Reducing agent for reductive alkylation of glycopeptide antibiotics

Abstract: This invention is concerned with improved processes for reductive alkylation of glycopeptide antibiotics, the improvement residing in employing pyridine·borane as reducing agent.

Description: The present invention is directed to improved methods for reductively alkylating glycopeptide antibiotics, the improvement residing in the portionwise addition of pyridine·borane complex as the reducing agent. The present invention relates to reductive alkylation of glycopeptide antibiotics. The glycopeptide antibiotics are a large class of substances either produced by microorganisms, or produced by microorganisms and thereafter subsequently modified in part. Two of these, vancomycin and teicoplanin, are sold as antibacterial products, but many others have been discovered and are being considered for development, especially since the emergence in the late 1980s of resistance to various antibiotics, including the glycopeptides themselves. The entire class of glycopeptide antibiotics is well described in "Glycopeptide Antibiotics", edited by Ramakrishnan Nagarajan (Marcel Dekker, Inc., New York, 1994). Among the more recently discovered glycopeptides are those known as A82846A (also called ereomomycin), A82846B (also known as chloroorienticin A), A82846C (also known as orienticin C), and orienticin A. The present invention is preferred for use with vancomycin type glycopeptide antibiotics, including vancomycin, A82846A, A82846B, A82846C, and orienticin A. The invention is especially preferred for use with A82846B. Many modifications of naturally-occurring glycopeptides have been made. Among the modifications are reductive alkylations of reactive amine(s) in glycopeptides. See, for example, U.S. 4,698,327 describing reductive alkylations of vancomycin, and EP0 435 503 A1 and EPO 667 353 A1, both of which describe reductive alkylations of a variety of glycopeptides including vancomycin, A82846A, A82846B, A82846C, and orienticin A. These references describe reductive alkylations which introduce into the parent glycopeptides a great variety of alkyl groups. The present invention can be utilized to make the alkylated glycopeptides described in these references. JP-A-5310772 relates to the production of anthracycline antibiotics. It indicates that desired compounds can be achieved by reacting a starting material and an amine borane complex in the presence of 1,4-dioxane-2,6-diol. Bomann et al. (1995) J. Org. Chem. 60, 5995-5996 relates to the use of pyridine borane as a reducing agent for reductive amination and as a substitute for cyanoborohydride. Wong et al. (1984) Anal. Biochem. 139, 58-67 relates to the use of pyridine borane as a reducing agent for proteins and as a substitute for sodium cyanoborohydride, which may cause problems at large scale preparations because of the release of cyanide during reaction. Cooper et al. (1996) J. Antibiotics, 575-581 concerns the reductive alkylation of glycoprotein antibiotics. It indicates that products obtained from N-alkylation of A82846B provide potent derivatives. N-alkylation is effected by refluxing the allcylating agent and the glycoprotein in methanol in the presence of sodium cyanoborohydride. Nagarajan et al (1989) J. Antibiotics 42, 63-72 relates to N-alkylation of vancomycin using cyanoborohydride in DMF. Cabacungan et al. (1982) Anal Biochem. 124, 272-27.8 relates to the use of boranes as alternative reducing agents. It compares the reductive activity of amino boranes, including pyridine borane, against sodium cyanoborohydride. Preferred alkylated glycopeptides which can be prepared by the present process include the following: N 4 -n-octylA82846B N 4 -n-decylA82846B N 4 -benzylA82846B N 4 -(p-chlorobenzyl)A82846B N 4 -(p-bromobenzyl)A82846B N 4 -(p-propylbenzyl)A82846B N 4 -(p-isopropylbenzyl)A82846B N 4 -(p-butylbenzyl)A82846B N 4 -(p-isobutylbenzyl)A82846B N 4 -(p-pentylbenzyl)A82846B N 4 -(p-isohexylbenzyl)A82846B N 4 -(p-octylbenzyl)A82846B N 4 -(p-propoxybenzyl)A82846B N 4 -(p-isopropoxybenzyl)A82846B N 4 -(p-butoxybenzyl)A82846B N 4 -(p-tert-butoxybenzyl)A82846B N 4 -(p-pentyloxybenzyl)A82846B N 4 -(p-hexyloxybenzyl)A82846B N 4 -(o-hexyloxybenzyl)A82846B N 4 -(p-heptyloxybenzyl)A82846B N 4 -(p-octyloxybenzyl)A82846B N 4 -phenethylA82846B N 4 -(4-phenylbenzyl)A82846B N 4 -(4-(4-chlorophenyl)benzylA82846B N 4 -(4-(4-methylbenzyloxy)benzyl)A82846B N 4 -(4-(4-ethylbenzyloxy)benzyl)A82846B N 4 -(4-(4-chlorophenethyl)benzyl)A82846B N 4 -(4-(2-(4-methoxyphenyl)ethynyl)benzyl)A82846B. US-A-4698327, EP-A-435503 and EP-A-667353 describe the reductive alkylation as comprising a first step, in which the glycopeptide is reacted with the respective aldehyde or ketone to form a Schiff's base, which in a second step is reduced to the desired alkylated product. In one variation of this procedure, EPO 667 353 A1 describes a process in which the reducing agent is added simultaneously with the glycopeptide and aldehyde or ketone. The references suggest a strong preference for sodium cyanoborohydride as reducing agent. While sodium cyanoborohydride is a successful reagent for small scale use, its use in large scale production is less satisfactory. This is due to safety and environmental issues posed by the cyanide ion. Accordingly, sodium cyanoborohydride is less than an ideal reducing agent for larger scale reactions. Reducing agents are legion, but many are unsatisfactory for the glycopeptides. One of the many reducing agents known for use in reductive alkylations is pyridine·borane (see J. Chem Soc. Perkin Trans. 1 (1984), pages 717-720). It has now been discovered that pyridine-borane is a uniquely acceptable reagent for alkylative reductions on glycopeptides, while presenting no safety or environmental hazards as is the case with sodium cyanoborohydride. Furthermore, it has been discovered that portionwise addition of the pyridine·borane increases yields. Thus, the present invention is directed to an improved process for reductively alkylating an amine-containing glycopeptide antibiotic, which process comprises reacting the glycopeptide antibiotic with an aldehyde or ketone in the presence of a reducing agent, characterised in that the reducing agent is pyridine·borane and that one or more portions of pyridine·borane are added thereafter. Thus, the glycopeptide antibiotic, aldehyde or ketone, and a portion of the reducing agent can be mixed together at the same time, and one or more additional portions of reducing agent are added thereafter. Typically, the ketone or aldehyde is 4'-chloro-4-biphenylcarboxaldehyde. In carrying out the present invention, standard conditions for reductive alkylations of glycopeptides are employed, other than the identity of the reducing agent and its portionwise addition. Thus, the glycopeptide and aldehyde or ketone are initially dissolved in a solvent which is at least predominantly methanol, and which is preferably only methanol. If only these reagents are supplied, some small amount of Schiff's base is produced, but the reaction equilibrium does not favor complete production of the Schiff's base. Addition of reducing agent shifts the equilibrium as Schiff's base is converted to alkylated product. As noted, EPO 667 353 Al teaches a preference for simultaneous addition of the reducing agent with the glycopeptide antibiotic and aldehyde or ketone. Thus, in the present invention, the glycopeptide, aldehyde or ketone, and pyridine·borane are typically added at essentially the same time. Further, it has been discovered that when employing pyridine·borane as reducing agent, even simultaneous addition of glycopeptide antibiotic, aldehyde or ketone, and reducing agent leads to only modest yields and that such yields can be increased by portionwise addition of the pyridine-borane, with no more than a portion being added initially to the glycopeptide antibiotic and the aldehyde or ketone. The exact number and timing of portions is not critical. The reaction is generally conducted over a period of time from 4 to 