, -NH((C1-C4)alkyl), -N((C1-C4)alkyl)2, -CHO, -CO((C1-C4)alkyl) and -- CO2((C1-C4)alkyl). The person skilled in the art can easily choose a suitable Deputy on the basis of stability and pharmacological and synthetic activity of the compounds of Formula I.

As used herein, the phrase "antimicrobial effective amount" of a compound comprising Formula I, appreciating what is the antimicrobial effectiveness of the connection. In some embodiments of the invention antimicrobial effective amount inhibits the growth of certain microbes to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. In some embodiments of the invention antimicrobial effective amount also means "therapeutically effective amount", thereby the compound reduces or eliminates at least one adverse effect of a microbe in a mammal.

The present invention provides compounds of Formula I

,

where:

each A is independently-C=O, C=S or-CH2;

each D is independently O or S;

each R1represents independently hydrogen, C1-3alkyl, C1-3alkoxy, halo, or Gialos1-3alkyl;

each R2represents independently hydrogen, C1-3alkyl, C1-3alkoxy, halo, or Gialos1-3alkyl;

each R3represents independently hydrogen, C1-4alkyl, C1-4alkoxy, halo, or Gialos1-4alkyl; and

each R4represents independently hydrogen, C1-3alkyl, C1-3alkoxy, halo, or halo is 1-3alkyl;

or their pharmaceutically acceptable salt.

In some embodiments of the invention, at least one A is a-C=O. In some embodiments of the invention each A represents-C=O.

In some embodiments of the invention at least one D is O. In some embodiments of the invention, each D represents O.

In some embodiments of the invention each R1represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R1represents independently hydrogen, methyl, methoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R1represents independently hydrogen, methyl or methoxy. In some embodiments of the invention at least one of R1represents hydrogen. In some embodiments of the invention each R1represents hydrogen.

In some embodiments of the invention each R2represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R2represents n is dependent hydrogen, methyl, methoxy or halo. In some embodiments of the invention at least one of R2represents hydrogen. In some embodiments of the invention each R2represents hydrogen.

In some embodiments of the invention each R3represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R3represents independently methyl, methoxy, halo, or Gialos1-3alkyl. In some embodiments of the invention each R3represents independently halo, or Gialos1-3alkyl. In some embodiments of the invention each R3represents independently Gialos1-3alkyl. In some embodiments of the invention at least one of R3represents trifluoromethyl. In some embodiments of the invention each R3represents trifluoromethyl.

In some embodiments of the invention each R4represents independently hydrogen, methyl, ethyl, methoxy, ethoxy or Gialos1-3alkyl. In some embodiments of the invention each R4represents independently hydrogen, methyl, methoxy, halo, or Gialos1-3alkyl. In some is, some embodiments of the invention each R 4represents independently hydrogen, methyl, methoxy or halo. In some embodiments of the invention at least one of R4represents hydrogen. In some embodiments of the invention each R4represents hydrogen.

In some embodiments of the invention each A is independently-C=O or C=S; each D is independently O or S; each R1represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl; each R2represents independently hydrogen, methyl, methoxy, halo, or halomethyl; each R3represents independently C1-3alkyl, C1-3alkoxy, halo, or haloalkyl; and each R4represents independently hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl.

In some embodiments of the invention each A is independently-C=O or C=S; each D is independently O or S; each R1represents independently hydrogen, methyl, methoxy, halo, or halomethyl; each R2represents independently hydrogen, halo, or halomethyl; each R3represents independently methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl; and each R4represents independently in the location, methyl, ethyl, methoxy, ethoxy, halo, halomethyl or haloacyl.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen, halo, or halomethyl; each R2represents independently hydrogen or halo; each R3represents independently methyl, methoxy, halo, or halomethyl; and each R4represents independently hydrogen, methyl, methoxy, halo, or halomethyl.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently methyl, halo, or halomethyl; and each R4represents independently hydrogen, methyl, halo, or halomethyl.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently halo, or halomethyl; and each R4represents independently hydrogen or halo.

In some embodiments, the implementation of the image is the shadow each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently methyl, halo, or halomethyl; and each R4represents independently hydrogen, methyl, halo, or halomethyl.

In some embodiments of the invention each A represents-C=O; each D represents O; each R1represents independently hydrogen or halo; each R2represents independently hydrogen or halo; each R3represents independently halo, or halomethyl; and each R4represents independently hydrogen, methyl, halo, or halomethyl.

In some embodiments of the invention the compound is a Compound of A

or its pharmaceutically acceptable salt.

Acceptable examples of salts include, for example, hydrochloric acid and triperoxonane acid.

The compounds of Formula I can contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as isomers with double bonds (i.e., geometric isomers), enantiomers or diastereomers. In accordance with the invention, the chemical structures presented herein, and therefore the unity of Formula I encompass all of the corresponding enantiomers and stereoisomers of the compounds, namely stereomono the correct form (e.g., geometrically correct, correct enantiomeric or diastereomeric right) and enantiomeric or stereoisomeric mixtures. Enantiomeric or stereoisomeric mixtures can be resolved by their component enantiomers or stereoisomers by using well-known techniques such as gas chromatography with chiral phase high-performance liquid chromatography with chiral phase, crystallization of the compound in the form of a complex of chiral salts or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can be obtained from stereomono or enantiomerically pure intermediates, reagents and kataliziruetsa using well-known methods of asymmetric synthesis.

The compounds of Formula I further include a hydrate and a solvate.

Compounds containing amine function may also form N-oxides. In this description reference is made to the compound containing an amine function, also including N-oxide. If the compound contains a few of amine functions, one or more than one nitrogen atom may be oxidized to obtain N-oxide. Examples of N-oxides include N-oxides of tertiary amine or a nitrogen atom containing a nitrogen heterocycle. N-oxides can be formed by suitable processing is an amine with an oxidizing agent, such as hydrogen peroxide or percolate (for example, percarbonate acid) (see Advanced Organic Chemistry, by Jerry March, 4thEdition, Wiley Interscience).

In some embodiments of the invention the compounds of Formula I is isolated and/or purified.

The present invention also provides pharmaceutical compositions comprising one or more compounds described above, or one or more salts, and a pharmaceutically acceptable carrier.

Acceptable compositions include, but are not limited to, orally is not absorbed by the composition. Acceptable compositions also include, but are not limited to, saline, water, solutions of cyclodextrin and buffered solutions with pH 3-9.

Compounds described herein, including a Compound A or its pharmaceutically acceptable salt, can be obtained using numerous excipients, including, but not limited to, purified water, propylene glycol, PEG 400, glycerol, DMA, ethanol, benzyl alcohol, citric acid/sodium citrate (pH 3), citric acid/sodium citrate (pH 5), Tris(hydroxymethyl)amino methane-HCl (pH 7.0), 0.9 percent saline solution and 1.2% saline, and any combination thereof. In some embodiments of the invention excipient selected from propylene glycol, purified water and glycerin.

In some vari is ntah embodiment of the invention excipient is a multi-component system, selected from 20% weight/volume of propylene glycol in saline, 30% weight/volume of propylene glycol in saline, 40% weight/volume of propylene glycol in saline, 50% weight/volume of propylene glycol in saline, 15% weight/volume of propylene glycol in purified water, 30% weight/volume of propylene glycol in purified water, 50% weight/volume of propylene glycol in purified water, 30% weight/volume of propylene glycol and 5 weight/volume of ethanol in purified water, 15% weight/volume of glycerol in purified water, 30% weight/volume of glycerol in purified water, 50% weight/volume of glycerol in purified water, 20% weight/volume Kleptos in purified water, 40% weight/volume Kleptos in purified water and 25% weight/volume of Captisol in purified water. In some embodiments of the invention excipient selected from 50% weight/volume of propylene glycol in purified water, 15% weight/volume of glycerol in purified water, 20% weight/volume Kleptos in purified water, 40% weight/volume Kleptos in purified water and 25% weight/volume of Captisol in purified water. In some embodiments of the invention excipient selected from 20% weight/volume Kleptos in purified water, 20% weight/volume of propylene glycol in purified water and 15% weight/volume of glycerol in purified water.

In some embodiments of the invention the composition comprises 50 mg/ml Compound A 20% weight/is the volume of Kleptos in purified water.

In some embodiments of the invention the composition can be dried into a solid substance and recovered, for example, water before using.

When administered to a mammal (e.g., animal for veterinary use or to a human for clinical use) the compounds of Formula I can be introduced in an isolated form. Alternative compounds of Formula I can be introduced together with (i.e., as a combined composition or as separate compositions) other antibiotics, such as, for example: 1) inhibitors of protein synthesis, including, but not limited to, amikacin, anisomycin, apramycin, azithromycin, blasticidin S, brefeldin a, butirosin, chloramphenicol, chlortetracycline, clindamycin, clotrimazole, cycloheximide, demeclocycline, dibekacin, dihydrostreptomycin, doxycycline, garamycin, emetine, erythromycin, fozilova acid, G418, gentamicin, Elvaloy acid, hygromycin B, jozamitsin, kanamycin, terramycin, lincomycin, meclocycline, mepartricin, midecamycin, minocycline, neomycin, netilmicin, nitrofurantoin, nourseothricin, oleandomitsin, oxytetracycline, paromomycin, puromycin, rapamycin, ribostamycin, rifampicin, rifamycin, rapamicin, sizomitsin, spectinomycin, spiramycin, streptomycin, tetracycline, triaminicol, thiostrepton, tobramycin, tunicamycin, tylosin, viomycin, virgi amicin; 2) means that prevents the synthesis of DNA, including, but not limited to, camptothecin, 10-deacetylbaccatin III, azacytidine, 7-aminooctanoic D, 8-hinolinol, 9-dihydro-13-acetylaceton III, aclarubicin, actinomycin D, actinomycin I, actinomycin V, bafilomycin A1, bleomycin, capreomycin, chromomycin, cinoxacin, ciprofloxacin, CIS-diaminopurine(II) dichloride, kumaresan A1, L(+)-lactic acid, cytochalasin B, cytochalasin D, dacarbazine, daunorubicin, distamycin A, doxorubicin, economizing, enrofloxacin, etoposide, flanagin, formazin, fumagillin, ganciclovir, gliotoxin, lomefloxacin, metronidazole, mithramycin A, mitomycin C, nalidixic acid, netropsin, nitrofurantoin, nogalamycin, nonactin, novobiocin, ofloxacin, oxolinic acid, paclitaxel, fenesin, bleomycin, piperidou acid, rebeccamycin, sinefungin, streptonigrin, streptozocin, succinylsulfathiazole, sulfadiazine, sulfadimetoksin, sulfaguanidine clean, sulfamethazine, sulfamonometoksin, sulfanilamide, sulfoxidation, sulfasalazin, sulfathiazole, trimethoprim, tubercidin, 5-azacytidine, korditsepin and formazin A; 3) funds preventing cell wall synthesis, including, but not limited to, (+)-6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid, amoxicillin, ampicillin, azlotillin, bacitracin, carbenicillin is, cefaclor, cefamandole, Cefazolin, cefmetazole, cefoperazone, Cefotaxime, cefsulodin, Ceftriaxone, cephalexin, cephalosporin C, cephalothin, cefradine, cloxacillin, D-cycloserine, dicloxacillin, D-penicillamine, econazole, ethambutol, lysostaphin, moxalactam, nafcillin, nikkomycin Z, nitrofurantoin, oxacillin, penicillin, penicillin G, penicillin, phenoxymethylpenicillin acid, fosfomicin, piperidou acid, piperacillin, ristomycin, and vancomycin; 4) means of preventing cell membrane permeability (ionophores), including, but not limited to, 2-mercaptopyridine, 4-bromellite A23187, alamethicin, amphotericin B, calcimining A23187, chlorhexidine, clotrimazole, colistin, econazole, hydrocortisone, filipin, gliotoxin, gramicidin A, gramicidin C, ionomycin, lasalocid a, ionomycin a, monensin, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonate, narasin, nigericin, lowlands, nonactin, nystatin, fenesin, pimaricin, polymyxin B, DL-penicillamine, polymyxin B, praziquantel, salinomycin, surfactin and valinomycin; 5) enzyme inhibitors, including, but not limited to, (+)-uninova acid, (±)-miconazole, (S)-(+)-camptothecin, 1-methoxybenzylamine, 2-heptyl-4-hydroxyquinoline N-oxide, cordycepin, 1,10-phenanthrolin, 6-diazo-5-oxo-L-norleucine, 8-hinolinol, antimycin, antipain, ascomycin, azaserine, bafilomycin, cerulenin, chlorine is hin cinoxacin, ciprofloxacin, mevastatin, concanamycins a, concanamycins C, kumaresan A1, L(+)-lactic acid, cyclosporine A, econazole, enrofloxacin, etoposide, flanagin, formazin A, furazolidone, husarova acid, geldanamycin, gliotoxin, gramicidin A, gramicidin C, herbimycin A, indomethacin, irgasan, lomefloxacin, mycophenolate acid, myxothiazol, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonate, nalidixic acid, netropsin, niclosamide, nikkomycin, N-methyl-1-deoxynojirimycin, nogalamycin, nonactin, novobiocin, ofloxacin, oleandomitsin, oligomycin, oxolinic acid, piericidin A, piperidou acid, radicial, rapamycin, rebeccamycin, sinefungin, staurosporin, stigmatella, succinylsulfathiazole, sulfadiazine, sulfadimetoksin, sulfaguanidine, sulfamethazine, sulfamonometoksin, sulfanilamide, sulfoxidation, sulfasalazin, sulfathiazole, triacsin C, trimethoprim and videomizer A1; and 6) the membrane modifiers, including, but not limited to, paracelsan.

