WO2020159389A1 - Compounds of vancomycin and tp10, methods of their preparation, composition and use in antibacterial treatment - Google Patents

Compounds of vancomycin and tp10, methods of their preparation, composition and use in antibacterial treatment Download PDF

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WO2020159389A1
WO2020159389A1 PCT/PL2020/000010 PL2020000010W WO2020159389A1 WO 2020159389 A1 WO2020159389 A1 WO 2020159389A1 PL 2020000010 W PL2020000010 W PL 2020000010W WO 2020159389 A1 WO2020159389 A1 WO 2020159389A1
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peg
van
group
solution
conjugate
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Izabela RUSIECKA
Ivan KOCIĆ
Jarosław RUCZYŃSKI
Piotr REKOWSKI
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Gdański Uniwersytet Medyczny
Uniwersytet Gdański
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
    • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the subject of the invention comprises conjugates of vancomycin (Van) and transportan 10 (TP10) as new anti-MRSA antibacterial compounds.
  • the subject of the invention also includes the method of their preparation, composition and antibacterial use against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
  • Vancomycin is a glycopeptide antibiotic, primarily active against Gram(+) aerobic and anaerobic bacteria. Vancomycin is often used for treating life-threatening infections caused by such bacteria as Staphylococcus aureus, Enterococcus spp. and Clostridium difficile. Infections with these microorganisms tend to be common in the society, which leads to a number of complications and mortality among patients. Particularly dangerous are infections with methicilin-resistant Staphylococcus aureus (MRSA). They are, e.g. bacterial meningitis, often located in the cerebral tissue and their treatment is the major challenge for contemporary medicine.
  • MRSA methicilin-resistant Staphylococcus aureus
  • Vancomycin and a method of its preparation are known from patent description
  • Vancomycin efficacy is insufficient due to a growing number of multi-drug resistant (MDR) strains occurring in hospitals.
  • MDR multi-drug resistant
  • BBB blood-brain barrier
  • Unfortunately there are no alternative drugs to vancomycin.
  • TP10 vancomycin conjugation with transportan 10 (CPP) group.
  • CPP cell-penetrating peptides
  • TP10 a member of cationic CPP family, is a short, amphipathic peptide (which stands for X-AGYLLGK(X)INLKALAALAKKIL-X-NH 2 ).
  • TP10 is known for its transport capabilities through cellular membrane but the transport mechanism is unknown. TP10 is also able to transport various particles, including drugs, into a cell. Moreover, TP10 has antibacterial properties.
  • Van-TP10 conjugates have resulted in improvement of pharmacokinetic and pharmacodynamic properties when compared to vancomycin alone, while maintaining low cell toxicity. Van-TP10 conjugates demonstrate much better antibacterial effects against MRSA, while the concentration of one of the conjugates in the brain is 200 times higher than the concentration of vancomycin alone, at their simultaneously low toxicity level.
  • the conjugates can be used for treating life-threatening MRSA infections, particularly those occurring in the brain.
  • the new (Van-TP10) compounds can be used as drugs in the pharmaceutical sector and become a therapy alternative to traditional vancomycin.
  • the objective of this invention is to provide a compound in a form of a conjugate, which can be used for treating life-threatening MRSA infections, including those occurring in the brain (a new antibiotic in the glycopeptide group).
  • a new antibiotic in the glycopeptide group a new antibiotic in the glycopeptide group.
  • X - stands for PEG 3 ; PEG 4 ; Ala- PEG 4 TP10 - stands for X-AGYLLGK(X)INLKALAALAKKIL-X-A3 ⁇ 4
  • a compound with V an- PEG 3 -TP10 formula A compound with V an- PEG 3 -TP10 formula.
  • TP10 chain synthesis occurs in two stages: a) TP10 chain synthesis TP10 and its analogues are synthesised using TentaGel S RAM resin, Fmoc- protected amino acids are attached as active derivatives using a 3-fold molar TBTU excess with an addition of HOBt and NMP at the molar ratio of 1 : 1 :2 in DMF for 2x30 min; Fmoc groups are removed using 20% piperidine solution in DMF in 2 cycles (2x3.5 min); furthermore an ivDde group which is labile in hydrazine environment is used to shield the Lys 7 residuee-ami ne group (conjugate IV and IVa synthesis); jV-terminal Ala residue (conjugate IV synthesis) is attached as a Boc- protected amino acid; in the synthesis of IVa conjugate 6-carboxy fluorescein ( Fl) is attached to N -terminal amino group using 3 -fold molar excess of 6- carboxyfluorescein A
  • conjugate I and II synthesis where A-terminal Fmoc group is removed with 20% piperidine solution in DMF (2x5 min), and then a propiolic (Prop) group is attached to A-terminal amine group using a 10-fold molar excess of propiolic acid anhydride in DMF for 1.5 hours; the mixture is stored at 0°C for 10 min; the sediment is drained, and after DCM evaporation the obtained anhydride is dissolved in 5 ml of DMF and added to a reaction vessel with peptidyl-resin; conjugate III modified with an alkyne group is obtained by attaching Fmoc-L-propargylglycine (Fmoc-Prg-OH) derivative to the resin, and then attaching subsequent amino acid residues in theTP10 sequence; conjugate IV and IVa synthesis, ivDde group is removed with 10% hydrazine monohydrate solution in DMF (3x20 min), and then an alkyne group is attached to Lys 7 residue
  • Peptides are cleaved from resin with a simultaneous removal of functional groups shields in the side chains by means of a mixture of trifluoroacetic (TFA) acid, phenol, triisopropylsilane and water (molar ratio: 88:5:2:5) for 2 hours; the peptides are precipitated from the reaction mixture by means of cold diethyl ether; then the sediment is drained, dissolved in water and lyophilised; raw peptides are analysed and purified by means of High Performance Liquid Chromatography in a reversed phase array; the identity of obtained products is confirmed with mass spectrometry.
