Therapeutic peptides

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a peptide, a peptide mimetic or an amino acid derivative, which contain di-substituted β-amino acid, wherein each of substituting group in the β-amino acid, which can be identical or different, contains at least 7 non-hydrogen atoms, is lipophilic and contains at least one cyclic group; one or more cyclic groups in the substituting group can be bound or condensed with one or more numbers of cyclic groups in the other substituting groups, and when the cyclic groups are fused so that an aggregate total number of non-hydrogen atoms for these two substituting groups makes at least 12, wherein the above peptide, peptide mimetic or amino acid derivative consist of 1-4 amino acids or length-equivalent sub-units.

EFFECT: preparing the peptide, peptide mimetic or amino acid derivative, which contain the di-substituted β-amino acid.

17 cl, 4 dwg, 10 tbl, 4 ex

 

The present invention relates to modified amino acids, peptides and peptidomimetics, as well as their use as cytolytic drugs, which are particularly widely used as antimicrobial and anticancer drugs.

Infections caused by multi-resistant bacteria have become a major social problem over the last 20-25 years. In particular, this problem arose in hospitals, where infections caused by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE), can lead to serious injuries, prolonged hospitalization and death, particularly among patients with a weakened immune system. Vancomycin used to be the drug for admission in exceptional circumstances, however, the reports from hospitals around the world demonstrate that vancomycin is currently used much more often.

As for the above-mentioned gram-positive strains, clinicians also have reported problems arising from gram-negative strains, including Pseudomonas aeruginosa and Eschericha coli. It is highly desirable for drugs-antibiotics had an effect against wide range of bacteria, including gram-positive and gram-negative strains.

Long time between major pharmaceutical companies ve�ve the struggle for the development of new classes of antimicrobial compounds, obviously, due to the huge development costs and the relatively limited duration of treatment patients compared with treatment of chronic diseases. However, the need for new antimicrobials is essential, because the number of deaths in the U.S. caused by infections acquired in the hospital now exceed deaths from HIV.

A promising class of Antimicrobial drugs is a cationic antimicrobial peptides (AMP), also known as the immunological protective peptides. AMP are characterized by a unique mechanism of action, focusing its influence on the inner and/or outer shell of the bacteria preceptor-specific way. The detailed mechanism of rupture of the shell AMP is still not fully understood. Suggest different models to explain the observed effects.

Due to the relative similarity between the components of the lipid cell membrane of prokaryotic and eukaryotic tumor cells, selective membrane destabilization and consequently lytic activity against tumor cells was also observed for these Antimicrobial peptides. For both types of target cells was identified effective class amphipathicity peptides and peptidomimetic molecules having a net positive charge, and a lipophilic group Il� group. Although the first generation of these molecules was assumed sporoobrazuyushchie mechanism of action, and they usually consisted of ten or more amino acids, recently it was proved that a much smaller molecule can maintain a therapeutically significant levels of activity and selectivity (M. B. Strøm et al., J. Med. Chem. 2003, 46, 1567-1570).

Despite it sometimes being contrary to the purpose of therapeutic activity, selectivity, toxicity, stability in vivo and in vitro, cost of production and ease of reception mean that there is a constant need to develop new drugs in this General class of molecules.

The present inventors have found that by applying a disubstituted β-amino acids, you can create a new class of cytolytic drugs, which have a potentially powerful effect and other desired characteristics, including a wide range of antibacterial activity, e.g., activity against gram-positive and gram-negative strains. These drugs are mainly intended for oral administration.

Thus, in one aspect, the present invention is a peptide, peptidomimetic or (modified) amino acid having a net positive charge of at least +2 and incorporating a disubstituted β amino acid, each of the substituting groups in the β-aminoxy�lot, which may be the same or different, comprises at least 7 novogorodnaya atoms, is a lipophilic or has at least one cyclic group, one or more cyclic groups in the replacement group may be associated or combined with one or more cyclic groups in the other substituting group, and if the cyclic group is mixed thus, the cumulative total number novogorodnaya atoms of the two substituting groups is at least 12, for use as therapeutic cytolytic drug. 2 substituting groups in the β-amino acid, preferably the same.

Not wanting to be limited by theory, it is likely that the inclusion of a disubstituted β-amino acids increases the stability of the drug, and although forced conformational expenses improve amphipathicity of the drug, as well as contribute to a powerful destructive action on the shell due to the mutual repulsion of two lipophilic fragments dibasic residue. This has a cytolytic effect, which may be cytotoxic.

Cytotoxic activity can be an antimicrobial, preferably an antibacterial effect and/or antitumor activity, and this medical application is the preferred embodiments of the present invention. Thus, the present image�eenie provides peptides, peptidomimetics and a modified amino acid as described above (and described in more detail below) for use as cytolytic antimicrobial or anticancer drugs. In considering alternative points of view, the present invention provides peptides, peptidomimetics and a modified amino acid as described above (and described in more detail below) for use in the treatment of microbial (especially bacterial) infection or for the treatment of tumor cells (particularly solid tumors).

Microbes against which can be the action or treatment, including bacteria (gram-positive and gram-negative), fungi, archaea and the protests. Bacteria are of particular interest due to their ability to infect humans and animals, causing harm to their health and life threatening.

Preferred bacterial targets include gram-positive bacteria, in particular Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). Gram-negative strains, for example, Pseudomonas aeruginosa and Eschericha coli, may also be subjected to treatment. Chronic wounds often become infected with gram-positive and gram-negative strains, and the treatment of the patient who has or may have multipathogen infection (e.g., chronic wounds), is� the preferred use is in accordance with the present invention.

Antimicrobial activity can also be used in non-therapeutic purposes, for example, as an antimicrobial agent. Another aspect of the present invention provides ex vivo using the peptide, peptidomimetic or modified amino acid, as shown and described herein as cytolytic agent.

The lipophilicity can be measured on the basis of distribution of the drug on a two-phase system, for example, "liquid-liquid", for example, 1-octanol/water). It is widely known that polar substituents, e.g., hydroxyl, carboxyl, carbonyl, amine groups and esters reduce the separation factor for two-phase systems, for example, 1-octanol/water because they reduce the lipophilicity; residual lipophilic groups, thus, should preferably contain no more than one or two, preferably one or not contain such polar groups.

β-amino acid contains an amino group, attached to the β-carbon atom; genetically encoded amino acids in which the amino group attached to the carbon atom. Such organization is extended by one atom on the β-amino acid based peptide containing one or more β-amino acids. In this structure α and/or β carbon atom may be substituted, α or β carbon atom may be disubstituted; �if the carbon atom is disodium, is obtained β2,2amino acid, and if the β carbon atom is disubstituted, the result is generated β3,3amino acid. One substitute in each group α or β carbon atom leads to the formation of β2,3amino acids, β2,2and β3,3dibasic amino acids preferred for use in accordance with the present invention, and β2,2disubstituted amino is especially preferred.

β amino acid is substituted with two groups containing at least 7 novogorodnaya atoms. Preferably one, more preferably both of the residual groups must contain at least 8, more preferably at least 10 novogorodnaya atoms. These groups are lipophilic in nature, and although they may be different, preferably they should be the same. Each of them contains at least one cyclic group, typically a 6-membered ring, which may be aliphatic or aromatic, preferably aromatic and may be substituted and the substituting group may include such atoms as oxygen, nitrogen, sulfur or halogen, in particular fluorine or chlorine. Preferred substituting groups include C1-C4alkyl (particularly, t-butyl), methoxyl, fluorine and formative group. Cyclic groups can be�ü Homo - or heterocyclic, preferably, they are homozygotes ring carbon atoms. Preferred residual lipophilic groups contain two or three cyclic groups, preferably two cyclic groups, which may be associated or connected, preferably, are connected. Particularly preferably, the substituting groups include naphthalene group.

Another preferred group of the lipophilic substituent groups should have one substituted or unsubstituted cyclic group, preferably phenyl or pirazinokarbazolovogo cyclohexyl group.

Cyclic group, or groups, usually separated from the peptide framework (i.e. α or β carbon atoms of the β-amino acid) chain of 1-4, preferably 1-3 atoms; these linking atoms may include nitrogen and/or oxygen, however, as a rule, are carbon atoms, preferably linking atoms unsubstituted. These separators are naturally a part of substituent groups, as described above.

Each replacement functional group disubstituted β-amino acids, typically includes 7-20 novogorodnaya atoms, preferably 7 to 13, more preferably 8 to 12, but is most preferably 9-11 novogorodnaya atoms.

Molecules for use in accordance with the invention, preferably, are peptides or peptidomimetics 1 or 2-12 amino acid�or the equivalent in length of pobjednici. Unless the context otherwise provides, the reference to "amino acid" herein includes equivalent polyadenine in peptidomimetic. In Antimicrobial purposes, the preferred molecules have 1-3 or 4 amino acids, anti-tumor purposes, the preferred molecules are longer, i.e. 3-12 amino acids, more preferably, 5 to 12 amino acids in length. As shown in the Examples, the molecules for use in accordance with the invention may include only one amino acid, but it will be "modified" amino acid for the purpose of fulfilling the requirements of a charge.

Individual amino acids and peptides and peptidomimetics are preferably incorporated into a modified C-terminal, C-terminal modifying group, usually leads to conversion of the charge, i.e. removes the negative charge of the carboxyl group and adds a positive charge, for example, by the presence of the amino group. This modification by itself, provided that the N-terminal modified, will provide an overall net charge of the molecule is equal to +2. If the C-terminal is modified to ensure the circulation of the charge, or just to relieve the negative charge of the carboxyl group, molecule, preferably also contain one or more cationic amino acids. Thus, the total charge of the molecule composition may�Yat +3, +4 or more for large molecules.

Suitable C-terminal groups, which are preferably cationic in nature, will typically have a maximum size of 15 novogorodnaya atoms. C-terminal preferably aminirovanie, and amide group may be further substituted to form an N-alkyl or N,N-dealkylase amide. Primary and secondary amide groups are preferred. The relevant groups for the substitution of amide groups include aminoalkyl, for example, aminoethyl or dimethylaminoethyl; the nitrogen atom of the amide group can form part of a cyclic group, for example pyrazolidine, piperidine, imidazolidine and piperazine, wherein the piperazine is preferred, and these cyclic groups may themselves be substituted, for example, alkyl aminoalkyl group.

Peptides for use in accordance with the invention should preferably include one or more cationic amino acids, lysine, arginine, ornithine and histidine are preferred, but must not contain any non-genetically encoded or modified amino acid carrying a positive charge at a pH of 7.0.

Corresponding non-genetically encoded cationic amino acids and modified cationic amino acid analogs include lysine, arginine and histidine, for example Somalis�n, ornithine, diaminobutane acid, diaminopimelic acid, diaminopropionic acid and homoarginine and trimethyltin and trimethylaniline, 4-aminopiperidin-4-carboxylic acid, 4-amino-1-carbamimidoyl-4-carboxylic acid and 4-guanidinopentanoic.

The dipeptides, usually include one cationic amino acid, and longer peptides, as a rule, will contain additional cationic amino acids, therefore, the peptides of 4 or 5 amino acids may have 2 or 3 cationic amino acids and peptides of 6-9 amino acids can include can have 3-6 cationic amino acids.

A preferred group of molecules includes β2,2dibasic amino acid attached to the remainder of the C-terminal L-argininemia, and the dipeptides with such a structure, are particularly preferred.

Peptides with three or more amino acids, typically have one or more additional lipophilic amino acids, i.e. amino acids with a lipophilic R group. Generally, lipophilic R group has at least one, preferably two cyclic groups which may be fused or connected. Lipophilic R group may contain atoms such as O, N or S, however, usually not more than one heteroatom, which is preferably nitrogen. This R-group booth will preferably be �e 2 more polar groups, more preferable not to have or to have one, it is most preferable not to have.