48 hours and preferably from 6 to 24 hours. In the preferred practice of the present invention, a first portion of pyridine·borane is added with the glycopeptide antibiotic and aldehyde or ketone, and the remainder of the pyridine·borane is added in one, two, or more subsequent portions. The ideal sequence of pyridine-borane addition appears to be five portions at 2 to 4 hour intervals (counting the initial addition as the first of the five). Devices can be employed to provide a continuous delivery of the pyridine·borane. In another preferred embodiment of the invention, a source of soluble copper is supplied to the reaction mixture, initially converting the glycopeptide to a copper complex, which becomes the reactive entity. The use of copper confers regioselectivity of reaction in those glycopeptides having multiple reactive amines. For example, in A82846B, the use of copper minimizes reaction on the N 1 (leucine) site and on the N 6 (monosaccharide) site, thereby providing higher yields of the product monoalkylated on the N 4 (disaccharide) amine. The identity of the copper source is not critical, so long as it is at least partially soluble and does not negatively impact the pH. Suitable copper salts are cupric acetate, cupric trifluoroacetate, cupric cyclohexanebutyrate, cupric 2-ethylhexanoate, cuprous chloride, cupric chloride, and cupric bromide. A preferred source of copper is copper (II) acetate, most conveniently employed as the hydrate. The reaction should be conducted at a pH of 6-8, and preferably at a pH of 6.3-7.0. The amounts of reactants and reagents to be employed are not critical; amounts to maximize the yield of product will vary somewhat with the identity of the reactants. The reaction consumes the glycopeptide antibiotic and the aldehyde or ketone in equimolar amounts. A slight excess of the aldehyde or ketone, e.g., 1.3 to 1.7:1, is preferred. The amount of the glycopeptide antibiotic to be used must be corrected for its purity. The reaction consumes an equimolar amount of the pyridine-borane. A slight excess is preferable. The amount of soluble copper, if used, is important. The process first results in the formation of a 1:1 copper complex with the glycopeptide antibiotic. Therefore, the copper is preferably present in an amount approximately equimolar with the glycopeptide antibiotic. However, amounts exceeding one molar equivalent are undesirable because excess copper decomposes the pyridine·borane. Summarizing the foregoing, the ideal amounts to be employed are a ratio of: glycopeptide:aldehyde or ketone:reducing agent:copper salt of: 1:1.3 to 1.7:1.5:0.9 to 1. The concentration of the reactants in the solvent has some bearing on the process. Methanol volume relative to mass of glycopeptide antibiotic can vary from 50:1 to 500:1; a 100:1 dilution appears to be a useful, practical ratio, although higher dilutions may give slightly higher yields. The temperature at which the process is carried out is important. Reaction mixtures in methanol boil at about 67°C., thereby setting the maximum temperature when employing straight methanol as the solvent. Higher temperatures are of course possible when employing mixtures of methanol or when operating under pressure. Lower temperatures can be tolerated, but preferably not lower than about 45°C. The ideal conditions depend upon whether or not copper is employed in the reaction. When copper is not added, it is preferred to conduct the reaction in straight methanol at temperatures of about 58-67°C. When employing copper, it is important to conduct the reaction at slightly lower temperatures of about 58-63°C; again, straight methanol is preferred. Upon the completion of the reaction, the reaction mixture is preferably quenched, as by the addition of sodium borohydride. This reagent consumes residual aldehyde or ketone and thereby prevents further undesired reactions. The product is isolated from the reaction mixture in conventional manner. When copper has been employed, the product is isolated from the reaction