  • TFA trifluoroacetic
  • iV-hydroxysuccinimide ester of 15-azide-4, 7,10,13- tetraoxapentadecanoic acid (N 3 -PEG 4 -NHS) is attached to vancomycin hydrochloride through a primary amine group in the sugar fragment, the reaction was carried out in an aqueous solution containing DIPEA (molar ratios of the reagents 1:1.5:2.5), the solution was mixed at 4°C for 30 min;
  • Van-PEG 3 -N 3 derivative by attaching 1 -amine- 11 -azide-3, 6, 9-trioxaundecane (N 3 -PEG 3 -NH 2 ) to vancomycin hydrochloride through a carboxyl group in the aglycone fragment, a reaction is carried out in a DMF solution using HATU ( 2-(1 H- 9-azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate) with DIPEA addition (molar ratio: 1 :0,8:1 :2), the solution is mixed at room temperature for 1 hour, and after the time period the reaction products are immediately separated by means of preparative RP-HPLC; eluates are fractioned and analysed by means of analytic RP-HPLC,
  • Van-PEG 4 -N 3 or Van-PEG 3 -N 3 fractions with purity over 98% are combined and lyophilised, and the identity of the obtained products is confirmed by means of mass spectrometry.
  • a pharmaceutical composition which contains the abovementioned new compound and a pharmaceutically acceptable carrier.
  • composition is used for antibacterial treatment, preferably against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
  • Fig. 1 shows a chemical structure of the synthesised conjugates.
  • Conjugate a) Van-PEG 3 -TP10 (conjugate I);
  • Fig. 2 shows antibacterial activity of [Lys 7 (PEG 4 -Van)]TP10 against intracellularly located MRS A 12673 strains
  • Fig. 3 shows antibacterial activity of [Lys 7 (PEG 4 -Van)]TP10 against intracellularly located MRSA h-VISA 6347 strains.
  • Fig.4 shows fluorescent images of Fl-[Lys 7 (PEG 4 -Van)]TP10 distribution in mouse brain sections (4x objective).
  • TOP Lower part of the right brain hemisphere with the olfactory tract.
  • BOTTOM Middle part of the right hemisphere with striatum. The images are a representative of three independent experiments.
  • the scale bar reflects 5 mm in the brain images.
  • Fig. 5 shows haemolytic activity of Van, Van-PEG3-TP20 and Van-PEG4-TP10 in the concentration range of 100-0.049 mM measured spectrophotometrically at 450 nm. ⁇ Van - 0% haemolysis in the whole concentration range
  • Table 1 shows a primary structure of prepared Van-TP10 conjugate
  • Table 2 shows the prepared conjugates, their molecular weights and conjugation reaction efficiency.
  • Table 3 shows antibacterial activity of Van-TP10 conjugates
  • Table 4 shows [Lys 7 (PEG 4 -Van)]TP10, Van and TP10 concentration in murine brains homogenates.
  • Van conjugates with TP10 were prepared, with structures presented in Table 1 and Fig.1.
  • TP10 and its analogues were synthesised using a standard procedure of peptide synthesis (SPPS) in solid phase, using an automatic peptide synthesiser (Quartet, Protein Technologies Inc.) and TentaGel S RAM resin (with 0.25 mmol/g amine seatability).
  • SPPS standard procedure of peptide synthesis
  • Fmoc-protected amino acids were attached as active derivatives using 3- fold molar excess of TBTU (2-( 1H -benzotri azole- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium tetrafluoroborate) with an addition of HOBt (1-hydroxybenzotriazole) and NMP (N - methylmorpholine) (molar ratio: 1:1:2) in DMF (TV,TV-dimethylformamide) for 2x30 min.
  • Fmoc (fluorenyl-9-methoxycarbonyl) groups were removed using 20% piperidine solution in DMF in 2 cycles (2x3.5 min).
  • ivDde (l-(4,4- dimethyl-2,6-dioxycyclohexadiene)-3-methylbutyl) group labile in hydrazine environment was used to shield the Lys 7 residue e-amine group instead of a standard Boc (tert-butyloxycarbonyl) group labile in acidic environment (conjugate IV and Iva synthesis).
  • Boc tert-butyloxycarbonyl
  • N - terminal Fmoc group was removed with 20% piperidine solution in DMF (2*5 min), and then a propiolic (Prop) group was attached to N - terminal amine group using a 10-fold molar excess of propiolic acid anhydride in DMF for 1.5 hours.
  • Propiolic acid anhydride was prepared by mixing TV,TV'-diisopropylcarbodiimide (DIC) with propiolic acid (molar ratio: 1 :2) in dichloromethane (DCM). The mixture was stored at 0°C for 10 min.
  • Conjugate III modified with an alkyne group was prepared by attaching Fmoc-L-propargylglycine (Fmoc-Prg-OH) derivative to the resin, and then by attaching subsequent amino acid residues in TP10 sequence.
