Derivatives of antibiotic A82846B (Chloroorienticin A)

Abstract: The present invention is directed to glycopeptides and more particularly to derivatives of the glycopeptide A82846B. In these derivatives, the leucyl has been removed to create "hexapeptides" of A82846B and its N DISACC variations. These hexapeptides are useful as antibacterials and also as starting materials from which further antibacterial compounds are prepared.

Description: The present invention is directed to glycopeptides and more particularly to derivatives of the glycopeptide A82846B. In these derivatives, the leucyl has been removed to create "hexapeptides" of A82846B and its N DISACC variations. These hexapeptides are useful as antibacterials and also as starting materials from which further antibacterial compounds are prepared. US Patent 5,534, 420 (Zmijewski et al ., 9 July 1996) and European Patent application 521408 (Hoechst AG, 7 January 1993) both concern glycopeptide hexapeptides DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to compounds of the formula wherein R 1 represents an epivancosaminyl radical of the formula wherein R 2 represents -CH 2 -R 3 ; and R 3 represents alkyl of C 1 -C 11 , alkyl of C 1 -C 11 -R 4 , or R 4 -(0 (0 or 1) -R 4 ) 0 or 1 , wherein each R 4 is independently phenyl or phenyl substituted by one or two substituents, each of which is independently halo, loweralkyl of C 1 -C 4 , loweralkoxy of C 1 -independently halo, loweralkyl of C 1 -C 4 , loweralkoxy of C 1 -C 4 , or loweralkylthio of C 1 -C 4 , and pharmaceutically acceptable salts thereof. The compounds of the present invention are prepared by an Edman degradation of a parent glycopeptide of the formula wherein R 2 is as defined above. The Edman degradation is a two-step process for the cleavage of the N-terminal residue of a peptide or protein. In the present invention, the above parent glycopeptide is first reacted with an isothiocyanate of the formula SCN-R 5 , to obtain an intermediate N LEU -(thiocarbamoyl)-A82846B compound of the formula In the foregoing formula, R 5 represents alkyl of C 1 -C 10 , phenyl, naphthyl, or phenyl substituted by one or two substituents, each of which is independently halo, loweralkyl of C 1 -C 4 , loweralkoxy of C 1 -C 4 , benzyloxy, nitro, or wherein each R 6 is independently loweralkyl of C 1 -C 4 . This reaction is conveniently carried out in water with pyridine, at a temperature of 25° to 30°C, employing a slight excess of the isothiocyanate reactant. The N LEU -(thiocarbamoyl)A82846B intermediate can be separated in conventional manner or can be employed after removal of reaction solvent in the second step of the Edman degradation. In the second step, the N LEU -(thiocarbamoyl)A82846B is reacted with an organic acid, preferably trifluoroacetic acid, in a non-polar solvent such as dichloromethane. The reaction proceeds at temperatures of from 0°C to 35°C but is preferably carried out at temperatures of from 0°C to 25°C. The reaction is generally complete in several hours. The resulting hexapeptide product is separated and purified if desired in conventional procedures. When it is desired to employ a salt, the product is reacted in standard procedures. This second step of the Edman degradation can in some instances result in loss of the disaccharide epivancosamine. Longer reaction times can be used to prepare the N DISACC -des-epivancosaminyl (R 1 =hydrogen) compound of the present invention. The following examples illustrate the preparation of the compounds of the present invention. Preparation of N DISACC -(p-(p-chlorophenyl)benzyl)-N LEU - (phenylthiocarbamoyl)A82846B N DISACC -(p-(p-Chlorophenyl)benzyl)A82846B trihydrochloride (100.0 mg, 0.0526 mmol) was dissolved in 10 ml H 2 O - pyridine (1:1 v/v) and treated with phenyl isothiocyanate (0.010 ml, 0.083 mmol). The resulting mixture was stirred at room temperature for 1 hr at which time HPLC analysis indicated complete consumption of the starting material. The reaction mixture was concentrated in vacuo and the crude product was purified by preparative HPLC to give 76.6 mg (76% yield) of