CHEMICAL DERIVATIVES OF ANTBIOTIKA LL-E19020 ALPHA AND BETA

CHEMICAL DERIVATIVES OF ANTIBIOTICS LL-E19020 ALPHA AND BETA

BACKGROUND OF THE INVENTION

This invention relates to novel antibacterial compounds and to the treatment of bacterial infections in animals in need of such treatment. The antibiotics LL-E19020 alpha and beta are disclosed and claimed by physical/chemical characteristics in U.S. Patent 4,705,680, the contents of which are hereby incorporated by reference. These antibiotics, derived from the microorganism streptomyces lydicus subspecies tanzanius NRRL 18036, are active as antibacterial agents. They are also growth promoters, anti-protozoan agents, and anthelmintic agents.

Another complex of antibiotics named Phenelfamycins has been disclosed by Abbott Laboratories, Abbott Park, Ill. at the 27th Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, NY in October, 1987.

The structures of LL-E19020 alpha and beta are reproduced below: EMI2.1

An object of the present invention is to provide chemical derivatives of the antibiotics LL-E19020 alpha and LL-E19020 beta which also have activity as antibacterial agents.

SUMMARY OF THE INVENTION

Accordingly, this invention is concerned with chemically prepared derivatives of LL-E19020 alpha and beta which are also antimicrobial agents and which are described below by their structures and physical/chemical characteristics together with methods for their preparation.

Because Chemical Abstract names have not as yet been assigned to these structures, they will be referred to as derivatives of LL-E19020 alpha and beta throughout the balance of this text.

Ammonolysis Product of LL-E19020 alpha and beta EMI3.1

The ammonolysis product of LL-E19020 alpha and beta has ultraviolet absorption spectra as shown in Figure I; an infrared absorption spectrum as shown in Figure II; a proton magnetic resonance spectrum as shown in Figure III; and a carbon-13-nuclear magnetic resonance spectrum as shown in Figure IV.

The ammonolysis product of LL-E19020 alpha and beta is prepared by dissolving LL-E19020 alpha or beta in acetonitrile, reacting with ammonium hydroxide for 12-24 hours, diluting with water and acidifying with hydrochloric acid to pH 1. This solution is extracted into ethyl acetate, evaporated and then purified by chromatography.

Methyl Esters of LL-E19020 alpha and beta EMI4.1

The methyl ester of LL-E19020 beta has a proton magnetic resonance spectrum as shown in Figure V.

The methyl esters of LL-E9020 alpha and beta are prepared by dissolving either LL-E19020 alpha or beta in a solvent such as ethyl acetate, acetonitrile or dichloromethane, or mixtures thereof, followed by reaction with ethereal diazomethane and purification by chromatography.

Monosaccharides of the Methyl Esters of LL-E19020 alpha and beta EMI5.1

The monosaccharide of the methyl ester of LL-E19020 alpha has an infrared spectrum as shown in Figure VI; a proton magentic resonance spectrum as shown in Figure VII; and a carbon-13-magnetic resonance spectrum sas shown in Figure VIII.

The monosaccharide of the methyl esters of LL-E19020 alpha and beta are prepared by dissolving the methyl ester of LL-E19020 alpha or beta in methanol, cooling to 0<o>C and reacting with dilute hydrochloric acid in methanol for several hours, followed by dilution with water, extraction into ethyl acetate and purification by chromatography.

Aglycone of the Methyl Esters of LL-E19020 alpha and beta EMI6.1

The aglycone of the methyl ester of LL-E19020 alpha has ultraviolet absorption spectra as shown in Figure IX; an infrared absorption spectrum as shown in Figure X; a proton magnetic resonance spectrum as shown in Figure XI; and a carbon-13-nuclear magnetic resonance spectrum as shown in Figure XII.

The aglycone of the methyl ester of LL-E19020 beta has ultraviolet absorption spectra as shown in Figure XIII; an infrared absorption spectrum as shown in Figure XIV; a proton magnetic resonance spectrum as shown in Figure XV; and a carbon-13-magnetic resonance spectrum as shown in Figure XVI.

The aglycone of the methyl esters of LL-E19020 alpha and beta are prepared by reacting the methyl ester of LL-E19020 alpha or beta with hydrochloric acid in methanol at reduced temperature for several hours, followed by extraction into ethyl acetate and chromatographic purification.