  • Peptides were cleaved from resin with a simultaneous removal of functional groups shields in the side chains by means of a mixture of trifluoroacetic (TFA) acid, phenol, triisopropylsilane and water (molar ratio: 88:5:2:5) for 2 hours.
  • TFA trifluoroacetic
  • the peptides were precipitated from the reaction mixture by means of cold diethyl ether. Then the sediment was drained, dissolved in water and lyophilised.
  • Raw peptides were analysed and purified by means of High Performance Liquid Chromatography in a reversed phase array (RP-HPLC). The identity of the obtained products was confirmed with mass spectrometry, MALDI-TOF method (Bruker Daltonics, HCT Ultra design) or ESI method (ABSciex, TripleTOF 5600+).
  • Van derivatives modified with an azide group were synthesised in a solution.
  • Van-PEG 4 -N 3 derivative 15-azide-4,7,10,13-tetraoxapentadecanoic acid A-hydroxysuccinimide ester (N 3 -PEG 4 -NHS) was attached to vancomycin hydrochloride through the primary amine group in the sugar fragment.
  • the reaction was carried out in an aqueous solution containing DIPEA (molar ratios of the reagents 1 :1.5:2.5). The solution was mixed at 4°C for 30 min.
  • Van-PEG 3 -N 3 derivative was in turn prepared by attaching 1 -amine- 11 -azide-3, 6, 9-trioxaundecane (N 3 -PEG 3 -NH 2 ) to vancomycin hydrochloride through a carboxyl group in the aglycone fragment.
  • the reaction was carried out in a DMF solution using HATU (2- ( 1 H-9-azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate) with DIPEA addition (molar ratio: 1 :0,8 : 1 :2).
  • the solution was mixed at room temperature for 1 hour.
  • Fig. 2 Overview of 1 ,3-dipolar Huisgen cycloaddition, catalysed with copper (I) ions ("click reaction"); where R - Van, R 1 - TP10 with amino acid sequence as presented in Fig. 1.
  • the synthesised compounds were purified in Reprosil 100 C18 column (Dr. Maisch GmbH, 40x250 mm, grain size 10 mm, mobile phase flow rate 25 mL/min) or reprosil 100 C18-XBD column (Dr. Maisch GmbH, 20x250 mm, grain size 10 mm, mobile phase flow rate 10 mL/min) using SpotPrep (Armen) chromatograph and various gradient methods.
  • the mobile phase consisted of 0.08% TFA in acetonitrile (ACN) (solution A) and 0.1% TFA in water (solution B).
  • the column was maintained at ambient temperature.
  • the eluates were fractio ned and analysed by means of analytic RP-HPLC.
  • MRSA N3 15 is a reference strain sensitive to Van
  • MRSA 12673 is clinical strain with moderate sensitivity to Van
  • MRSA 6347 is clinical h-VISA strain with moderate resistance to Van. All results were compared to the Van alone.
  • the tested conjugates were checked for their ability to penetrate into eukaryotic cells.
  • a human cell line HEK293 was used; it was infected with MRS A 12673 strain and then the intracellular antibacterial efficacy of one of the tested conjugates was checked (Fig. 2).
  • the conjugate concentration in the brain was over 200 times higher than Van concentration, which is testimony to a very high potential of penetrating through the BBB.
  • the synthesised conjugates revealed higher antibacterial activity against clinical MRSA strains, both those located extra- and intracellularly. Moreover, the conjugates are capable of penetrating through the BBB (tested on a selected conjugate), reaching 200 times higher concentrations in the brain than Van alone. This fact is particularly important for MRS A infections located in the brain. It should be emphasised that the toxicity of the tested conjugates in low (therapeutic) concentrations is similar to Van toxicity.

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Abstract

The subject of the invention comprises new compounds forming Van-TPlO conjugates, method of their preparation, composition and use in antibacterial treatment against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.

Description

COMPOUNDS OF VANCOMYCIN AND TP10, METHODS OF THEIR PREPARATION, COMPOSITION AND USE IN ANTIBACTERIAL
TREATMENT
The subject of the invention comprises conjugates of vancomycin (Van) and transportan 10 (TP10) as new anti-MRSA antibacterial compounds. The subject of the invention also includes the method of their preparation, composition and antibacterial use against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
Vancomycin is a glycopeptide antibiotic, primarily active against Gram(+) aerobic and anaerobic bacteria. Vancomycin is often used for treating life-threatening infections caused by such bacteria as Staphylococcus aureus, Enterococcus spp. and Clostridium difficile. Infections with these microorganisms tend to be common in the society, which leads to a number of complications and mortality among patients. Particularly dangerous are infections with methicilin-resistant Staphylococcus aureus (MRSA). They are, e.g. bacterial meningitis, often located in the cerebral tissue and their treatment is the major challenge for contemporary medicine.
Figure imgf000003_0001
Fig.l. Vancomycin structure
Vancomycin and a method of its preparation are known from patent description
US 3067099 B. Vancomycin efficacy is insufficient due to a growing number of multi-drug resistant (MDR) strains occurring in hospitals. There are many constraints related to using vancomycin for treating MRSA infected patients— besides the previously mentioned insufficient efficacy as a result of quickly developing resistance to the antibiotic— including its poor blood-brain barrier (BBB) penetration, which limits its use for treating brain infections, such as bacterial meningitis. Unfortunately, there are no alternative drugs to vancomycin.