N DISACC -(p-(p-chlorophenyl)benzyl)-N LEU -(phenylthiocarbamoyl)A82846B. FAB-MS : calc. For C 93 H 102 Cl 3 N 11 O 26 S 1925.5, obtained 1928.5 (M+3). Preparation of Compound of Example 6, from Isolated Thiourea A sample of purified N DISACC -(p-(p-chlorophenyl)benzyl)-N LEU -(phenylthiocarbamoyl)A82846B (63.3 mg, 0.0327 mmol) was suspended in 10 ml CH 2 Cl 2 , cooled to 0 °C, then treated with trifluoroacetic acid (0.10 ml). After 1 hr the reaction mixture was warmed to room temperature and stirred an additional 2 hr. The solvent was removed in vacuo and the crude product was purified by preparative HPLC to give 25.3 mg (46% yield) of N DISACC -(p-(p-chlorophenyl)benzyl)desleucyl-A82846B as a white powder. FAB-MS: calc. for C 79 H 84 Cl 3 N 9 O 25 1663.5, obtained 1666.4 (M+3). Preparation of Compound of Example 4, from Parent Antibiotic without Isolation of Thiourea Intermediate N DISACC -(p-Phenylbenzyl)A82846B (41.0 mg, 0.0233 mmol) was dissolved in 4 ml H 2 O - pyridine (1:1 v/v) and treated with phenyl isothiocyanate (0.0040 ml, 0.033 mmol). The resulting mixture was stirred at room temperature for 3 hr at which time HPLC analysis indicated complete consumption of the starting material. The reaction mixture was concentrated in vacuo to give the crude thiourea intermediate as a white solid. The thiourea derivative was then suspended in 10 ml CH 2 Cl 2 , cooled to 0 °C, then treated with trifluoroacetic acid (0.25 ml). After 30 minutes the reaction mixture was warmed to room temperature and stirred an additional 1 hr. The solvent was removed in vacuo and the crude product was purified by preparative HPLC to give 14.0 mg (37% yield) of N DISACC -(p-phenylbenzyl)desleucyl-A82846B as a white powder. FAB-MS: calc. for C 79 H 85 Cl 2 N 9 O 25 1629.5, obtained 1632.5 (M+3). The HPLC procedures reported in these examples were as follows: Analytical: Reactions were monitored by analytical HPLC using a Waters C 18 µBondapak or Novapak C 18 column (3.9x300 mm) and UV detection at 280 nm. Elution was accomplished with a linear gradient of 5% CH 3 CN - 95% buffer to 80% CH 3 CN - 20% buffer over 30 minutes. The buffer used was 0.5% triethylamine in water, adjusted to pH with H 3 PO 4 . Preparative: Crude reaction mixtures were purified by preparative HPLC using a Waters C 18 Nova-Pak column (40x300 mm) and UV detection at 280 nm. Elution was accomplished with a linear gradient of 5% CH 3 CN - 95% buffer to 80% CH 3 CN - 20% buffer over 30 minutes. The buffer used was 0.5% triethylamine in water, adjusted to pH 3 with H 3 PO 4 . The desired fractions were subsequently desalted with a Waters C 18 Sep-Pak (35 cc) followed by lyophilization. Compounds were desalted as follows. A Waters Sep-Pak cartridge was pre-wet with methanol (2-3 column volumes) then conditioned with water (2-3 column volumes). The sample, dissolved in a minimum volume of water, was loaded onto the Sep-Pak column which was then washed with water (2-3 column volumes) to remove the unwanted salts. The product was then eluted with an appropriate solvent system, typically 1:1 CH 3 CN/H 2 O, CH 3 CN, and/or methanol. The organic solvent component was removed in vacuo and the resulting aqueous solution lyophilized to give the final product. Representative compounds of the present invention are listed in the following table. Ex # Name FAB-MS M+X Analytical HPLC, min 1 DESLEUCYL-A82846B 1466.7 3 4.6 2 N DISACC -(p-CHLOROBENZYL)-DESLEUCYL-A82846B 1590.5 3 14.3 3 N DISACC -(p-PHENOXYBENZYL)-DESLEUCYL-A82846B 1648.3 3 17.4 4 N DISACC -(p-PHENYLBENZYL)-DESLEUCYL-A82846B 1632.5 3 17.1 5 N DISACC -(p-n-BUTYLBENZYL)-DESLEUCYL-A82846B 1612.5 3 13.6 6 N DISACC -(p-(p-CHLOROPHENYL)-(BENZYL)DESLEUCYL-A82846B 1666.4 3 14.2 7 N DISACC -DES-(EPIVANCOSAMINYL-DESLEUCYL-A82846B 1323.4 2 8.5 8 N DISACC -(8-PHENYL-n-OCTYL)DESLEUCYL-A82846B 1654.6 3 16.1 The compounds of the present invention are useful for the treatment of bacterial infections. Therefore, in another embodiment, the present invention is directed to the use of a compound of the invention in the preparation of a medicament for controlling a bacterial