In some embodiments of the invention, the term "pharmaceutically acceptable" means approved by a regulatory Agency of the Federal or government or listed in the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia used in animals, and more specifically for men. The term "carrier" apply the to the diluent, adjuvant or excipient, which introduced a compound of Formula I. Such pharmaceutical carriers can be a liquid, such as water and oils, including oils, petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Farmacevticheskikh media can also be a physiological solution, Arabian gum, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. May be optionally used auxiliary, stabilizing, thickening, lubricating and coloring agents. With the introduction of the man of the compounds of Formula I and pharmaceutically acceptable carriers can be sterile. Water is acceptable carrier, when the compound of the Formula I is administered intravenously. Saline solutions and aqueous solutions dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Acceptable pharmaceutical carriers include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. In the present composition, if it is desired, can also contain a small amount of moisturizer or emulsifying agents, or sautereau pH agents.

The compositions described herein, may take the form of solution, suspension, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, composition with delayed release, suppository, aerosol, spray or any other form acceptable to use. Examples of acceptable pharmaceutical carriers are described in Remington's Pharmaceutical Sciense, A. R. Gennaro (Editor) Mack Publishing Co.

In one of the embodiments of the invention the compounds of Formula I receive in accordance with normal procedures as a pharmaceutical composition adapted for administration to humans. Usually the compounds of Formula I are solutions in sterile isotonic aqueous buffer. If necessary, the connection may also include solubilizers agent. Compositions for intravenous administration can optionally include a local anesthetic such as lidocaine to ease pain at the injection site. Usually the ingredients are available separately or mixed in a standard dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of outdoor activities the agent. If the compound of the invention should be administered by infusion, it can be prepared, for example, using the bottle for infusion, containing pure pharmaceutical grade sterile water for infusion or saline. If the compound of Formula I is administered by injection, may be granted an ampoule of sterile water for injection or physiological saline so that the ingredients can be mixed prior to injection.

The compounds of Formula I and compositions comprising the same, can be administered orally. Compounds and compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Oral introduced compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of gaultheria or cherry; coloring agents; and preserving agents, to obtain the pharmaceutically palatable preparation. In addition, when there is a form of tablet or pill form, the compositions can be coated to slow raspadaemosti and absorption in the gastrointestinal tract, thereby providing a continuous action over a long period is Yes time. Selectively permeable membranes surrounding osmotically active moving connection is also acceptable for oral insertion compounds of the Formula I. Oral compositions can include standard bases, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such bases are respectively pharmaceutically pure.

The pharmaceutical compositions can be in a standard dosage form. In this form, the composition may be divided into single doses containing appropriate quantities of the active component. Standard dosage form can be in the form of a packaged preparation, the package contains a discrete number of drugs, such as packaged tablets, capsules, and powders in vials or ampoules. Standard dosage form may also be in the form of capsules, sachets or tablets, or it can represent an appropriate number of any of these packaged forms.

The present invention also provides methods of obtaining Compound A, including:

1A) the interaction of (R)-(-)-N-Boc-3-pyrrolidinone with a strong base to obtain a mixture; then the interaction of the mixture with 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene with obtaining compounds having the Formula II

1b) the interaction of the compounds of Formula II with an alcohol and a catalyst of transition metal in the presence of hydrogen to obtain the compounds of Formula III

1c) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine with obtaining the compounds of Formula IV

1d) the interaction of the compounds of Formula IV with N-Boc-guanidine butyric acid to obtain the compounds of Formula V

1e) removing the protective groups of the compounds of Formula V with Connection A.

In some embodiments of the invention in (a) the strong base is an NaH; and (b) the transition metal catalyst is a Pd/C, and alcohol is an ethanol. In particular, this method is described below more in detail in Example 1.

The present invention also provides additional methods of obtaining Compound A, including:

a) deprotonation of tert-butyl methyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid and the interaction of the compounds with 2-chloro-1,3-dinitro-5-triftorperasin obtaining tert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid;

b) the restoration of the tert-butyl ester (R)-3-(2,6-dinitro-4-trift is methylphenoxy)pyrrolidin-1-carboxylic acid in the presence of alcohol, the transition metal catalyst and hydrogen to obtain tert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid;

c) a combination oftert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid with pyrimidine-4,6-dicarboxylic acid in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride with obtaining bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;

d) the interaction of bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid ({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanol acid in the presence of phosphorus oxychloride to obtain bis-{[3-(5-({[(tert-butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)pentanediamine)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;

e) removing the protective groups of the bis-{[3-(5-({[(tert-butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)-pentanediamine)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid to obtain crude bis-{[3-5-guanidino-pentanediamine)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid; and

f) purification of the crude bis-{[3-(5-guanidino-pentanediamine)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid using chromatography with reversed phase.

In some embodiments of the invention in b) the transition metal catalyst is a Pd/C, and alcohol is an ethanol. In particular, this method is described below in more detail in Example 2.

The present invention also provides additional methods of obtaining Compound A, including:

a) deprotonation of tert-butyl methyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid and further interaction of the compounds with 2-chloro-1,3-dinitro-5-triftorperasin obtaining tert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid;

b) the restoration of the tert-butyl ester (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid in the presence of alcohol, the transition metal catalyst and hydrogen to obtain tert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid;

c) a combination of tert-butyl methyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid with pyrimidine-4,6-dicarboxylic acid in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide the hydrochloride with obtaining bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;

d) the interaction of bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid with N-Cbz acid in the presence of tonila chloride;

e) recovering the obtained compound (d) in the presence of alcohol, the transition metal catalyst and hydrogen;

f) the interaction of the obtained compound (e) with di-Boc-pyrazole; and

g) removing the protective group of the obtained compound (f) with Connection A.

In some embodiments of the invention in (b) and (e) the transition metal catalyst is a Pd/C, and alcohol is an ethanol. In particular, this method is described below in more detail in Example 3.

The person skilled in the art will be able to replace an acceptable reagents reagents listed in the methods described herein, getting A Connection, as well as additional compounds of Formula I.

The present invention also provides methods of obtaining pharmaceutically acceptable salt of Compound A, including:

(a) the interaction of (R)-(-)-N-Boc-3-pyrrolidinone with a strong base to obtain a mixture; then the interaction of the mixture with 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene with obtaining compounds having the Formula II

b) interaction of the compounds of Formula II with an alcohol and a catalyst switched the aqueous metal in the presence of hydrogen to obtain the compounds of Formula III

c1) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine with obtaining the compounds of Formula IV

c2) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC1) and anhydrous pyridine to obtain the compounds of Formula IV

d) adding a compound of Formula IV with N-Cbz acid in a solution comprising anhydrous pyridine, dimethylaminopropylamine and any one of tonila chloride, POCl3, (EtO)2POCl or oxalyl chloride to obtain the compounds of Formula Va

e) hydrogenolysis of the Cbz group of compounds of Formula Va with obtaining the compounds of Formula VI

f) protection of the compounds of Formula VI to obtain the compounds of Formula VII

g) removing the protective groups of the compounds of Formula VII to obtain pharmaceutically acceptable salt of Compound A.

Obtaining compounds of Formula I may include protecting or Unprotecting various chemical groups. The need to protect and unprotect and the selection of appropriate protective groups can be easily determined by the person skilled in the art. Chemistry protective the x groups can be found, for example, in T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rdEd. Wiley & Sons, Inc. New York (1999), which is incorporated in this description by reference in its entirety.

The present invention also provides methods of inhibiting growth of a microbe comprising contacting the microbe with one or more compounds described above, or its pharmaceutically acceptable salt. In some embodiments of the invention the compound of Formula I can act as an antiseptic for cleaning surfaces, such as, for example, in kitchens and bathrooms. In these versions of the invention, the compound of the Formula I can be obtained for such use by using techniques well known specialist in this field.

The present invention also provides methods of treating a mammal suffering from a microbial infection, comprising the administration to a mammal in need of this, an antimicrobial effective amount of any of the compounds described above, or its pharmaceutically acceptable salt. In some embodiments of the invention, the mammal can be diagnosed in the presence of a microbial infection prior to treatment. In some embodiments of the invention the official diagnosis cannot be made; in such VA is iontach the invention, the mammal may be suspected in the presence of a microbial infection, for which treatment is recognized as desirable.

In one embodiment of the invention, "treatment" or "treatment" refers to the weakening of microbial infection or at least her one visible symptom; or to a weakening of the at least one measurable physical parameter, not necessarily noticed by the patient; or inhibition of progression of microbial infection; or delaying the start of the development of microbial infection.

In some embodiments of the invention, the microbe is a or microbial infection is caused by gram-negative aerobe, gram-positive aerobe, gram gone anaerobic, gram-positive gone anaerobic or yeast. In some embodiments the invention, the gram-negative aerobe selected from, but not limited to, Escherichia coli, Citrobacter freundii, Citrobacter diverus, Citrobacter koseri, Enterobacter cloacae, Enterobacter faecalis, Klebsiella pneumoniae, Klebsiella oxytoca, Morganella morganii, Providencia stuartii, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter lwoffii, Haemophilus influenzae, Stenotrophomonas maltophilia and Pseudomonas aeruginosa. In some embodiments of the invention are gram-positive aerobe selected from, but not limited to, Enterococcus faecalis, Enterococcus faecium, Mycobacterium tuberculosis, Staphylococcus aureus, Staphylococcus pneumoniae, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus colmii, Staphylococcus sciuri, Staphylococcus warneri, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus aninosus, Streptococcus mitis and Streptococcus oralis. In some embodiments of the invention gone anaerobic gram-negative represents Bacteroides fragilis. In some embodiments of the invention are gram-positive gone anaerobic represents Clostridium difficile or Clostridium perfringens. In some embodiments of the invention Mycobacterium is a Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti or Mycobacterium microti. In some embodiments of the invention, the yeast is selected from, but not limited to, Candida albicans and Candida krusei.

In some embodiments of the invention, the microbe is a resistant to the antibiotic bacterial strain, such as strain of the bacteria listed in the Examples below.

The compounds of Formula I or their pharmaceutically acceptable salt and compositions comprising them, can be introduced in various ways, such as, for example, infusion or bolus injection, and can be put together with another biologically active agent, such as another antibiotic. The administration can be systemic or local. There are various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and they can be used to administer the compounds of Formula I. routes of administration include, but are not limited to, nutritionally, intramuscular, Nutripro is ion battery, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, pulmonary, by inhalation or tapicerki, especially in the ears, nose, eyes, or skin. In some embodiments of the invention acceptable routes of administration include intravenous, topical, or subcutaneous. The desired route of administration is left to the discretion of the practitioner, and will depend, in part, on the localization of microbial infection and the health of the mammal or human, which is subjected to treatment. In most cases, the introduction can lead to the release of the compounds of Formula I in the bloodstream.

In some embodiments of the invention it may be desirable to apply one or more compounds of the Formula I or their pharmaceutically acceptable salt topically to the area requiring treatment. This can be achieved, for example, and without limitation, by local infusion during surgery, topical application, for example, together with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, where the implant has a porous, non-porous, or gelatinous material, including membranes, such as selecticve m is mpany or fiber.