Tryptophan is preferred lipophilic amino acid and peptides preferably include tryptophan residues 1-3. Additional genetically encoded lipophilic amino acids that can be incorporated is phenylalanin and tyrosine.

Lipophilic amino acids can be non-genetically coded, including, genetically encoded amino acids with modified R-group.

Peptidomimetic as a rule, characterized by polarity, three-dimensional size and function (biological effects), its peptide equivalent, however, if the peptide bond has been replaced as a rule, more stable compounds. By "stable" is understood to be more resistant to enzymatic degradation by hydrolytic enzymes. As a rule, substituting an amide bond (amide bond surrogate) retains many of the properties of the amide bond, for example, structure, steric volume, electrostatic in nature, the possibility of linking with hydrogen, etc. In Chapter 14, "Drug Design and Development, Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pub, provide a General description sposoby design and synthesis peptidomimetics. In this case, where the molecule reacts with the shell and not with �pacificism active site of the enzyme, some describes the accurate simulation of affinity and efficacy, or features of the substrate are not significant, and peptidomimetic can be easily prepared on the basis of this peptide structure or motive of the required functional groups. Suitable amide bond surrogates include the following groups: N-alkylate (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46, 47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res, 1993, 26, 266), thioamide (Sherman D. and B. Spatola, A. F. J. Am. Chem. Soc., 1990, 112, 433), a thioether, Fofana, catameran (X Hoffman, R. V. and Kim, H. O. J. Org. Chem., 1995, 60, 5107), hydroxymethylene, Porvenir (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl, methylamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 1997, 45, 13), metileno (Spatola, A. F., Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamide (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391).

Molecules of the present invention contain a disubstituted β amino acid, and the various molecules described in the examples that contain only one additional amino acid, i.e., molecules having 2 amino acids, United amide bond.

Such molecules can be considered dipeptides due to amide bond; however, the amide bond in these molecules is actually pressalexey with the distribution of β-amino acids and, as such, these molecules should be considered as peptidomimetics. For the purposes of the present invention, such molecules (and more cu�Phnom molecules with large amino acids) peptides are considered (and not a peptidomimetic), because of the presence of the amide bond. This allows to clarify the nomenclature without testing whether a given cleaving an amide bond and to what extent. In other words, if all the amino acids in the molecule are connected by amide bonds, the molecule is considered to be a peptide, even if one or more of the amide linkages are not fissile.

Peptidomimetics compounds in the present invention typically will have identified pobjednici which are approximately equivalent in size and function amino acids. Peptidomimetics, typically have a group equivalent to the R-groups of amino acids, and a description of suitable R groups and N and C-terminal modifying groups applies, mutatis mutandis, to peptidomimetics connections.

As described in the manual mentioned above, along with replacement of the amide linkages, peptidomimetics can participate in the replacement of large structural groups distributionsystem structures, and in this case, the mimetic group containing the peptide bond, such as azole derivatives, mimetics, can be used as a dipeptide replacements. Peptidomimetic and, therefore, the basis of peptidomimetics, which were only substituted amide bond as described above are, however, preferred.

Suitable peptidomimetics include restore�branched peptides, in which an amide bond was restored to methyleneimine by processing such a reducing substance, for example a borane hydride or reagent, such as alumoweld sodium. Such restoration has the additional benefit of increasing the total content of cations of the molecules.

Other peptidomimetics are formed peptide, for example, by stepwise synthesis amino-functionalityand of polyglycine. Some of the basics of peptidomimetics will be available from their peptide precursors, such as peptides, which were subject dermatillomania. Corresponding methods are described Ostresh, J. M. et al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strong-base conditions will contribute to the predominance of M-metelli over O-metalasia and will lead to metelli some or all of the nitrogen atoms in the peptide bond and the N-terminal nitrogen.

Preferred substitutes for peptide bonds include esters, palaminy and derivatives, as well as substituted alkanes and alkenes, in particular aminomethyl and kilometre. Peptidomimetics will be preferable to have N and C-terminals, which can be modified as described in the present work.

In the following aspect the present invention describes a method for the treatment or prophylaxis of microbial infections, preferably bacterial infections, which provides a basic introduction to�of the patient a peptide, peptidomimetic or modified amino acid as described above.

In the following aspect the present invention describes a method of treating tumor cells, or prevent or inhibit the growth, proliferation or metastasis of a tumor, which involves introducing the patient a peptide of peptidomimetic or modified amino acid as described above.

As in the present invention, treatment of a microbial/bacterial infection would be preferable to indicate a decrease in the number of viable microbial/bacterial cells, but also may provide a bacteriostatic mechanism of action, in which the cells are held in amounts that are less harmful to the patient than if the infection proceeded without interference. "Prevention" involves the suppression of growth of microbial/bacterial cells so that measurable and/or harmful, the population has not been installed in a patient who received treatment.

Treated tumor cells may be circulating, however, as a rule, are part of a solid tumor; and as for microbial cells, the treatment will be preferable to provide cell death by cell lysis. Cell lysis may lead to the appearance of antigens of tumor cells and formation of acquired immunity, which can �to Omusati or inhibit the development of secondary tumors.

In the following aspect the present invention describes a product containing (a) a peptide, peptidomimetic or a modified amino acid, as indicated above, and (b) an additional anti-microbial agent in the form of a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of microbial infections.

In another aspect of the present invention describes a product containing (a) a peptide, peptidomimetic or a modified amino acid, as indicated above, and (b) an additional antitumor agent as a combined preparation for separate, simultaneous or sequential use in the treatment of tumor cells.

In the following aspect the present invention describes the use of a peptide of peptidomimetic or modified amino acid as described above, in the manufacture of a medicament for the treatment of microbial infections, preferably bacterial infections.

In the following aspect the present invention describes the use of a peptide of peptidomimetic or modified amino acid as described above, in the manufacture of a medicament for the treatment of tumor cells, or prevent or slow the growth, proliferation or metastasis of tumors.

Of the above class of molecules there is a new group� highly effective molecules. These molecules are suitable for various applications and methods specified in this document. Thus, in the following aspect the present invention is a peptide, peptidomimetic or (modified) amino acid having a net positive charge of at least +2, which incorporates a group of the formula I

where any 2 of R1, R2, R3and R4are hydrogen atoms, and 2 - substituting groups that can be identical or different, comprise at least 7 novogorodnaya atoms, lipophilic and comprise a cyclic group, wherein the above-mentioned cyclic group is not attached directly to the α or β carbon atom, and optionally linked or fused with a cyclic group in the other substituting group, where the cyclic groups are connected with a combined total novogorodnaya atoms for the two substituting groups is at least 12, and where X displays O, C, N or S,

however, with the exception of the compounds N-methyl-L-phenylalanyl-L-lysyl-L-propyl-2,2-bis(phenylmethyl)-β-alanyl-D-arginine and N-methyl-L-phenylalanyl-L-lysyl-L-propyl-D-cyclohexylethyl-2,2-bis(phenylmethyl)-β-alanyl-D-arginine.

Using the nomenclature of Chemical abstracts service, not the IUPAC system, the above two molecules will be referred to as D-arginine, N2-[N-[1-[N2-(N-methyl-L-phenylalanyl)-L-lysyl]--propyl]-2,2-bis(phenylmethyl)-β-alanyl] and D-arginine, N2-[N-[3-cyclohexyl-N-[1-[N2-(N-methyl-L-phenylalanyl)-L-lysyl]-L-propyl]-D-alanyl]-2,2-bis(phenylmethyl)-β-alanyl] and their numbers in accordance with the database CAS-145149-42-4 and 145149-43-5, respectively.

Cancelled the compounds described in WO 92/12168 as ligands anaphylatoxin receptor useful for the treatment of inflammatory disease States, and as such this document does not describe the problem resolved by the present invention.

It is desirable that the minimum number of 12 for the combined total number novogorodnaya atoms of the two groups R1-4that code is a cyclic group of each of the linked fragment was achieved by adding a minimum number of unconnected groups (7+7=14) and subtracting 2 because two novogorodnaya atom effectively contribute to the formation of rings in each group. Preferably, the combined total number novogorodnaya of atoms in the two groups R1-4that code is a cyclic group of each fragment is connected, is 14. Challenging United and associated groups can be analyzed if the two groups attached to Cαor Cβmay contain more than one pair of connected cyclic groups, with or without additional connections between the residual groups. Despite this two substituting groups, preferably not connected not connected�nye molecules, in which these groups are characterized by the highest flexibility of movement, preferred.

The nitrogen atom a group of formula (I) is preferably not attached to any atom of the group R1-4except, of course, indirect through Withβorα. Preferably 5 atoms in the above basis (N-Cβ-Cα-C-X) are related to each other only linear, not a cyclical manner. Preferably, X and N in formula (I) have its usual valence and therefore are additionally substituted, as they relate to other parts of the connection, for example with additional amino acids or the N - or C-terminal blocking groups.

The residual group R1-4generally lipophilic in nature and preferably do not carry any charge and preferably have no more than two, more preferably no more than one polar group. One or both of the residual group, R1-4preferably contain at least 8, more preferably at least 9 or 10 novogorodnaya atoms, for example 7-13, 7-12, 8-12 or 9-11 novogorodnaya atoms. The two substituting groups are preferably identical for the sole purpose of ease of synthesis. Preferably, the two substituting groups are R1and R2or R3and R4, R3and R4the most preferable.

As indicated above, the cycle�ical group, R 1-4not attached directly to the α or β carbon atom, since they are separated from it by a chain of 1-4, preferably 1-3 atoms; these linking atoms may include nitrogen and/or oxygen, but, as a rule, will be carbon atoms, preferably linking atoms unsubstituted. The preferred separating the fragments shown in the Examples and form part of the replacement group R1-4as indicated in this document.

X may be substituted or unsubstituted and preferably should be an N-atom, and is preferably substituted. If X is N, X can form part of the amide bond in an additional amino acid, as shown in molecules in Example 1. Alternatively, the N-atom may be substituted, for example aminoacylase group, for example, aminoethyl or aminopropyl or dimethylaminoethyl. These molecules are shown in Example 2. In the further alternative to the N-atom may be part of a cyclic group, for example, piperazine, which may be itself substituted alkyl or aminoacylase group, which is also shown in Example 2.

Peptides or peptidomimetics containing the group of formula I will preferably be a modified C-terminal, which preferably imidiwan and described above in connection with molecules for use as therapeutic cytolytic drugs.

<> In the previous paragraphs define preferred substituting groups of β-amino acids are applied, mutatis mutandis, two substituting groups R1-4. Peptides and peptidomimetics containing the group of formula I are preferred subclass of molecules, described earlier in this application for use as therapeutic cytolytic drugs, and so all previous paragraphs describing the preferred characteristics of the molecules, for example, their length and other amino acids they contain, are also applied to these molecules, identified by their incorporation of the group of formula I and Vice versa. Particularly preferred molecules are molecules long 1-7 or 8 (for example 1-5), more preferably 1, 2, 3 or 4 amino acids. Molecule peptidomimetic will include such number of pobjednici, however, pobjednici will, as a rule, associated simulators amide bond; preferred communication described earlier and include esters and aminomethyl and kilometre.

The examples show that the structural motif of the present invention in the form of di-peptides (Example 1) and modified by individual amino acids and gepta-peptides (Examples 2 and 4). The molecules in the Examples represent preferred molecules and their use in the present invention.

Peptides, peptidomimetics and amino acids of the invention can be be salt, cyclic or esterification, and preferred amidarone derivatives described above.

