WO1988000950A2

WO1988000950A2 - Production of covalent-linked conjugates from an antibiotic and a non-toxic derivative of polymyxine b

Info

Publication number: WO1988000950A2
Application number: PCT/CH1987/000095
Authority: WO
Inventors: Jean-Luc Fauchere; Klaus Mosbach
Original assignee: Fauchere Jean Luc; Klaus Mosbach
Priority date: 1986-08-06
Filing date: 1987-08-03
Publication date: 1988-02-11
Also published as: WO1988000950A3; AU7753787A

Abstract

The invention concerns the known resistance of Gram-negative bacteria to hydrophobic antibiotics. It makes use of the property of the cyclic heptapeptide part of polymyxine B to act in a membrane-permeabilizing manner, without being toxic. It demonstrates, using the example of rifamycin B, that conjugates of hydrophobic antibiotics with the cyclic heptapeptide part of polymyxine B have a bacteriostatic effect and that this effect can be increased if a suitable spacer molecule is inserted between the two associated substances. A description is also given of a synthetic strategy for producing these conjugates in a chemically specific manner.

Description

PRODUCTION OF COVALENTLY CONJUGATES FROM A SINGLE

ANTIBIOTIC AND A NON-TOXIC DERIVATIVE OF POLYMYXIN B

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). For this purpose, 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 use of a mere mixture of two such agents, namely the polymyxin derivative and the antibiotic, is known to have the following disadvantages: 1) The simultaneous use of two therapeutic agents is required

2) When used, this requires the simultaneous dissolution of two agents, for example in water, with a very different polar character, namely the hydrophilic polymyxin derivative and the hydrophobic antibiotic,

3) The mix requires the determination of new dose ratios for each new pair,

4) The possible mixture gives little insight into the synergistic effect of the two agents, for example, it is not clear whether a jointly parallel increase in the doses of the two partners would be suitable to increase the effect or whether only a threshold concentration of the polymyxin should be reached, or whether local enrichments of one or the other partner arise due to different solubility, and other problems more,

5) Finally, a mixture of two such agents does not lead to the lowest achievable concentration of the partners for a specific target effect, even with an optimal dose ratio (as will be shown below by the use of conjugates).

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.

The inventive production of 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. For this purpose, 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). Since classic digestion with ficin (Chihara et al., Agric. Biol. Chem. 37, 24555 (1973)) fails because of the insolubility of the substrate, the protease Nagarse (subtilisin B, EC 3.4.21.14) is used. 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.

The coupling of this key connection with rifamyein B is described step by step as an example according to the invention. It is carried out by simple activation with dicyclohexylcarbodiimide (III). Acid treatment of the conjugate (IV) then gives compound A (cf. FIG. 1) in which the two partners are bonded directly to one another. A spacer between the two molecules is introduced by a first coupling of rifamyein B activated with dicyclohexylcarbodiimide with, for example, the tetrapeptide ester H-Leu-Ala-Leu-Ala-OMe, then by cleavage of the methyl ester by mildly alkaline treatment ( VII). The structure of 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). In one example, the chemically synthetic part of the invention will now be described with reference to the drawings below.

1 shows the reaction scheme which, starting from polymyxin B, on the one hand, from a generally designated antibiotic ROH (for example rifamyein B), on the other hand, leads to the end products (compounds A and B). The abbreviations for amino acids are: Dab, 2,4-diamino-butyric acid ,; Leu, Leucine; Thr, Threonine; D-Phe, D-phenylalanine; Ala, alanine. Other abbreviations: Boc, tert. Butyloxycarbonyl; FS, fatty acid residue from Polymyxin B; R, residue of rifamyein B (or generally a hydrophobic antibiotic); DCCI; Dicyclohexylcarbodiimide; HC1 / HCOOH, hydrogen chloride in formic acid. 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.

A variant of the introduction of the spacer (not shown in FIG. 1) consists in the coupling of the N-protected tetrapeptide Z-Leu-Ala-Leu-Ala-OH (Z = benzyloxycarbonyl) with the tri-boc-polymyxin activated with dicyclohexylcarbodiimide. Heptapeptide (key compound) and in the subsequent Z-cleavage by catalytic hydrogenation. The product is then coupled to the rifamyein B activated by dicyclohexylcarbodiimide and converted into compound B by acid treatment.

The detailed description of the individual synthesis steps follows.