The amount of the compounds of Formula I or its pharmaceutically acceptable salt, which will be effective for the treatment of specific microbial infection will depend on the nature of the violation or pathological conditions and can be determined by standard clinical techniques. Additionally, if necessary, may be applied testsin vitroorin vivoto help identify the optimal dose range. The exact dose for use in the compositions will also depend on the route of administration and the severity of infection, and should be decided in accordance with the medical assessment practitioner and patient care. However, an appropriate range of doses for injection is usually from about 0.001 milligram to about 200 milligrams per kilogram of body weight. In some embodiments of the invention, the dose is from about 0.01 milligram to about 70 milligrams per kilogram of body weight, or from about 0.1 milligram to about 50 milligrams per kilogram of body weight, or from about 0.5 milligrams to about 20 milligrams per kilogram of body weight, or from about 1 milligram to about 10 milligrams per kilogram of body weight. In some embodiments of the invention, the dose is about 5 milligrams per kilogram of body weight. The number of doses of sannich in this description, refers to the total entered amount; that is, if you enter more than one compound of Formula I, dosages correspond to the total amount of the compounds of Formula I. the Compositions may contain from 10% to 95% active ingredient by weight. Effective doses may be extrapolated from the curve dose-response receivedin vitroor animal model test systems. Such animal models and systems are well known in this field.

The present invention also provides one or more compounds described above, or pharmaceutically acceptable salt, or a pharmaceutical composition comprising one or more compounds described above, for the treatment of microbial infection.

The present invention also provides one or more compounds described above, or pharmaceutically acceptable salt, or a pharmaceutical composition comprising one or more compounds described above for use in the manufacture of a medicine for the treatment of microbial infections.

The present invention also provides the use of one or more compounds described above, or their pharmaceutically acceptable salts, or pharmaceutical compositions comprising one or more compounds described above, for inhibiting growth of a microbe.

Present from Britanie also provides the use of one or more compounds described above, or their pharmaceutically acceptable salts, or pharmaceutical compositions comprising one or more compounds described above, for the treatment of microbial infection of a mammal.

In order that the invention disclosed herein, could be better understood, the following examples. It should be understood that these examples are for illustrative purposes only and shall not be construed as limiting the invention in any way. In these examples, the completed reaction molecular cloning and other standard techniques based on recombinant DNA in accordance with the methods described in Maniatis with al., Molecular cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

Examples

In short, the results obtained on the basis of the examples given below indicate that the Connection A is active againstSusceptible spp. and other gram-positive and gram-negative organisms. For example, the screening sensitivity against 150 isolates ofS. aureusand negative for coagulase staphylococci with certain antibacterial susceptibility to other antimicrobial agents. In General, were obtained MIC values90from 0.5 to 2.0 µg/ml at screening, 150 organisms Staphylococcus, and was absent Chu is stateliest phenotypes to other antibiotics. Serial passage methicillin-sensitive (MSSA ATCC 29213) and resistant (MRSA ATCC 33591) strains ofS. aureusat concentrations of 0.5 x MIC for 17 passages did not cause any changes in MIC values. In General, the Compound A was bactericidal against time-eradication in the range from 30 minutes to 6 hours.

Compound a was effectivein vivoin the mouse model of loads on the femur against MSSA 29213 and MRSA 33591 and in the mouse model of peritonitis/sepsis against MSSA 27660. In the mouse model of loads on the femur using MSSA 27660 compound a was lowered through 24 hours after infection up to 410CFU/thigh relative to untreated infected mice at doses that were well tolerated in repeated toxicity studies dose. Thus, sustainable effective against MSSA and MRSA was observed in the mouse model of loads on the femur and against MSSA in a rat model of loads on the femur and a murine model of peritonitis. Compound a was stable in the presence of plasma and isolated hepatocytes of many species.

Compound a was more tolerant in studies of acute toxicity when administered by the IV [intravenous] infusion. MTD [maximum tolerant dose] (IV bolus [jet] introduction to Connect A mouse to 30 mg/kg) was significantly higher than static effective dose to model the load on the femur (2-4 mg/kg).

Compound a is currently in Phase 1 clinical trials in humans for the development of the IV pan-staphylococcal agent.

Example 1: synthesis of Compound A

Stage 1:

Sodium hydride (1.12 g, 60% in mineral oil, 28 mm) was added in anhydrous DMF (24 ml) solution of (R)-(-)-N-Boc-3-pyrrolidinone (5.0 g, 27,6 mm) at room temperature. The resulting mixture was stirred another 15 minutes. This mixture was then added dropwise to DMF (20 ml) solution of 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene (7,45 g, 27,6 mm) at 0°C. the Solution is dark red was stirred at room temperature for 4 hours. The reaction was stopped with ice water and was extracted with ethyl acetate. The organic layer was washed with salt solution and water and dried over Na2SO4. After removal of solvent the residue was purified using a column for distillation of light fractions (ethyl acetate/hexane = ¼ volume/volume). The yield was 54%.

Stage 2:

(R)-tert-butyl-3-(4-trifluoromethyl)-2,6-dinitrophenoxy)pyrrolidin-1-carboxylate (4,84 g, 9.8 mm) and Pd/C (0,78 g, 10% on carbon) and ethanol (140 ml) was placed in a Parr flask. The mixture is evaporated under hydrogen three times and stirred at 40 kPa of hydrogen at room temperature in ECENA night. The mixture was filtered through celite. The filter cake was washed twice with ethanol (2×20 ml). The filtrate is evaporated under vacuum. Got a grayish-white solid and used as such for further reaction. The yield was 100%.

Stage 3:

2-Chloro-4,6-dimethoxy-1,3,5-triazine (5,97 g, 34 mm) was stirred in anhydrous THF (200 ml). Was added N-methylmorpholine (7.5 ml, 68 mm). The resulting mixture was stirred at room temperature for 30 minutes. Then was added (R)-tert-butyl-3-(2,6-diamino-4-(trifluoromethyl)phenoxy)pyrrolidin-1-carboxylate (10,84 g, 30 mm) and pyrimidine-4,6-dicarboxylic acid (2,48 g, 14,8 mm). The mixture was stirred at room temperature for 24 hours. The solvent is evaporated completely in vacuo. Was added water (250 ml) and the mixture was stirred for 4 hours. After filtration of the yellow precipitate was washed with water (3×100 ml) and was stirred in water (250 ml) for 4 hours. The procedure of filtration and washing were repeated twice. The solid was air-dried and stirred in dichloromethane (20 ml) for 30 minutes, followed by ultrasonic treatment for 1 hour. After filtration of the yellow precipitate on the filter was quickly washed with cold dichloromethane (2×10 ml). Product (10.0 g, output: 79,1%) was used as such for further reaction.

Stage 4:

Source material (6.5 g, 7.6 mm), N-Boc-guanidine butyric acid (10,9 g, 30,4 mm) was stirred in anhydrous pyridine (40 ml) at 0°C. POCl3(2,78 ml, 30,4 mm) in pyridine (4 ml) was added dropwise. The resulting mixture was stirred at 0°C for 1.5 hours. The reaction mixture is evaporated under vacuum. Water (140 ml) was added to the residue. The mixture was extracted using ethyl acetate (260 ml). The organic layer was washed brine (100 ml) and dried over Na2SO4. After evaporation the residue was purified using column (eluent: ethyl acetate/hexane/dichloromethane = 1/1/1, volume/volume/volume, then 2%~4% methanol in dichloromethane). The output amounted to 29.1%. Rfwas the same as in the standard sample, which was characterized by NMR.

Stage 5:

Source material (3.4 g, 2.3 mm) was stirred in 4 BC HCl (34 ml) at room temperature over night. The solvent was removed under vacuum. The residue was titrated in the air. The solid was filtered and purified using C18 reversed-phase C18 columns. The quality of the product received light yellow solid with a purity of 98% (HPLC); LC-MS (M+1): 937. Yield: 51%.

Example 2: synthesis of Compound A

Stage 1:tert-butyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid deprotonated using potassium tert-bout is xida (KOtBU) in tetrahydrofuran (THF). The resulting anion interacts with 2-chloro-1,3-dinitro-5-triftorperasin intert-butyllithium ether (MTBE)/THF. When the reaction ends, the reaction mixture was quenched with water and divide by extension MTBE. The organic layer was washed with saline solution and water and concentrated on a rotary evaporator. Solid concentrate is re-dissolved in methanol and re-precipitated with water. The precipitate is filtered and dried to providetert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid, which can be used in the next stage without further purification.

Stage 2: the product of stage 1 is dissolved in methanol and hydrogenizing at 100-200 kPa and 30-50°C in the presence of 10% Pd/C until then, when the restoration will be completed using HPLC. The reaction mixture was filtered through calit. The filtrate is concentrated and dried to providetert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid, which can be used in the next stage without further purification.

Stage 3: the product of stage 2 is connected with the pyrimidine-4,6-dicarboxylic acid in the approximate ratio 2 M diamine:1 M DIACID in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride (EDCI) in pyridine under an inert atmosphere is at ambient temperature. When the reaction ends, the reaction mixture is dissolved in the water. The precipitate is isolated and re-dissolved in MTBE. The MTBE solution was washed with water, and 0.2 N. HCl and brine, dried over anhydrous sodium sulfate, isolated and dissolved in heptane. The precipitate isolated by filtration and dried to provide bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid, which can be used in the next stage without further purification.

Stage 4: product stage 3 interacts with 2.5 to 3 molar equivalents ({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanol acid in pyridine in the presence of phosphorus oxychloride at a temperature of from about -5 to -10°C. the Reaction is quenched with water at a temperature of 15°C. the Supernatant separated from the amorphous precipitate is re-dissolved in MTBE, washed with water and brine, dried over anhydrous sodium sulfate, isolated and dissolved in heptane. The precipitate isolated by filtration and dried to provide bis-{[3-(5-({[(tert-butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)pentanediamine)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid, to the which is used in the next stage without further purification.

Stage 5: the product of stage 4 are deprived of the protective groups (removing sixtert-butoxycarbonyl groups) using 4M HCl/1,4-dioxane in formic acid at ambient temperature. The reaction mixture is dissolved in 1,4-dioxane. The precipitate is filtered, washed with 1,4-dioxane and dried to provide the crude bis-{[3-(5-guanidino-pentanediamine)-2-((R)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid (crude Compound A). The crude product is further purified by re-precipitation from a solution of methanol using THF (50°C to the ambient temperature) and/or re-precipitated from the water/solution of methanol using THF at ambient temperature.

Stage 6 (chromatographic purification): the final purification of Compound A is achieved by chromatography with reversed phase (RP-HPLC) using phase YMC ODS-AQ, 50 micron, 120 angstroms, the suspension was Packed in a column with dynamic axial compression ProChrom. The mobile phase is a gradient of solvent B in solvent A, where solvent A is water with 0.05% of triperoxonane acid (TFA) and solvent B is acetonitrile with 0.05% of TFA. The fractions containing the purified product, concentrated using a rotary evaporator to provide the Compound as A salt of triptoreline the Final form of the monohydrochloride salt reacquire by passing aqueous/methanolic solution of salts trifenatate through the ion exchange column of Dowex 1×2-400 (Cl-form), collect API-containing eluate, concentrated and dried.

Substance bulk dosage form of Compound A store at 2-8°C, protected from light and air, in a yellow HDPE containers or in bags of double polyethylene in a cardboard drum.

Example 3: synthesis of Compound A

Stage 1:tert-butyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid (compound 20) deprotonated using potassium tert-butoxide (KOtBU) in tetrahydrofuran (THF). The resulting anion is reacted with 2-chloro-1,3-dinitro-5-triftorperasin (compound 2) intert-butyllithium ether (MTBE)/THF. When the reaction ends, the reaction mixture was quenched with water and divide by extension MTBE. The organic layer was washed with saline solution and water and concentrated on a rotary evaporator. Solid concentrate is re-dissolved in methanol and re-precipitated with water. The precipitate is filtered and dried to providetert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid, which can be used in the next stage without further purification. This reaction will be in scale with the use of 4.2 kg of compound 2.

Stage 2: compound 21 was dissolved in methane is e and hydrogenizing at 100-200 kPa and 30-50°C in the presence of 10% Pd/C as long when the restoration will be completed using HPLC. The reaction mixture was filtered through celite. The filtrate is concentrated and dried to providetert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid (compound 22) with HPLC purity of 92.2%. The reaction is performed in four series in the scale of 1.64 kg connection 21 for each series.

Stage 3: Connection 22 connects with the pyrimidine-4,6-dicarboxylic acid (compound 8), in the approximate ratio 2 M diamine:1 M DIACID in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride (EDCI) in pyridine under an inert atmosphere at ambient temperature. When the reaction ends, the reaction mixture is diluted with water. The precipitate is isolated and re-dissolved in MTBE. The MTBE solution was washed with water, and 0.2 N. HCl and brine, dried over anhydrous sodium sulfate, isolated and dissolved in heptane. The precipitate allocate by filtration and dried to provide bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid (compound 23), which can be used in the next stage without further purification. The reaction is performed in scale with the use of 3.15 kg of compound 22.

