DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to alkylated A82846B hexapeptides of the formula

1 wherein R represents alkyl of C1-C11 la alkyl of C1-C11-R, or 1)-R1a)0or1'R1a-(linker(0or wherein each R1a is indepenently phenyl or phenyl substituted by one or two substituents, each of which is independently halo, hydroxy, loweralkyl of C1-C8, loweralkoxy of C1-Cg, loweralkylthio of C1-C4, or trifluoromethyl, and"linker"is-0-,-CH2-, or-O- (CHZ) n- wherein n is 1-3; R represents hydrogen or an epivancosaminyl radical of the formula

wherein R2a represents hydrogen or-CH2-R1 wherein R1is defined as above and may be the same or different than the R1R1on the N1 position; and wherein anrepresents epivancosaminyl radical of the formula

wherein R3a is hydrogen, or, when anepivancosaminylis and Rza thereon is-CHz-R, R can also represent-CH2-R identical to that on the N1-position ; and the pharmaceutically acceptable salts thereof.

The alkylated A82846B hexapeptides of the present invention are in general prepared by reductive alkylation of the corresponding A82846B hexapeptides of the formula:

wherein R is as defined above. In carrying out the reductive alkylation, the A82846B hexapeptide is first reacted with an aldehyde of the formula R-CHO, wherein R is as defined above. This results in the formation of a Schiff's base, which is thereafter reduced to obtain the desired alkylated A82846B hexapeptide. Both reaction steps are carried out in a polar solvent, such as DMF, methanol, or a mixture of the same, and at temperatures of from 25° to 100°C, preferably 60° to 70°C. Preferred reducing agents are sodium borohydride and especially sodium cyanoborohydride.

In a further embodiment, the hexapeptide, aldehyde, and reducing agent, especially sodium cyanoborohydride, are all mixed together at one time. This embodiment is preferred for the reaction with nonbenzylic aldehydes, but may be used as well for the reaction with benzylic aldehydes.

Reductive alkylation of the A82846B hexapeptide can result in alkylation of more than one site. The N-position reacts preferentially, but alkylation may also occur at the

DISACC MONOSACC N@ and/or N@ sites in the molecule. Different alkyl groups on the N1-position and the location are conveniently achieved by starting with an A82846B DISACC hexapeptide with the desired N@ group already present, and thereafter alkylating the N1-position.

The starting A82846B hexapeptides are themselves synthesized from the parent glycopeptides:

wherein R2a is as defined above. This synthesis is by the "Edman degradation", 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 4 reacted with an isothiocyanate of the formula SCN-R, to obtain an intermediate NLEU-(thiocarbamoyl)-A82846B compound of the formula

In the foregoing formula, R represents alkyl of Cl-cl0, phenyl, naphthyl, or phenyl substituted by one or two substituents, each of which is independently halo, loweralkyl of C1-C4, loweralkoxy of C1-C4, benzyloxy, nitro, or wherein each R4a is independently loweralkyl of C1-C4.

This reaction is conveniently carried out in water with pyridine, at a temperature of 25°-30°C, employing a slight

excess of the isothiocyanate reactant. The NLEU- (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.

LEU In the second step, the N- (thiocarbamoyl) A82846B is reacted with an organic acid, preferably trifluoroacetic acid, in a non-polar solvent such a 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.

The second step of the Edman degradation can in some instances result in loss of the disaccharide epivancosamine.

DISACC Longer reaction times can be used to obtain the N-des- 2 epivancosaminyl compound (R =hydrogen).

The compounds of the present invention readily form salts, which can be prepared in conventional manner.

The following examples illustrate the preparation of the compounds of the present invention.

Preparation of N- (henvlthiocarbamool)-N (D- (D-chloroDhenvl) benzvl) A82846B DISACC N- (p- (p-Chlorophenyl) benzyl) A82846B trihydrochloride (100.0 mg, 0.0526 mmol) was dissolved in 10 ml H2O-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 the title compound. FAB-MS: calc. for C93HmzCl3NllO26S 1925.5, obtained 1928.5 (M+3).

DISACC PreDaration of N- (r- (n-chlorophenyl) benzyl)- desleucvl-A82846B from isolated thiourea A sample of the purified N- (phenylthiocarbamoyl)- DISACC N- (p- (p-chlorophenyl) benzyl) A82846B (63.3 mg, 0.0327 mmol) was suspended in 10 ml CH3Cl2, 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 the title compound as a white powder.

FAB-MS: calc. for C79H84Cl3NgO2s 1663.5, obtained 1666.4 (M+3).

DISACC Preparation of N-tn-Dhenvlbenzvl) desleucvl-A82846B without isolation of thiourea intermediate DISACC N- (p-Phenylbenzyl) A82846B (41.0 mg, 0.0233 mmol) was dissolved in 4 ml H2O - 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 CH2Cl2, 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 the title compound as a white powder.