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. Then 3.8 g di-tert butyl dicarbonate was added and the reaction mixture was stirred at the same pH and at room temperature for 48 hours, followed by neutralization to pH 6 with 5N HCl, evaporation of the solvents, taking up in ethyl acetate and water 2: 1, after which the insoluble residue was filtered off and Yield: 5.15 g of tri-Boc -Pol ym yxi n -hep peptide / step II 3.14 g of penta-Boc-polymyxin B are dissolved in 960 ml of 1,4-butanediol over 14 hours 640 ml of a 0.125 M phosphate buffer are added to the solution over 2 hours, then a total of 150 mg of Nagarse (pre-dissolved in 1 ml of buffer in each case) are added in 150 mg portions over 150 hours Hrs. At 37 ° C. 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 μl 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-polymγxin 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.

Rifamycyl-B-leucyl-alanyl-leucyl-alanyl-polymyxin heptapeptide (compound B) / stages V and VI 206 mg of rifamycyl BLeucyl-alanyl-leucyl-alanine (preparation: stage VII) are mixed with 204 mg of tri-boc-polymyxin Heptapeptide dissolved in 10 ml of dimethylformamide in the presence of 24 ul of triethylamine. The coupling is carried out by adding 24 mg of hydroxybenztriazole and 46 mg of dicyclohexylcarbodiimide during 16 hours at room temperature. After the same processing as in stage III 112 mg of intermediate are obtained. Treatment of 80 mg in

0.4 M HCl in formic acid for 30 min. at room temperature and further purification by column chromatography on silica gel in a chloroform / methanol / acetic acid mixture gives compound B. Yield: 42 mg.

Ri fam yc yl B-Leuc yl -Al an yl -Leuc yl -A l ani n / Stage VII 328 mg rifamyein B together with 54 mg hydroxybenztriazole, 202 mg H-Leu-Ala-Leu-Ala-OMe, 28 μl Triethylamine and 102 mg of dicycloethyl carbodiimide dissolved in 10 ml of dimethylformamide and stirred for 16 hours at room temperature. After working up as in stage II, the product is dissolved in 2 ml of 0.8 N KOH / dioxane 1: 1 for 15 min. left at room temperature. The solution is then brought to pH 2 with 2N HCl and the product is extracted into ethyl acetate and passed through. Evaporation of the solvent isolated. Yield: 270 mg.

As already mentioned, 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. Microbiological results:

The growth-inhibiting effect on gram-negative bacteria is chosen as a measure of the biological activity. For example, 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 μg 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. Using the example of the polymyxin conjugates with rifamyein B, it is shown here that compounds A and B (cf. FIG. 1) are microbiologically active and that compound II (cf. FIG. 2) is considerably more active than rifamyein alone and as unconjugated 1 : 1 Mixtures of polymyxin B-heptapeptide and rifamyein B. If the MIC values are compared, the most active conjugate, compound B (FIG. 2), is in the growth test with E. coli

127 B, wild type, under the specified conditions

30 to 100 times more active than a 1: 1 mixture of rifamyein B and polymyxin heptapeptide, 100 to 500 times more active than rifamyein B alone. The cyclic polymyxin B-heptapeptide has no measurable activity in the control experiments.

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. In principle, the method can be applied to any hydrophobic antibiotics and any gram-negative strains.

INTERNATIONAL APPLICATION PUBLISHED AFTER THE INTERNATIONAL COOPERATION AGREEMENT IN THE PATENT AREA (PCT)

(51) International Patent Classification 4 (11) International publication number: WO 88/0 C07K 7/62, A61K 37/02 C07D 498/08 // C07D 498/08 A3 (43) International 307/00, 267/00 publication date 11. February 1988 (11

(21) International file number: PCT / CH87 / 00095 (81) Destination countries: AT (European patent), BE (European patent), CH (European pa

(22) International filing date; DE (European patent), DK, FR (European

August 3, 1987 (Aug 3, 1987) tent), GB (European patent), IT (European tent), JP, LU (European patent), NL (European patent), SE (European patent), SU, US.

(31) Priority number: 3151 / 86-6

(32) Priority date: August 6, 1986 (Aug 6, 1986) Published

With international research report.

(33) Priority country: CH Approved prior to change of claims

Deadline. Publication will be repeated if changes occur.

(71X72) Applicant and inventor: FAUCHERE, Jean-Luc [CH / CH]; Gartenstrasse 13b, CH-8902 Urdorf (CH). MOSBACH, Klaus [SE / CH]; Rebbergstrasse 83, CH- (88) Date of publication of the international research 8102 Oberengstringen (CH). Judge: March 24, 1988 (March 24.

(74) Lawyer: FREI PATENTANWARTSBÜRO; Hedwigsteig 6, P.O.Box 95, CH-8029 Zurich (CH).