A preferred class of molecules and their use in the present invention are derivatives of β, preferably β2,2-amino acids, which have separate β2,2-amino acid containing two lipophilic side chains, as described above, disubstituted β-amino acid, surrounded by two cationic groups. As described above, the two substituting groups are preferably identical and include 6-membered cyclic group and at least 8, preferably at least 10 novogorodnaya atoms. These molecules are particularly suitable as Antimicrobial and acceptable for oral administration.

The molecules described in this invention can be synthesized by any convenient method. Typically, the reactive group (e.g., amine, tialavea and/or carboxyl) will be protected throughout the synthesis process. The final step in the synthesis would, therefore, remove protection from a protected derivative of the invention. Methods of synthesis of compounds represent another aspect of the present invention. For example, in one embodiment of the invention discloses a method of synthesizing a peptide, peptidomimetic or modified amino acid having a net positive for�poison at least +2, and which incorporates a group of formula I, as described herein, wherein the method includes removing the protective group from the specified peptide, peptidomimetic or modified amino acid.

When you build the peptide can, in principle, start with C-terminal or N-terminal, although the procedure of the beginning of the C-terminal preferred.

Methods peptide synthesis is widely known in science, however, the present invention may be particularly useful to carry out the synthesis using the solid phase, and these bases are widely known in science.

There are a large number of protective groups for amino acids, and the corresponding aminosidine group may include carbobenzoxy (also referred to as Z) t-butoxycarbonyl (also referred to as Boc), 4-methoxy-2,3,6-trimethylbenzoyl sulfonyl (Mtr) and 9 toradolethex-carbonyl (also referred to as Fmoc). Desirably, the construction of peptide-based end of the C-terminal, was present aminosidine group in α-amino group of each added new surplus, which must be extracted selectively to the next phase of the connection.

Carboxyl protecting groups which may be used include, for example, ready to split the ether group, benzyl (Bzl), p-nitroaniline (ONb), t-butyl (OtBu) groups, and linking groups� on solid foundations for example Rink amide associated with polystyrene.

Tiolive protective groups include p-methoxybenzyl (Mob), trityl (Trt) and atsetamidometil (Acm).

Preferred peptides of the invention can be easily prepared by using t-butyloxycarbonyl (Boc) protecting groups for amide side chains of Lys, Orn, Dab and Dap, as well as to protect tryptophan residues indole nitrogen. Fmoc can be used to protect the alpha-amino groups. For peptides containing Arg, 2,2,4,6,7-pentamethylcyclopentadiene-5-sulfonyl can be used to protect the guanidine side chain.

There are many procedures to extract Aminovich and carboxyl protective groups. However, they must comply with the applicable synthetic strategies. Side chain protecting groups must be stable relative to the conditions used in the extraction of the temporary α-amino protective group to the next phase of the connection.

Amine protective group, e.g. Boc, and a carboxyl protective group, for example, tBu, should be removed simultaneously by acid treatment, for example using trifluoroacetic acid. Tiolive protective group, for example, Trt, can be extracted selectively by using an oxidizing agent, for example iodine.

Scientific literature and methods of synthesis peptidomimetics compounds described above are known and experienced scientists.

Recipe�ry, containing one or more compounds of the invention, mixed with a suitable solvent, carrier or auxiliary substance, constitute another aspect of the present invention. Such formulations can be used, inter alia, in the pharmaceutical (including veterinary) purposes and therefore suitable solvent, carrier or excipient will preferably be acceptable from a pharmaceutical point of view. Appropriate diluents, excipients and carriers known to experienced scientists.

The molecules described in this document are cytolytic in nature and are particularly useful as antimicrobial, e.g. antibacterial or antifungal drug, and the use of antibacterial purposes it is preferable. The specificity of the molecules also provides their use as anticancer drugs. Thus, in the following aspect the present invention describes peptide, peptidomimetic or (modified) amino acid having a net positive charge of at least +2, and incorporarse group of the formula I

where any 2 of R1, R2, R3and R4atoms of hydrogen and 2 - substituting groups which may be the same or different, include, men�her least 7 novogorodnaya atoms, lipophilic and comprise a cyclic group, a cyclic group is not attached directly to the α or β carbon atom, however, selectively connected or fused with a cyclic group in the other substituting group, where cyclic groups are fused, and the cumulative total number novogorodnaya atoms for the two substituting groups is at least 12, and where X displays O, C, N or S,

however, with the exception of the compounds N-methyl-L-phenylalanyl-L-lysyl-L-propyl-2,2-bis(phenylmethyl)-β-alanyl-D-arginine and N-methyl-L-phenylalanyl-L-lysyl-L-propyl-D-cyclohexylethyl-2,2-bis(phenylmethyl)-β-alanyl-D-Arginine, for use in therapy.

The invention is also a peptide, peptidomimetic or a modified amino acid having a net positive charge of at least +2, which incorporates a group of the formula I

where any 2 of R1, R2, R3and R4atoms of hydrogen and 2 is the residual group, which may be identical or different, contain at least 7 novogorodnaya atoms, lipophilic and comprise a cyclic group, a cyclic group is not attached directly to the α or β carbon atom, but is selectively connected or fused with a cyclic group in the other substituting group, where cyclic groups are fused, and scoop�Phnom total number novogorodnaya atoms of the two substituting groups is, at least 12, and where X displays O, C, N, or S, for use as therapeutic cytolytic drug. Preferred areas of use is antimicrobial, especially antibacterial agents or anticancer drugs.

In the following aspect the present invention describes a method for the treatment or prophylaxis of microbial infections, preferably bacterial infections, which provides for the introduction of patients peptide, peptidomimetic or modified amino acid as described above.

In the following aspect the present invention describes a method of treating tumor cells, or prevention, or suppression of growth, spreading or metastasis of the tumor, which provides for the introduction of patients peptide, peptidomimetic or modified amino acid as described above.

In the following aspect the present invention describes the use of a peptide of peptidomimetic or modified amino acid as described above, in the manufacture of a medicament for the treatment of microbial infections, preferably bacterial infections.

In the following aspect the present invention describes the use of a peptide of peptidomimetic or modified amino acid as described above, in the manufacture of medicaments for treating tumor cells or prevent lipocalin growth the spread or metastasis of the tumor.

Microbial infection may be present or cause suspicion multipathogen infection, and the treatment of such infections (e.g., in the hearth of chronic wounds, for example infections, including gram-positive and gram-negative bacterial strains, represent a preferred target of use and methods in accordance with the present invention.

The quantity of the drug should be effective from the point of view of loss of all or parts of the target cells, or prevent or slow down their reproduction, or inhibition of metastasis or reduce the harmful effects of tumors on the patient. Clinician or the patient should see improvement in one or more parameters or symptoms associated with the tumor. The introduction can also be preventive. The patient must be human, but also allowed the treatment of non-human animals, such as Pets or livestock.

Unlike most drugs, which are potential target molecules of the present invention can provide a targeted effect against wide range of cancers. Preferred types of cancer - lymphoma, leukemia, neuroblastoma and glioblastoma (e.g. in the brain), carcinoma and adenocarcinoma (particularly in the breast, colon, kidney, liver, lung, ovaries, pancreas, prostate and skin) and melanoma.

Compositions in accordance with the invention can be represented, for example, in a form suitable for oral, local, nasal, parenteral, intravenous, intratumoral, rectal or focal (such as isolated limb perfusion) introduction. Introduction as a rule, is carried out parenterally, preferably by injection subcutaneously, intramuscularly, vnutricapsulino, intraspinal, nutripure or intravenously.

The active compounds described herein may be presented in traditional pharmacological forms of administration, for example in the form of pills, tablets, coated tablets, nasal sprays, solutions, emulsions, liposome, powder, capsules or forms of continuous releases. Traditional pharmaceutical excipients, as well as standard methods of production can be used in the preparation of these forms.

Can also be applied organ-specific system-carriers.

Solutions for injection can, for example, be produced in the traditional way, e.g. by adding preservatives such as p-hydroxybenzoates, or stabilizers, for example, EDTA. The solutions were then filled�safety in vials or ampules.

Preferred formulations are those in which the peptides are dissolved in the salt solution. Such formulations are suitable for use in the preferred methods of administration, especially the local application, i.e., intratumoral, for example, by injection or perfusion/infusion preferably in an isolated (including partially isolated) extremities, body area or organ.

Unit dose containing the active molecule, preferably contains 0.1-10 mg, for example 1-5 mg antineoplastic agent. Pharmaceutical compositions can additionally contain other active ingredients, including other cytotoxic drugs, such as anticancer peptides. Other active ingredients may include various kinds of cytokines, such as IFN-y, TNF, CSF, and growth factors, immunomodulators, chemotherapeutic, for example, cisplatin or antibodies or cancer vaccines.

When applying such compositions systemically, the active molecule is present in an amount sufficient to achieve the level of bioactive molecules in the serum of at least about 5 μg/ml. basically, the levels in serum should not exceed 500 µg/ml. the Preferred level in serum is about 100 μg/ml. Such levels in serum can be achieved when the content of bioactive molecules in the composition, which BBO�GSI systemically in a dose of from 1 to about 10 mg/kg. Typically, the drug (s) cannot be entered in a dose exceeding 100 mg/kg.

In the following aspect the present invention describes a product containing (a) a peptide, peptidomimetic or a modified amino acid having a net positive charge of at least +2 and containing a group of formula (I) as described herein, and (b) an additional anti-microbial agent as a combined preparation for separate, simultaneous or sequential use, or prevent microbial infections.

In another aspect of the present invention describes a product containing (a) a peptide, peptidomimetic or a modified amino acid having a net positive charge of at least +2 and containing a group of formula (I) as described herein, and (b) an additional antitumor agent as a combined preparation for separate, simultaneous or sequential use in the treatment of tumor cells.

Compounds of the invention and compounds suitable for the methods and uses of the invention include salt forms and corresponding pharmaceutically acceptable salts of peptides and similar molecules, and is widely known for the experienced scientist.

The invention is also described in the following Examples, which include some reference molecules, vyho�the above boundaries of the present invention, and with reference to figures.

FIG.1 shows the compounds 4a-n in Example 1.

FIG.2 shows a schematic overview of the synthesis of compounds 4a-n, as described in Example 1.

FIG.3 shows compounds 4a-d, 5a-d, 6a-d and 7a-d in Example 4.

FIG.4 shows a schematic overview of the synthesis of compounds in Example 4; (a) NaOMe (1 EQ.), R-Br (1 equiv.), held twice 78°C s: MeOH; (b) Ra/Ni, H2(g), 45°C, 5 days, s: MeOH containing 2% acetic acid; (C) tea, pH 8, 15 Vos2O (1.2 EQ.), room temperature, 18 hours, s: H2O: dioxane (1:5); (d) LiOH (6 equiv.), 18 hours, 100°C, s: H20: dioxane (1:3); (e) DIPEA (3 EQ.), TFFH (1.5 EQ.), room temperature, for 2 hours, after that add the desired amine (2 EQ.), within 7 days max, s: DMF; (f) TFUCK:TIS:H2O (95:2.5:2.5), room temperature, 2 hours, s: was held. All derivatives of β2,2amino acids were isolated in the form of di-trifenatate salts.

Examples

Example 1

The sequence of molecules on the basis of the achiral backbone lipophilic 3-amino-2,2-disubstituted propionic acid (β2,2-amino acid), which is connected with the amide residue C-terminal of L-arginine, has been created and tested for antimicrobial effect. These di-peptides have the functions of the side chain of a tri-peptide for a derivative of β2,2-amino acids. Was explored a wide range of lipophilic substituents of β2,2-amino acids, as pokazanova FIG.1. An overview of the synthesis of compounds 4a-n shown in FIG.2.