Triacetate of the Aglycone Methyl Ester of LL-E19020 alpha and beta EMI7.1 EMI8.1

The triacetate of the aglycone methyl ester of LL-E19020 alpha has an infrared absorption spectrum as shown in Figure XVII; a proton magnetic resonance spectrum as shown in Figure XVIII; and a carbon-13-nuclear magnetic resonance spectrum as shown in Figure XIX.

The triacetate of the aglycone methyl ester of LL-E19020 beta has a proton magnetic resonance spectrum as shown in Figure XX; and a carbon-13-nuclear magnetic resonance spectrum as shown in Figure XXI.

The triacetate of the aglycone methyl ester of LL-E19020 alpha and beta is prepared by reacting the aglycone methyl ester of LL-E19020 alpha or beta with a solution of 4-dimethylamino pyridine and acetic anhydride in dichloromethane for several hours followed by purification by chromatography.

DESCRIPTION OF THE DRAWINGS

The above noted and other features of the invention will be best understood from the following detailed description, when considered with reference to the accompanying drawings mentioned above, in which: Fig. I is an ultraviolet absorption spectrum of the ammonolysis product in acidic, basic and alcoholic solutions; Fig. II is an infrared absorption spectrum of the ammonolysis product of LL-E19020 beta dispersed in potassium bromide; Fig. III is a proton magnetic resonance spectrum of the ammonolysis product; Fig. IV is a carbon- 13-nuclear magnetic resonance print-out of the ammonolysis product of LL-E19020 beta ; Fig. V is a proton magnetic resonance spectrum of the methyl ester of LL-E19020 beta ; Fig. VI is an infrared absorption spectrum of the monosaccharide of the methyl ester of LL-E19020 alpha dispersed in potassium bromide; Fig.VII is a print-out of the proton magnetic spectrum of the monosaccharide of the methyl ester of LL-E19020 alpha ; Fig. VIII is a print-out of the carbon-13-magnetic resonance spectrum of the monosacchoride of the methyl ester of LL-E19020 alpha ; Fig. IX is an ultraviolet absorption spectrum of the aglycone of the methyl ester of LL-E19020 alpha ; Fig. X is an infrared absorption spectrum of the aglycone of the methyl ester of LL-E19020 alpha in potassium bromide; Fig. XI is a proton magnetic resonance spectrum print-out of the aglycone of the methyl ester of LL-E19620 alpha ; Fig. XII is a carbon-13-nuclear magnetic resonance spectrum of the aglycone of the methyl ester of LL-E19020 alpha ; Fig. XIII is the ultraviolet absorption spectrum of the aglycone of the methyl ester of LL-E19020 beta ; Fig.XIV is the infrared absorption spectrum of the aglycone of the methyl ester of LL-E19020 beta in potassium bromide; Fig. XV is the proton magnetic resonance spectrum of the aglycone of the methyl ester of LL-E19020 beta ; Fig. XVI is the carbon-13-nuclear magnetic resonance spectrum of the aglycone of the methyl ester of LL-E19020 beta ; Fig. XVII is the infrared absorption spectrum of the triacetate of the aglycone methyl ester of LL-E19020 alpha in potassium bromide; Fig. XVIII is the proton magnetic resonance spectrum of the triacetate of the aglycone methyl ester of LL-E19020 alpha ; Fig. XIX is the carbon-13-nuclear magnetic resonance spectrum of the triacetate of the aglycone methyl ester of LL-E19020 alpha ; Fig. XX is the proton magnetic resonance spectrum of the triacetate of the aglycone methyl ester of LL-E19020 beta ; Fig.XXI is the carbon-13-nuclear magnetic resonance spectrum of the triacetate of the aglycone methyl ester of LL-E19020 beta .

DESCRIPTION OF THE INVENTION

The in vitro antibacterial activity of these compounds was determined against a spectrum of gram-positive and gram-negative bacteria and anaerobes by a standard agar dilution method. Mueller-Hinton agar containing 5% sheep blood or Wilkins Chalgren agar and two-fold decreasing concentrations of the test compounds were poured into petri dishes. The agar surfaces were inoculated with 1 to 5 x 10<4> colony forming units of bacteria by means of the Steers replicating device. The lowest concentration of antibiotic that inhibited growth of a bacterial strain after 18 hours incubation was recorded as the minimal inhibitory concentration (MIC) for that strain. The results are given in Table I.