That is why it has become a challenge to create modified vancomycin with higher antibacterial efficacy and good penetration into the brain tissue.
The improved properties have been achieved by vancomycin conjugation with transportan 10 (TP10), a member of cell-penetrating peptides (CPP) group. TP10, a member of cationic CPP family, is a short, amphipathic peptide (which stands for X-AGYLLGK(X)INLKALAALAKKIL-X-NH2).
TP10 is known for its transport capabilities through cellular membrane but the transport mechanism is unknown. TP10 is also able to transport various particles, including drugs, into a cell. Moreover, TP10 has antibacterial properties.
The development of Van-TP10 conjugates has resulted in improvement of pharmacokinetic and pharmacodynamic properties when compared to vancomycin alone, while maintaining low cell toxicity. Van-TP10 conjugates demonstrate much better antibacterial effects against MRSA, while the concentration of one of the conjugates in the brain is 200 times higher than the concentration of vancomycin alone, at their simultaneously low toxicity level.
The conjugates can be used for treating life-threatening MRSA infections, particularly those occurring in the brain.
The new (Van-TP10) compounds can be used as drugs in the pharmaceutical sector and become a therapy alternative to traditional vancomycin.
The objective of this invention is to provide a compound in a form of a conjugate, which can be used for treating life-threatening MRSA infections, including those occurring in the brain (a new antibiotic in the glycopeptide group). Unexpectedly, the problem was solved to a great extent by this invention. The subject of the invention includes new compounds with the following general formula:
Figure imgf000005_0001
where:
X - stands for PEG3; PEG4; Ala- PEG4 TP10 - stands for X-AGYLLGK(X)INLKALAALAKKIL-X-A¾
A compound with V an- PEG3-TP10 formula.
A compound with Van-PEG4-TP10 formula.
A compound with TP10-Ala(PEG4-Van) formula.
A compound with [Lys7(PEG4-Van)]TP10 formula.
A method of preparing the abovementioned new compounds, where the synthesis ofTP10 chain and vancomycin structure modification occur, followed by conjugation of both compounds.
A method where TP10 chain synthesis occurs in two stages: a) TP10 chain synthesis TP10 and its analogues are synthesised using TentaGel S RAM resin, Fmoc- protected amino acids are attached as active derivatives using a 3-fold molar TBTU excess with an addition of HOBt and NMP at the molar ratio of 1 : 1 :2 in DMF for 2x30 min; Fmoc groups are removed using 20% piperidine solution in DMF in 2 cycles (2x3.5 min); furthermore an ivDde group which is labile in hydrazine environment is used to shield the Lys7 residuee-ami ne group (conjugate IV and IVa synthesis); jV-terminal Ala residue (conjugate IV synthesis) is attached as a Boc- protected amino acid; in the synthesis of IVa conjugate 6-carboxy fluorescein ( Fl) is attached to N -terminal amino group using 3 -fold molar excess of 6- carboxyfluorescein A-hydroxysuccinimide ester (Fl-NHS) with an addition of DIPEA, in a molar ratio of 1 : 1 in DMF for 1.5 hours. b) TP10 modification with alkyne group
conjugate I and II synthesis where A-terminal Fmoc group is removed with 20% piperidine solution in DMF (2x5 min), and then a propiolic (Prop) group is attached to A-terminal amine group using a 10-fold molar excess of propiolic acid anhydride in DMF for 1.5 hours; the mixture is stored at 0°C for 10 min; the sediment is drained, and after DCM evaporation the obtained anhydride is dissolved in 5 ml of DMF and added to a reaction vessel with peptidyl-resin; conjugate III modified with an alkyne group is obtained by attaching Fmoc-L-propargylglycine (Fmoc-Prg-OH) derivative to the resin, and then attaching subsequent amino acid residues in theTP10 sequence; conjugate IV and IVa synthesis, ivDde group is removed with 10% hydrazine monohydrate solution in DMF (3x20 min), and then an alkyne group is attached to Lys7 residue f-amine group using 10-fold molar excess of propiolic acid anhydride; c) Peptides cleaving from resin
Peptides are cleaved from resin with a simultaneous removal of functional groups shields in the side chains by means of a mixture of trifluoroacetic (TFA) acid, phenol, triisopropylsilane and water (molar ratio: 88:5:2:5) for 2 hours; the peptides are precipitated from the reaction mixture by means of cold diethyl ether; then the sediment is drained, dissolved in water and lyophilised; raw peptides are analysed and purified by means of High Performance Liquid Chromatography in a reversed phase array; the identity of obtained products is confirmed with mass spectrometry.
A method in which vancomycin structure modification and preparation of its derivatives modified with an azide group will be synthesised in a solution,
- for Van-PEG4-N3 derivative, iV-hydroxysuccinimide ester of 15-azide-4, 7,10,13- tetraoxapentadecanoic acid (N3-PEG4-NHS) is attached to vancomycin hydrochloride through a primary amine group in the sugar fragment, the reaction was carried out in an aqueous solution containing DIPEA (molar ratios of the reagents 1:1.5:2.5), the solution was mixed at 4°C for 30 min;
- for Van-PEG3-N3 derivative, by attaching 1 -amine- 11 -azide-3, 6, 9-trioxaundecane (N3-PEG3-NH2) to vancomycin hydrochloride through a carboxyl group in the aglycone fragment, a reaction is carried out in a DMF solution using HATU ( 2-(1 H- 9-azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate) with DIPEA addition (molar ratio: 1 :0,8:1 :2), the solution is mixed at room temperature for 1 hour, and after the time period the reaction products are immediately separated by means of preparative RP-HPLC; eluates are fractioned and analysed by means of analytic RP-HPLC,
- Van-PEG4-N3 or Van-PEG3-N3 fractions with purity over 98% are combined and lyophilised, and the identity of the obtained products is confirmed by means of mass spectrometry.