Stage 4: the solution 3,66 g DMAP in 60 ml of anhydrous pyridine was cooled to 0°C in an ice bath. Was slowly added of 3.60 g of tonila chloride. The resulting solution was stirred for 10 minutes. The source material of N-Cbz acid (7,53 g, 30 mm), the compound (Cpd) 23 (8,54 g, 10 mm) was added to the solution, respectively. The resulting mixture was stirred at room temperature (RT) for 4 hours. Was added water (500 ml). After vigorous stirring the mixture at room temperature for 2 hours, the solid was filtered and washed with 250 ml of water. The solid was dissolved in ethyl acetate (300 ml). The organic layer was washed with 10% citric acid solution (100 ml) and brine (100 ml) and dried over Na2SO4. After evaporation the residue was dissolved in 40 ml of DCM was then added 250 ml of hexane. The precipitate was collected and dried under vacuum. Got 13,20 g of the product from 95% purity. Yield: 100%.

Stage 5: compound 26 (13,20 g) was dissolved in MeOH with 2 equiv. 1 N. HCl was added 1.0 g of Pd/C catalyst (10%). The reaction mixture was injected into hydrogenator Parra and was shaken for 2 hours at 60 kPa of hydrogen. If LCMASS showed a lack of progress, was added 1.0 g of catalyst. The reaction mixture was injected into hydrogenator Parra and was shaken for 3 hours at 60 kPa of hydrogen. The mixture was filtered through celite to remove kata is Isadora. The filtrate was concentrated until dry in a rotary evaporator at 30°C. Received 11,50 g of the product from 95% purity. Yield: 100%.

Stage 6: compound 27 (11,50 g, 10 mm) was dissolved in 60 ml of methanol and DCM (1:1). Then added of 4.04 g of triethylamine (40 mm). Was added di-Boc-pyrazole of 9.3 grams (30 mm) and the resulting mixture was stirred at room temperature for 1 hour. After removal of 95% of the solvent was added 300 ml of water and the mixture was vigorously stirred for 2 hours. The solid was filtered and washed with 300 ml of water. The solid was dissolved in 300 ml ethyl acetate and dried over Na2SO4. After evaporation of the solvent, the solid was dissolved in 40 ml of DCM, then with 500 ml of hexane was used for precipitation of the product. The solid was collected and dried under vacuum. Got 13,0 grams of product with 85% yield (90% purity).

Step 7: compound 28 (1.5 g) was purified on a column of 80 g of silica gel using a gradient 10-88% EtOAc in DCM. Fractions with purity higher than about 95% were collected, evaporated under vacuum and dried. The output amounted to 50-60%. The connection 28 with 95% purity (0.3 g) was dissolved and stirred in ethyl acetate (3 ml) at room temperature (22°C) under argon. The HCl gas was barbotirovany the solution for 20 minutes. The color of the solution changed to dark yellow when providentially. Solid began to break up within 15 minutes. The solution was stirred at room temperature for another 1 hour. An additional 4 ml of ethyl acetate was introduced into the reaction mixture due to the loss of ethyl acetate. The mixture was barbotirovany HCl gas for 10 minutes. The mixture was stirred for 2.5 hours. One third of the mixture was filtered and washed with ethyl acetate. Two-thirds of the mixture stirred at room temperature overnight. Then to the mixture was added 30 ml of ethyl acetate. After filtration of the precipitate on the filter was washed with ethyl acetate twice (2×140 ml) and dried. The solid is immersed in ethyl acetate (8 ml) and kept in the freezer. The reaction process was performed for 4 hours. Mixing during the night gave no significant changes. The purity of the final product was 98% with one primary admixture of 1.2%.

Example 4: synthesis of Compound A

Stage 1: purged with nitrogen 4-necked 12 l RBF was added 305,32 g of compound 2, 700 ml MTBF with stirring and the mixture was cooled in an ice/water bath. Compound 20 (212,43 g) was dissolved with potassium tert-piperonyl (1,31 l 1 M solution in THF) to obtain a slightly turbid mixture. This mixture was added to a solution of compound 21 in RBF within 86 minutes with stirring, at the same time maintaining an internal temperature <9,0°C. the Reaction was removed is from a cold bath after 30 minutes and provided stirring at ambient temperature for a 15.5 hours. When mixing several small pieces of ice were added to reduce the temperature from 21,4°C to 18.4°C. Then was added water (1.5 l) MTBF (1.5 l) and the mixture was stirred for 10 minutes. The mixture was a split phase was separated in a 6-l separating funnel. The aqueous layer was re-extracted with MTBF (500 ml). The organic layers were combined and washed with 2:1 water/saturated salt solution (3×900 ml) and concentrated in solid reddish/reddish color under reduced pressure. This solid was dissolved in 2,45 l MeOH and the solution was transferred into a 4-l Erlenmeyer flask. Then added water (1 l) with stirring parts with the formation of a thick suspension. The mixture was sealed and placed in a refrigerator (1-5°C) for 16 hours. The solid is collected by filtration and dried under vacuum. The dried product was a bright yellow powder. Output: 395,3 g, HPLC purity 94%.

Potassium tert-piperonyl can be replaced, for example, on any alkoxide, sodium hydride, potassium hydride, or any basis that can deprotonate hydroxyl compound 20. Compound 2 may be substituted in position 2 by any halide.

Stage 2: in a mixer Parra stainless steel 2 gallon was added Pd/C catalyst (10% weight/volume, 20 g), compound 21 from stage 1 (394,37 g) and then gently with premesis is of 2 l of MeOH. The vessel was loaded with hydrogen and twice ventolinbuy. The mixture was then stirred, starting at 82 kPa hydrogen pressure was lowered to 0 kPa. The vessel was re-filled to 62 kPa 28 kPa 36 kPa, respectively, each time providing a return to 0 kPa (total absorption of 208 kPa for 51 minutes). The internal temperature began with 16°C, and the mixture showed a smooth, but rapid exothermic effect with a maximum at 38°C. the Internal temperature was maintained at 33-38°C. the Vessel was subjected to a pressure of 49 kPa with the absorption of 23 kPa. The vessel was subjected to a pressure of 90 kPa with the absorption of 12 kPa for 1 hour. The vessel was subjected to pressure 120 kPa with the absorption of 82 kPa for 6 hours. The vessel is then subjected to pressure 51 kPa with the absorption of 37 kPa for 14,33 hours (total absorption of hydrogen was 362 kPa). The reactor was dismantled and the mixture was filtered through a pad celite 545, pre-moistened MeOH (Buchner funnel 11 cm diameter). The reactor and the pillow was rinsed MeOH and cushion the filtrate was pumped to a state of slow drip drops and colorless filtrate (~3.0 l total volume). The filtrate was further filtered through fluted paper disk to remove a certain amount of fine dark powder. Clear filtrate was transferred into a 5-l RBF and concentrated to painted orange/brown thick oil, coloroado up to 3-4°C during the night, during this time the material is partially hardened/crystallized. The material was heated and was then pumped under reduced pressure to obtain a solid reddish/brown sticky/hard waxy solid agglomerate. In RBF was added heptane (2×700 ml) and MTBF (700 ml). The mixture was stirred using verhneprivodnaya mechanical stirrer for 3.5 hours. Liquid layer decantation from the solids, the sinter is crushed into small pieces, the liquid was placed back in RBF and the suspension was vigorously stirred for x 16.75 hours. Then left small pieces were then crushed using the end of a glass rod stirrer and the mixture was vigorously stirred for 70 minutes. The suspension was filtered through a funnel of bagged mitterolang glass using filtrate for a full migration. The funnel was closed and vacuum dried at low heat (41°C) for 4 hours to provide the product as a faint peach/beige powder. Output: 270,60 g, HPLC purity of 98.5%.

The Pd/C catalyst can be replaced, for example, any catalyst suitable for hydrogenation of the nitro group.

Stage 3 (Option 1): 2-chloro-4,6-dimethoxy-1,3,5-triazine (4.0 g) was stirred with anhydrous THF (60 ml). Was added N-methylmorpholine (4.4 g). The resulting mixture was stirred at room t is mperature within 30 minutes. Then was added the compound 22 (7.2 g) and pyrimidine-4,6-dicarboxylic acid (1.68 g). The mixture was stirred at room temperature for 24 hours. The solvent is then evaporated completely in vacuo. Was added water (250 ml) and the mixture was stirred for 4 hours. After filtration of the yellow precipitate on a filter, washed with water (3×100 ml) and again stirred in water (250 ml) for 4 hours. The procedure of filtration and washing were repeated twice. Then the solid was air-dried. The solid was dissolved in 15 ml DCM:hexane:acetone (5:5:1). The mixture was stirred at room temperature for 2 days. The solid was filtered and washed with 10 ml DCM:hexane (1:1) twice. The recrystallization procedure was repeated one more time with the provision of a light-yellow solid. Yield: 70%, HPLC purity 100%.

Stage 3 (Option 2): EDCI (6.0 g) was stirred in anhydrous pyridine (60 ml). Then was added the compound 22 (7.2 g) and compound 9 (0.56 g). The mixture was stirred at room temperature for 2 hours. Then added another part pyrimidine-4,6-dicarboxylic acid (0.56 g). After the mixture was mixed at room temperature for another 2 hours, was added to the third part of the pyrimidine-4,6-dicarboxylic acid (0.56 g). The resulting mixture was stirred at room temperature for 24 hours. Then R is storytell completely evaporated in vacuum. Was added water (250 ml) and the mixture was stirred for 4 hours. After filtration of the yellow precipitate on a filter, washed with water (3×100 ml) and was stirred in water (250 ml) over 4 hours. The procedure of filtration and washing were repeated twice. Then the solid was air-dried. The solid was dissolved in 15 ml DCM:hexane:acetone (5:5:1). The mixture was left at room temperature for 2 days. The solid was filtered and washed with 10 ml DCM:hexane (1:1) twice. The recrystallization procedure was repeated one more time with the provision of a light-yellow solid. Yield 70%, HPLC purity 100%.

EDCI can be replaced, for example, any amide binding reagents that produce acid anhydride or activated ester, such as CDI, DCC, HOBt, HOAt, POCl3.

Stage 4: a solution of DMAP (3,66 g) in 60 ml of anhydrous pyridine was cooled to 0°C in an ice bath. Was slowly added thionyl chloride (of 3.60 g). Then the resulting solution was stirred for 10 minutes. The source material of N-Cbz acid (7,53 g, 30 mm), Cpd 5 (8,54 g, 10 mm) was added to the solution, respectively. The resulting mixture was stirred at room temperature for 4 hours. Then added water (500 ml). After the mixture was vigorously stirred at room temperature for 2 hours, the solid was filtered and washed and 250 ml of water. The solid was dissolved in ethyl acetate (300 ml). The organic layer was washed with 10% citric acid solution (100 ml) and brine (100 ml) and dried over Na2SO4. After evaporation the residue was dissolved in 40 ml of DCM was then added 250 ml of hexane. The precipitate was collected and dried under vacuum. Got 13,20 g of the product from 95% purity. Yield: 100%.

Thionyl chloride can be replaced, for example, POCl3,(EtO)2POCl or oxalyl chloride.

Stage 5: compound 26 (13,20 g) was dissolved in MeOH with 2 equiv. 1 N. HCl was added 1.0 gram of catalyst Pd/C (10%). The reaction mixture was injected into hydrogenator Parra and was shaken for 2 hours at 60 kPa of hydrogen. If LCMASS showed a lack of progress, was added 1.0 gram of catalyst. The reaction mixture was injected into hydrogenator Parra and was shaken for 3 hours at 60 kPa of hydrogen. The mixture was filtered through celite to remove the catalyst. The filtrate was concentrated until dry in a rotary evaporator at 30°C. Received 11,50 g of the product from 95% purity. Yield: 100%.

The Pd/C catalyst can be replaced, for example, any catalyst suitable for hydrogenation of the CBZ group.

Stage 6: compound 27 (11,50 g, 10 mm) was dissolved in 60 ml of methanol and DCM (1:1). Then added of 4.04 g of triethylamine (40 mm). Was added di-Boc-pyrazole 9, g (30 mm) and the resulting mixture was stirred at room temperature for 1 hour. After removal of 95% of the solvent was added 300 ml of water and the mixture was vigorously stirred for 2 hours. The solid was filtered and washed with 300 ml of water. The solid was dissolved in 300 ml ethyl acetate and dried over Na2SO4. After evaporation of the solvent, the solid was dissolved in 40 ml of DCM, then with 500 ml of hexane was used for precipitation of the product. The solid was collected and dried under vacuum. Received 13.0 grams of product with 85% yield (90% purity).

di-Boc-pyrazole can be replaced, for example, samochowiec or di-Boc-smokeview.