Reagents and analytical methods

The spectra of1H and13C NMR were recorded using a Varian spectrometers at 400 or 600 MHz. Mass spectra were obtained on Micromass Quattro LC (Micromass, Manchester, UK). Mass spectra with high resolution were obtained on a Waters Micromass LCT Premier (Micromass, Manchester, UK). Commercially available compounds and solvents were purchased from Sigma-Aldrich and were used without further purification. Preparative RP-HPLC was carried out on the system of Waters, equipped with a RP column BondaPak C18 125 Å, 10 µm, 25×100 mm, and-eluted with acetonitrile and water containing 0.1% TFUK. Analytical HPLC was carried out on the system Waters 2695 HPLC equipped with a water column RP-HPLC Delta Pak C18.100 Å, 5 μm, 3.9×150 mm and analyzed at a wavelength of 214 nm using PDA detector with wavelengths from 210 to 310 nm. All compounds were prepared using the carousel parallel reactions system Radleys®.

General procedures for dialkylamide of metalloelastase (GP1) 1a n.

Sodium methoxide (20 mmol) was diluted in methanol (0.2 M) and added medicinehat (20 mmol). After 5 minutes of stirring at room temperature was added the desired methyl benzyl (20 mmol) and the solution was heated to delegirovali. After 15 minutes the solution was cooled to room temperature. Added second p�rciu of sodium methoxide (20 mmol) and after 5 minutes of mixing at room temperature was added a second portion of the desired benzyl bromide (20 mmol), then again spent delegirovano for 15 minutes. Volume of reaction mixture was reduced to approximately 1/3 in vacuum and extracted with water/ethyl acetate. The organic phase is dried over MgSO4and evaporated to dryness. The product was used in the subsequent synthesis without any further purification.

The General procedure of recovery of NITRILES to amines using Ra/Ni and subsequent Boc protection (GP2) 2a-n).

Ra/Ni (approximately 2 ml/g of nitrile) were washed three times with methanol under argon, and then added the desired nitrile (3,5 mmol) dissolved in methanol (0.1 M), together with acetic acid (about 1 ml/g of the nitrile). The reaction mixture was hydrogenosomal at a temperature of 45°C for 5 days under a pressure of 1 bar H2. Thereafter, the reaction mixture was filtered through a filter to remove celite may Ra/Ni before evaporation to dryness. The crude methyl ester of β2,2-amino acids (0,35 mmol) was diluted in a mixture of 1,4-dioxane and water 5:1 (-0,35 M), and pH adjusted to 8 with tea, Vos2O (0,42 mmol) was dissolved in the smallest possible amounts of 1,4-dioxane. The solution was stirred at room temperature for approximately 18 hours prior to its oxidation to pH 2-3 with 10% citric acid and extracted three times with ethyl acetate. The organic phase is dried over MgSO4evaporated to dryness. Cont�CT was used in the subsequent synthesis without any further purification.

The General procedure of virginalis (GP3) 3a-n).

The BOC-protected methyl ester of β2,2-amino acids (0,35 mmol) was diluted in a mixture of 1,4-dioxane and water 3:1 (1,17 mm) and added lithium hydroxide (2.1 mmol), dissolved in the smallest possible amount of water. The reaction mixture was stirred at the reflux condenser under N2 for 18 hours until reduced to about 1/5 under vacuum. Water (10 ml) was added to the reaction mixture and pH adjusted pocaply way to 1-2 with 0.1 M HCl. This aqueous solution was extracted three times with equal volume of ethyl acetate. The organic phase is dried over MgSO4and evaporated to dryness. The product was used in the subsequent synthesis without any further purification.

General procedure for compounds of L-arginine BOC-protected β2,2-amino acids (GP4) 4a-n).

The BOC-protected β2,2-amino acid (0.2 mmol) was diluted in DMF (0.02 M) and added DIPEA (0.6 mmol) together with TFFH (0,2 mmol). Amino acid pre-activated 2 hours before the addition of H-Arg-NH2×2HCl (0,3 mmol). The reaction mixture was stirred at room temperature for 7 days before I diluted with ethyl acetate and washed with salt solution. The organic phase is dried over MgSO4and evaporated to dryness. With crude BOC-protected product removed protection by diluting it in was held (-0,4 M) and adding volun�and, equivalent TFUK:TIS:water (95:2.5:2.5). The mixture was stirred at room temperature for 2 hours, evaporated to dryness. The crude product was purified by the method of preparative RP-HPLC. The purity of the peptides was checked by analytical method by RP-HPLC before the solution was evaporated to dryness and the residue re-dissolved in water and liofilizirovanny. All compounds were characterized by a purity above 95%.

Tests in a

Antimicrobial tests were carried out TosLab A/S (Tromso, Norway). Each compound was diluted to 1 mg/ml in water and tested in duplicate at 200, 100, 50, 35, 15, 10, 5, 2,5, 1, 0,5 μg/ml, except 4n that is experienced when 50, 35, 15, 10, 5, 2,5, 1, 0,5 µg/ml because of problems with solubility. All tested compounds were di-TFUK salts.

Bacterial strains were grown in 2% of Bacto-peptone to achieve exponential growth. MIC was determined by nocturnal incubation in 1% Bacto-peptone at 37°C. Used a bacterial concentration of 2×106CFU/ml of Both a negative control (without peptide) and positive control (gentamicin) was used for all bacterial strains. The growth or absence of growth was determined based on the turbidity in the wells. The MBC was determined by inoculation on the agar plates all concentrations with the values of MIC and above by incubating at 37°C overnight and defined�I the growth or lack of growth.

All compounds showed MIC values less than 50 ág/ml, were tested in the same procedure.

The values of MIC and MBC are shown in Tables 1A and B.

Hemolytic tests on human erythrocytes was carried out in Lytix Biopharma A/S (Tromso, Norway). Each compound was tested on the basis of a concentration of 1 mg/ml and below, except 4k and 4n, which were tested only at a maximum of 0.5 mg/ml because of problems with solubility. 8 ml of blood was obtained from adult healthy donors were men. The blood was divided into equal portions and distributed by a commercially available test tube containing EDTA (BD blood collection tubes, 7.2 mg K2 EDTA) and in the reaction vial with a volume of 10 ml containing 40 μl of heparin solution (1000 units/ml in 0.9% sodium chloride). After 30 minutes determined the hematocrit of blood treated with EDTA. Heparinized blood was centrifuged for 10 minutes at 1500 rpm and the supernatant removed. Subsequently, RBC were washed with pre-warmed PBS three times and was diluted to 10% hematocrit. Compounds were diluted in PBS (at a concentration of 1 μg/ml to 1000 μg/ml), and the erythrocytes were incubated with constant shaking at 37°C for one hour. Added as positive control, with a final concentration of 0.1% Triton X-100 and a negative control containing pure PBS-buffer. The samples were centrifuged (4000 rpm) for 5 minutes and the absorbance�tion of the supernatant was measured at 405 nm. The values presented in the Table 1A, correspond to 50% hemolysis.

Analysis compliance on the basis of α-chymotrypsin

The stability tests based on α-chymotrypsin was carried out by diluting the desired connection to 1 mg/ml in water. α-chymotrypsin was diluted to 0.1 mg/ml in 1 mm HCl, containing 2 mm CaCl2. Enzymatic digestion was carried out in 100 mm TRIS HCl containing 10 mm CaCl2. The final enzyme concentration was 2 μg/ml, and the final peptide concentration was 100 μg/ml. Total volume was 0.5 ml.

15 μl samples were obtained after 0, 15, 30, 60, 120 and 240 minutes in addition to the samples collected after 24 and 48 hours. To samples added external standard (atenolol hydrochloride) and 100 ál of 10% acetic acid to stop the digestion before dilute to 1 ml with water.

For each test performed negative controls without enzyme to ensure that degradation occurred due to the action of enzymes, and not other factors. Succinyl-Ala-Ala-Pro-PHE-pairnitroaniline was used as a positive control. All tests were carried out in triplicate.

Results

Table 1A
Minimum inhibitory concentration (MIC) against S. aureus, MRSA, MRSE and E. col and values of the EU 50against human RBC for antimicrobial dipeptides prepared in this study
MICa(μm)EC50b(μm)
ConnectionS, aureuscMRSAdMRSEeE colifRBCg
4A-----
4b15015075--
4C7,27,27,2144-
4d----n.t.
4th14714774 --
4f494935--
4g6,65,06,6266470
4h1477474--
4i12,73,212,7254n, t,
4j10,26,86,8136386
4k6,53,35,046-
4l604935--
4m259259129--
4n2,92,110,15,799
andThe biggest tested concentrations were 200 μg/ml.bThe largest concentrations were tested 1000 μg/ml.CStaphylococcus aureus (ATTC 25923).dmethicillin-resistant Staphylococcus aureus (ATCC 33591).emethicillin-resistant Staphylococcus epidermidis (ATCC 27626).fEscherichia coli (ATTC 25922) and9red blood cells of man. The symbol "-" indicates no detectable activity (MIC or EC50) in the tested range of concentrations, n.t.: not tested.

Table 1B
The minimum bactericidal concentration (MBC) against S. aureus, MRSA, MRSE and E. coli. The strains are the same as those presented in Table 1A above
MBCe(nM)
ConnectionS, aureusaMRSAb MRSEcE. colid
4A>360>360>360>360
4b30115075>301
4C7,27,27,2289
4d>307>307>307>307
4th14714774>294
4f494949>282
4g6,65,36,6266
4h147110110>294
4i12,76,312,7>254
4j6,86,86,8136
4k6,56,55,246
4l494921>282
4m>259259129>259
4n4,64,63,45,7

A selection of six compounds (4c, 4g, 4h, 4i, 4k and 4m) was investigated on the proteolytic stability of α-chymotrypsin. The results showed that the degradation could not be set for any connection within a 48-hour period. We also found that all the investigated compounds were chemically stable in aqueous solutions at a pH of 7.4, for at least 48 hours.

The results demonstrated�Ali is a strong correlation between antimicrobial capacity and overall lipophilicity of the prepared compounds. This can be illustrated by comparing the capacity of the compounds against S. aureus and their retention time (Rt) in an analytical C18 column for RP-HPLC, demonstrated that the affinity of the compounds from the hydrophobic stationary phase column (results not shown).

Hemolytic activity of the prepared compounds was used to measure toxicity, and except for compounds 4g, 4j and 4n, the compounds were nonhemolytic in the tested range of concentrations (<1000 µg/ml). The most powerful hemolytic activity was shown by compound 4n despite this, however, this concentration EC50were 10-50 times higher than the MIC values against gram-positive and gram-negative bacteria. It should be noted that the compounds 4j and 4k, which reflected the effect of antimicrobial actions differed greatly regarding hemolytic action. This indicated that the hemolytic activity and antimicrobial effects were determined on the basis of the same structural properties.

Example 2

Additional preparations of the invention were made and tested for antibacterial and anticancer effect.

Materials and methods

Bacterial strains

Staphylococcus aureus (ATTC 25923)

Methicillin-resistant Staphylococcus aureus (ATCC 33591)

Methicillin-resistant Staphylococcus epidermidis (ATCC 27626)

Eschericha coli (ATTC 25922)

Cancer cell lines

A20 or MethA

Cells for toxicity studies

MRC-5 and human RBC

The highest tested concentration in different samples were:

Antimicrobial samples: 200 mcg/ml

Anti-cancer samples: 500 mcg/ml

Samples of MRC-5: 500 mcg/ml

The RBC samples: 1000 mcg/ml

Chem Draw Ultra, version 11.0 was used to calculate the physico-chemical properties Log P, tPSA and CLogP.

Synthesis of molecules

Swelling resin: amide resin Rinca, MUNA (loading of 0.64 mmol/g) was placed into the wells in 7 ml of DMF for 1 hour before was washed five times with 7 ml of DMF.

Removing Fmoc: 7 ml of 20% piperidine in DMF was added to the reaction tube, and the suspension was stirred for 10 minutes to extract the solution. This procedure was repeated twice, shaking for 1 minutes before the resin was washed five times with 7 ml of DMF.

