Lipooligourea, pharmaceutical compositions, and lipooligourea for use as a medicament

Abstract

The subject of the present invention is lipooligourea of formula (1): FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u In —NH2(1), (FA) is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain, and Xaa1 u Lys u or Dab u or Arg u and Xaa2 u Lys u or Dab u or Leu u or Arg u and Xaa3 u Lys u or Dab u or Leu u or Arg u The present invention also relates to a pharmaceutical composition comprising the lipooligourea of formula 1.

Description

[Technical Field]

[0001] The present invention relates to lipooligourea, pharmaceutical compositions comprising lipooligourea, and lipooligourea for use as a medicament in the treatment of bacterial infections. [Background technology]

[0002] The increasing incidence of bacterial drug resistance is currently a significant clinical problem, spurring interest in compounds that offer effective alternatives while exhibiting mechanisms of action distinct from those of conventional antibiotics. An entirely new group of compounds that may fulfill these requirements are the lipooligoureas, described below.

[0003] Conventional antibiotics on the market mostly exert specific effects based on disrupting biochemical processes occurring within bacterial cells. For example, they affect the activity of enzymes involved in cell wall synthesis, disrupt metabolic pathways, or damage DNA. Peptide- and lipopeptide-based antibiotics are also known, but their effects are less specific and are based on the ability of such compounds to penetrate and irreversibly disrupt the structure of bacterial cell membranes (see, for example, Alborn et al., Antimicrob. Agents Chemother. 1991, 35, 2282, and Kang et al., Journal of Microbiology vol. 2017, 55, 1). The membrane-lytic properties of antimicrobial peptides and lipopeptides, precisely because of their nonspecific mechanism of action, significantly reduce the likelihood of bacterial resistance to such compounds. Currently, there are pharmaceuticals on the market that use lipopeptides as active antibiotic components. This class includes polymyxins and daptomycin. The latter is a naturally occurring branched cyclic antibiotic lipopeptide of nonribosomal origin. This lipopeptide exhibits potent bactericidal activity in vitro and in vivo against Gram-positive bacteria that can cause serious and life-threatening diseases, as described, for example, in Tally, FP et al., "Daptomycin: a Novel Agent for Gram-positive Infections," Exp. Opin. Invest. Drugs 8:1223-1238 (1999). Most of the lipopeptides described in the literature, such as daptomycin, are naturally occurring and derived from bacteria or other organisms, making their isolation from biological materials costly. Furthermore, most of the lipopeptides currently used in pharmaceuticals, such as daptomycin and polymyxins, have cyclic and relatively complex structures, complicating their potential synthesis and further modification.However, examples are known from the literature of synthetic compounds belonging to the group of lipopeptides that show great potential as useful antibiotics and at the same time are acyclic, i.e., based on a linear structure (see, for example, US Pat. No. 6,911,525, US Patent Application Publication No. 2015 / 0080292, WO 2021 / 064593). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 6,911,525 [Patent Document 2] US Patent Application Publication No. 2015 / 0080292 [Patent Document 3] International Publication No. 2021 / 064593 [Non-patent literature]

[0005] [Non-Patent Document 1] Alborn et al., Antimicrob. Agents Chemother. 1991, 35, 2282 [Non-patent document 2] Kang et al., Journal of Microbiology vol. 2017, 55, 1 [Non-patent document 3] Tally, FP et al., “Daptomycin: a Novel Agent for Gram positive Infections,” Exp. Opin. Invest. Drugs 8:1223-1238 (1999) Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide an entirely new class of compounds, lipooligoureas, with an acyclic structure that exhibit antibacterial activity against Gram-positive and Gram-negative bacteria. [Means for solving the problem]

[0007] This goal was achieved through the development of a new class of lipooligoureas with antibacterial properties.

[0008] The subject of the present invention is a lipooligourea of general formula 1: FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1) (FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain, Xaa1 u Lys u or Dab u or Arg u is either Xaa2 u Lys u or Dab u or Leu u or Arg u is either Xaa3 u Lys u or Dab u or Leu u or Arg u It is either

[0009] Preferably, the lipooligourea is LOU1 [ka] LOU2 [ka] LOU3 [ka] LOU4 [ka] is selected from the group consisting of:

[0010] A subject of the present invention is also the above-mentioned lipooligourea for use as a medicament.

