WO1988000950A2 - Fabrication de conjugues a liaisons de covalence a partir d'un antibiotique et d'un derive non toxique de la polymyxine b - Google Patents

Fabrication de conjugues a liaisons de covalence a partir d'un antibiotique et d'un derive non toxique de la polymyxine b Download PDF

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Publication number
WO1988000950A2
WO1988000950A2 PCT/CH1987/000095 CH8700095W WO8800950A2 WO 1988000950 A2 WO1988000950 A2 WO 1988000950A2 CH 8700095 W CH8700095 W CH 8700095W WO 8800950 A2 WO8800950 A2 WO 8800950A2
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WO
WIPO (PCT)
Prior art keywords
polymyxin
antibiotic
hydrophobic
derivative
coupling
Prior art date
Application number
PCT/CH1987/000095
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German (de)
English (en)
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WO1988000950A3 (fr
Inventor
Jean-Luc Fauchere
Klaus Mosbach
Original Assignee
Fauchere Jean Luc
Klaus Mosbach
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fauchere Jean Luc, Klaus Mosbach filed Critical Fauchere Jean Luc
Publication of WO1988000950A2 publication Critical patent/WO1988000950A2/fr
Publication of WO1988000950A3 publication Critical patent/WO1988000950A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related peptides

Definitions

  • the invention is in the field of molecular pharmacology and microbiology of bacteriostatic and bacteriocidal antibiotics and relates to conjugated antibiotics.
  • the invention aims to abolish the resistance of gram-negative bacteria to hydrophobic antibiotics.
  • Bacterial resistance is a common difficulty in combating bacterial infections caused by gram-negative germs. It is due to the special structure of the bacterial membrane, which acts as an impermeable barrier to these therapeutic agents.
  • Polymyxin B a well-known polypeptide antibiotic, has the property of binding to the bacterial membrane and making it partially permeable through structural changes in the molecular order (disorganization).
  • Vaara and Vaara (Nature 303, 526 (1983)) showed that a derivative of polymyxin B, which no longer contained the fatty acid residue and the terminal diaminobutyric acid, was no longer toxic to eukaryotic cells, but was able to do so Resistance of gram-negative bacteria for various antibiotics, among others Erythromycin, clindamycin, rifampicin, fusidic acid, novobiocin and cloxacillin to a large extent if used together with any of these substances.
  • the object of the invention is therefore to produce preparations with increased bacteriostatic activity which do not have these disadvantages.
  • This goal is achieved by the synthesis of conjugates, each containing a polymyxin B derivative as the membrane-permeabilizing part and a hydrophobic antibiotic as the antibiotic part.
  • the invention is based on the idea that combining these two active substances in the same molecule will reduce the minimum inhibitory concentration (MIC) of the individual active substance.
  • a further improvement is achieved by the chemical incorporation of a connecting molecule (spacer, spacer molecule) between the two active substances, which are reactants. This is intended to obtain additional degrees of freedom, namely by varying the spacer length and spacer structure, the interaction with the cell membrane and the release of the antibiotic can be modulated within the cell.
  • an antibiotic with a polymyxin derivative that is chemically conjugated is then described, with both components not losing their characteristic effect.
  • a suitable choice of the spacer for example the spacer length and spacer sequence in the case of a peptide-like spacer, can further increase their effect.
  • the membrane permeabilizing agent is in any case a derivative of polymyxin B, while the antibiotic can vary.
  • Antibiotic and polymyxin derivative can be chemically bound either directly or via a spacer.
  • the chemically specific synthesis of the conjugates requires a strategy that enables the coupling of the antibiotic to a single free amino group of the polymyxin.
  • this amino function should preferably be chosen in the linear chain (tail) of the polymyxin B, in order in this way to free the cyclic part of the molecule (head) for the Recognize the bacterial membrane preserve.
  • This procedure is explained using a multi-stage process in FIG. 1 using the example of rifamycin B (denoted by ROH).
  • the five free amino functions of polymyxin B are with the tert. Butyloxycarbonyl (Boc) group by treatment with di-tert. Butyld ica rbonat protected at alkaline pH, which creates Penta-Boc-Polymyxin B (I). The fatty acid residue is then split off by enzymatic hydrolysis (II).
  • the penta-boc-polymyxin B is split in a water / 1,4-butanediol mixture into two characterized fragments: first, the linear polymyxin B-tripeptide (tail) substituted with the fatty acid and with two Boc groups, and second, the cyclic polymyxin B-heptapeptide (head) substituted with three Boc groups.
  • the cyclic heptapeptide contains a single free amino function to which it can be chemically specifically substituted, making it the key link in the general coupling strategy.
  • rifamycin B remains intact under these conditions according to the control test.