Step 7: compound 28 (1,17 kg, 0.76 M) was dissolved in 24 l of EtOAc and then adding 281 ml of water. The HCl gas was added into the solution while the reaction temperature was maintained below 45°C by regulating the speed of adding. The total reaction time was 5 hours, of which 1.5 hours represents the time of adding HCl. HPLC showed that the source material is less than 1%, and the precipitated product was collected by filtration under nitrogen. The solid was rinsed using EtOAc, was titrated with MeOH/THF (1:1) and dried under vacuum. Yield 84%.

HCl/EtOAc can be replaced, for example, HCl/dioxane.

Example 5: antimicrobial activity against gram-positive clinical isolates (table 1A) and gram-negative clinical isolates (the figure 1B)

Compound a was evaluated byin vitroin accordance with the CLSI documents [Institute for clinical and laboratory standards] specific organisms (aerobic, anaerobic or yeast), tested in this study. Ampicillin, ceftazidime, cefuroxime, ciprofloxacin, linezolid and vancomycin were tested in the same row as agents for aerobic bacteria, clindamycin and metronidazole were tested as agents for anaerobes; uconazole tested as agent for comparison of isolates of yeast. Basic solutions of Compound A was obtained in dimethyl sulfoxide (DMSO). Ampicillin, ceftazidime, cefuroxime, ciprofloxacin, linezolid, vancomycin, metronidazole, clindamycin and uconazole received each in accordance with its guidance for production.

Aerobes (M7-A7)1

The minimum inhibiting concentration (MIC) in μg/ml was determined in accordance with the guide CLSI M7-A7 by microdesmidae in the broth. All aerobes tested using broth medium Mueller-Hinton, exceptStreptococcus spp., which was tested with adjustable cations broth Mueller-Hinton with the addition of 2-5% lysed horse blood. The results are shown in table 1A and table 1B.

Compound a showed a broad with whom CTR activity against gram-positive pathogens with S. aureusand negative on the coagulase of Staphylococcus species showing the lowest MIC. Compound a was active against gram-negative pathogens, but the full range of activity was less than for gram-positive organisms.

Example 6: MIC with Staphylococcus species with defined phenotypes of resistance

Evaluation of the sensitivity profiles of Compound A against selected isolates was performedin vitrousing techniques microdesmidae in broth using a bouillon medium Mueller Hinton in accordance with CLSI document M7-A7. Applied interpreted the limits of sensitivity to the antibiotic CLSI when applicability is in accordance with the requirements of the document M100-S17 CLSI. The results are shown in table 2.

Table 2
Generic-
public
feelings.
OXA-RTo the VRSA/VISA
OXA-R
LZD-NS
OXA-R
DAP-NS
OXA-R
To the VRSA/VISA
DAP-NS
OXA-R
isolates5969755Connecting a MIC range0.25 to 10.25 to 20,5-10,5-10,5-20,5-1
* Analysis of microdesmidae in broth were performed in accordance with standard CLSI guidelines.

OXA-R: oxacillin-resistant; to the VRSA: vancomycin-resistantS. aureus; VISA: intermediate sensitive to vancomycinS. aureus; LZD-NS: not sensitive to linezolid; DAP-NS: not sensitive to daptomycin.

Compound a was the activein vitroagainst all isolates ofS. aureusand negative on the coagulase negative staphylococci, including isolates ofS. aureuswith characterized resistance to daptomycin, linezolid and vancomycin (the new therapeutic tool for the treatment of multidrug-resistantS. aureussuch as MRSA). Against isolates ofS. aureusthere were no changes in activity against resistant isolates relatively sensitive isolates. In relation to the negative on the coagulase Staphylococcus aureus activity was not changed as a result of resistance to methicillin.

Example 7: cytotoxicity and selectivity

The cytotoxicity of the Compounds was evaluated in A colorimetric analysis using the receiving transformed cell lines human liver (HepG2, HB-8065) and embryonic cell line of mouse cells NIH/3T3, CRL-1658). Using this analysis were measured bioremediation new connection of tetrazole in soluble product formazan by viable cells. The HepG2 cells were seeded in 96-well tablets with a density of 2×104cells/well in MEM medium with 10% fetal bovine serum (FBS) for 24 hours before use. Cells NIH/3T3 were seeded in 96-well tablets with a density of 2×104cells/well in DMEM with 10% calf serum (BSC) for 24 hours before use. Cell monolayers were rinsed serum-free medium and incubated for one hour with Compound A in serum-free medium. After incubation, the medium was replaced by medium with the addition of serum and living cells was measured using a set of Cell Titer 96 Aqueous Non-Proliferation Assay (Promega, Madison, items of Wisconsin). The values of EC50was determined using a four parameter logistic equation: Y=bottom+(top-level-lower level)/(1+10^)((LogEC50-X)*gentle slope)).

Cytotoxicity of Compound A was also evaluated in the analysis of hemolysis using human erythrocytes. Mixed whole human blood was centrifuged for separation of red blood cells [RBCs] (PNC). Isolated RBC were rinsed and diluted in Tris-buffered saline (TBS, pH 7,4) obtaining 0,22% RBC suspension. 5 μl of Compound A base solution was added to 45 μl of the RBC suspension, and incubated with the pump for 1 hour at 37°C. After the incubation time the samples were centrifuged and 30 μl of the supernatant was added to 100 μl of water. The results of measuring OD414filmed for haemoglobin concentration. As a positive control was used peptide poison bee melittin. The values of EC50was determined as described above. The results are shown in table 3.

Table 3
ConnectionMIC or MIC90(ág/ml)Cytotoxicity
(EC50µg/ml)
Selectivity
(EC50/MIC)
S. aureusRBC3T3HepG2RBC3T3HepG2
Connection A1,0*>5004301,031>500430 1,031
melittin2241120,5

The connection shows A larger overall selectivity.

Example 8: time-eradication againstS. aureus(ATCC 27660)

Study the curve of the time-eradication of Compound A againstE coliATCC25922,E coliD31 (lab. strain) andS. aureusATCC27660 defined in the standard Protocol by measuring the time required to reduce the initial inoculum of 3 log units. Three ml adjustable cation environment Mueller Hinton was inoculable 20 ál of frozen bacterial strain and incubated at 37°C on a rocking platform (250 rpm) overnight. The suspension was diluted to approximately 5×105CFU/ml and treated 2x, 5x, 10x and 20×MIC (MIC=1 µg/ml). Got a basic solution of Compound A 10 mg/ml in DMSO. At certain points in time bacteria were selected and the number of viable bacteria was counted on cups with MH agar after 18 hours of incubation. Studies on the analysis of the kinetics curve time-eradication of Compound A againstS. aureusATCC 27660 at concentrations of 2x MIC showed that a reduction of 3 log10units in the initial inoculum is 5 hours. The absence of the re-growth was observed in cultures after 72 hours at concentrations of 1x MIC. Cm. figures 1A and 1B.

Example 9: the resistance of serial passage in MSSA (ATCC 29213) and MRSA (ATCC 33591)

Frozen bacterial strains (20 µl)S. aureusATCC29213 or methicillin-resistantS. aureus(MRSA ATCC 33591) were inoculable in 3 ml adjustable cation environment Mueller-Hinton and incubated at 37°C on a rocking platform (250 rpm) overnight. The suspension was diluted to approximately 5×105CFU/ml and was inoculable in polypropylene (Costar) 96-well round-bottom tablet (volume 90 ál). The combination of basic solutions of Compound A and norfloxacin (Sigma Aldrich, St. Louis, mo; Catalog #N9890) was obtained in DMSO and did a serial twofold dilution of compounds in 0.01% acetic acid, and 0.2% bovine serum albumin directly in the wells of polypropylene tablet 10 μl/well. The final concentration of the Compound A was 50; 25; 12,5; 6,25; 3,13; 1,56; 0,78; 0,39; 0,19; 0,098; 0,049 and 0.024 mg/ml, the final concentration Ranges norfloksatsina was 100; 50; 25; 12,5; 6,25; 3,13; 1,56; 0,78; 0,39; 0,19; 0,098 and 0,049 µg/ml concentration of DMSO did not exceed 1% in the analysis. All samples were made in three replications. After 24 hours incubation at 37°C, cell growth was assessed by monitoring the presence of "allowable growth" defined by CLSI as ≥2 mm clot or a certain turbidity. MIC hole was defined as the lowest concentration at which no observed acceptable growth. For eringo passage received aliquots of 50 µl of 2 of 3 re holes at 0.5× MIC and mixed with 900 μl of fresh regulated cation environment Mueller Hinton. Measured OD600and the cell suspension was inoculable in polypropylene 96-well round-bottom plates (volume 90 ál) at about 5x105CFU/ml. Ten μl of compounds basic solutions previously added to the wells to obtain a range of concentrations for each compound described above. All samples were made in three replications. The wells were incubated for 24 hours at 37°C. This process was repeated for all passages 17 and the MIC values were recorded in each passage.

The passage of theS. aureuswith norfloxacin was associated with a significant increase in MIC values in passage 3 (4 double dilution) for MSSA and MRSA, which reached 128-fold and 64-fold increase, respectively, in the passage 15. On the other hand, there was no change in MIC for Compound A against MSSA ATCC 29213 or MRSA ATCC 33591 in the dynamics of the full 17 passages. Cm. figure 2.

Example 10:in vitrothe metabolic stability of Compound A - plasma

Mixed samples of human plasma (mixed gender), rats (mixed by breed and sex) and dogs (mixed by breed and sex) were incubated with Compound A (5 μm) at 37°C for 0 and 60 minutes (duplicate samples). The incubation was completed by the addition of ice-cold solvent to precipitate (acetonitrile:glacial acetic acid, 9:1 volume/volume). Supernatant was diluted with an equal volume of 0.1% formic is islote and analyzed using HPLC-MS/MS. The stability of the plasma is represented as % of starting compound for 60 minutes relative to the number of input connections for 0 minutes. The results are shown in table 4.

Table 4
TypesThe stability of the plasma (%)
People96
Rat (mixed breed)102
Dog (mixed breed)100

In the plasma of humans, rats and dogs have little or no Connection after A 1 hour incubation at 37°C, indicating high stability of the plasma. There were also a small number or no Connection A in humans, cynomolgus macaque and rabbits (data not shown).

Example 11: effect of Compound A on a murine model of loads on the femur

Females 6-7 weeks of age mice CD-1 caused neutropenia cyclophosphamide (150 mg/kg/b) on days 4 and 1 before/m inoculationS. aureus(ATCC 13709). The inoculateS. aureusreceived by transferring colonies from 18-20-hour cultures on trypticase soy agar (TSA) in sterile PBS. The density was adjusted to approximately 106 CFU/ml for the spectrophotometer and the concentration of the inoculum was determined using the method of dilutions count on the Cup. Mice were inoculable injected into each of the rear part of the femur 0.1 ml inoculum. Compound a was given to separate groups of mice (4 females/group) via/bolus doses of 1 or 2 mg/kg/dose 1 and 5, 1 and 9 or 1 and 13 hours after inoculation, as shown in table 5. A separate control group of 4 mice received the inoculum without antibiotic treatment. Compound a was dissolved in 50%/50% volume/volume of sterile USP purified water/PBS. The femur was isolated in 25 hours after inoculation. The muscle of the femur and the bone tissue homogenized, and aliquots of serial dilutions were placed on TSA and incubated at 37°C for 20 hours, and received the number of colonies to calculate CFU/thigh. The parameters shown in table 5.

Table 5
Group # ProcessingDose (mg/kg/
dose)
Total dose (mg/kg)Volume
(ml/kg)
Processing
(h after
inoculation)
Collection
hip
bones
(h after
inoculation)
No.
The mouse is
1A monk.
Control
NANANANA254
2Connection
A
1241 and 5254
3Connection
A
2441 and 5254
4Connection
A
1241 and 9254
5Connection
A
2441 and 9254

6Connection
A
1241 and 13254
7Connection
A
2441 and 13254
NA - no data

Compound a was more effective in reducing bacterial populations in inoculated thighs when administered 2 mg/kg/dose at 1 and 5 or 1 and 9 hours after inoculation. Reducing the number of bacteria in these 2 groups was by 3.96 and 3,93 log lower, respectively, than the same in inoculated control group. Cm. figure 3.