mixture as a copper complex of the alkylated glycopeptide. Isolation is achieved by concentration of the reaction mixture and precipitation of the complex by addition of an antisolvent such as ethyl acetate, acetone, 1-propanal, isopropyl alcohol, or preferably acetonitrile. The complex can be broken by aqueous treatment at pH 2 4, freeing the simple alkylated glycopeptide product, which can, if desired, be purified in conventional manner. The following examples illustrate the present invention and will enable those skilled in the art to practice the same. EXAMPLE 1 (Comparative) A82846B (0.50 g, 84.3% potency, 0.42 bg, 0.26 mmol) was stirred in 50 mL methanol and 4'-chloro-4-biphenylcarboxaldehyde (72 mg, 0.33 mmol) and pyridine·borane complex (0.033 mL, 0.33 mmol) were added. The mixture was heated at reflux for 6 hours before being cooled to ambient temperature. HPLC analysis of a reaction aliquot afforded a yield of 0.25 g (53.2%) of N 4 -(4-(4-chlorophenyl)benzyl)A82846B. EXAMPLE 2 (portionwise addition of pyridine·borane) A82846B (0.50 g, 83.9 % potency, 0.26 mmol) and 4'-chloro-4-biphenylcarboxaldehyde (98 mg, 0.45 mmol) were stirred in 50 mL methanol and pyridine·borane complex (0.015 mL, 0.15 mmol) was added. The reaction mixture was heated at reflux for 4 hours and an additional aliquot of pyridine-borane complex (0.015 mL, 0.15 mmol) was added. After heating at reflux for 4 hours longer a final addition of pyridine·borane complex (0.015 mL, 0.15 mmol) was made. The reaction mixture was heated at reflux for another 20 hours. After cooling to ambient temperature HPLC analysis of a reaction aliquot afforded a yield of 0.27 g (58.0 %) of N 4 -(4-(4-chlorophenyl)benzyl)A82846B. EXAMPLE 3 (Comparative) (with copper) A82846B (0.50 g, 84.3% potency, 0.42 bg, 0.26 mmol) was stirred in 50 mL methanol and cupric acetate (45 mg, 0.25 mmol) was added. After stirring at ambient temperature for 10 min, 4'-chloro-4-biphenylcarboxaldehyde (84 mg, 0.39 mmol) and pyridine·borane complex (0.039 mL, 0.39 mmol) were added. The mixture was heated at 57° C for 24 hours before being cooled to ambient temperature. HPLC analysis of a reaction aliquot afforded a yield of 0.34 g (72.3%) of N 4 -(4-(4-chlorophenyl)benzyl)A82846B. EXAMPLE 4 (with copper + portionwise addition of pyridine·borane) A82846B (0.50 g, 76.3% potency, 0.38 bg, 0.24 mmol) and cupric acetate monohydrate (43 mg, 0.216 mmol) were stirred in 50 mL methanol and 4'-chloro-4-biphenylcarboxaldehyde (84.5 mg, 0.39 mmol) and pyridine-borane complex (0.011 mL, 0.11 mmol) were added. The mixture was heated at 63°C for 2 hours and an additional portion of pyridine·borane was added (0.01 mL, 0.1 mmol). After 2 hours more at 63°C a third portion of pyridine·borane (0.005 mL, 0.05 mmol) was added. A fourth portion of pyridine-borane (0.005 mL, 0.05 mmol) was added 2 hours later followed by a fifth portion of pyridine·borane (0.005 mL, 0.05 mmol) after another 5 hours at 63°C. The mixture was heated at 63°C for another 11 hours before being cooled to ambient temperature. HPLC analysis of a reaction aliquot afforded a yield of 0.34 g (79.2%) of N 4 -(4-(4-chlorophenyl)benzyl)A82846B. The reactions reported in Examples 1, 3, and 4 were also evaluated (1) for the amount of the remaining starting glycopeptide, (2) for the amount of products alkylated on amine sites other than the N 4 -position, and (3) for the amount of multiply-alkylated products. The results are set forth in the following table and are expressed as a percentage relative to the intended product monoalkylated on the N 4 -amine; yields of the intended product are actual yields as recited in the foregoing examples. Ex. No. % Mono-alkylated at N 4 % A82846B % Mono-alkylated at N 6 % Mono-alkylated at N 1 % Di-alkylated at both N 4 and N 6 % Di-alkylated at both N 1 and N 4 % Tri-alkylated 1 53.2 47.6 9.9 1.3 21.7 7.8 1.8 3 72.3 17.8 2.1 0.7 6.2 2.5 0.4 4 79.2 9.2 1.4 0.3 7.4 3.4 0.3 These data show that portionwise addition of pyridine·borane, accompanied by the use of