[0011] Preferably for use in the treatment or prevention of bacterial infections.

[0012] Preferably, the bacterial infection is caused by either a gram-positive or a gram-negative bacterium.

[0013] Preferably, the bacterial infection is caused by Staphylococcus aureus

[0014] Preferably, the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.

[0015] Preferably, the bacterial infection is caused by a methicillin-resistant Staphylococcus aureus (MRSA) strain.

[0016] A subject of the present invention is lipooligourea of formula LOU2 or LOU3 for use in the treatment or prevention of bacterial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains.

[0017] Another subject of the invention is a pharmaceutical composition, characterized in that it contains a lipooligourea of general formula 1: FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1) (FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain, Xaa1 u Lys u or Dab u or Arg u is either Xaa2 u Lys u or Dab u or Leu u or Arg u is either Xaa3 u Lys u or Dab u or Leu u or Arg u It is either

[0018] Preferably, the pharmaceutical composition comprises: LOU1 [ka] LOU2 [ka] LOU3 [ka] LOU4 [ka] is selected from the group consisting of:

[0019] Preferably, the pharmaceutical composition comprises 0.1% to 99% by weight of at least one lipooligourea.

[0020] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or diluent and / or adjuvant and / or excipient.

[0021] Preferably, the pharmaceutical composition comprises corn starch and / or gelatin and / or lactose and / or sucrose and / or microcrystalline cellulose and / or kaolin and / or mannitol and / or dibasic calcium phosphate and / or sodium chloride and / or alginic acid.

[0022] Preferably, the pharmaceutical composition is in a form suitable for oral and / or intravenous and / or intramuscular and / or subcutaneous and / or parenteral and / or rectal and / or topical administration.

[0023] Preferably, the pharmaceutical composition is in a form suitable for tablet and / or capsule and / or elixir and / or suspension and / or syrup and / or gel and / or cream and / or ointment and / or suppository and / or aerosol and / or drops.

[0024] Preferably, the pharmaceutical composition further comprises at least one other antibiotic. [Brief explanation of the drawings]

[0025] [Figure 1] 1 shows the time-dependent change in the amount of residual lipooligourea after proteolysis in human serum. DETAILED DESCRIPTION OF THE INVENTION

[0026] As mentioned above, unlike most conventional antibiotics that act specifically, lipooligourea exhibits the ability to incorporate into bacterial cell membranes and irreversibly damage their structure. Because their action is less specific than conventional drugs, the likelihood of resistance to this class of compounds is low. Another important feature and advantage of lipooligourea over antimicrobial peptides and lipopeptides is that the oligourea chain is less susceptible to proteolysis. This process significantly reduces the activity of peptides and lipopeptides, whereas synthetic peptidomimetics such as lipooligourea largely eliminate the activity. Therefore, the solution presented herein relates to an entirely new class of antimicrobial compounds, namely lipooligoureas. The lipooligoureas consist of an oligourea moiety comprising five urea residues linked to linear or branched carboxylic acid residues. For the purposes of this description, the letter "U" will be used to indicate the urea nature of the residue, to refer to urea residues that are analogs of individual amino acids. Superscript Standard nomenclature is used in the form of three-letter amino acid abbreviations followed by letters. The structure of the described lipooligourea can be represented by the general formula: FA-Phe u -Xaa1 u -Xaa2 u -Leu u -Xaa3 u -NH2(1) where FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain; Xaa1 u Lys u or Dab u or Arg u is either Xaa2 u Lys u or Dab u or Leu u or Arg u is either Xaa3 u Lys u or Dab u or Leu u or Arg u Either of the following.

[0027] The developed lipooligourea has an amphiphilic structure and can be incorporated into the lipid bilayer that constitutes the backbone of the cell membrane. u , Dab u or Arg u Due to the presence of positively charged urea residues such as , they have an excess positive charge under physiological pH conditions, which further facilitates their interaction with bacterial cell membranes, which normally have an excess negative charge due to their specific lipid composition (i.e., the presence of lipids from phosphatidylglycerol groups).