Combining amino acids with unprotected resin: Fmoc-Lys(Boc)-OH or Fmoc-Trp(BOC)-HE (4 EQ.), hydrate HOBt (4 EQ.) and HBTU (3,92 EQ.) was dissolved in 5 ml of DMF, added DIPEA (8 EQ.), and the mixture left to pre-activate for 15 minutes before adding it to the resin. After agitation for 1 hour a compound mixture was removed and the resin was washed five times with 7 ml of DMF.

The compound β-amino acids with unprotected resin: Fmoc-β-aa-OH (2 EQ.) and TFFH (1,96 EQ.) was dissolved in 5 ml of DMF, added DIPEA (8 EQ.), and CME�ü left to pre-activate for 15 minutes prior how to add it into the resin. After shaking for 48 hours compound mixture is removed and the resin was washed five times with 7 ml of DMF.

Compound amino acid after connecting β-amino acids in the unprotected resin: Fmoc-Trp(Boc)-OH (4 EQ.) and TFFH (3.92 EQ.) diluted in 5 ml of DMF, added DIPEA (8 EQ.), and the mixture left to pre-activate for 15 minutes before adding it to the resin. After shaking for 24 hours the joined mixture was removed and the resin was washed five times with 7 ml of DMF.

After the last connection, the resin was washed five times with was held before leave it to dry overnight.

Cleavage from the resin and extracting the Boc: 10 ml TFUK:TIS:water 95:2.5:2.5 was added to the reaction and the suspension was stirred for 2 hours before assembled peptide solution. This procedure was repeated twice with shaking for 10 minutes. The collected peptide solutions were evaporated to dryness under reduced pressure, besieged diethyl ether and was washed with diethyl ether. The peptides were purified by HPLC and liofilizirovanny.

Cell testing

Corresponding methods are described in Example 1.

Results

Table 2
Biological activity of derivatives of 1,2-diaminoethane � 2,2-amino acids (All values submitted in µg/ml)
123
Name in accordance with IUPACN-(2-aminoethyl)-2-(aminomethyl)-2-propylpentanoic Log P: 0,53 tPSA: 81,14 CLogP: 1,243-amino-N-(2-aminoethyl)-2,2-bis(cyclohexylmethyl)propanamide Log P: 2,7 tPSA: 81,14 CLogP: 4,424N-(2-aminoethyl)-2-(aminomethyl)-4-cyclohexyl-2-(2-cyclohexylethyl)butanamide Log P: 3,54 tPSA: 81,14 CLogP: 5,482
Formula Molecular weightC11H25N3O Exact mass: 215,1998 Molecular weight: 215,3357C19H37N3O Exact mass: 323,2937 Molecular weight: 323,5166C21H41N3O Exact mass: 351,3250 Molecular weight: 351,5697
Antimicrobial activity(MIC)
S. aureus>2002007,5/10
MRSA>2001005/15
MRSE>200352,5/5
E. coli>200>20015/10
Anticancer activity (IC50)
A20n.t.n.t.<3,35/<3,35/1,7
MethAn.t.n.t.n.t.
Toxicity
MRC-5n.t.18,5<10/<10
RBC (EC50)n.t.>100062*
Anticancer selectivity
MRC-5/A20n.t.n.t.(<5,9)
RBC/A20n.t.n.t.36,5
* Low solubility in the test medium
n.t.=not tested

Table 2
Continued (All values are in µg/ml)
456
Name in accordance with IUPAC3-amino-N-(2-aminoethyl)-2,2-dibenzylamine amide Log P: 2,08 tPSA: 81,14 CLogP: 2,11N-(2-aminoethyl)-2-(aminomethyl)-2-phenethyl-4-phenylbutane Log P: 2,91 tPSA: 81,14 CLogP: 3,018N-(2-aminoethyl)-2-(aminomethyl)-5-phenyl-2-(3-phenylpropyl)pentanone Log P: 3,75 tPSA: 81,14 CLogP: 4,076
Formula Molecular weightC19H25N3O
Exact mass: 311,1998 Molecular weight: 311,4213
C21H29N3O Exact mass: 339,2311 Molecular weight: 339,4745C23H33N3O Exact mass: 367,2624 Molecular weight: 367,5276
Antimicrobial activity (MIC)
S. aureus>200 10035/50
MRSA>20010035
MRSE>2005015
E. coli>200>200200
Anticancer activity (IC50)
A2068,6n.t.8,3
MethAn.t.n.t.n.t.
Toxicity
MRC-526121.53
RBC (EC50)>1000>1000820
Anticancer selectivity
MRC-5/A203,8n.t.4,2
RBC/A20n.t.n.t.98,8
n.t. = not tested

Table 2
Continued (All values are in µg/ml)
7 89
Name in accordance with IUPAC3-amino-N-(2-aminoethyl)-2,2-bis-naphthalene-2-ylmethyl)propanamide Log P: 4,07 tPSA: 81,14 CLogP: 4,4583-amino-N-(2-aminoethyl)-2,2-bis 4-methylbenzyl)propanamide Log P: 3,05 tPSA: 81,14 CLogP: 3,1083-amino-N-(2-aminoethyl)-2,2-bis(4-trifluoromethyl)benzyl) propanamide Log P: 4,02 tPSA: 81,14 CLogP: 4,106
Formula Molecular weightC27H29N3O Exact mass: 411,2311 Molecular weight: 411,5387C21H29N3O Exact mass: 339,2311 Molecular weight: 339,4745C21H23F6N3O Exact mass: 447,1745 Molecular weight: 447,4172
Antimicrobial activity(MIC)
S. aureus2,5/520010
MRSA51005
MRSE 2,5/510010
E. coli15>20035
Anticancer activity (IC50)
A20<3,35/0,7*n.t.<5/1,0*
MethAn.t.n.t.n.t.
Toxicity
MRC-513,5/<1425/44<10/12
RBC (EC50)292*>1000287
Anticancer selectivity
MRC-5/A2019,3-20n.t.10-12
RBC/A20417n.t.287
* Low solubility in the test medium
n.t. = not tested

Table 2
Continued (All values are in µg/ml)
101112
Name in accordance with IUPAC 3-amino-N-(2-aminoethyl)-2,2-bis(4-isopropylbenzyl)propanamide Log P: 4,55 tPSA: 81,14 CLogP: 4,9643-amino-N-(2-aminoethyl)-2,2-bis(4-tert-butylbenzyl)propanamide Log P: 5,49 tPSA: 81,14 CLogP: 5,7623-amino-N-(2-aminoethyl)-2,2-bis(3,5-dimethylbenzyl)propane amide Log P: 4,02 tPSA: 81,14 CLogP:= 4,106
Formula Molecular weightC25H41N3O Exact mass: 395,2937 Molecular weight: 395,5808C27H41N3O Exact mass: 423,3250 Molecular weight: 423,6339C23H33N3O Exact mass: 367,2624 Molecular weight: 367,5276
Antimicrobial activity (MIC)
S. aureus5/102,535
MRSA5/102,535
MRSE52,55/10
E. coli12,5/35550/100
Anticancer activity (IC50)
A20<5/<5/1,4*<5/<1.65 V/<1.65 V/1,1<5/<10/<10
MethAn.t.n.t.n.t.
Toxicity
MRC-5<6,67/<6,67410
RBC (EC50)4712*n.t.
Anticancer selectivity
MRC-5/A20(<4,8)3,6(>2)
RBC/A2033,610,9n.t.
* Low solubility in the test medium
n.t. = not tested

Table 2
Continued (All values are in µg/ml)
1314
Name in accordance with IUPAC3-amino-N-(2-aminoethyl)-2,2-bis(3,5-dimethoxybenzyl)propanamide Log P: 1,57 tPSA: 118,06 CLogP: 2,1263-amino-N-(2-aminoethyl)-2,2-bis(3,5-di-tert-butylbenzyl)propanamide Log P: 8,89 tPSA: 81,14 CLogP: 9,414
Formula Molecular weightC23H33N3O5Exact mass: 431,2420 Molecular weight: 431,5252C35N57N3O Exact mass: 535,4502 Molecular weight: 535,8466
Antimicrobial activity (MIC)
S. aureus2003,75/5
MRSA2002,5
MRSE1002,5/5
E. coli>2005/>20
Anticancer activity (IC50)
A20n.t.n.t.
MethAn.t.n.t.
Toxicity
MRC-562,5/687,5/8,5
RBC (EC50)>1000>62,52*
Anticancer selectivity
MRC-5/A20n.t.n.t.
RBC/A20n.t.n.t.
* Low solubility in the test medium
2* The highest tested concentration due to reduced solubility in water.
n.t. = not tested

td align="justify"> MRC-5/A20
Table 3
Biological activity gepta-β-peptides (All values decree�cies in m kg/ml)
1516
Name in accordance with IUPAC(R)-N-((2R,9S, 12S, 15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-15-carbamoyl-6,6-bis(2-cyclohexylethyl)-1-(1H-indol-3-yl)-3,7,10,13-tetraoxo-4,8,11,14-tetragenococcus-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-6,6-dibenzyl-15-carbamoyl-1-(1H-indol-3-yl)-3,7,10,13-tetraoxo-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide
Formula Molecular weightC65H104N14O7Exact mass: 1192,8212 Molecular weight: 1193,6109C63H88N14O7Exact mass: 1152,6960 Molecular weight: 1153,4624
Antimicrobial activity (MIC)
S. aureusn.t.n.t.
MRSAn.t.n.t.
MRSEn.t.n.t.
E. colin.t.n.t.
Anticancer activity(IC50)
A2015,0263
MethAn.t.n.t.
Toxicity
MRC-545,7>500
RBC (EC50)n.t.n.t.
Anticancer selectivity
3,0(>1,9)
RBC/A20n.t.n.t.
n.t. = not tested

Table 3
Continued (All values are in µg/ml)
1718
Name in accordance with IUPAC(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-15-carbamoyl-1-(1H-indol-3-yl)-3,7,10,13-tetraoxo-6,6-bis(3-phenylpropyl)-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-15-carbamoyl-1-(1H-indol-3-yl)-6,6-bis(naphthalene-2-ylmethyl)-3,7,10,13-tetraoxo-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide
The Molecular formula in� C67H96N14O7Exact mass: 1208,7586 Molecular weight: 1209,5687C71H92N14O7Exact mass: 1252,7273 Molecular weight: 1253,5798
Antimicrobial activity (MIC)
S. aureusn.t.n.t.
MRSAn.t.n.t.
MRSEn.t.n.t.
E. colin.t.n.t.
Anticancer activity (IC50)
A20of 26.5/40*24
MethAn.t.n.t.
Toxicity-
MRC-5241 /221*103
RBC (ECjo)n.t.n.t.
Anticancer selectivity
MRC-5/A209,1/5,5*4,3
RBC/A20n.t.n.t.
* Two sets of results obtained due to two separate screenings (and the average of three Parallels each screening)
n.t. = not tested

Table 3
Continued (All values are in µg/ml)
1920
Name in accordance with IUPAC(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-15-carbamoyl-6,6-bis(4-terbisil)-1-(1H-indol-3-yl)-3,7,10,13-tetraoxo-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-15-carbamoyl-1-(1H-indol-3-yl)-3,7,10,13-tetraoxo-6,6-bis(4-(trifluoromethyl)benzyl)-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide
Formula Molecular weightC63H86F2N14O7Exact mass: 1188,6772 Molecular weight: 1189,4433C65H86F6N14O7Exact mass: 1288,6708 Molecular weight: 1289,4584
Antimicrobial activity (MIC)
S. aureusn.t.n.t.
MRSAn.t. n.t.
MRSEn.t.n.t.
E. colin.t.n.t.
Anticancer activity (IC50)
A2019737
MethAn.t.n.t.
.'
Toxicity
MRC-5>500260
RBC (ECS0)n.t.n.t.
Anticancer selectivity
MRC-5/A20(>2.5 m)7,
RBC/A20n.t.n.t.
n.t. = not tested