  • the resulting product can then be coupled to tri-boc polymyxin heptapeptide (the key compound) via dicyclohexylcarbodiimide activation again (V).
  • the compound B (cf. Fig. 1 and 2) is formed by simple acid treatment (VI).
  • VI simple acid treatment
  • R-Leu-Ala-Leu-Ala-OH is the intermediate product that is created by coupling rifamyein B with H-Leu-Ala-Leu-Ala-OMe and subsequent basic cleavage (see text below).
  • Fig. 2 represents the chemical structural formula of an end product (compound B), in this example the polymyxin conjugate with rifamyein B.
  • Pen ta -Boc -Pol ym yxin B / Stu fe I 5.05 g polymyxin B, sulfate are suspended in 150 ml water / dioxane (1: 2 and brought to pH 10.5 with 2N NaOH.
  • the solution is then diluted to 4 l with water, as a result of which a large part of the starting material still present fails and can be recovered by centrifugation.
  • the combined supernatants are mixed with 1 l of methylene chloride and the product extracted into the organic phase.
  • the oily residue obtained after evaporation of the solvent is treated with water and the precipitate formed is filtered off.
  • the mixture is further separated by silica gel chromatography in a chloroform / methanol / acetic acid mixture, as a result of which the tri-boc-polymyxin heptapeptide can be isolated from di-boc-acyldiaminobutyryl-threonyl-diaminobutyric acid and from other components. Yield: 550 mg. Rifamycyl B-Tri-Boc-Polymyxin heptapeptide / Stage III 75 mg
  • Rifamyein B are dissolved in 10 ml of dimethylformamide with 107 mg of tri-boc polymyxin heptapeptide. After adding 14 ⁇ l of triethylamine, 13 mg of hydroxybenztriazole and 25 mg of dicyclohexylcarbodiimide, the reaction mixture is stirred at room temperature for 24 hours. Work-up: filter off the insoluble by-product, evaporate the solvent, take up in ethyl acetate and shake out three times each against aq. 5% NaHCO 3 , ag. NaHSO 4 (pH 2) and aq. NaCl. The residue obtained after evaporation of the solvent is purified by column chromatography on silica gel in a chloroform / methanol / acetic acid mixture. Yield: 91 mg.
  • Rifamycyl B-polym ⁇ xin heptapeptide (compound A) / stage IV 80 mg of the product of stage III are dissolved in 1 ml of 0.4 M HCl in formic acid for 30 min. left at room temperature. Adding the solution to 10 ml of diethyl ether leads to the precipitation of the product, which can be isolated as a centrifugate. Yield: 62 mg.
  • the invention is not limited to the combination of polymyxin compounds with rifamyein using a spacer or without. Rather, other combinations within the meaning of the invention can be selected, such as: polymyxin (B, B 1 , B 2 , D 1 , D 2 E) with hydrophobic antibiotics such as erythromycin, clindamycin, rifampicin, fusidic acid, novobiocin, cloxacillin and others.
  • Spacers are linear molecules that are known from the fields of ligand immobilization in affinity chromatography and enzyme immobilization in enzyme technology. In particular, peptide-like spacers of any length are suitable.
  • the spacer length that determines the partner distance in the conjugate is a size that can be optimized. It is to be expected that if the spacer is too short, the entire surface of the polymyxin derivative and the hydrophobic antibiotic will no longer be free to interact with the cell membrane and the cell organelles.
  • the sequence of a peptide-like spacer can also be optimized with regard to its selective hydrolytic cleavage. The ideal would be a spacer structure that is sufficiently stable to penetrate the cell without being split and yet sufficiently labile to release the hydrophobic antibiotic in the cell.
  • the growth-inhibiting effect on gram-negative bacteria is chosen as a measure of the biological activity.
  • an E. coli strain (127B, wild type, 10 -4 cells / ml) is used, its growth in a minimal Davis medium (with as additives per ml: 1 mg glucose, 1 mg caso amino acids and 20 ⁇ g tryptophan ) is observed.
  • the minimum concentration (MIC) of the conjugates for total growth inhibition for 14 hours at 37 ° C. is determined by measuring the optical density at 600 nm.
  • compounds A and B cf. FIG. 1 are microbiologically active and that compound II (cf. FIG.
  • Compound A (conjugate without spacer, FIG. 1) has approximately the same growth-inhibiting activity as the 1: 1 mixture of rifamyein B and polymyxin heptapeptide.
  • the results show that a method was invented to increase the effect of rifamyein B on an E. coli strain.
  • the method can be applied to any hydrophobic antibiotics and any gram-negative strains.
  • Deadline Publication will be repeated if changes occur.
  • the invention concerns the known resistance of Gram-negative bacteria to hydrophobic antibiotics. It makes u the property of the cyclic heptapeptide part of polymyxine B to act in a membrane-permeabilizing manner, without b toxic. It demonstrates, using the example of rifamyein B, that conjugates of hydrophobic antibiotics with the cyclic h peptide part of polymyxine B have a bacteriostatic effect and that this effect can be increased if a suitable spacer mole is inserted between the two associated substances. A description is also given of a synthetic strategy for producing t conjugates in a chemically specif ⁇ c manner.