[0028] The pharmaceutical composition of the present invention contains any lipooligourea selected from the group of lipooligoureas represented by general formula (1). Preferably, the composition contains 0.1 to 99% by weight of a lipooligourea selected from the group comprising lipooligoureas represented by general formula (1). The pharmaceutical composition may also contain a pharmaceutically acceptable carrier and / or diluent and / or auxiliary agent and / or excipient. For example, but not limited to, the pharmaceutical composition may contain corn starch and / or gelatin and / or lactose and / or sucrose and / or crystalline cellulose and / or kaolin and / or mannitol and / or dicalcium phosphate and / or sodium chloride and / or alginic acid. Preferably, the pharmaceutical composition is in a form suitable for oral and / or intravenous and / or intramuscular and / or subcutaneous and / or parenteral and / or rectal and / or topical administration (e.g., cutaneous, intranasal, intraaural), such as a tablet and / or capsule and / or elixir and / or suspension and / or syrup and / or gel and / or cream and / or ointment and / or suppository and / or aerosol and / or drops.

[0029] The present invention also includes lipooligourea of general formula (1) for use as a medicament. Preferably, the lipooligourea of general formula (1) is intended for the treatment of bacterial infections caused by Gram-positive bacteria, in particular Gram-positive bacteria of the genus Staphylococcus.

[0030] The lipooligourea of the present invention can be obtained by any synthetic method known in the art, including, for example, either liquid phase or solid phase synthesis.

[0031] The lipooligourea of the present invention has an amphiphilic structure and contains Lys u , Dab u or Arg u Due to the presence of urea residues, it has an excess positive charge at physiological pH conditions (approximately 7.4) or below, which promotes interaction with bacterial cell membranes.

[0032] The lipooligourea of the present invention has growth inhibitory activity against strains of Gram-positive bacteria such as Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, as well as Gram-negative bacteria such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Yersinia enterocolitica, and can therefore be used to treat bacterial infections. The lipooligourea of the present invention exhibits enhanced activity against staphylococci. Importantly, the lipooligourea of the present invention exhibits significant growth inhibitory activity against strains of methicillin-resistant Staphylococcus aureus (MRSA).

[0033] The advantage of the lipooligoureas according to the present invention is their mechanism of action, which is based on direct interaction with bacterial cell membranes, thereby reducing the risk of drug resistance. Furthermore, the lipooligoureas described according to the present invention have a relatively simple structure, which greatly simplifies their synthesis. Their sufficient solubility in aqueous media is also ensured, which significantly expands the range of possible application forms for potential pharmaceutical compositions. [Example]

[0034] To illustrate the present invention, the following examples are included, which should not be construed as in any way limiting the scope of the invention.

[0035] Bacterial strains and media All bacterial strains were obtained from the Polish Collection of Microorganisms (PCM) or the American Type Culture Collection (ATCC). Gram-positive bacterial strains were Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, and Enterococcus faecalis ATCC 14506. Methicillin-resistant Staphylococcus aureus (MRSA) strains were S. aureus 401 / 5, S. aureus 4811 / 13, and S. aureus 5288 / 13. Gram-negative bacterial strains: Escherichia coli ST2-8624 O157:H7, Pseudomonas aeruginosa PAO1 PCM 499, Klebsiella pneumoniae PCM 1, Salmonella sv. typhimurium TT622, and Yersinia enterocolitica PCM 2081. All strains were cultured in lysogenic broth (LB medium).

[0036] Synthesis of Tested Lipooligoureas According to the Invention Oligoureas were obtained from succinimidyl carbamate derivatives of 2-azidoethylamine substituted at the 2-position. These building blocks were obtained from amino acids according to a method known from the literature (C. Douat-Casassus, K. Pulka, P. Claudon, G. Guichard, Org. Lett., 2012, 14, 3130-3133). Oligoureas were prepared in the solid phase on NovaPEG-type Rink amide resin with a loading of 0.45 mmol / g. 300 mg of resin was used per synthesis. The synthesis was performed under microwave irradiation, and each step was verified by chloranil test. For the coupling steps of the individual building blocks, 1.5 equivalents of each block, 3 equivalents of DIPEA, and 4 mL of DMF were used. The reaction was carried out in a microwave reactor at 50 °C, 50 W, and for 30 min. This step was repeated (once or twice) until the appropriate color of the resin particles was obtained by the chloranil test. The reduction of the azide group to an amine group was carried out using a 1 M solution of trimethylphosphine (PMe3) in THF. The reaction was carried out in a 7:3 1,4-dioxane:HO solution using 10 equivalents of PMe3. The reaction was carried out in a microwave reactor under the same conditions as the coupling step. The reduction reaction was repeated until the appropriate color of the resin particles was obtained by the chloranil test. After synthesis of the oligomer portion, carboxylic acid (FA) residues were attached. 3.5 equivalents of acid, 3.5 equivalents of HBTU, and 9 equivalents of DIPEA were used. The reaction was carried out in DMF at room temperature for 24 hours. The final compound was cleaved from the resin using a stripping mixture of TFA:TIS:HO in a ratio of 95:2.5:2.5. The lipooligourea was purified by semi-preparative HPLC using acetonitrile and water containing 0.1% TFA as the phases. For each compound, a gradient was selected to obtain the best possible distribution and yield. After HPLC purification, the oligoureas were obtained as salts, i.e., trifluoroacetate salts. The trifluoroacetate ions were then exchanged for chloride ions using a Dowex 1X8-100 type ion exchange resin, and the resulting solution was lyophilized to obtain the final compounds.