A method in which compounds are conjugated through reaction of TP10 analogues (0.8 mM) modified with an alkyne group with a modified azide group of (0.4 mM) Van-PEG3-N3 (conjugate I) or Van-PEG4-N3 (conjugates II-IV) derivatives was carried out in 1.5 ml of water/tert-butanol (1:1 v/v) mixture in the presence of 8 pL of 0.1 M CuSO4xH20 solution and 4 mL of fresh 0.5 M solution of ascorbic acid (molar ratio: 2: 1 :2:5); the solution is mixed at a room temperature for about 24 hours; once 1,2, 3 -triazole formation is complete, the solvent is evaporated and synthesis products are lyophilised; the obtained raw conjugates are purified with preparative or semi-preparative RP-HPLC; the purity of the compounds is checked by means of analytic RP-HPLC, while the molecular weight of the obtained compounds is confirmed by means of mass spectrometry. The abovementioned new compounds for use in antibacterial treatment, preferably against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
An application where antibacterial treatment has intracellular action.
A pharmaceutical composition which contains the abovementioned new compound and a pharmaceutically acceptable carrier.
The composition is used for antibacterial treatment, preferably against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
Description of drawings and tables:
Fig. 1 shows a chemical structure of the synthesised conjugates.
Conjugate: a) Van-PEG3-TP10 (conjugate I);
b) Van-PEG4-TP10 (conjugate II);
c) TP10-Ala(PEG4-Van) (conjugate III);
d) [Lys7(PEG4-Van)]TP10 (conjugate IV);
e) Fl[Lys7(PEG4-Van)]TP10 (conjugate IVa)
Fig. 2 shows antibacterial activity of [Lys7(PEG4-Van)]TP10 against intracellularly located MRS A 12673 strains
* statistical significance ( p<0.05) as compared to control (without treatment) and Van
Fig. 3 shows antibacterial activity of [Lys7(PEG4-Van)]TP10 against intracellularly located MRSA h-VISA 6347 strains.
* statistical significance ( p<0.05) as compared to control (without treatment) and Van
Fig.4 shows fluorescent images of Fl-[Lys7(PEG4-Van)]TP10 distribution in mouse brain sections (4x objective). (TOP) Lower part of the right brain hemisphere with the olfactory tract. (BOTTOM) Middle part of the right hemisphere with striatum. The images are a representative of three independent experiments. The scale bar reflects 5 mm in the brain images.
Fig. 5 shows haemolytic activity of Van, Van-PEG3-TP20 and Van-PEG4-TP10 in the concentration range of 100-0.049 mM measured spectrophotometrically at 450 nm. ^Van - 0% haemolysis in the whole concentration range
* statistical significance ( p<0.05) as compared to Van
Table 1 shows a primary structure of prepared Van-TP10 conjugate
Table 2 shows the prepared conjugates, their molecular weights and conjugation reaction efficiency.
Table 3 shows antibacterial activity of Van-TP10 conjugates
Table 4 shows [Lys7(PEG4-Van)]TP10, Van and TP10 concentration in murine brains homogenates.
The following, non-limiting examples illustrate various embodiments of the invention.
Example 1
Synthesised conjugates
4 Van conjugates with TP10 were prepared, with structures presented in Table 1 and Fig.1.
Table.1. Primary structure of the prepared TP10 conjugates with Van
Figure imgf000009_0001
FI - fluorescein, PEG4 - 4,7,10,13-tetraoxapentadecanoic linker, PEG3 - linker 3,6,9-trioxaundecane linker, Tra - 1,2,3-triazol ring TP10 chain synthesis
TP10 and its analogues were synthesised using a standard procedure of peptide synthesis (SPPS) in solid phase, using an automatic peptide synthesiser (Quartet, Protein Technologies Inc.) and TentaGel S RAM resin (with 0.25 mmol/g amine seatability). Fmoc-protected amino acids were attached as active derivatives using 3- fold molar excess of TBTU (2-( 1H -benzotri azole- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium tetrafluoroborate) with an addition of HOBt (1-hydroxybenzotriazole) and NMP (N - methylmorpholine) (molar ratio: 1:1:2) in DMF (TV,TV-dimethylformamide) for 2x30 min. Fmoc (fluorenyl-9-methoxycarbonyl) groups were removed using 20% piperidine solution in DMF in 2 cycles (2x3.5 min). Moreover, ivDde (l-(4,4- dimethyl-2,6-dioxycyclohexadiene)-3-methylbutyl) group labile in hydrazine environment was used to shield the Lys7 residue e-amine group instead of a standard Boc (tert-butyloxycarbonyl) group labile in acidic environment (conjugate IV and Iva synthesis). With regard to the fact that hydrazine removes the Fmoc group, the N - terminal Ala residue (conjugate IV synthesis) was attached as Boc-protected amino acid. In the synthesis of conjugate IVa 6-carboxyfluoroscein ((FI) was attached to N - terminal amine group using a 3 -fold molar excess of 6- carboxyfluoroscein TV-hydroxysuccinimide ester (F1-NHS) with DIPEA (N,N- diisopropylethylamine) addition (molar ratio: 1 : 1) in DMF for 1.5 hours.