Example 12: efficiency versus vancomycin in rat model of loads on the femur

For each experiment in female rats, 8-9 weeks of age with kanilirovannoy femoral vein Crl:CD(SD) caused neutropenia cyclophosphamide (150 mg/kg/b) on days 4 and 1 before/m inoculationS. aureus(ATCC 13507). SuspensionS. aureusreceived from the colonies obtained from the overnight culture, placed in PBS and adjusted to approximately 107CFU/mlds spectrophotometer. Each rat was injected with 0.2 ml of inoculum into the thigh muscle of the right hind limb. The femur was collected after 25 hours after inoculation and processed for determination of CFU/thigh. Compound a was given in/in bolus injection into the tail vein or 1-hour/infusion, or 4-hour on/in infusion through kanilirovannoy the femoral vein at different time intervals after inoculation. A separate control group of inoculation were included in each experiment, groups of vancomycin were included as agents of the comparison in the first and second experiments. Each group, including the controls and the agent comparison, consisted of 4 rats.

To Connect A/bolus injection (10 mg/kg/dose, 20 mg/kg total dose) and 1 hour/infusion (10 mg/kg/dose, 20 mg/kg total dose) reduced the bacterial load of 3.2 and 3.0 log, respectively, compared to inoculated controls. The relatively lower levels of inoculum within 1 hour after infection was approximately 2.2 to 2.0 log respectively. The efficacy was compared with vancomycin. Cm. figure 4.

Example 13: effect of Compound A on murine models of sepsis: infectionS. aureus

Sterile saline solution, vancomycin or Compound A was administered to separate groups of 8-week-old female mice CD-1 (8 mice/group) after 1 and 7 hours after the/b injectionsS. aureus(ATCC 13709, 5×107CFU/ml in 5% mucin, 0.5 ml/mouse). Compound a was dissolved in 50%/50% volume/volume of sterile USP purified water/TBS. SuspensionS. aureusreceived from the colonies, transferred from a Cup with TSA in sterile PBS. An aliquot of the base of the suspension was added in 5% mucin to a final concentration of approximately 5×107CFU/ml. Planning studies and doses are shown in table 6. Mice were observed for 6 days after inoculation to death.

Table 6
ProcessingDose
(mg/kg/
dose)
General
dose
(mg/kg)
The way of Testino-
imago
connection
Volume
(ml/kg)
Diagram
injection
astrologo
connection
(h after inoculation)
No.
mice
A monk.
control
NANANANANA8
Vancomycin1010n/a101 8
Connection A36in/at41&78
Connection A510in/at41&78
Connection A1020in/at41&78

Dose-dependent efficacy was observed with Compound A, which was comparable with group treatment with vancomycin. All untreated mice died during the first day of treatment. At doses of 2×5 and 2×10 mg/kg Compound A complete protection was achieved with Compound A. See. figure 5.

Example 14: a study of the acute toxicity maximum tolerated dose

Determine the maximum tolerated dose (MTD) was obtained in studies of the ascending/descending doses in mice and rats. Compound a was administered by/bolus injection into the tail vein of rats and mice or by I.V. infusion through a catheter in the femoral vein of rats. Each dose of two or three animals enter and connection, and clinical signs were recorded over a period of 4 to 7 days.

In conclusion, the studies were carried out General necropsy. The results are shown in table 7.

Table 7
The dose of Compound AMTD (mg/kg)
Mouserat
/bolus30N. D.
/infusion 1 hourN. D.>24
N. D. - not determined

MTD for Compound A in rats was >24 mg/kg with introduction by I.V. infusion over 1 hour.

Example 15: the pharmacokinetics of Compound A in rats

Rats Crl:CD(SD) were administered Compound A by/bolus injection in the dose. Plasma was obtained from blood samples taken at 9 time points (n=3) within 28 hours. The levels of the compounds were determined using HPLC-MS/MS. All animals were equipped with two needle in the jugular vein (JVC) one for dose and blood sampling. Each route of administration has dosaged as N=3. Animals were provided a commercial diet for rodents and water indefinitely. Each rat received a metered bolus through choosing the proper route of administration at time zero a day dose. Time of collection of blood samples is shown in table 8.

Each sample of blood was collected from rats through JVC and were placed into chilled polypropylene tubes containing sodium EDTA as an anticoagulant. Samples were centrifuged at 4°C and the speed of 13000 rpm for 5 minutes. Samples kept chilled throughout processing. Each plasma sample is then transferred into labeled polypropylene tubes were placed on dry ice and kept in the freezer installation temperature from -60°C to -80°C.

The samples of the studied plasma were extracted and analyzed using a previously developed method. Samples for one standard curve and six repetitions quality control samples at three concentrations were extracted using DMSO containing 0.1% formic acid. Plasma samples (50 μl) was added to 150 μl of solvent and centrifuged. Supernatant analyzed using LC/MSMS [liquid chromatography with tandem mass spectrometry] using a Perkin Elmer series 200 Micronase and mass spectrometer with elektrorazpredelenie PE Sciex API4000. Standard curves were obtained at concentrations of 10000, 5000, 1000, 500, 250, 100, 50 and 25 ng/ml of the quality control Samples were obtained at concentrations of 5000, 500 and 50 ng/ml of the Samples to the standard curve and quality control recip is whether independently received from the base solutions. At least 5/8 standards must be accurate to within ±15% except LLOQ, where ±20% is acceptable. Two-thirds of the party QC must be accurate to within ±15% of nominal value, and at least one third of the QC must pass each level to ensure that the mileage is accepted.

Individual plasma concentration versus time data for Compound A were analyzed without regard to compartments using pharmacokinetic program WinNonlin v4.1. The plasma concentration is below the limit of quantitative analysis (25 ng/ml) was evaluated by the value of zero for pharmacokinetic analysis. Nominal dose concentration was used in all calculations.

The results are shown below in table 9.

Table 9
The parameter PKCompound a (5 mg/kg/bolus)
Cmax(ág/ml)89,2
T½ (watch)3
Vd(ml/kg)110
CLml/h/kg)28

The period of existence in the plasma of Compound A in plasma cu the si was significantly greater but the ground below.

Example 16: the composition

The before saturation solubility of Compound A in various excipients was investigated at 25°C, and the results are presented in table 10 (before saturation solubility of Compound A as a pure basis).

Table 10
ExcipientFunctional
Category
The before saturation solubility of Compound A (clean basis) at 25°C (mg/ml)
Purified waterControl65
Propylene glycolThe cosolvent90
PEG40018,5
Glycerin53
DMA0,60
Ethanol1,13
Benzyl alcohol:1,83
Citric acid/sodium citrate (pH 3)Buffers65,5
Citric acid/sodium t is spending (pH 5) 11,2

Tris(hydroxymethyl)
aminomethane HCl (pH 7.0)
61,4
a 0.9% saline solutionSolventN/A
1,2% salineN/A
N/A: not applicable as the composition, forming a viscous yellow gel.

Preliminary studies have shown that benzyl alcohol, ethanol and DMA was bad bearing solutions for Connection with A value of solubility to saturation 1,83; 1.13 and of 0.60 mg/ml, respectively. On the other hand, a good solubility to the saturation of 90 mg/ml, 65 mg/ml and 53 mg/ml was achieved in propylene glycol, purified water and glycerin, and they were therefore investigated further. Good value solubility was achieved at pH 3 and 7.4 with values 65,5 and to 61.4 mg/ml, respectively. The solubility, however, was drip at pH 5 with a value of 12.1 mg/ml solubility to the saturation of Compound A in 0.9% and 1.2% solution of sodium chloride could not be determined, because the composition was formed into a viscous yellow gel.

The before saturation solubility of Compound A in different megacolon ntih systems investigated at 25°C, and the results are presented in table 11 (before saturation solubility of Compound A as a pure basis)

Table 11
ExcipientSolventThe before saturation solubility of Compound A (clean basis) at 25°C (mg/ml)
20% weight/volume of propylene glycolSalineN/A
30% weight/volume of propylene glycolSalineN/A
40% weight/volume of propylene glycolSalineN/A
50% weight/volume of propylene glycolSalineN/A
15% weight/volume of propylene glycolPurified water64,9
30% weight/volume of propylene glycolPurified water59,1*
50% weight/volume of propylene glycol Purified water74,7
30% propylene glycol and 5 weight/volume ethanolPurified water43,9
15% weight/volume glycerolPurified water63,5
30% weight/volume glycerolPurified water63,1

50% weight/volume glycerolPurified water56,8
20% weight/volume of KleptuzaPurified water79,7
40% weight/volume of KleptuzaPurified water102,0
25% weight/volume CaptisolPurified water64,3
N/A: not applicable as the composition, forming a viscous yellow gel
*: composition generoulsy during centrifugation, but oijala in the process of aging.

The compounds that showed acceptable results included a 50% weight/volume of propylene glycol and 1% weight/volume of glycerol in purified water with a solubility of up to saturation at 25°C for 74.7 mg/ml and 63.5 mg/ml, respectively. The good solubility before saturation was also achieved with different complexing agents with values 79,7; 102,0 and 64,3 mg/ml to 20% mass/volume Kleptos, 40% weight/volume Kleptos and 25% weight/volume of Captisol respectively. The results also showed that the addition of Compound A in 20-50% weight/volume propylene glycol in saline solution resulted in the formation of a viscous yellow gel and, therefore, could not be analyzed using UV. However, the phenomenon of gelation concentration dependent and was observed in compounds when the concentration of Compound A in the composition has reached a value before saturation solubility of the drug. In addition, the gelation process can be easily reversed by adding a small amount of the composition of excipient or a few drops of ethanol. The addition of 5% weight/volume of ethanol in the composition does not inhibit the phenomenon of gelation, but he was easily reversible and could be analyzed using UV spectrophotometry. After the evaluation of the preliminary data screening of excipients selected three acceptable composition for further formulation development. These compounds represent a 20% weight/volume solution Kleptos, 20% weight/volume propylene glycol in purified water and 15% weight/volume of glycerol in purified water. The composition of 50 mg/ml Compound A 20% weight/volume of Clept what we chose in phase I clinical trials. In addition, can be taken aliquots of solutions of Compound A in water, Claptone or Mannitol and dried into a solid substance. The solid can be recovered by application of water prior to use.

Various modifications of the invention, in addition to those described herein, will be obvious to experts in this field from the foregoing description. Such modifications are also within the attached claims. This application claims the priority of provisional application U.S. serial No. 61/108595, filed October 27, 2008, which is described in this description by reference in its entirety. The present invention was supported by funds from the U.S. government (NIH(Grant No. AI4866 and 1R43AI058407), and the U.S. government may therefore have certain rights in this invention.

1. The method of producing drug containing 50 mg/ml compound of the following formula:

where the method includes:
(a) the interaction of (R)-(-)-N-Boc-3-pyrrolidinone with a strong base to obtain a mixture; and subsequent interaction of the mixture with 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene with obtaining compounds having the Formula II

b) interaction of the compounds of Formula II with an alcohol and a catalyst of transition metal in the presence of odor is Yes with obtaining the compounds of Formula III

(C) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine with obtaining the compounds of Formula IV

d) the interaction of the compounds of Formula IV with N-Boc-guanidine butyric acid to obtain the compounds of Formula V

e) removing the protective groups of the compounds of Formula V to obtain compounds of the following formula:

f) the formulation of the compounds obtained in the amount of 50 mg/ml with 20% weight/volume of β-cyclodextrin or 40% weight/volume of β-cyclodextrin in water.

2. The method of producing drug containing 50 mg/ml compound of the following formula:

where the method includes:
a) deprotonation of tert-butyl methyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid and the interaction of the compounds with 2-chloro-1,3-dinitro-5-triftorperasin obtaining tert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid;
b) the restoration of the tert-butyl ester (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid in the presence of alcohol, the transition metal catalyst and hydrogen to obtain tert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)Pyrrhus is lidin-1-carboxylic acid;
(C) a combination of tert-butyl methyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid with pyrimidine-4,6-dicarboxylic acid in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride with obtaining bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}pyrimidine-4,6-dicarboxylic acid;
d) the interaction of bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}pyrimidine-4,6-dicarboxylic acid ({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanol acid in the presence of phosphorus oxychloride to obtain bis-{[3-(5-({[(tert-butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)pentanediamine)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}pyrimidine-4,6-dicarboxylic acid;
e) removing the protective groups of the bis-{[3-(5-({[(tert-butoxycarbonyl)amino][tert-butoxycarbonyl)imino]methyl}amino)-pentanediamine)-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid to obtain crude bis-{[3-(5-guanidino-pentanediamine)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid; and
f) purification of the crude bis-{[3-(5-guanidino-pentanediamine)-2-((R)-pyrrolidin--yloxy)-5-trifluoromethyl-phenyl]amide}tetrahydrochloride pyrimidine-4,6-dicarboxylic acid using chromatography with reversed phase;
(g) the formulation of the compounds obtained in the amount of 50 mg/ml with 20% weight/volume of β-cyclodextrin or 40% weight/volume of β-cyclodextrin in water.