[0037] [ka]

[0038] The following section of the Examples describes the biological activity of exemplary lipooligoureas represented by the following formula: LOU1 [ka] LOU2 [ka] LOU3 [ka] LOU4 [ka]

[0039] Determination of minimum inhibitory concentration (MIC) Ten milliliters of LB medium was inoculated with material from a single bacterial colony and incubated overnight at 30°C with shaking. The overnight culture was diluted with a fresh portion of LB medium to an optical density (600 nm) of 0.05. Test compounds were dissolved in water. Serial dilutions of the compounds were performed in LB medium ranging from 5 to 50 μg / mL (final concentrations). Experiments were performed by adding 100 μL of each dilution to 100 μL of diluted overnight bacterial culture in wells of a 96-well titer plate. The MIC was defined as the lowest concentration of test compound required to inhibit bacterial growth, assessed after 24 hours of incubation at 30°C with shaking (final optical density at 600 nm of 0.05 or less). Optical density measurements were performed using a TECAN Sunrise plate reader. Data were obtained from three independent experiments. The results are summarized in Table 1.

[0040] [Table 1]

[0041] The results, summarized in Table 1, suggest that all tested LGUs exhibit varying levels of activity against the bacterial strains tested. LGUs according to the present invention are active against both Gram-positive and Gram-negative strains. The results also indicate that LGUs according to the present invention are somewhat selective against the Gram-positive strains Staphylococcus aureus ATCC 29213 and Staphylococcus epidermidis ATCC 12228, for which they exhibited by far the highest activity, i.e., the lowest MIC values, of all LGUs tested. This trend is also reflected in their activity against methicillin-resistant Staphylococcus aureus (MRSA) strains, where LOU2 and LOU3 exhibited particularly high activity.

[0042] Exemplary Pharmaceutical Compositions According to the Invention Antibacterial ointments contain the following ingredients: - 40.0 g of 1% boric acid solution - 29.9g lanolin - 29.9g of Eucerin - 0.2 g of Lipooligourea according to the present invention.

[0043] Susceptibility of lipooligourea to proteolysis Lipooligourea stability testing in serum was performed for compounds LOU1-LOU4 using factor-free human serum (obtained from Innovative Research, Inc., USA). Lipooligourea stock solution (0.168 M) was diluted 1:69 with human serum to an incubation concentration of 2.4 μmol / ml for each compound. Samples were then incubated at 37°C, and 50 μL aliquots were withdrawn at designated time intervals (0 min, 24 h, 48 h, 72 h, and 96 h).

[0044] To precipitate serum proteins, 200 μL of ACN / HO / FA (89:10:1, v / v) was added to the collected samples, resulting in a cloudy mixture. These mixtures were vortexed at 3,000 rpm for 1 minute, cooled in a refrigerator for 20 minutes, and then centrifuged at 4°C (14,000 rpm) for 15 minutes to precipitate proteins. 150 μL of supernatant was collected from each sample, and 300 μL of water was added before lyophilization. The lyophilized samples were lyophilized in 150 μL. The samples were redissolved in ACN / HO / FA (10:90:0.01, v / v) and analyzed using RP-HPLC. A gradient of 10% solvent B in A was used for 5 min, followed by a gradient of 10% to 97% solvent B in A for 30 min. Here, solvent A was 0.1% TFA in HO, and solvent B was 0.1% TFA in CHCN. H-Trp-OH and Z-Lys-OH internal standards were used for the analysis. Each experiment was performed once, and two independent experiments were performed for each sample.