/tr>
Table 3
Continued (All values are in µg/ml)
2122
Name in accordance with IUPAC(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-6,6-bis(4-tert-butylbenzyl)-15-carbamoyl-1-(1H-indol-3-yl)-3,7,10,13-petrocco-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide(R)-N-((2R,9S,12S,15S)-9-((1H-indol-3-yl)methyl)-19-amino-12-(4-aminobutyl)-15-carbamoyl-6,6-bis(3,5-dimethylbenzyl)-1-(1H-indol-3-yl)-3,7,10,13-tetraoxo-4,8,11,14-tetrathionate-2-yl)-6-amino-2-((R)-2,6-diaminohexanoic)hexanamide
Formula Molecular weightC71H104N14O7Exact mass: 1264,8212 Molecular weight: 1265,6751 C67H96N14O7Exact mass: 1208,7586 Molecular weight: 1209,5687
Antimicrobial activity (MIC)
S. aureusn.t.n.t.
MRSAn.t.n.t.
MRSEn.t.n.t.
E. colin.t.n.t.
Anticancer activity (IC50)
A2014,5/17,5*32/40*
MethAn.t.n.t.
Toxicity
MRC-542/55,2*237/205*
RBC (EC50)n.t.n.t.
Anticancer
selectivity
MRC-5/A202,9/3,2*7,4/5,1*
RBC/A20n.t.n.t.
* Two sets of results obtained due to two separate screenings (and the average of three Parallels each screening)
n.t. = not tested

Table 4
The biological activity of dimethylaminoethyl derivatives of β2,2-amino acids (All values are in µg/ml)
23242526
Name in accordance with IUPAC3-amino-2,2-dibenzyl-N-(2-dimethylamino)-ethyl)-propanamide Log P: 2,97 tPSA: 58,36 CLogP: 3,00783-amino-2,2-bis(4-tert-butylbenzyl)-N-(2-(dimethylamino)-ethyl)-propanamide Log P: 6,38 tPSA: 58,36 CLogP: 6,65983-amino-N-(2-(dimethylamino)-ethyl)-2,2-bis(3,5-dimethylbenzyl)-propanamide Log P: 4,92 tPSA: 58,36 CLogP: 5,00382-(aminomethyl)-N-(2-(dimethylamino)ethyl)-5-phenyl-2-(3-phenylpropyl)-pentanone Log P: a 4.64 tPSA: 58,36 CLogP: 4,9738
Formula Molecular weightC21H29N3O Exact mass: 339,2311 Molecular weight: 339,4745C29H45N3O Exact mass: 451,3563 Molecular weight: 451,6871C25H37N3O Exact mass: 395,2937 Molecular weight: 395,5808C25H37N3O Exact mass: 395,2937 Molecular weight: 395,5808
Antimicrobial activity (MIC)
S. aureus>100510035
MRSA>10055035
MRSE>1002,51035
E. coli>100550100
Anticancer activity (IC50)
A20n.t.n.t.n.. n.t.
MethAn.t.n.t.n.t.n.t.
Toxicity
MRC-5n.t.n.tn.t.n.t.
RBC (ECso)>1000185820316
Anticancer selectivity
MRC-5/A20n.t.n.t.n.t.n.t.
RBC/A20n.t.n.t.n.t.n.t.
n.t. = not tested

Table 4
Continued (All values are in µg/ml)
2728
Name in accordance with IUPAC3-amino-N-(2-(dimethylamino)ethyl)-2,2-bis(naphthalene-2-imetroproperty Log P: 4,97 tPSA: 58,36 CLogP: 5,35583-amino-N-(2-(dimethylamino)ethyl)-2,2-bis(4-(trifluoromethyl)benzyl) -propanamide Log P: 4,82 tPSA: 58,36 CLogP: 4,7738
Formula Molecular weightC29H33N3O Exact mass: 439,2624 Molecular weight: 439,5918C23H27F6N3O Exact mass: 475,2058 in Molecular�with: 475,4704
Antimicrobial activity(MIC)
S. aureus510
MRSA55
MRSE515
E. coli1535
Anticancer activity (IC50)
A20n.t.n.t.
MethAn.t.n.t.
Toxicity
MRC-5n.t.n.t.
RBC (EC50)145329
Anticancer selectivity
MRC-5/A20n.t.n.t.
RBC/A20n.t.n.t.
n.t. = not tested

Table 5
Biological activity of N-methyl piperazine derivative β2,2-amino acids (All values are in µg/ml)
293031
Name in accordance with IUPAC3-amino-2,2-bis(tert-butylbenzyl)-1-(4-methylpiperazin-1-yl)propane-1-he LogP: 6,37 tPSA: 49,57 CLogP: 7,0724 3-amino-2,2-bis(3,5-dimethylbenzyl)-1-(4-methylpiperazin-1-yl)propan-1-one LogP: 4,91 tPSA: 49,57 CLogP: 5,41642-(aminomethyl)-1-(4-methylpiperazin-1-yl)-5-phenyl-2-(3-phenylpropyl)pentane-1-he LogP: 4,63 tPSA: 49,57 CLogP: 5,3864
Formula Molecular weightC30H45N3O Exact mass: 463,36 Molecular weight: 463,70C26H37N3O Exact mass: 407,29 Molecular weight: 407,59C26H37N3O Exact mass: 407,29 Molecular weight: 407,59
Antimicrobial activity(MIC)
S. aureus10>200200
MRSA10200200
MRSE535200
E. coli10200 200
Anticancer activity (IC50)
A20n.t.n.t.n.t.
MethAn.t.n.t.n.t.
Roxicet
MRC-5n.t.n.t.n.t.
RBC (EC50)241>1000>1000
Anticancer selectivity
MRC-5/A20n.t.n.t.n.t.
RBC/A20n.t.n.t.n.t.
n.t. = not tested

td align="justify"> C24H27F6N3O Exact mass: 487,21 Molecular weight: 487,48
Table 5
Continued (All values are in µg/ml)
3233
Name in accordance with IUPAC3-amino-1-(4-methylpiperazin-1-yl)-2,2-bis(naphthalene-2-ylmethyl)propane-1-he LogP: 4,96 tPSA: 49,57 CLogP: 5,76843-amino-1-(4-methylpiperazin-1-yl)-2,2-bis(4-(trifluoromethyl)benzyl)propane-1-he LogP: 4,81 tPSA: 49,57 CLogP: 5,1864
Formula Molecular weightC30H33N3O Exact mass: 451,26 Molecular weight: 451,60
Antimicrobial activity(MIC)
S. aureus1035
MRSA510
MRSE1050
E. coli1550
Anticancer activity (IC50)
A20n.t.n.t.
MethAn.t.n.t.
Toxicity
MRC-5 n.t.n.t.
RBC (EC50)225376
Anticancer selectivity
MRC-5/A20n.t.n.t.
RBC/A20n.t.n.t.
n.t. = not tested

10
Table 6
Biological activity of N,N-dimethylamino-ethyl-piperazine derivatives of β2,2-amino acids (All values are in µg/ml)
343536
Name in accordance with IUPAC3-amino-2,2-bis(4-tert-butylbenzyl)-1-(4-(2-dimethylamino)ethyl)piperazine-1-yl)propane-1-he LogP: 6,37 tPSA: 52,81 CLogP: 7,44753-amino-1-(4-(2-(dimethylamino)ethyl)piperazine-1-yl)-2,2-bis(3,5-dimethylbenzyl)propane-1-he LogP: 4,91 tPSA: 52,81 CLogP: 5,79152-(aminomethyl)-1-(4-(2-dimethylamino)ethyl)piperazine-1-yl)-5-phenyl-2-3-phenylpropyl)pentane-1-it Log P: 4,63 tPSA: 52,81 CLogP: 5,7615
Formula Molecular weightC33H52N4O Exact mass: 520,41 Molecular weight: 520,79C29H44N4O Exact mass: 464,35 Molecular weight: 464,69C29H44N4O Exact mass: 464,35 Molecular weight: 464,69
Antimicrobial activity (MIC)
S. aureus1050200
MRSA105050
MRSE3550
E. coli10100200
Anticancer activity (IC50)
A20n.t.n.t.n.t.
MethAn.t.n.t.n.t.
Toxicity
MRC-5n.t.n.t.n.t.
RBC (EC50)252n.t.>1000
��
Anticancer selectivity
MRC-5/A20n.t.n.t.n.t.
RBC/A20n.t.n.t.n.t.
n.t. = not tested

Table 7
Continued (All values are in µg/ml)
3738
IUPAC-name3-amino-1-(4-(2-(dimethylamino)ethyl)piperazine-1-yl)-2,2-bis(naphthalene-2-ylmethyl)propane-1-he LogP: 4,96 tPSA: 52,81 CLogP:6,14353-amino-1-(4-(2-(dimethylamino)ethyl)piperazine-1-yl)-2,2-bis(4-(triptoreline)propane-1-he LogP: 4,81 tPSA: 52,81 CLogP: 5,5615
Formula Molecular weightC33H40N4O Exact mass: 508,3202 Molecular weight: 508,6969C27H34N4O Exact mass: 544,2637 Molecular weight: 544,5755
Antimicrobial activity(MIC)
S. aureus1535
MRSA2,55
MRSE1050
E. coli35100
Anticancer activity (IC50)
A20n.t.n.t.
MethAn.t.n.t.
Toxicity
MRC-5n.t.n.t.
RBC (EC50)301862
Anticancer selectivity
MRC-5/A20n.t.n.t.
RBC/A20n.t.n.t.

Example 3

Compound 7 described in Example 2 was analyzed for two normal cell lines and nine cancer cell lines.

All cell lines were of human origin. The basic solution of compound 7 was dissolved in the sample containing 10% DMSO. Spent three parallel experiments on each cell line.

Previous results for compound 7 on cancer cells A20 showed IC500.7 µ g/ml and RBC EU50292 µg/ml table 2). Previous tests of compound 7 on cells MRC-5 showed IC5013.5 and 14 μg/ml, and screening diagnostics showed IC508,21 μg/ml.

Table 8
The results of the screening diagnostics of compound 7 in two normal cell lines (MRC-5 and HUV-EC-C) and nine cancer cell lines
The cell lineBodyDiseaseView of a cellTAR IC50mcg/ml
MRC-5SLightNormalFibroblast8.21
HUV-EC-CUmbilical veinNormalEndothelial3.81
DU-145ProstateCarcinomaEpithelial15.21
OVCAR-3OvariesAdenocarcinoma Epithelial18.05
MDA-MBMammary glandsCarcinomaNo data6.54
FEMXLeatherMelanomaNo data17.1
UT-SCC-24ALanguageSquamous cell carcinomaPrimary cancer cells of the oral cavity14.19
UT-SCC-34ANadjezjda larynxSquamous cell carcinoma of the oral cavityPrimary cancer cells of the oral cavity16.18
HT-29Large intestineColorectal adenocarcinomaEpithelial15.45
RamosNo dataBurkitt lymphomaB-lymphocytes2.61
KellyNo dataNeuroblastomaNo data3.90

Example 4

Derivatives of β2,2- amino acids of the invention were made and analyzed for antimicrobial action and investigated for their compliance for oral administration.