FAB-MS: calc. for C79HssCl2NgO2s 1629.5, obtained 1632.5 (M+3).

Preparation of Example 19 A sample of purified desleucyl-A82846B (141 mg, 0.0962 mmol), 8-phenyloctanal (28 mg, 0.137 mmol), and sodium cyanoborohydride (35 mg, 0.556 mmol) were dissolved in 20 ml DMF-MeOH (1: 1 v/v). The resulting mixture was heated to 65°C and stirred for 1 hour at which time HPLC analysis revealed complete consumption of the starting material. The reaction mixture was cooled to room temperature, concentrated in vacuo, and the crude product purified by preparative HPLC to give 20 mg (13% yield) of Example 19.

Preparation of Example 3 A sample of purified desleucyl-A82846B (140 mg, 0.0956 mmol) and 4-phenylbenzaldehyde (30 mg, 0.165 mmol) was dissolved in 20 ml DMF-MeOH (1: 1 v/v). The resulting mixture was heated to 65 °C and stirred for 1.5 hours, sodium cyanoborohydride (27 mg, 0.429 mmol) was added and the reaction stirred for an additional 1.5 hours at which time HPLC analysis revealed consumption of the starting material. The reaction mixture was cooled to room temperature, concentrated in vacuo, and the crude product purified by preparative HPLC to give 38 mg (24% yield) of Example 3.

The HPLC procedures reported in these examples were as follows: Analytical: Reactions were monitored by analytical HPLC using a Waters C18, uBondapak or Novapak C18 column (3.9x300 mm) and W detection at 280 nm. Elution was accomplished with a linear gradient of 5% CH3CN-95% buffer to 80% CH3CN-20% buffer over 30 minutes. The buffer used was 0.5% triethylamine in water, adjusted to pH 3 with H3P04.

Preparative: Crude reaction mixtures were purified by preparative HPLC using a Waters C18 Nova-Pak column (40x300 mm) and UV detection at 280 nm. Elution was accomplished with a linear gradient of 5% CH3CN-95% buffer to 80% CH3CN -20% buffer over 30 minutes. The buffer used was 0.5% triethylamine in water, adjusted to pH 3 with H3PO4. The desired fractions were subsequently desalted with a Waters C18 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 CH3CN/H20, CH3CN, 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: TABLE I EX NAME FAB-MS M + X Analytical min#HPLC*, NI- (12-PHENYL-n- 1710.5 3 21.1 DODECYL) DESLEUCYL- A82846B N1-(12-PHENYL-n- 1876. 1 2 22.9 DISACC DODECYL)-N-(p- PHENYLBENZYL)- DESLEUCYL-A82846B N1-(p-PHENYLBENZYL)- 1632. 5 3 14.1 DESLEUCYL-A82846B N1, NDISACC-BIS(p- 1798. 4 3 17.4 PHENYLBENZYL)- DESLEUCYL-A82846B N1-BENZYL-NDISACC-(p- 1722. 7 3 14.9 PHENYLBENZYL)- DESLEUCYL-A82846B 1812.9316.5N1,NMONOSACC-DIBENZYL- DISACC _ PHENYLBENZYL)- DESLEUCYL-A82846B NDISACC-1633114.2@N1, DIHEXYLDESLEUCYL- A82846B 1 DISACC MONOSACC N, N,- TRI-n- HEXYLDESLEUCYL- A82846B 9 1 DISACC S (1679. 1 4 9.9 HYDROXYBENZYL)- DESLEUCYL-A82846B 10 N-n-HEXYLDESLEUCYL-1549.6 2 11.8 A82846B 11 N-n-HEXYL-NDIs"cc- (p-1716.8 3 16.2 PHENYLBENZYL)- DESLEUCYL-A82846B 12 N-BENZYLDESLEUCYL-1556.3 3 10.1 A82846B 13 N1-(p-HYDROXYBENZYL)- 1572. 1 3 9.0 DESLEUCYL-A82846B 14 N- (6-PHENYL-n- 1626.1 3 15.5 HEXYL) DESLEUCYL- A82846B 15 N, , NDISACC-BIS(6- 1785. 4 2 19.1 PHENYL-n-HEXYL)- DESLEUCYL-A82846B 16 N, NDISACC-BIS(10- 1898.7 3 24.5 PHENYL-n-DECYL)- DESLEUCYL-A82846B N1-(p-HYDROXYBENZYL)- 1737. 3 2 14.1 NDISACC-(p- PHENYLBENZYL)- DESLEUCYL-A82846B 18 N1-(10-PHENYL-n- 1682. 6 3 19.7 DECYL) DESLEUCYL- A82846B 19 N- (8-PHENYL-n- 1653.6 2 17.6 OCTYL) DESLEUCYL- A82846B 20 N1-(6-PHENYL-n- 1792. 5 3 18.4 DISACC HEXYL)-N- (p- PHENYLBENZYL)- DESLEUCYL-A82846B 21 N- (p- (3-PHENYL-n- 1690. 3 3 15.9 PROPOXY) BENZYL) DESLE UCYL-A82846B 22 N1 (p- (3, 5-BIS- 1768.2 3 17.5 (TRIFLUOROMETHYL)- PHENYL) BENZYL)- DESLEUCYL-A82846B 23 N1-(p-(n-OCTYLOXY)- 1683. 5 2 18.3 BENZYL) DESLEUCYL- A82846B 24 N1-(p-(METHYLTHIO)- 1602. 1 3 13.6 BENZYL) DESLEUCYL- A82846B 25 N1 N BIS (P- 1738.1 3 11.3 (METHYLTHIO)- BENZYL) DESLEUCYL- A82846B 26 N- (p- (3, 5-BIS-1934.6 3 19.4 (TRIFLUOROMETHYL)- PHENYL) BENZYL)- NDISACC -(p- PHENYLBENZYL)- DESLEUCYL-A82846B 27 N- (p- 3 21.2 (TRIFLUOROMETHYL)- PHENYL) BENZYL)- DISACC N- (P- (P- CHLOROPHENYL) BENZYL-