(54) Title: PRODUCTION OF COVALENT-LINKED CONJUGATES FROM AN ANTIBIOTIC AND A NON XIC DERIVATIVE OF POLYMYXINE B

(54) Name: MANUFACTURE OF COVALENTLY CONJUGATES FROM AN ANTIBI KUM AND A NON-TOXIC DERIVATIVE OF POLYMYXIN B

(57) Abstract

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 specifϊc manner.

(57) Summary

Known resistance of gram-negative bacteria to hydrophobic antibiotics. It uses the properties of the cyclic heptapeptide part of polymyxin B to have a membrane-permeabilizing effect without being toxic. Using the example of rifamyein B, she demonstrates that conjugates of hydrophobic antibiotics with the cyclic heptape part of polymyxin B have a bacteriostatic effect and that this effect can be increased if a suitable spacer molecule is incorporated between the two partners. She also gives a synthetic strategy to make conjugates in a chemically specific way.

EEDIGEL ^ IJRJNFORMAπON

Code used to identify PCT contracting states on the headers of the publications that publish international applications in accordance with the PCT.

AT Austria ER France MR Mauritania

AU Australia GA Gabon MW Malawi

BB Barbados GB United Kingdom NL Netherlands

BE Belgium HU Hungary NO Norway

BG Bulgaria rr Italy RO Romania

BJ Benin JP Japan. SD Sudan

BR Brazil. SB _ Democratic ^" Vblksrepublil_ Korea SE Sweden

CF Central African Republic KR Republic of Korea SN Senegal

CG Congo LI Liechtenstein SU Soviet Union

CH Switzerland LK Sri Lanka TD Chad

CM Cameroon LU Luxembourg TG Togo

DE Germany, Federal Republic of MC Monaco US United States

DK Denmark MG Madagascar m. Finland ML Mali

Claims

PATENT CLAIMS

1. Method for increasing the growth-inhibiting effect of hydrophobic antibiotics on (gram-negative) bacteria, characterized by the chemical conjugation of a hydrophobic antibiotic to a polymyxin derivative.

2. The method according to claim 1, characterized in that the hydrophobic antibiotic is chemically conjugated to a polymyxin derivative via a connecting molecule (spacer).

3. The method according to claim 2, characterized in that the connecting molecule is a peptide compound.

4. The method according to claim 1, characterized by the process steps: a) protecting the amino group of the polymyxin b) cleaving off the fatty acid residue of the polymyxin c) using the cyclic polymyxin portion d) chemical activation of a hydrophobic antibiotic x) coupling the polymyxin portion to the activated antibiotic.

5. The method according to claim 2, characterized by the process steps: a) protecting the amino group of the polymyxin b) cleaving off the fatty acid residue of the polymyxin c) using the cyclic polymyxin portion d) chemical activation of a hydrophobic antibiotic e) coupling of the activated antibiotic to a connecting molecule x) coupling of the polymyxin portion from step c with the antibiotic from step e coupled to the connecting molecule.

6. The method according to claim 3, characterized by the process steps: a) protecting the amino group of the polymyxin b) cleaving off the fatty acid residue of the polymyxin c) using the cyclic polymyxin portion d) chemical activation of a hydrophobic antibiotic e) coupling of the activated antibiotic to a peptide. x) Coupling the polymyxin fraction from step c with the antibiotic from step e coupled to the connecting peptide.

7. The method according to any one of claims 4,5 or 6, characterized in that the cleavage of the fatty acid portion (process step b) is carried out by means of enzymatic hydrolysis.

8. The method according to any one of claims 4, 5 or 6, characterized in that the steps: a) the protective group tert. Butyloxycarbonyl (Boc) is used, b) the fatty acid fraction is split off by means of enzymatic hydrolysis.

9. The method according to claim 6, characterized in that for the steps: d) a hydrophobic antibiotic is activated by means of dicyclocarbodiimide, and e) is coupled with a polypeptide (n-peptide, n = 1,2,3, ...).

10. Product from the process according to claim 1, characterized by a hydrophobic antibiotic conjugated to a polymyxin derivative.

11. Product according to claim 10, characterized by a hydrophobic antibiotic conjugated to a polymyxin derivative via a connecting molecule.

12. Product according to claim 10, characterized by a conjugated to a polymyxin derivative rifamycin derivative.

13. Product according to claim 11, characterized by a rifamycin derivative conjugated to a polymyxin derivative via a connecting polypeptide.

14. Use of the products from claim 1 in a pharmaceutical preparation.