Synthesis

Derivatives of β2,2-amino acids (FIG.3) were synthesized in accordance with the strategies shown in FIG.4. To explore the wide range of ways to prepare β-amino acids, see Abele, S. et al. Eur. J. Org. Chem. [2000] 2000, 1-15. The BOC-protected β2,2-amino acids 3A-d was combined with four different C-terminal cationic groups (FIG.4). For better results, 1.5 EQ. linking reagent TFFH used in the final stage of connection, and β2,2-amino acids were pre-activated for 2 hours with TFFH instead of the standard 10 minutes. After reaction of the compounds was carried out mass spectrometric analysis and terminated by adding aqueous Na2CO3. However, due to steric constraints, the time needed to increase to 7 days to attach the C-terminal cationic groups.

General procedure of synthesis of 1a-d.

The synthesis was based on the previous synthesis of previously conducted Cronin et al. Anal. Biochem. [1982] 124, 139-149. Briefly, sodium methoxide (20 mmol) was dissolved in methanol to achieve an approximate concentration of 0.2 M before added methyl cyanoacetate (20 mmol) and the reaction mixture was stirred for 5 minutes at room temperature. Added the desired methyl benzyl (20 mmol) and the solution was heated to delegirovali for 15 minutes, after which the solution was cooled to room temperature and added to a second portion of sodium methoxide (20 mmol). After agitation for 5 minutes at room temperature was added another portion of the desired benzyl bromide (20 mmol), and then spent delegirovano for 15 minutes. Approximately 2/3 of the methanol was removed in vacuo, then the reaction mixture was diluted with ethyl acetate and washed with water. The organic phase is dried over MgSO4, filtered and evaporated to dryness. The product 1a-d were used in the subsequent synthesis without any further purification.

General procedure for synthesis of 2a-d.

The synthesis was based on the previous synthesis conducted by Cronin et al. (see above) and Bodanszky et al. "The practice of peptide synthesis" Springe-Verlag 1994. Briefly, Ra/Ni (typically 2 ml) was washed with methanol to remove water, then add the required derivative metalloelastase (1a-d) (3,5 mmol) along with acetic acid (typically 1 ml) and methanol to obtain the final to�ncentratio 1a-d, component of approximately 0.1 M. the Reaction mixture was hydrational at a temperature of 45°C with a reverse condenser installed for 5 days at a pressure of 1 bar H2, then Ra/Ni filtered, and the reaction mixture was evaporated to dryness. The crude methyl ester of β2,2-amino acid was dissolved in a mixture of 1,4-dioxane and water 5:1 (0.35 M), and pH adjusted to 8 with tea, and then added Boc2O (1.5 EQ.), dissolved in the minimum amount of 1,4-dioxane, and the solution was stirred at room temperature for 18 hours. The reaction mixture is oxidized to pH 4-5 using 0.1 M HCl and extracted three times with ethyl acetate, then the organic phase is dried over MgSO4, filtered and evaporated to dryness. The crude product 2a-d were used in the subsequent synthesis without any further purification.

General procedure for synthesis of 3A-d.

The synthesis was based on the publication of Seebach et al. Helv. Chim. Acta [1998] 81, 2218-2243. Briefly, the crude BOC-protected methyl ester of β2,2-amino acids 2a-d (0,35 mmol) was dissolved in a mixture of 1,4-dioxane and water 3:1 to achieve a final concentration of approximately 1.2 mm, after which the lithium hydroxide (2.1 mmol) was diluted in the minimum amount of water. The reaction mixture was heated to defragmentierung and left for 18 hours, after which the volume has been reduced by approximately 1/5 in vacuo, a pH adjusted d� 1-2 by slow addition of 0.1 M HCl. The aqueous solution was extracted three times with ethyl acetate, then the organic phase is dried over MgSO4, filtered and evaporated to dryness. The crude product 3A-d were used in the subsequent synthesis without any further purification.

General procedure for synthesis of 4a-d, 5a-d, 6a-d and 7a-d

The synthesis was based on the manual by Chan and White, "Fmoc solid phase peptide synthesis: a practical approach", Oxford University Press 2000; p346. Briefly, Boc-protected β2,2-amino acids (3A-d) (typically, 0.2 mmol) was diluted in DMF (0.02 M), then add DIPEA (3 EQ.) together with TFFH (1 EQ.). β2,2-amino acids are activated for 2 hours before adding the desired amine (2 EQ.). After reactions add MS, and reaction takes place within a maximum of 7 days, then diluted with ethyl acetate and washed with brine. The organic phase is dried over MgSO4, filtered and evaporated to dryness. Removed the protected derivative Boc-protected β2,2-amino acids by dissolution in was held, adding the equivalent volume TFUK:TIS:water (95:2.5:2.5) and stirred at room temperature for 2 hours, then evaporated to dryness. The crude products were purified by the method of preparative RP-HPLC and liofilizirovanny. Compound purity was checked by analytical method HPLC with PDA detectors in the range from 210 nm to 310 nm. All compounds had a purity above 95%.

Proteomika�th action

Each compound was tested in duplicate at 200, 100, 50, 35, 15, 10, 5, 2,5, 1, 0,5 μg/ml. All tested compounds were salts of di-triperoxonane acid.

Hemolytic action

The fraction of plasma heparinized human blood was first removed by centrifugation method and three additional washing stages at a temperature of 37°C in pre-warmed phosphate-buffered saline (PBS). After that human RBC was diluted to 10% hematocrit, and derivatives of β2,2-amino acid was dissolved in PBS, providing a concentration in the range of 1-1000 μg/ml. Diluted RBC added in connective solutions to achieve a final concentration of erythrocytes with 1% o/O. PBS and Triton X-100 at a final concentration of 0.1% o/o were used as negative and positive controls. After 1 hour of incubation with constant shaking at 37°C the samples were centrifuged at 4000 rpm for 5 minutes. The release of hemoglobin was determined by absorbance of the supernatant at 405 nm. Hemolytic activity was calculated as the ratio of treated sample with a derivative of β2,2-amino acids and the sample treated with tension in accordance with the formula

25
Table 9
Minimum inhibitory concentration (MIC in µm) against MRSA, MRSE, S. aureus, E. coli, and p. aeruginosa, and hemolytic activity (EC50in µm) against human RBC for a small sequence of β2,2-amino acids. therapeutic index was calculated by dividing the values of EC50RBC on the MIC against each strain of bacteria
MIC3EC50bRBChTherapeutic index
PositionMTRSACMRSEdS. aureuseE. colifP. aeruginosa9MRSAMRSES. aureusE. coliP. aeruginosa
4A7272289289
4b6.56545129111617217258.6
4C3.414204840912029208.5
4d13131313255337262626261.3
5A315315315315
5b14704970525387.5117.5
5C7.4151522-30341202014
5d157.21515289348234823231.2
6A565656160 5079.19.19.13.2
6b7.12114504686622339.4
6C7.57.57.5233001171616165.10.4
6d7.43.77.47.474274377437373.7
7a597133613772355194.1
7b7.41515524255728288
7C7.83.93.9245545759117117198.3
7d3.83.83.87.723184.74.74.72.3 0.8
The highest tested concentrations werea200 µg/mlb1000 µg/ml. All derivatives of β22amino acids were isolated in the form of their di-triptoreline salts, and their molar concentrations were calculated in this form.cMethicillin-resistant Staphylococcus aureus (ATCC 33591),dMethicillin-resistant Staphylococcus epidermidis (ATCC 27626),eStaphylococcus aureus (ATCC 25923),fEscherichia coli (ATCC 25922),sPseudomonas aeruginosa (ATCC 27853) and red blood cells of man. The symbol "-" indicates no detectable activity (MIC or EC50) in the tested range of concentrations.

The similarity of drugs and oral absorption

Using the applicator schrödinger QikProp, which comes with software Schrodinger Maestro v9.1, the assessment of similarity of drug derivatives of β2,2-amino acids relative to the "rule of five" Opinsky was carried out together with an assessment of the percentage of oral absorption in humans. The rule States that the active drug to be taken orally should not violate more than one of these four criteria; 1) the logarithm of the P partition coefficient octanol-water should be less than 5, 2) molecular mass (molecular weight) should not exceed 500 daltons, 3) is allowed a maximum of 5 groups ogorodnicheskih relations (HBD), and 4) should be no more than 10 groups hydrogen donor-acceptor bonds (NCA). The results presented in the Table 10 below.

6C
Table 10
The assessment of similarity of drug prepared derivatives of β2,2-amino acids relative to the "Rule of five" Opinski and calculation of potential oral absorption in humans
Comp.The "rule of five" Opinsky (maximum)To the AML. peror. ASB.e(%)
Mwa(500)HBDb(5)HBAc(10)The logarithm of βd(5)
4A464.7173.872
4b544.6174.262
4C508.717 4.363
4d520.8174.866
5A407.6153.986
5b487.5154.488
5C451.6154.387
5d463.7155.179
6A395.624.53.884
6b475.524.54.689
439.624.54.789
6d451.724.55.380
7a367.543.52.973
7b447.443.53.471
7C411.543.53.472
7dTaiyuan 423.643.53.773
Molecular weight calculated for uncharged derivatives of β-amino acids.bIt is estimated the number of groups of a hydrogen donor relations (HBD).cCounted the number of hydrogen groups Dono�but-acceptor bonds (NCA). Values were averaged for a number of configurations; therefore, the values are not integers.dCalculated the logarithm of the P partition coefficient octanol-water.eEstimated percentage of oral absorption in humans. All the estimates have been calculated using the applicator QikProp schrödinger, supplied with the software Schrodinger Maestro v9.1.

The results of the calculations show that all the derivatives of β2,2-amino acids correspond to the "Rule of five" Opinski, since no connection is not violated more than one of the four rules. One rule violated 5 of 16 prepared derivatives of β2,2-amino acids, three violations of which arose as a result of the fact that the molecular weight exceeds 500 (4b, 4C and 4d), and the other two violations resulted from the fact that the logarithm of P exceeded the allowed value is 5 (5d and 6d).

The calculation of the percentage of oral absorption in humans using the software demonstrated that derivatives of β2,2-amino acids may well enough be absorbed, and estimated oral absorption ranged from 62% to 89%. The largest percentage of oral absorption was calculated for the derivatives of β2,2-amino-5A, 5b, 5C, 6b and 6C, all of which were in the range of 86%-89% oral absorption.

Permeability

On the basis of theoretical calculations, the permeability of β2,2-amino acids was also investigated using the newly installed barrier model based on phospholipid vesicles (Flaten et al., Eur. J. Pharm. Sci. [2006] 27, 80-90). Were investigated four derivatives of β2,2-amino acids (4C, 5C, 6C and 7C), which showed similar activity against MRSA and against E. coli. Classification-based absorption model, all four compounds showed a permeability equivalent to a moderate absorption in humans. From the point of view of experimental values of permeability, compound 5 showed the highest concentration, after it is compound 6C, 4C and 7C.

1. A method of treating a microbial infection, comprising administering to the patient a peptide, peptidomimetic or derivative of an amino acid having a net positive charge of at least +2 at pH 7.0 and containing a disubstituted β amino acid, each of the substituting groups in the β amino acid, which may be identical or different, comprises at least 7 novogorodnaya atoms, is lipophilic and includes at least one cyclic group, and one or more cyclic groups in the replacement group may be linked or condensed with one or more cyclic groups in the other substituting group, and when the CEC�algebraic groups merged so total total number novogorodnaya atoms for the two substituting groups is at least 12, where the specified peptide, peptidomimetic or derived amino acids are composed of 1-4 amino acids or equivalent in length subunits, and the introduction is carried out at a dose of from 1 to 10 mg/kg.

2. A method according to claim 1, in which two substituting groups are the same.

3. A method according to claim 1, wherein the β-amino acid is a β2,2or β3,3dibasic amino acid.

4. A method according to claim 1, wherein each lipophilic substituent groups may include substituted phenyl or pirazinokarbazolovogo cyclohexyl group.