3. The method of producing drug containing 50 mg/ml compound of the following formula:

where the method includes:
a) deprotonation of tert-butyl methyl ether (R)-3-hydroxypyrrolidine-1-carboxylic acid followed by the interaction of the compounds with 2-chloro-1,3-dinitro-5-triftorperasin obtaining tert-butyl ether (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid;
b) the restoration of the tert-butyl ester (R)-3-(2,6-dinitro-4-triptoreline)pyrrolidin-1-carboxylic acid in the presence of alcohol, the transition metal catalyst and hydrogen to obtain tert-butyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid;
c) a combination of tert-butyl methyl ether (R)-3-(2,6-diamino-4-triptoreline)pyrrolidin-1-carboxylic acid with pyrimidine-4,6-dicarboxylic acid in the presence of 1-[(3-(dimethylamino)propyl)]-3-ethylcarbodiimide hydrochloride with obtaining bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide} pyrimidine-4,6-dicarboxylic acid;
d) the interaction of bis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-enyl]amide} pyrimidine-4,6-dicarboxylic acid with N-Cbz acid in the presence of tonila chloride;
e) recovering the obtained compound (d) in the presence of alcohol, the transition metal catalyst and hydrogen;
f) the interaction of the obtained compound (e) with di-Boc-pyrazole;
g) removing the protective group of the obtained compound (f) to obtain the compound of the following formula:

h) the formulation of the compounds obtained in the amount of 50 mg/ml with 20% weight/volume of β-cyclodextrin or 40% weight/volume of β-cyclodextrin in water.

4. The method of producing drug containing 50 mg/ml compound of the following formula:

in the form of pharmaceutically acceptable salts, where the method includes:
(a) the interaction of (R)-(-)-N-Boc-3-pyrrolidinone with a strong base to obtain a mixture; followed by the interaction of the mixture with 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene with obtaining compounds having the Formula II

b) interaction of the compounds of Formula II with an alcohol and a catalyst of transition metal in the presence of hydrogen to obtain the compounds of Formula III

c1) adding the compounds of Formula III and pyrimidine-4,6-dicarboxylic acid in a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine and N-methylmorpholine with obtaining the compounds of Formula IV

c2) adding the compounds of Formula III and pyrimidine-4,6-decarb the new acid in a mixture of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC1) and anhydrous pyridine to obtain the compounds of Formula IV

d) adding a compound of Formula IV with N-Cbz acid in a solution comprising anhydrous pyridine, dimethylaminopropylamine and any one of tonila chloride, POCl3, (EtO)2POCl or oxalyl chloride to obtain the compounds of Formula Va

e) hydrogenolysis of the Cbz group of compounds of Formula Va with obtaining the compounds of Formula VI

f) protection of the compounds of Formula VI to obtain the compounds of Formula VII

g) removing the protective groups of the compounds of Formula VII to obtain pharmaceutically acceptable salts of the compounds of the following formula:

h) the formulation obtained in the form of pharmaceutically acceptable salts of compounds in a quantity of 50 mg/ml with 20% weight/volume of β-cyclodextrin or 40% weight/volume of β-cyclodextrin in water.

5. The drug is designed to inhibit the growth of a microbe, comprising 50 mg/ml compound of the following formula:

or its pharmaceutically acceptable salt and 20% weight/volume of β-cyclodextrin (Kleptuza) in purified water, 40% weight/volume of β-cyclodextrin (Kleptuza) in purified water or 25% weight/volume Captisol in purified water.

6. The drug under item 5, comprising a compound of the following formula:

or its pharmaceutical is Eski acceptable salt 20% weight/volume of β-cyclodextrin (Claptone) in purified water or 40% weight/volume of β-cyclodextrin (Cleese) in purified water.

7. The drug under item 5 or 6, comprising a compound of the following formula:

or its pharmaceutically acceptable salt in 20% weight/volume of β-cyclodextrin (Claptone) in purified water.

8. The drug is designed to inhibit the growth of a microbe, obtained by the method according to any of paragraphs.1-4.

9. Method of inhibiting growth of a microbe comprising contacting the microbe with a preparation according to any one of paragraphs.5-8.

10. The method according to p. 9, where the microbe is a gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic or yeast.

11. The method of treatment of a subject suffering from a microbial infection, comprising the administration to a subject in need, an antimicrobial effective amount of a preparation according to any one of paragraphs.5-8.

12. The method according to p. 11, where the microbial infection is a gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic or yeast.

13. The drug according to any one of paragraphs.5-8, where the microbe is a gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic or yeast.

14. The drug according to any one of paragraphs.5-8, intended for the treatment of microbial infection.

15. The drug under item 14, where the microbial infection is a gram-negative aerobe, gram the aerobe, gone anaerobic gram-negative, gram-positive gone anaerobic or yeast.

16. The use of the drug according to any one of paragraphs.5-8 to obtain drugs for the treatment of microbial infection.

17. Application under item 16, where the microbial infection is a gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic or yeast.

18. The use of the drug according to any one of paragraphs.5-8 to obtain drugs for inhibiting growth of a microbe.

19. Application under item 18, where the microbe is a gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic or yeast.

20. The use of the drug according to any one of paragraphs.5-8 for the treatment of microbial infection.

21. Application on p. 20, where the microbial infection is a gram-negative aerobe, gram-positive aerobe, gram-negative gone anaerobic, gram gone anaerobic or yeast.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to heterocyclic compound - 6-methyl-5-morpholynomethyl-1-(thiethan-3-yl)pyrimidine-2,4(1H,3H)-dione of formula 6-methyl-5-morpholynomethyl-1-(thiethan-3-yl)pyrimidine-2,4(1H,3H)-dione of formula: .

EFFECT: novel compound, possessing antioxidant activity, is obtained.

2 cl, 6 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to heterocyclic compounds of general formula I

or to pharmaceutically acceptable salts or solvates or stereoisomers thereof, where R and R* are each independently -CR1R2R3, C1-4alkylamino, benzylamino, C6-10arylamino, heteroC4-7cycloalkyl containing 1 heteroatom selected from O; where R1 is selected from C1-4alkyl; phenyl, optionally substituted with 1, 2 or 3 substitutes independently selected from halogen, C1-4alkyl, C1-4alkoxy, trifluoromethoxy or 2 substitutes at neighbouring ring atoms, which form a 1,3-dixolane group; benzyl, optionally substituted with a halogen or methoxy; phenylsulphonylmethyl; C3-5heteroaryl containing 1 to 2 heteroatoms independently selected from N and O; C3-5heteroarylmethyl containing 1 to 2 heteroatoms selected from N and C3-6cycloalkyl; R2 is selected from hydrogen, hydroxyl, di-C1-4alkylamino, C1-4alkylcarbonylamino, C1-4alkyloxycarbonylamino, C1-4alkylaminocarbonylamino, piperidin-1-yl or imidazol-1-yl; R3 is hydrogen or, alternatively, R2 and R3 together form an oxo group; or R1 and R3 together form cyclopropyl; under the condition that if one of R and R* is -CH(C6H5)N(CH3)2, the other cannot be -CH(C6H5)NHC(=O)OCH3; and if R and R* are identical, R1 is not phenyl, when R2 is hydroxyl, acetylamino, methoxycarbonylamino or tert-butoxycarbonylamino, and R3 is hydrogen; and R1 is not C1-4alkyl, when R2 is C1-4alkyloxycarbonylamino, and R3 is hydrogen. The invention also relates to a pharmaceutical composition based a compound of formula I and use thereof.

EFFECT: obtaining novel compounds which are useful in preventing or treating HCV infection.

9 cl, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula

,

where R2 is a heteroaryl group and where said monocyclic heteroaryl group is unsubstituted or substituted with one or more groups selected from F, Cl, Br, I, -NR10R11 and C1-C12 alkyl; and groups selected from F, -NH2, -NHCH3, -N(CH3)2, -OH, -OCH3, -C(O)CH3, -NHC(O)CH3, -N(C(O)CH3)2, -NHC(O)NH2, -CO2H, -CHO, -CH2OH, -C(=O)NHCH3, -C(=O)NH2, and -CH3; R3x, R3y, R3z and R3p is hydrogen; R4x, R4y, R4z and R4p are independently selected from a group consisting of: hydrogen, F, Cl, Br, I, and -C(C1-C6 alkyl)2NR10R11; and R10 and R11 are hydrogen, which are phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors.

EFFECT: high effectiveness of compounds.

7 cl, 7 tbl, 50 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of structural formula

possessing inhibitory activity on BTK, TEC, BMX, ITK, ErbB1, ErbB4 and/or JAK3 kinases. In formula (I-b), ring A and ring B represents phenyl; Ry represents -CN, -CF3, C1-4 aliphatic group, C1-4 halogenaliphatic group, -OR, -C(O)R or -C(O)N(R)2; each group R independently represents hydrogen or a group specified in C1-6 aliphatic group optionally containing a substitute presented by halogen, -(CH2)0-4R°, -(CH2)0-4OR°, -(CH2)0-4N(R°)2, -(CH2)0-4N(R°)C(O)OR°, -(CH2)0-4C(O)R°, -(CH2)0-4S(O)2R°, or 5-6-merous substituted or aryl ring containing 1-2 heteroatoms independently specified in nitrogen or oxygen optionally substituted by group =O, -(CH2)0-4R°, -(CH2)0-4N(R°)2 or -(CH2)0-4OR°; phenyl; 5-6-merous heterocyclic ring containing 1-2 heteroatoms independently specified in nitrogen, oxygen or sulphur optionally substituted by group -(CH2)0-4R°, -(CH2)0-4OR° or =O; or 6-merous monocyclic heteroaryl ring containing 1 nitrogen atom; W1 and W2 represent -NR2-; R2 represents hydrogen, C1-6aliphatic group or -C(O)R; m and p are independently equal to 0, 1, 2, 3 or 4; Rx is independently specified in -R, -OR, -O(CH2)qOR or halogen, wherein q=2; Rv is independently specified in -R or halogen; R1 and R° radical values are presented in the patent claim. The invention also refers to a pharmaceutical composition containing the above compounds.

EFFECT: preparing the compounds possessing the inhibitory activity on BTK, TEC, BMX, ITK, ErbB1, ErbB4 and/or JAK3 kinases.

17 cl, 25 dwg, 20 tbl, 286 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of organic chemistry, namely to heterocyclic compound of formula (I) or its racemate, enantiomer, diastereoisomer and their mixture, as well as to their pharmaceutically acceptable salt, where A is selected from the group, consisting of carbon atom or nitrogen atom; when A represents carbon atom, R1 represents C1-C6-alkoxyl; R2 represents cyano; when A represents nitrogen atom, R1 hydrogen atom or C1-C6-alkoxyl; where said C1-C6-alkoxyl is optionally additionally substituted with one C1-C6-alkoxyl group; R2 is absent; R3 represents radical, which has the formula given below: or , where D represents phenyl, where phenyl is optionally additionally substituted with one or two halogen atoms; T represents -O(CH2)r-; L represents pyridyl; R4 and R5 each represents hydrogen atom; R6 and R7 each is independently selected from hydrogen atom or hydroxyl; R8 represents hydrogen atom; R9 represents hydrogen atom or C1-C6-alkyl; r equals 1 and n equals 2 or 3. Invention also relates to intermediate compound of formula (IA), method of obtaining compound of formulae (I) and (IA), pharmaceutical composition based on formula (I) compound and method of its obtaining and to application of formula (I) compound.

EFFECT: novel heterocyclic compounds, inhibiting activity with respect to receptor tyrosine kinases EGFR or receptor tyrosine kinases HER-2 are obtained.

18 cl, 12 ex, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are described new isatin-5-sulphonamide derivatives of general formula or their physiologically acceptable salts, wherein R represents phenyl, 3-fluorophenyl, 2,4-difluorophenyl, 3,5-difluorophenyl, tetrahydropyranyl, diazine or triazolyl methyl optionally substituted by one C1-6alkyl, which can be additionally substituted by one halogen; R' represents phenyl optionally substituted by one or two halogens, or triazolyl optionally substituted by one C1-6alkyl which can be additionally substituted by one halogen; provided R means phenyl, R' represents optionally substituted triazolyl, pharmaceutical compositions containing the above derivatives, using them as molecular imaging agents, using them in diagnosing or treating diseases or disorders related to apoptosis dysregulation, methods for synthesis of the above derivatives, methods for molecular imaging of caspase activity and apoptosis, and methods for assessing the therapeutic exposure of the analysed compound on caspase activity.

EFFECT: new isatin-5-sulphonamide derivatives are described.