copper, maximizes yields of the product monoalkylated on N 4 , while minimizing yields of other alkylated products. EXAMPLE 5 (with copper + portionwise addition of pyridine·borane by syringe pump) A82846B (0.50 g, 83.4 % potency, 0.26 mmol) and cupric acetate (47 mg, 0.26 mmol) were stirred in 50 mL methanol and 4'-chloro-4-biphenylcarboxaldehyde (98 mg, 0.45 mmol) and pyridine·borane complex (0.015 mL, 0.15 mmol) were added. The reaction mixture was heated at 63°C for 2 hours. Additional pyridine·borane complex (0.03 mL, 0.30 mmol) in 2 mL methanol was added to the reaction mixture at a rate of 400 uL/hour using a syringe pump. The temperature was maintained at 63°C during the addition. After the addition was complete, heating was continued to afford a total reaction time of 24 hours. After cooling to ambient temperature, HPLC analysis of a reaction aliquot afforded a yield of 0.35 g (74.3 %) of N 4 -(4-(4-chlorophenyl)benzyl)A82846B. The following HPLC System was used for in situ reaction monitoring and yield calculation: HPLC system Waters 600E with HP3395 integrator and Applied Biosystems 757 detector set at 230 nm, sensitivity 0.1 absorption units, 1 sec. filter rise time. Column: DuPont Zorbax SB-Phenyl, 4.6 mm x 25 cm. Eluant A: 10% acetonitrile, 90% buffer (0.2% triethylamine, 0.25% H 3 PO 4 ). Eluant B: 60% acetonitrile, 40% buffer (0.2% triethylamine, 0.25% H 3 PO 4 ). Gradient profile at 1 mL/min: initialize 100% A, gradient to 80% A, 20% B over 5 minutes, hold 5 minutes, gradient to 100% B over 20 minutes, gradient to 100% A over 5 minutes, hold 20 minutes. Sample preparation: 0.5 - 1.0 g of reaction mixture diluted to 25 mL in acetonitrile - buffer. Hold at ambient temperature about 30 minutes until the purple color of the copper complex is discharged. The desired glycopeptide alkylation product elutes at 16-18 minutes, the starting glycopeptide nucleus at 3-4 minutes, the site N 6 (monosugar) alkylation product at 18-19 minutes, the site N 1 (methyl leucine) alkylation product at 19-21 minutes, dialkylated impurities at 24-26 minutes, and aldehyde at 35-36 minutes. In situ yield is determined by correlation to standards prepared with a reference sample of the product.

Claims: A process for reductively alkylating an amine-containing glycopeptide antibiotic, which process comprises reacting the glycopeptide antibiotic with an aldehyde or ketone in the presence of a reducing agent, characterised in that the reducing agent is pyridine.borane and that one or more portions of pyridine.borane are added thereafter. A process of claim 1, conducted in methanol at a temperature of about 58 to 67°C. A process of any of claims 1 and 2, in which a source of soluble copper is additionally supplied. A process of claim 3, conducted at a temperature of about 58 to 63°C. A process of claim 3 or 4, wherein the pyridine.borane is added in five portions at two to four hour intervals. A process of any of claims 3 to 5, wherein the soluble source of copper is selected from cupric acetate, cupric trifluoroacetate, cupric cyclohexanebutyrate, cupric 2-ethylhexanoate, cuprous chloride, cupric chloride and cupric bromide. A process of any of claims 3 to 5, wherein the source of soluble copper is a hydrate of copper (II) acetate. A process of any of claims 3 to 7, wherein the source of soluble copper is present in an amount approximately equimolar with the glycopeptide antibiotic. A process of any of claims 3 to 8, further comprising a step of adding sodium borobydride. A process of any of claims 1 to 9, in which the glycopeptide antibiotic is vancomycin, A82846A, A82846B, A82846C, or orienticin A. A process of any of claims 1 to 10, in which the glycopeptide antibiotic is A82846B. A process of any of claims 1 to 11, in which the ketone or aldehyde is 4'-chloro-4-biphenylcarboxaldehyde.

Inventors: BERGLUND RICHARD ALAN, ZHENG HUA, BERGLUND, RICHARD ALAN, ZHENG, HUA
Applicants: LILLY CO ELI, ELI LILLY AND COMPANY
Application year: 1997
Main classification: C07K9
IPC codes: C07H17, C07K9