Because of the lengthy nomenclature, the compounds in Table I have been coded as follows: Compound I: Ammonolysis Product of LL-E19020 alpha and beta . Compound II: Methyl Ester of LL-E19020 alpha . Compound III: Methyl Ester of LL-E19020 beta . Compound IV: Monosaccharide of Methyl Ester of LL-E19020 alpha . Compound V: Monosaccharide of Methyl Ester of LL-E19020 beta . Compound VI: Aglycone Methyl Ester of LL-E19020 beta . Compound VII: Triacetate Aglycone Methyl Ester of LL-E19020 beta . <tb><TABLE> Columns=7 In Vitro Antibacterial Activity of LL-E19020 a and b Derivatives <tb>Head Col 1: Organism <tb>Head Col 2 to 7: Minimal Inhibitory Concentration (mcg/ml) Compounds <tb>SubHead Col 1: <tb>SubHead Col 2: I <tb>SubHead Col 3: II <tb>SubHead Col 4: III <tb>SubHead Col 5: IV <tb>SubHead Col 6: V <tb>SubHead Col 7:VI <tb>Streptococcus b-hemolyticus C 203 <SEP>8<SEP>0.12<SEP>2<SEP>4<SEP>8 (SEP)<> 128 <tb>Streptococcus b-hemolyticus VGH 84-60 <SEP>4<SEP>0.5<SEP>2<SEP>2<SEP>8 (SEP)<> 128 <tb>Streptococcus b-hemolyticus VGH 84-61 <SEP>16<SEP>2<SEP>8<SEP>8<SEP>32 (SEP)<> 128 <tb>Streptococcus b-hemolyticus VGH 84-62 <SEP>16<SEP>2<SEP>8<SEP>8<SEP>32 (SEP)<> 128 <tb>Streptococcus pneumoniae SV-1<SEP>64<SEP>4 <SEP>8<SEP>16<SEP>32 (SEP)<> 128 <tb>Streptococcus pneumoniae K 84-21 (SEP)<> 128 <SEP>8<SEP>32<SEP>16<SEP>64 (SEP)<> 128 <tb>Bacteroides vulgaris ATCC 29327<SEP>128 <SEP>128<SEP>16 (SEP)<> 128 (SEP)<> 128 (SEP)<> 128 <tb>Clostridium perfringens ATCC 13124 (SEP)<> 128 <SEP>64 (SEP)<> 128 (SEP)<> 128 (SEP)<> 128 (SEP)<> 128 <tb>Clostridium difficile ATCC 17858<SEP>4 <SEP>2<SEP>8<SEP>8<SEP>64<SEP>NT <tb>Peptococcus magnus ATCC 29328<SEP>0.25 <SEP></=0.06<SEP></=0.06<SEP></=0.06 <SEP></=0.06<SEP>0.5 <tb>Peptococcus magnus ATCC 14956<SEP></= 0.06 <SEP></=0.06<SEP></=0.06<SEP></=0.06 <SEP></=0.06<SEP>0.5 <tb>Peptococcus asaccharolyticus ATCC 29743<SEP>0.25 <SEP></=0.06<SEP></=0.06<SEP></=0.06 <SEP></=0.06<SEP>0.5

NT = Not tested <tb></TABLE>

In therapeutic use, the compounds of this invention find utility in the suppression of bacterial infections and as general disinfectants for laboratories. As such they may be administered in the form of conventional pharmaceutical or veterinary compositions appropriate for the intended use. Such compositions may be formulated so as to be suitable for oral, parenteral, or topical administration. The active compounds may be combined in admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms, depending on the form of preparation desired for administration, i.e., oral, parenteral or topical.

The invention will be described further by the following non-limiting examples.

Example 1

Ammonolysis Product of LL-E19020 alpha

A 9.69 g portion of LL-E19020 alpha was dissolved in 250 ml of acetonitrile and 125 ml of ammonium hydroxide was addedl After 14 hours, the mixture was diluted with 1 liter of water and acidified to pH 1 with 6N hydrochloric acid. The aqueous portion was separated and extracted with three 600 ml portions of ethyl acetate. The extracts were combined, washed with 200 ml of saturated sodium chloride solution, dried and then evaporated. The 9.0 g of residue was dissolved in 20 ml of methanol and injected onto a Waters Prep 500 column which had been equilibrated with 3 liters of 32% acetonitrile in 0.1N ammonium acetate pH 4.5. The column was eluted with the same solvent system at the rate of 100 ml per minute, collecting 300 ml fractions.After fraction 55, the eluting solvent was switched to 35% acetonitrile in 0.1N ammonium acetate pH 4.5 and elution continued for a total of 90 fractions. Fractions 70-79 were combined, extracted with ethyl acetate and the extract dried and concentrated. The 1.5 g of residue was purified by HPLC, eluting with 35% acetonitrile in 0.1M ammonium acetate pH 4.5 at a flow rate of 50 ml per minute. Fractions of 300 ml each were collected. Fractions 48-56 were combined and evaporated, giving 818 mg of the desired product.