TP 10 modification with alkyne group
In the case of conjugate I and II synthesis, N - terminal Fmoc group was removed with 20% piperidine solution in DMF (2*5 min), and then a propiolic (Prop) group was attached to N - terminal amine group using a 10-fold molar excess of propiolic acid anhydride in DMF for 1.5 hours. Propiolic acid anhydride was prepared by mixing TV,TV'-diisopropylcarbodiimide (DIC) with propiolic acid (molar ratio: 1 :2) in dichloromethane (DCM). The mixture was stored at 0°C for 10 min. The sediment was drained, and after DCM evaporation the obtained anhydride was dissolved in 5 ml of DMF and added to the reaction vessel with peptidyl-resin. Conjugate III modified with an alkyne group was prepared by attaching Fmoc-L-propargylglycine (Fmoc-Prg-OH) derivative to the resin, and then by attaching subsequent amino acid residues in TP10 sequence. In the case of conjugate IV and IVa synthesis, ivDde group was removed with a 10% hydrazine monohydrate solution in DMF (3 x20 min), and then an alkyne group was attached to Lys7 residue famine group using 10-fold molar excess of propiolic acid anhydride (prepared as described above).
Peptides cleaving from resin
Peptides were cleaved from resin with a simultaneous removal of functional groups shields in the side chains by means of a mixture of trifluoroacetic (TFA) acid, phenol, triisopropylsilane and water (molar ratio: 88:5:2:5) for 2 hours. The peptides were precipitated from the reaction mixture by means of cold diethyl ether. Then the sediment was drained, dissolved in water and lyophilised. Raw peptides were analysed and purified by means of High Performance Liquid Chromatography in a reversed phase array (RP-HPLC). The identity of the obtained products was confirmed with mass spectrometry, MALDI-TOF method (Bruker Daltonics, HCT Ultra design) or ESI method (ABSciex, TripleTOF 5600+).
Modification of Van structure
Van derivatives modified with an azide group were synthesised in a solution. In the case of Van-PEG4-N3 derivative, 15-azide-4,7,10,13-tetraoxapentadecanoic acid A-hydroxysuccinimide ester (N3-PEG4-NHS) was attached to vancomycin hydrochloride through the primary amine group in the sugar fragment. The reaction was carried out in an aqueous solution containing DIPEA (molar ratios of the reagents 1 :1.5:2.5). The solution was mixed at 4°C for 30 min. Van-PEG3-N3 derivative was in turn prepared by attaching 1 -amine- 11 -azide-3, 6, 9-trioxaundecane (N3-PEG3-NH2) to vancomycin hydrochloride through a carboxyl group in the aglycone fragment. The reaction was carried out in a DMF solution using HATU (2- ( 1 H-9-azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate) with DIPEA addition (molar ratio: 1 :0,8 : 1 :2). The solution was mixed at room temperature for 1 hour. After the time period the reaction products were immediately separated by means of preparative RP-HPLC. The eluates were fractioned and analysed by means of analytic RP-HPLC. Van-PEG4-N3 or Van-PEG3-N3 fractions with purity over 98% were combined and lyophilised. The identity of the obtained products was confirmed by means of mass spectrometry, MALDI-TOF or ESI method. Van conjugation with TP 10
A specific 1,3-dipolar reaction of Huisgen cycloaddition, also known as a "click reaction", catalysed with copper Cu(I) ions, was used for covalent conjugation of TP10 with vancomycin (Van). (Fig. 2). The method is quick, highly effective as well as regio- and chemoselective in mild reaction conditions.
Figure imgf000012_0001
Fig. 2. Overview of 1 ,3-dipolar Huisgen cycloaddition, catalysed with copper (I) ions ("click reaction"); where R - Van, R1 - TP10 with amino acid sequence as presented in Fig. 1.
Reactions of TP10 analogues modified with an alkyne group (0.8 mM each time) with a modified azide group deriving from (0.4 mM) Van-PEG3-N3 (conjugate I) or Van-PEG4-N3 (conjugates II-IV) were carried out in 1.5 ml of water /tert- butanol (1 :1 v/v) mixture in the presence of 8 mL of 0.1 M CuSO4xH2O solution and 4 mL of fresh 0.5 M solution of ascorbic acid (molar ratio: 2: 1:2: 5). The solution was mixed at a room temperature for about 24 hours. Once 1,2, 3 -triazole formation was complete, the solvent was evaporated and synthesis products were lyophilised. The obtained raw conjugates were purified with preparative or semi-preparative RP- HPLC. The purity of the compounds was checked by means of analytic RP-HPLC (using different acetonitrile-water gradient methods), and their purity was estimated to exceed 98%. The molecular weights of the obtained compounds were confirmed by means of mass spectrometry, ESI or MALDI-TOF. Table 2 presents a comparison of molecular weights of the prepared conjugates, calculated and determined experimentally, as well as conjugation capacity. Table 2. Comparison of molecular weights of the prepared conjugate and conjugation capacity
Figure imgf000013_0001
* obtained with MALDI-TOF mass spectrometry
** only fractions with HLPC purity over 98% were taken into consideration
Analysis and purification of reaction products
The synthesised compounds were purified in Reprosil 100 C18 column (Dr. Maisch GmbH, 40x250 mm, grain size 10 mm, mobile phase flow rate 25 mL/min) or reprosil 100 C18-XBD column (Dr. Maisch GmbH, 20x250 mm, grain size 10 mm, mobile phase flow rate 10 mL/min) using SpotPrep (Armen) chromatograph and various gradient methods. The mobile phase consisted of 0.08% TFA in acetonitrile (ACN) (solution A) and 0.1% TFA in water (solution B). The column was maintained at ambient temperature. The eluted solutions were monitored with an UV detector at l = 220 and l = 254 nm. The eluates were fractio ned and analysed by means of analytic RP-HPLC.