5. A method according to claim 4, where the specified phenyl or pirazinokarbazolovogo cyclohexyl group is separated from the α or β carbon atom of β-amino acids with 1-4 bridging atoms.

6. A method according to claim 1, in which each residual lipophilic group contains 8-12 novogorodnaya atoms.

7. A method according to claim 1, in which the C-end peptide of peptidomimetic or derived amino acid amidases and possibly replaced.

8. A method according to claim 1, wherein the peptide or peptidomimetic includes a cationic amino acid, preferably arginine or lysine.

9. Peptide, peptidomimetic or derivative of an amino acid having a net positive charge of at least +2 at pH 7.0, which includes a group of the formula I

�de any 2 of R 1, R2, R3and R4atoms of hydrogen and 2 is the residual group, which may be identical or different,
includes at least 7 novogorodnaya atoms, is lipophilic and includes a cyclic group, and mentioned cyclic group is not attached directly to the α or β carbon atom, and possibly linked or condensed with a cyclic group in the other substituting group, and when condensed cyclic group, the cumulative total number novogorodnaya atoms for the two substituting groups is at least 12, and
where X denotes O, S, N, or S, where the specified peptide, peptidomimetic or derived amino acids are composed of 1-4 amino acids or equivalent in length subunits.

10. Peptide, peptidomimetic or derivative of an amino acid according to claim 9, in which two substituting groups are the same.

11. Peptide, peptidomimetic or derivative of an amino acid according to claim 9, wherein the β-amino acid is a β2,2or β3,3dibasic amino acid.

12. Peptide, peptidomimetic or derivative of an amino acid according to claim 9, wherein the residual lipophilic group may include substituted phenyl or pirazinokarbazolovogo cyclohexyl group.

13. Peptide, peptidomimetic amino acid or derivative according to claim 12, where the specified phenyl or pirazinokarbazolovogo cyclohexyl group is separated from the α or β carbon β-amino acids with 1-4 bridging atoms.

14. Peptide, peptidomimetic or derivative of an amino acid according to claim 9, in which each residual lipophilic group contains 8-12 novogorodnaya atoms.

15. Peptide, peptidomimetic or derivative of an amino acid according to claim 9, in which the C-end derived peptide substances, peptidomimetic or amino acids amidines and possibly replaced.

16. Peptide, peptidomimetic or derivative of an amino acid according to claim 9, wherein the peptide or peptidomimetic includes a cationic amino acid, preferably arginine or lysine.

17. Pharmaceutical composition having antimicrobial activity comprising an effective amount of a molecule according to any one of claims.9-16, and a diluent, carrier or excipient.



 

Same patents:

Antibiotic peptides // 2472805

FIELD: chemistry.

SUBSTANCE: peptides and peptide derivatives have general formula Sub1-X1NX2X3PVYIPX4X5RPPHP-Sub2, where Sub1, X1, X2, X3, X5, Sub2 are given in the claim. The disclosed peptides or peptide derivatives have at least one of the following advantages compared to natural apidecine peptides: (i) longer half-life in mammal serum owing to higher protease stability, (ii) high antimicrobial activity with respect to one or more bacterial strains, particularly human pathogens or fungi, or other microbial infections, (iii) demonstrate a wide range of antimicrobial activity, (iv) cause slower development of resistance in microorganisms and (v) are not toxic for human cells, including erythrocytes.

EFFECT: improved properties of peptides.

21 cl, 8 dwg, 7 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to methods for producing protease inhibitors, particularly serine protease inhibitors.

EFFECT: producing protease inhibitors applicable for treating HCV infections.

43 cl, 14 ex

FIELD: chemistry.

SUBSTANCE: invention relates to amide group-containing bactericidal macrocycles of formula in which R26 denotes hydrogen or methyl, R7 denotes a group of formula or where R1 denotes hydrogen or a hydroxy group and * denotes the bonding position of the carbon atom. R2 denotes hydrogen or methyl.

EFFECT: high bactericidal activity of the compounds.

13 cl, 22 tbl, 281 ex

FIELD: medicine.

SUBSTANCE: invention relates to novel peptide compounds, which possess ability to inhibit protease of hepatitis C virus (HCV), their pharmaceutical compositions and application of compounds for obtaining medication for diseases associated with HCV.

EFFECT: improvement of compound properties.

48 cl, 6 tbl, 65 ex

FIELD: medicine.

SUBSTANCE: invention refers to chelating agents and their technetium complexes to be used as radiopharmaceuticals and characterised by formula I where X is -NR-, -CO2-, -CO-, -NR(C=S)-, -NR(C=O)-, -CONR- or Q; Y represents amino acid, -CH2-, -CH2OCH2-, -OCH2CH2O- or X; Z is an aggregation from peptides, their analogues, substrata, antagonists or enzyme inhibitors, receptor-bonding compounds, oligonucleotides, oligo-DNA- or oligo-RNA-fragments; n is a number 1 to 8; m is a number 0 to 30; R represents H, C1-4alkyl, C2-4alkoxyalkyl, C1-4hydroxyalkyl or C1-4fluoroalkyl; Q represents remains of succinimide , A is a pharmaceutically acceptable anion.

EFFECT: production of new chelating agents applicable for making the technetium complexes.

22 cl, 12 ex, 3 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention claims novel compounds with inhibition effect on HCV (hepatitis C virus) protease, and methods of obtaining the claimed compounds.

EFFECT: pharmaceutical compositions including these compounds, and methods of compound application in treatment of HCV protease related diseases.

23 cl, 5 tbl, 39 ex

FIELD: chemistry.

SUBSTANCE: agent is an N- and C-substituted peptide selected from n-decyl ether (1-tetradecyl-1,4-diazoniabicyclo[2.2,2.]octan-4-yl)-acetyl-glutamyl-glycyl lysyl-glycine (1), n-decyl ether (1-tetradecyl-1,4-diazoniabicyclo[2.2,2.]octan-4-yl)-acetyl-glutamyl-β-alanyl-arginyl-glycine (2) and n-decyl ether glutamyl-β-alanyl-lysyl-glycine (3).

EFFECT: high antiviral activity of the agent.

3 dwg, 2 tbl, 6 ex

Rgd-like peptides // 2396271

FIELD: chemistry.

SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.

EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.

2 tbl, 1 ex

Caspase inhibitors // 2274642

FIELD: medicine, biochemistry, pharmacy.

SUBSTANCE: invention relates to new compounds that represent inhibitors of caspases, in particular, inhibitors of interleukin-1β-converting enzyme and their pharmaceutical compositions. Proposed compounds can be used successfully as agents directed against diseases mediated by interleukin-1, apoptosis and factor inducing interferon-γ or by interferon-γ.

EFFECT: valuable medicinal and biochemical inhibitors.

35 cl

The invention relates to new compounds which are inhibitors of interleukin-1-converting enzyme (IAP), is characterized by a specific structural formula; to pharmaceutical compositions having the ability to inhibit interleukin-1-converting enzymes, method of treatment and prophylaxis of diseases selected from the group consisting of IL-1-mediated autoimmune inflammatory, neurodegenerative diseases, as well as the selection method of the IAP inhibitor

The invention relates to new compounds of General formula I

< / BR>
where a is Gly; the remainder of the formula II

< / BR>
m= 0 or 1; n= 2 or 3; R1and R2each independently of the other represents H, R1and R2both together represent also

< / BR>
or

< / BR>
where IS -(CO)-(CH2)q-(CO)rwhere q=1, 2, or 3, r=0 or 1, or-CO-CH=CH-CO-; X IS H, Cl or1-C6alkyl; and if the mean remains optically active amino acids and derivatives of amino acids, are included as D-and L-forms, and their salts, process for the preparation of compounds of formula I and their salts; pharmaceutical composition having the ability to inhibit integrin containing in its structure at least one compound of the formula I and/or one of its physiologically acceptable salts

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a water-soluble salicyl morpholide of formula .

EFFECT: derivative possesses nootropic activity in a combination with antidepressant action.

6 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to N-hydroxylsulphonamide derivatives of formula or , where R1 is H; R2 is H; n is 0; b is an integer in the range of 1-4; R3, R4, R5, R6 and R7 are independently selected from H, halogen, carboxyl, carboxyl ester selected from a group including -C(O)O-morpholino, -C(O)O-C1-C8alkyl and -C(O)O-substituted C1-C8alkyl, where the substitute is morpholino; acylamino, which is a -C(O)NRaRb group, where Ra and Rb are independently C1-C8alkyl, or Ra and Rb together with a nitrogen atom to which they are bonded form morpholino; and sulphonylamino, which is a SO2NR2 group, where two groups R, together with a nitrogen atom to which they are bonded, form morpholino; R8 is selected from halogen and carbonylamino, selected from a -CONH-substituted C1-C8alkyl, where the substitute is morpholino; and -CONR2, where two groups R, together with a nitrogen atom to which they are bonded, form morpholino; C is a heteroaromatic ring which contains cyclic fragments Q9, Q10, Q11, Q12, Q13 and Q14, which are independently selected from C, CH and S, under the condition that at least one of the fragments Q9, Q10, Q11, Q12, Q13 and Q14 is S. The invention also relates to a method of modulating nitroxyl levels, a method of treating diseases which respond to treatment with nitroxyl, a treatment set and a pharmaceutical composition containing compounds of formula (I) or (III).

EFFECT: compounds of formula (I) or (III) for treating diseases which respond to treatment with nitroxyl.

20 cl, 5 ex, 4 tbl, 7 ex

Aromatic compound // 2416608

FIELD: chemistry.

SUBSTANCE: invention describes a novel compound of general formula (1), where radicals R1, R2, X1, Y and A are as described in claim 1 of the invention. The invention also describes a method of obtaining compounds of formula (1), as well as a pharmaceutical composition based on said compounds, for treating fibrosis.

EFFECT: novel compounds with excellent collagen formation suppression, cause fewer side-effects and which are safer are obtained.

62 cl, 2717 ex, 432 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel naphthalene derivatives of general formula I , as well as to their pharmaceutically acceptable salts, which can be used for treating and/or preventing diseases associated with H-3 receptor modulation. In formula I, R1 is selected from hydrogen, lower alkyl, phenyl, phenyl-lower alkyl and lower alkoxyalkyl; R2 is selected from hydrogen, lower alkyl, C3-C7-cycloalkyl, lower alkoxyalkyl or lower alkylsuphanylalkyl (all values of R1 and R2 are given in the formula of inventions); or R1 and R2 together with the nitrogen atom to which they are bonded form a 4-7-member saturated or partially unsaturated heterocyclic ring which can contain one more heteroatom selected from nitrogen, oxygen and sulphur atoms, where the said heterocyclic ring can be unsubstituted or substituted with 1-2 groups, or can be condensed with an unsubstituted phenyl ring; A is selected from (values of R3-R7, R9, R10, X, m, n, t, p, q and s are given in the formula of invention). Invention also pertains to a pharmaceutical composition containing formula I compounds.

EFFECT: increased effectiveness of application.

30 cl, 2 tbl, 188 ex

FIELD: chemistry.

SUBSTANCE: invention claims compounds of the formula (I) with radicals as described in the claim, and medicine with inhibition effect on glycine absorption, based on compound of the formula (I) .

EFFECT: medicine for diseases treatment where glycine absorption inhibition can be effective.

21 cl, 1 tbl, 173 ex

FIELD: chemistry.

SUBSTANCE: there are disclosed 1-(2-aminobenzol)piperazine derivatives of formula (I) and pharmaceutically acceptable acid-additive salts with radical values specified in patent claim. The compounds are characterised with inhibiting effect on glycine I carrier. There is also disclosed medical product based on the compounds of formula (I).

EFFECT: compound can be used for treatment of the diseases associated with glycine uptake inhibition.

12 cl, 5 tbl, 396 ex

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