DESLEUCYL-A82846B 28 N- (6-PHENYL-n-1826.6 3 19.3 DISACC HEXYL)-N- (p- (p- CHLOROPHENYL) BENZYL) DESLEUCYL-A82846B *Waters C18 Bondapak The compounds of the present invention are useful for the treatment of bacterial infections. Therefore, in another embodiment, the present invention is directed to a method for controlling a bacterial infection in a host animal, typically a warm-blooded animal, which comprises administering to the host animal an effective, antibacterial amount of a compound of the present invention. In this embodiment, the compounds can be used to control and treat infections due to various bacteria, but especially gram- positive bacteria. In a preferred embodiment, the compounds are 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 Table II. The minimal inhibitory concentrations (MICs) were determined using a standard broth micro-dilution assay.

TABLE II: Antibacterial Activity, Minimal Inhibitory Concentration (MIC) against Various Organisms* EX RESISTANT SENSITIVE SA SA SA SH SH SE SPY SPN 446 489 447 105 415 270 C203 Pi 1 13 9. 2 8 2 2 4 8 4 0. 125 NO GROWTH 243264>64>64>64324#.06245 218888168#.06#.063>128 4 53 21 4 2 2 2 2 2 <. 06 06 9.22222220.1250.5523 6 16 6. 1 2 2 2 0. 5 1 0. 5 0. 125 0.5 11116884816887>128 8 76 55 16 8 8 4 16 8 1 2 9 >128 >128 16 16 16 32 32 32 16 32 10 >128 >128 32 16 32 64 64 32 16 32 11 27 11 1 1 0. 5 2 1 0. 5 0. 125 0.125 12 >128 128 >64 64 >64 >64 >64 >64 2 2 1354416832>64>64320. 25. 06 371688888#.06#.0614>50 15 8 6 4 2 2 1 2 2 0. 125 0.5 16 >128 >11 >64 64 >64 >64 >64 >64 8 16 2.6110.50.510.5#.06#.061727 18 19 12 2 2 2 4 2 4 0. 25 0.5 19 45 25 2 1 1 2 2 4 0. 5 0.5 11444111#.06#.062064 32444484#.06#.0621>128 22 9. 5 4. 6 2 1 2 1 2 2 06 <. 06 23 11 9. 2 8 4 4 4 8 4 0. 25 1 24 >128 >128 32 16 32 32 64 32 8 8 25 6. 7 2. 6 8 4 4 4 8 8 4 1 26 5. 7 6. 1 8 4 4 2 4 4 0. 25 06 27 9. 5 6. 1 64 32 32 8 32 8 64 32 28 6. 7 7 8 8 8 4 2 4 4 16

* ABBREVIATIONS ORGANISM RESISTANT Enterococcus faecium and faecalis (geometric mean of 4-6 isolates) SENSITIVE Enterococcus faecium and faecalis (geometric mean of 4-6 isolates) SA446 Staphylococcus aureus 446 SA489 Staphylococcus aureus 489 SA447 Staphylococcus aureus 447 SH 105 Staphylococcus haemolyticus 105 SH 415 Staphylococcus haemolyticus 415 SE 270 Staphylococcus epidermidis 270 SPY C203 Streptococcus pyogenes C203 SPN P1 Streptococcus pneumoniae. P1