Analytic separations were performed in Kinetex XB-C18 column (Phenomenex, 4.6x150 mm, grain size 5 mm, mobile phase flow rate 1 mL/min) using Shimadzu Prominence chromatograph and different gradient methods. The mobile phase, column temperature and eluate monitoring parameters were the same as in preparative separation. Example 2
Checking antibacterial activity of the prepared conjugates
In order to check antibacterial action of the prepared conjugates, a MIC in vitro test was performed on three MRS A bacteria strains (N315, 12673 and 6347). MRSA N3 15 is a reference strain sensitive to Van; MRSA 12673 is clinical strain with moderate sensitivity to Van; MRSA 6347 is clinical h-VISA strain with moderate resistance to Van. All results were compared to the Van alone.
Table 3.
Figure imgf000014_0001
1 reference strain
2 clinical strain
* statistical significance ( p<0.05) as compared to Van
A statistically significant improvement in antibacterial action was achieved in the case of clinical strains for all tested conjugates, as compared to traditional Van. Example 3
Evaluation of intracellular antibacterial action efficacy of the tested conjugates
In relation to the fact that S. aureus is an intracellular pathogen, the tested conjugates were checked for their ability to penetrate into eukaryotic cells. To that end a human cell line HEK293 was used; it was infected with MRS A 12673 strain and then the intracellular antibacterial efficacy of one of the tested conjugates was checked (Fig. 2).
This test revealed 70% improvement in MRSA 12673 intracellular bacteria inactivation as compared to Van alone, at a concentration of 32xMIC. The other clinical strain, MRSA h-VISA 6347, also demonstrated a significant antibacterial improvement at 8xMIC (Fig.3).
Example 4
Qualitative evaluation of Fl-[Lvs7(PEG4-VanilTP10 conjugate penetration through the blood-brain barrier (Ί3BB)
In relation to the fact that life-threatening MRA infections are localised in the brain, and Van alone penetrates poorly through the BBB, it was essential for the prepared conjugates to penetrate into the brain tissue. To that end [Lys7(PEG4- Van)]TP10 conjugate was selected and combined with a fluorescent dye - fluorescein (FI). The tested conjugate was injected in the tail vein in mice, and after two hours the animals' brains were isolated to test the conjugate presence by means of fluorescent microscopy. The conjugate presence was revealed by the green colour in the brain tissue preparations (Fig. 4).
Example 5
Qualitative evaluation of [Lys7(PEG4- V anllTP10 conjugate penetration through the blood-brain barrier (BBB)
In order to evaluate the conjugate amount in the brain tissue quantitatively, as compared to Van alone, a murine model was used. The tested conjugate was injected to the tail vein and after 2 hours the animals' brains were isolated, homogenised and analysed by means of LC-MS. The results are presented in Tab. 4.
Table 4.
Figure imgf000016_0001
ND - not detected
* statistical significance ( p<0.05) as compared to Van
The conjugate concentration in the brain was over 200 times higher than Van concentration, which is testimony to a very high potential of penetrating through the BBB.
Example 6
Testing the toxicity of selected conjugates
In order to check the safety of use of the tested conjugates, an erythrocyte lysis test was performed. Two conjugates: Van-PEG3-TP10 and Van-PEG4-TP10 were selected for the test. According to the literature data they could prove to be most toxic due to orthogonal arrangement of substituents. Both conjugates turned out to be non-toxic in the applied antibacterial concentration range, whereas the conjugate with PEG4 linker demonstrates higher safety of use (Fig. 5).
Conclusions
The synthesised conjugates revealed higher antibacterial activity against clinical MRSA strains, both those located extra- and intracellularly. Moreover, the conjugates are capable of penetrating through the BBB (tested on a selected conjugate), reaching 200 times higher concentrations in the brain than Van alone. This fact is particularly important for MRS A infections located in the brain. It should be emphasised that the toxicity of the tested conjugates in low (therapeutic) concentrations is similar to Van toxicity.

Claims

Claims:
1. A new compound with the general formula:
Figure imgf000018_0001
where:
X - stands for PEG3; PEG4; Ala-PEG4
TP10 - stands for X-AGYLLGK(X)INLKALAALAKKIL-X-NH2
2. A compound according to Claim 1, characterised in that its formula is Van-PEGs-TPlO.
3. A compound according to Claim 1, characterised in that its formula is Van-PEG4-TP10.
4. A compound according to Claim 1 , characterised in that its formula is TP10- Ala(PEG4-Van) .
5. A compound according to Claim 1, characterised in that its formula is [Ly s7 (PEG4-Van)]TP10.
6. A method of preparing new compounds, defined in Claim 1 , characterised in that a synthesis of TP10 chain and modification of vancomycin structure occur, followed by conjugation of both compounds.