27 cl, 26 dwg, 4 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compounds of general formula [1] or their pharmaceutically acceptable salts, which possess properties of an inhibitor of the JAK2 thyrokinase activity. In general formula radicals are selected from group (I) or (II). In group (I) X represents CH or N; R1 represents a halogen atom and R2 represents H, a halogen atom, CN, or is selected from the groups of formulas

,

or a group -ORP or 5-6-membered heteroaryl, containing 1-4 nitrogen atoms and optionally additionally containing an oxygen or sulphur atom or containing an oxygen atom as a heteroatom, optionally substituted; or (II) X represents -CRA; and RA represents a group of formula , RB represents (a) amino, optionally substituted with one or two groups, selected from the group, consisting of C1-6alkyl, C3-6cycloalkyl, (C3-6cycloalkyl)C1-6alkyl and C1-3alcoxyC1-3alkyl, (b) C1-3alcoxy, (c) hydroxy or (d) a 5-6-membered saturated cyclic amino group, which additionally can contain a heteroatom, selected from an oxygen atom; R1 represents a halogen atom and R2 represents H; R3 -R5 have values given above. Other values of the radicals are given in the invention formula.

EFFECT: compounds can be applied for the prevention or treatment of cancer, for instance hematologic cancer disease or a solid form of cancer, inflammatory disorder, for instance, rheumatoid arthritis, inflammatory intestinal disease, osteoporosis or multiple sclerosis and angiopathy, for instance, pulmonary hypertension, arteriosclerosis, aneurism or varicose veins.

14 cl, 19 tbl, 234 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, namely to a heterocyclic compound of formula I and its pharmaceutically acceptable salt, wherein if a chemical valency permits, i represents 1 or 2, R1 represents H; a linear (C1-C4) alkyl group, R2 represents H, Cl or F, X represents either N, or CR3, R3 represents H; halogen; a linear (C1-C4) alkyl or (C1-C4) alkoxyl group, Y represents Z represents O or NRx, Rx represents H or a linear or branched (C1-C4) alkyl, k is equal to 2, 3 or 4, n and p independently represents 2, and a sum of n+p cannot exceed 4, T represents H or a linear (C1-C4) alkyl group; T′ represents a linear C1-C3 alkyl chain substituted by either (C1-C6)-dialkylaminogroup, or a 5-6-merous saturated heterocycle containing one nitrogen atom and optionally containing the second heteroatom specified in O, such a heterocyclic ring is optionally substituted by a (C1-C4) alkyl chain at nitrogen atoms; or a 5-merous saturated heterocycle containing one nitrogen atom, such a heterocyclic ring is optionally substituted by a (C1-C4) alkyl chain at nitrogen atoms; r represents zero, 1; R′ represents di(C1-C4)alkylamino, (C1-C4)alkoxy; except for the compounds specified in the clause. The invention also refers to a pharmaceutical composition based on the compound of formula (I), using the compound of formula (I) and to a method of treating diseases, in which the hedgehog signalling pathway modulation is effective.

EFFECT: there are prepared new heterocyclic compounds possessing t effective biological properties.

20 cl, 193 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula (I), wherein A means morpholinyl, 1,4-oxazepamyl, piperidinyl, pyrrolidinyl or azetidinyl which is bound to N; R1 means C1-C6-alkyl group; R2 means bicyclic aryl group specified in 1H-indolyl, 1H-pyrrolo[3,2-b]pyridyl, quinolyl, naphthyl, 1H-pyrrolo[2,3-b]pyridyl, 5H-pyrrolo[3,2-d]pyrimidinyl, 7H-pyrrolo[2,3-d]pyrimidinyl, benzo[b]thiophenyl, imidazo[1,2-a]pyridyl, benzo[b]thiazolyl, 5H-pyrrolol[2,3-b]pyrazinyl and quinoxalinyl which can be substituted by R4; R3 means hydrogen or halogen atom; R4 means C1-C6-alkyl group, C1-C6-halogenalkyl group, OR1A, halogen, -(CH2)aOH, CN, NHCOR1A, SO2R1A or NHSO2R1A; R5 means C1-C6-alkyl group, -(CH2)aOH, -(CH2)aOR1B, halogen or CONH2; provided p is a plural number, R5 can be identical or different, or R5 can be combined with another R5; each of R1A and R1B independently means C1-C6-alkyl group; a is equal to 0, 1 or 2; n is equal to 1 or 2; p is equal to 0, 1, 2, 3, 4 or 5. Besides, the invention refers to intermediate compounds of formulas (IA) and (IB) for preparing the compounds of formula (I), to a preventive or therapeutic agent containing the compounds of formula (I), pharmaceutical compositions, using the compounds of formula (I) and to a method for preventing or treating diseases.

EFFECT: compounds of formula (I) as selective 5-HT2B receptor antagonists.

11 cl, 1 dwg, 18 tbl, 88 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the derivatives of pyrimidine with formula I, where Z is a carbon atom or a nitrogen atom; Y is a carbon atom or a nitrogen atom, where one of the Z and Y is a nitrogen atom; A, D and E are chosen from the carbon atom and nitrogen atom; R1 is hydrogen or methyl, when D is a carbon atom; R2 is hydrogen or an amine group; R3 is hydrogen, methyl, trifluoromethyl or (C0-C1) alkylaryl; R5 is hydrogen or methyl; L-R4 is chosen from: and R6 is chosen from hydrogen and methyl; R7 is chosen from hydrogen, methyl, (C1-C4)alkyl-OH and (CO)OCH3, R7a and R7b are independently chosen from hydrogen and methyl; R8 is chosen from halogen, hydrogen, hydroxy, (CO)OH, (CO)OCH3, O(C1-C4) alkyl, O(C1-C4)alkyl(C6-C10)aryl, O(C1-C4)alkyl(C2-C9)heterocyclyl, O(EtO)1-3(C1-C4)alkyl and OCF3; and R11 is chosen from hydrogen, methyl and O(C1-C4) alkyl. The invention also relates to a pharmaceutical formulation for curing of cancer which contains the compounds with the formula I, to the usage of the compounds with the formula I to produce a medicinal agent and pharmaceutical formulation and to the cancer curing method.

EFFECT: compounds with the formula I aimed at cancer curing.

20 cl, 3 tbl, 109 ex

FIELD: veterinary medicine.

SUBSTANCE: pharmaceutical composition in the form of ointments for treatment of mastitis in cows comprises n-tetradecyltributylphosphonium bromide as active ingredient and petrolatum as an excipient at the ratio of 1:2000.

EFFECT: invention provides a pharmaceutical composition in the form of an ointment, which is new in mechanism of action, effective when applied topically at low therapeutic concentration.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel hexasubstituted para-aminophenols: where R=C6H5, C6H4Cl, C6H4Br, C6H4CH3, with arylamide groups in 2,6 positions with respect to hydroxyl, which demonstrate antibacterial activity.

EFFECT: hexasubstituted para-aminophenols with arylamide groups in 2,6 positions with respect to hydroxyl, possessing high bactericidal and bacteriostatic activity.

1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to veterinary, in particular to obstetrics, gynecology and reproduction biotechnology, and can be used for treating animals in case of clinical mastitis. Medication for treating animals with clinical mastitis includes: trivit - 10.0 ml, cefotaximum - 750 mg, prednisolone - 10.0 mg, nystatin - 325.0 ml. Medication is introduced to ill animals intracycternally in dose 10.0 ml with interval 12 hours after milking.

EFFECT: invention provides increased efficiency of treating clinical mastitis by complex impact on all sides of pathological process.

4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to new dosage forms of cephem compounds effective for treating bacterial infections.

EFFECT: dosage forms are stable; they show the improved solubility and are especially applicable for parenteral administration.

9 cl, 29 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of veterinary and is intended for treatment and prevention of postpartum acute mastitis in cows. Medication contains antimicrobial, anti-inflammatory preparations and distilled water. Medication additionally contains licorice root. As antimicrobial preparation it contains metronidazole. as anti-inflammatory preparation it contains ciprofloxacin, with the following component ratio, g/l: licorice root 80-85 g; metronidazole 3.5-4.0 g; ciprofloxacin 2.0-2.5 g; distilled water - the remaining part.

EFFECT: application of claimed medication provides expressed antimicrobial and anti-inflammatory effect with simultaneous reduction of organism's response to histamine, release of spasm of smooth muscles and reduction of permeability of capillaries.

4 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: method involves prescribing proton pump inhibitor with one antibiotic in a dual therapy and with two antibiotics with a triple therapy twice a day within the course of 7-10 days. Helicobacter is eradicated by the separate administration of drugs. The proton pump inhibitor (omeprazole or pantoprazole) is administered two hours before the antibiotic in the dual therapy and four hours before the second antibiotic in the triple therapy. The drugs are taken for the second time 12 hours after the first one. The antibiotics (clarithromycin, amoxicillin or fromilid) are taken with water 250ml.

EFFECT: invention enables providing the more effective eradication of Helicobacter by the exposure of total therapeutic bioavailability of each preparation with underlying permanent gastric alkaline environment.

2 cl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and represents a cream for medical application for local treatment of bacterial infections and for wound healing, which contains fusidic acid in an amount of 0.1 wt % to 25 wt % and a biopolymer, preferentially chitosan; fusidic acid is formed in situ in an oxygen-free medium; the above cream contains fusidic acid formed in situ by transforming sodium fusidate at adding the acid slowly, with a particle size of an active agent of the substance of 2.33 mcm to 16.3 mcm, while the biopolymer is introduced into a cream base containing at least one ingredient of each type: primary and secondary emulsifying agents specified in a group containing ketostearyl alcohol, ketomacrogol-1000, polysorbate-80, Span-80, paraffin as a wax product, a combined solvent specified in a group containing propylene glycol, hexylene glycol, polyethylene glycol-400, nitric acid or lactic acid and water.

EFFECT: invention provides the high stability of the active ingredient during the whole shelf life.

12 cl, 18 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and represents a composition for inducing an immune response against Staphylococcus aureus (versions), a method for inducing the immune response against Staphylococcus aureus, and a method for eliciting passive immunity against Staphylococcus aureus in an individual. The composition contains an effective amount of the recovered capsular polysaccharide 5 S. aureus conjugated with the CRM197 carrier protein, an effective amount of the recovered capsular polysaccharide 8 S. aureus conjugated with the CRM197 carrier protein, and at least one ingredient specified in a group consisting of an effective amount of the recovered polypeptide of the clumping factor A (ClfA) of S. aureus, an effective amount of the recovered polypeptide of the clumping factor B (ClfB) of S. aureus (ClfB) and an effective amount of the recovered protein MntC of S. aureus. The capsular polysaccharide type 5 represents a capsular polysaccharide of high molecular weight from 70 to 300 kD. The capsular polysaccharide type 8 represents a capsular polysaccharide of high molecular weight from 70 to 300 kD, wherein from 5 to 23 lysines in CRM197 are conjugated. The CP5 and CP8 conjugates have the molecular weight from approximately 200 kD to approximately 5000 kD.

EFFECT: presented invention enables eliciting the improved immune response against Staphylococcus aureus.

25 cl, 29 dwg, 26 tbl, 32 ex

FIELD: medicine.

SUBSTANCE: method involves immunising with 5×107 microbial cells Vibrio cholerae 5879; 10 days later, a common pool of anticholeraic Ig is recovered from enteric washing liquid. That is combined with producing the primary enterocyte culture from intact mice. Thereafter, the doubly-diluted anticholeraic Ig are applied in an amount of 3 ml on a monolayer of erythrocytes formed in wells of culture panels. The latter are added with alive choleraic vibrions of the strain Vibrio cholerae 5879, 40 m.c. per 1 erythrocyte and incubated for 3 hours. Then, the panels are washed three times, fixed with ethanol for 20 minutes and stained by Romanowsky-Giemsa. Local humoral anticholeraic immunity is assessed under the microscope by the number of vibrions stained in dark blue and attached to one erythrocyte.

EFFECT: method enables assessing specific local humoral immunity and intensifying the anti-adhesive activity of the common pool of anticholeraic immunoglobulin.

4 cl, 2 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new bacitracin compounds with antibiotic activity

,

wherein at least one of R1, R2 and R3 represents -CH=CH2 and wherein R1, R2 and R3 independently represent -H, -CH3 or -CH=CH2.

EFFECT: preparing the new bacitracin compounds.

13 cl, 8 dwg, 7 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to heterocyclic compound - 6-methyl-5-morpholynomethyl-1-(thiethan-3-yl)pyrimidine-2,4(1H,3H)-dione of formula 6-methyl-5-morpholynomethyl-1-(thiethan-3-yl)pyrimidine-2,4(1H,3H)-dione of formula: .

EFFECT: novel compound, possessing antioxidant activity, is obtained.

2 cl, 6 tbl, 7 ex

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