Abstract

La présente invention concerne la résistance connue des bactéries gram-négatives vis-à-vis des antibiotiques hydrophobes. Elle fait appel à la propriété de perméabilisation membranaire de la partie heptapeptidique cyclique de la polymyxine B qui agit sans être toxique. Elle démontre, par exemple à partir de la rifamycine B, que des conjugués d'antibiotiques hydrophobes avec la partie heptapeptidique cyclique de la polymyxine B possèdent un effet bactériostatique et que cet effet peut être accru si on incorpore entre les deux substances associées une molécule d'espacement appropriée. Est également décrite une stratégie de synthèse pour fabriquer ces conjugués de manière chimiquement spécifique.
PCT/CH1987/000095 1986-08-06 1987-08-03 Fabrication de conjugues a liaisons de covalence a partir d'un antibiotique et d'un derive non toxique de la polymyxine b WO1988000950A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH315186 1986-08-06
CH3151/86-6 1986-08-06

Publications (2)

Publication Number Publication Date
WO1988000950A2 true WO1988000950A2 (fr) 1988-02-11
WO1988000950A3 WO1988000950A3 (fr) 1988-03-24

Family

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PCT/CH1987/000095 WO1988000950A2 (fr) 1986-08-06 1987-08-03 Fabrication de conjugues a liaisons de covalence a partir d'un antibiotique et d'un derive non toxique de la polymyxine b

Country Status (2)

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AU (1) AU7753787A (fr)
WO (1) WO1988000950A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6380356B1 (en) 1999-12-07 2002-04-30 Advanced Medicine, Inc. Multivalent polymyxin antibiotics
EP1453837A2 (fr) * 2001-11-21 2004-09-08 Activbiotics, Inc. Agents therapeutiques cibles et leurs utilisations
WO2014188178A1 (fr) 2013-05-22 2014-11-27 Cantab Anti-Infectives Limited Dérivés polymyxine et leur utilisation dans le cadre d'une thérapie combinée en association avec d'autres antibiotiques
US9234006B2 (en) 2011-11-18 2016-01-12 Novacta Biosystems Limited Compounds
US11459357B2 (en) 2018-06-25 2022-10-04 Spero Therapeutics, Inc. Polymyxin compounds

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW274552B (fr) * 1992-05-26 1996-04-21 Hoechst Ag

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nature, Band 303, 9. Juni 1983, Macmillan Publ., (London, GB), M. Vaara et al.: "Sensitization of Gramnegative bacteria to antibiotics and complement by a nontoxic oligopeptide", Seiten 526-528 siehe das ganze Dokument in der Anmeldung erw{hnt *
Naturwissenschaften, Band 64, 1977, Springer-Verlag, (DE), V. Braun: "Membranpermeation und Antibiotika-Resistenz bei Bakterien", Seiten 126-132 siehe Seite 130, rechte Spalte, 2. Absatz; Seite 131, rechte Spalte *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6380356B1 (en) 1999-12-07 2002-04-30 Advanced Medicine, Inc. Multivalent polymyxin antibiotics
EP1453837A2 (fr) * 2001-11-21 2004-09-08 Activbiotics, Inc. Agents therapeutiques cibles et leurs utilisations
EP1453837A4 (fr) * 2001-11-21 2007-11-07 Activbiotics Inc Agents therapeutiques cibles et leurs utilisations
US9234006B2 (en) 2011-11-18 2016-01-12 Novacta Biosystems Limited Compounds
WO2014188178A1 (fr) 2013-05-22 2014-11-27 Cantab Anti-Infectives Limited Dérivés polymyxine et leur utilisation dans le cadre d'une thérapie combinée en association avec d'autres antibiotiques
US10407467B2 (en) 2013-05-22 2019-09-10 New Pharma Licence Holdings Limited Polymyxin derivatives and their use in combination therapy together with different antibiotics
US11459357B2 (en) 2018-06-25 2022-10-04 Spero Therapeutics, Inc. Polymyxin compounds

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Publication number Publication date
WO1988000950A3 (fr) 1988-03-24
AU7753787A (en) 1988-02-24

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