[0045] The susceptibility of LOU1-LOU4 compounds to proteolysis was assessed by incubating LOU1 in human serum at 37°C for a total of 96 hours. The progress of enzymatic degradation was monitored for each sample at 24-hour intervals by RP-HPLC, and the change in LOU1 peak area on the chromatogram was measured. The initial area at time t0 was defined as 100%. After 96 hours, the greatest decrease in peak area (approximately 15%) was observed for LOU1. However, since no new peaks corresponding to proteolytic products were detected, this decrease may be due to coprecipitation of LOU1 with serum proteins. In contrast, the peak areas of the other compounds did not change significantly (maximum 8% variation), confirming the expected high resistance of LOU1 to proteolysis in serum. Figure 1 shows the time-dependent change in LOU1 concentration.

Claims

1. Lipooligourea of general formula 1. FA-Phe u -No1 u -No2 u -Leo u -No3 u -NH 2 (1) (FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain, Xaa1 u Lys u or Dab u or Arg u is either Xaa2 u Lys u or Dab u or Leu u or Arg u is either Xaa3 u Lys u or Dab u or Leu u or Arg u Either

2. LOU1 【Chemical 1】 LOU2 【Chemistry 2】 LOU3 【Chemistry 3】 LOU4 【Chemistry 4】 .

2. The lipooligourea of claim 1, selected from the group consisting of:

3. 3. Lipooligourea according to claim 1 or 2 for use as a medicament.

4. 3. The lipooligourea of claim 1 or 2 for use in the treatment or prevention of bacterial infections.

5. 5. The method of claim 4, wherein the bacterial infection is caused by either a gram-positive or a gram-negative bacterium.

6. 6. The method of claim 4, wherein the bacterial infection is caused by Staphylococcus aureus.

7. 7. Lipooligourea for use according to claim 4, 5 or 6, characterized in that the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.

8. 6. Lipooligourea for use according to claim 4 or 5, characterized in that the bacterial infection is caused by a methicillin-resistant Staphylococcus aureus (MRSA) strain.

9. Lipooligourea of formula LOU2 or LOU3 for use in the treatment or prevention of bacterial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains.

10. A pharmaceutical composition comprising a lipooligourea of general formula 1. FA-Phe u -No1 u -No2 u -Leo u -No3 u -NH 2 (1) (FA is a linear or branched carboxylic acid residue containing 6 to 15 carbon atoms in the alkyl chain, Xaa1 u Lys u or Dab u or Arg u is either Xaa2 u Lys u or Dab u or Leu u or Arg u is either Xaa3 u Lys u or Dab u or Leu u or Arg u Either

11. LOU1 【Chemistry 5】 LOU2 【Chemistry 6】 LOU3 【Chemistry 7】 LOU4 【Chemistry 8】 .

11. The pharmaceutical composition according to claim 10, comprising the lipooligourea according to claim 1 selected from the group consisting of:

12. 12. Pharmaceutical composition according to claim 10 or 11, characterized in that it contains from 0.1% to 99% by weight of at least one lipooligourea.

13. 13. Pharmaceutical composition according to any of claims 10 to 12, characterized in that it comprises a pharmaceutically acceptable carrier and / or diluent and / or adjuvant and / or excipient.

14. 14. The pharmaceutical composition according to claim 13, characterized in that it contains corn starch and / or gelatin and / or lactose and / or sucrose and / or crystalline cellulose and / or kaolin and / or mannitol and / or dibasic calcium phosphate and / or sodium chloride and / or alginic acid.

15. 15. Pharmaceutical composition according to any one of claims 10 to 14, characterized in that it is in a form suitable for oral and / or intravenous and / or intramuscular and / or subcutaneous and / or parenteral and / or rectal and / or topical administration.

16. 16. Pharmaceutical composition according to any one of claims 10 to 15, characterized in that it is in a form suitable for tablets and / or capsules and / or elixirs and / or suspensions and / or syrups and / or gels and / or creams and / or ointments and / or suppositories and / or aerosols and / or drops.

17. 17. A pharmaceutical composition according to any one of claims 10 to 16, further comprising at least one other antibiotic.

Images