7. A method according to Claim 6, characterised in that TP10 chain synthesis includes the following stages:
a) TP10 chain synthesis
TP10 and its analogues are synthesised using TentaGel S RAM resin, Fmoc-protected amino acids are attached as active derivatives using a 3-fold molar TBTU excess with an addition of HOBt and NMP at the molar ratio of 1 : 1 :2 in DMF for 2x30 min; Fmoc groups are removed using 20% piperidine solution in DMF in 2 cycles (2x 3.5 min); furthermore an ivDde group which is labile in hydrazine environment is used to shield the Lys7 residue e-amine group (conjugate IV and IVa synthesis); N - terminal Ala residue (conjugate IV synthesis) is attached as a Boc-protected amino acid; in the synthesis of IVa conjugate 6-carboxyfluorescein (FI) is attached to N - terminal amino group using 3 -fold molar excess of 6-carboxyfluorescein TV- hydroxysuccinimide ester (Fl-NHS) with an addition of DIPEA, in a molar ratio of 1 : 1 in DMF for 1.5 hours; b) TP10 modification with alkyne group
conjugate I and II synthesis where N - terminal Fmoc group is removed with 20% piperidine solution in DMF (2*5 min), and then a propiolic (Prop) group is attached to N - terminal amine group using a 10-fold molar excess of propiolic acid anhydride in DMF for 1.5 hours; the mixture is stored at 0°C for 10 min; the sediment is drained, and after DCM evaporation the obtained anhydride is dissolved in 5 ml of DMF and added to a reaction vessel with peptidyl-resin; conjugate III modified with an alkyne group is obtained by attaching Fmoc-L- propargylglycine (Fmoc-Prg-OH) derivative to the resin, and then attaching subsequent amino acid residues in the TP10 sequence; conjugate IV and IVa synthesis, ivDde group is removed with 10% hydrazine monohydrate solution in DMF (3x20 min), and then an alkyne group is attached to Lys7 residue famine group using 10-fold molar excess of propiolic acid anhydride; c) Peptides cleaving from resin
peptides are cleaved from resin with a simultaneous removal of functional groups shields in the side chains by means of a mixture of trifluoroacetic (TFA) acid, phenol, triisopropylsilane and water (molar ratio: 88:5:2:5) for 2 hours; the peptides are precipitated from the reaction mixture by means of cold diethyl ether; then the sediment is drained, dissolved in water and lyophilised; raw peptides are analysed and purified by means of High Performance Liquid Chromatography in a reversed phase array; the identity of the obtained products is confirmed with mass spectrometry.
8. A method according to claim 6 characterised in that modification of vancomycin structure and preparation of its derivatives modified with an azide group will be synthesised in a solution,
- for Van-PEG4-N3 derivative, A-hydroxysuccinimide ester of 15-azide- 4,7,10,13-tetraoxapentadecanoic acid (N3-PEG4-NHS) is attached to vancomycin hydrochloride through a primary amine group in the sugar fragment, the reaction was carried out in an aqueous solution containing DIPEA (molar ratios of the reagents 1:1.5:2.5), the solution was mixed at 4°C for 30 min;
- for Van-PEG3-N3 derivative, by attaching 1 -amine- 11 -azide-3 ,6,9- trioxaundecane (N3-PEG3-NH2) to vancomycin hydrochloride through a carboxyl group in the aglycone fragment, a reaction is carried out in a DMF solution using HATH ( 2-(lA-9-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate) with DIPEA addition (molar ratio: 1 :0, 8:1:2), the solution is mixed at room temperature for 1 hour, and after the time period the reaction products are immediately separated by means of preparative RP-HPLC; eluates are fractioned and analysed by means of analytic RP-HPLC, - Van-PEG4-N3 or Van-PEG3-N3 fractions with purity over 98% are combined and lyophilised, and the identity of the obtained products is confirmed by means of mass spectrometry.
9. A method according to claim 6 characterised in that compounds are conjugated through reaction ofTP10 analogues (0.8 mM) modified with an alkyne group with a modified azide group of (0.4 mM) Van-PEG3-N3 (conjugate I) or Van-PEG4-N3 (conjugates II-IV) derivatives was carried out in 1.5 ml of water/ tert-butanol (1:1 v/v) mixture in the presence of 8 pL of 0.1 M CUSO4xH2O solution and 4 pL of fresh 0.5 M solution of ascorbic acid (molar ratio: 2: 1 :2:5); the solution is mixed at a room temperature for about 24 hours; once 1 ,2,3-triazole formation is complete, the solvent is evaporated and synthesis products are lyophilised; the obtained raw conjugates are purified with preparative or semi -preparative RP-HPLC; the purity of the compounds is checked by means of analytic RP-HPLC, while the molecular weight of the obtained compounds is confirmed by means of mass spectrometry.
10. New compounds defined in Claim 1 to be used in antibacterial treatment, preferably against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
11. Application according to Claim 10, characterised in that antibacterial treatment has intracellular action.
12. A pharmaceutical composition which comprises a new compound defined in Claims 1-5 and a pharmaceutically acceptable carrier.
13. Composition according to Claim 12, characterised in that it is used in antibacterial treatment, preferably against Staphylococcus aureus, Enterococcus spp. and Neisseria spp.
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