infection in a host animal, typically a warm-blooded animal. In this embodiment, the medicament can be used to control and treat infections due to various bacteria, but especially gram-positive bacteria. In a preferred embodiment, the medicament is used to control and treat infections due to bacteria resistant to existing antibacterials. For example, certain bacteria are resistant to methicillin, and yet others are resistant to vancomycin and/or teicoplanin. The present compounds provide a technique for controlling and treating infections due to such resistant bacterial species. In carrying out this embodiment of the invention, the compounds of the present invention can be administered by any of the conventional techniques, including the oral route and parenteral routes such as intravenous and intramuscular. The amount of compound to be employed is not critical and will vary depending on the particular compound employed, the route of administration, the severity of the infection, the interval between dosings, and other factors known to those skilled in the art. In general, a dose of from about 0.5 to about 100 mg/kg will be effective; and in many situations, lesser doses of from about 0.5 to about 50 mg/kg will be effective. A compound of the present invention can be administered in a single dose, but in the known manner of antibacterial therapy, a compound of the present invention is typically administered repeatedly over a period of time, such as a matter of days or weeks, to ensure control of the bacterial infection. Also in accordance with known antibacterial therapy, a compound of the present invention is typically formulated for convenient delivery of the requisite dose. Therefore, in another embodiment, the present invention is directed to a pharmaceutical formulation comprising a compound of the present invention, in combination with a pharmaceutically-acceptable carrier. Such carriers are well known for both oral and parenteral routes of delivery. In general, a formulation will comprise a compound of the present invention in a concentration of from about 0.1 to about 90% by weight, and often from about 1.0 to about 3%. The antibacterial efficacy of the present compounds is illustrated by the table. The minimal inhibitory concentrations (MICs) were determined using a standard broth micro-dilution assay.

Claims: A compound of the formula wherein R 1 represents an epivancosaminyl radical of the formula wherein R 2 represents -CH 2 -R 3 ; and wherein R 3 represents alkyl of C 1 -C 11 , alkyl of C 1 -C 11 -R 4 , R 4 -(0 (0 or 1) -R 4 ) 0 or 1 , wherein each R 4 is independently phenyl or phenyl substituted by one or two substituents, each of which is independently halo, loweralkyl of C 1 -C 4 , loweralkoxy of C 1 -C 4 , or loweralkylthio of C 1 -C 4 , or a pharmaceutically acceptable salt thereof. A compound of Claim 1 in which -CH 2 -R 3 is p-(p-chlorophenyl)benzyl. A pharmaceutical formulation comprising a compound of Claim 1 in combination with a pharmaceutically-acceptable diluent or carrier. A compound of Claims 1 -2 for use as a pharmaceutical. The use of a compound of Claims 1 -2 in the preparation of a medicament for the treatment of bacterial infections. The use of Claim 5 wherein said bacterial infection is attributable to a vancomycin-resistant-enterococcus. A process for the preparation of a compound as claimed in Claim 1 which comprises treating a compound of the formula wherein R 2 is as defined in Claims 1-2, and R 5 represents alkyl of C 1 -C 10 , phenyl, naphthyl, or phenyl substituted by one or two substituents, each of which is independently halo, loweralkyl of C 1 -C 4 , loweralkoxy of C 1 -C 4 , benzyloxy, nitro, or wherein each R 6 is independently loweralkyl of C 1 -C 4 , with an organic acid in a non-polar solvent, and if desired, thereafter forming a pharmaceutically acceptable salt.

Inventors: THOMPSON RICHARD CRAIG, WILKIE STEPHEN CHARLES, THOMPSON, RICHARD CRAIG, WILKIE, STEPHEN CHARLES
Applicants: LILLY CO ELI, ELI LILLY AND COMPANY
Application year: 1998
Main classification: C07K9
IPC codes: C07K9, A61P31, A61K38