Example 2

Methyl Ester of LL-E19020 alpha

A 20 g portion of LL-E19020 alpha was dissolved in a mixture of 100 ml of dichloromethane and 100 ml of acetonitrile. A 75 ml portion of cold ethereal diazomethane was added. After 15 minutes, the entire reaction was chromatographed on silica gel, eluting with ethyl acetate. Fractions 1-6 were 250 ml each. Fractions 7-16 were 500 ml each. Fractions 5-11 were combined and evaporated, giving 12.5 g of the desired product.

Example 3

Methyl Ester of LL-E19020 beta

A 30 g portion of LL-E19020 beta was slurried in 200 ml of ethyl acetate. Diazomethane was added in portions until the reaction was complete. The material was then filtered and evaporated. The residue was dissolved in 40 ml of ethyl acetate and purified by chromatography, eluting with 70% ethyl acetate in hexane, then after 15 liters the eluent was step-graded from 80% to 90% to 100% ethyl acetate. Fractions 27-54 were combined and evaporated, giving 12.0 g of the desired product.

Example 4

Monosaccharide of the Methyl Ester of LL-E19020 alpha

A 5.582 g portion of the methyl ester of LL-E19020 alpha was dissolved in 75 ml of methanol and cooled to 0<o>C. A 15 ml portion of 5% hydrochloric acid in methanol was added and this mixture was stirred for 5 hours. The mixture was added to 200 ml of water and extracted with three 100 ml portions of ethyl acetate. The extracts were combined and lyophilized giving 4.97 g of solid. This solid was purified by chromatography eluting with the system buffered acid:acetonitrile (7:3). Fractions 67-70 were combined, concentrated, dissolved in t-butanol and lyophilized, giving 1.37 g of the desired product.

Example 5

Monosaccharide of the Methyl Ester of LL-E19020 beta

A 4.7 g portion of the methyl ester of LL-E19020 beta was dissolved in 50 ml of methanol and cooled to 0<o>C. A 25 ml portion of 3% hydrochloric acid in methanol was added and the reaction was stirred for 5 hours. The reaction was added to 100 ml of water and extracted with three 100 ml portions of ethyl acetate. The extracts were combined, washed with 25 ml of concentrated brine, dried and purified by chromatography, eluting with 50% acetonitrile/buffer. The appropriate fractions were combined and evaporated, giving 0.9 g of the desired product.

Example 6

Aglycone of the Methyl Ester of LL-E19020 beta

A 20.6 g portion of the methyl ester of LL-E19020 beta was dissolved in 150 ml of methanol, cooled to about 0 DEG C and 50 ml of 5% hydrochloric acid in methanol was added. The reaction was stirred for 4 hours, then added to 500 ml of water and extracted with three 250 ml portions of ethyl acetate. The extracts were combined and lyophilized giving 18.06 g of crude product. A 1 g portion of this product was purified by reverse phase liquid chromatography, eluting 25 ml fractions with 50% acetonitrile in 0.05M ammonium acetate pH 4.5 at the rate of 10 ml per minute. Fractions 51-71 were combined and evaporated, giving 230 mg of the desired product.

Example 7

Triacetate of the Aglycone Methyl Ester of LL-E19020 beta

A 200 mg portion of 4-dimethylaminopyridine was dissolved in 100 ml of dichloromethane with stirring. A 2 ml portion of acetic anhydride was added and after 5 minutes 1.5 g of the aglycone methyl ester of LL-E19020 beta was added with stirring. After 2 hours, 100 mg of 4-dimethylaminopyridine and 1 ml of acetic anhydride were added. After 4 hours, 150 mg of 4-dimethylaminopyridine and 1 ml of acetic anhydride were addedl After stirring overnight the reaction was chromatographed on silica gel, eluting with hexane:-ethyl acetate (7:3). Fractions 13-19 were combined and evaporated, giving 1.34 g of the desired product.