Boxazomycin A and B, new antibiotics containing benzoxazole nucleus

FIELD OF THE INVENTION
This invention relates to novel antibiotic compounds, their production and recovery, their use in pharmaceutical compositions, and their use in treating microbial infections. This invention also relates to a novel microorganism that produces said novel antibiotic compounds when cultivated.
BACKGROUND OF THE INVENTION
The present invention relates to novel antibiotic compounds designated as boxazomycin A and B, to pharmaceutically acceptable derivatives thereof, to pharmaceutical compositions containing said compounds, to a process for the production of said compounds, and to a method for the treatment of infectious diseases caused by Gram-positive bacteria.
More particularly, the process for producing boxazomycin A and B relates to an aerobic fermentation process using a novel actinomycete strain No. G495-11 isolated from a soil sample collected in Taiwan. The morphological, cultural and physiological characteristics and the cellular chemical compositions described hereinafter indicate that strain No. G495-11 is classified as a species of the genus Pseudonocardia.
SUMMARY OF THE INVENTION
The new antibiotic compounds of this invention, boxazomycin A and B, have the following structures: ##STR2## wherein R is --CH.sub.2 OH for boxazomycin A; and R is --CH.sub.3 for boxazomycin B.
Boxazomycin A and B, which are produced by cultivating actinomycete strain No. G495-11 (ATCC 53205), inhibit growth of aerobic Gram-positive bacteria and anaerobes with enhanced activity observed in acidic medium.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the .sup.13 C magnetic resonance spectrum of the sodium salt of boxazomycin A in deuterium oxide.
FIG. 2 shows the proton magnetic resonance spectrum of the sodium salt of boxazomycin A in deuterium oxide.
FIG. 3 shows the infrared spectrum of boxazomycin A in the free acid form.
FIG. 4 shows the infrared spectrum of boxazomycin B in the free acid form.

DETAILED DESCRIPTION OF THE INVENTION
The novel antibiotic compounds boxazomycin A and B, named for their benzoxazole necleus, are fermentation products of actinomycete strain No. G495-11, which is classified as a species of the genus Pseudonocardia. A biologically pure culture of the organism has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and added to its permanent collection of microorganisms as ATCC 53205. The morphological, cultural and physiological characteristics and the cellular chemical compositions of strain No. G495-11 (ATCC 53205) indicate that the strain is classified as a species of the genus Pseudonocardia as described below:
Morphology
Strain No. G495-11 develops an abundance of long branched aerial mycelium (0.5 to 0.7 .mu.m in width). The fragmentation of branched substrate mycelium occurs after one to three days, and the resulting elements are in the form of rods of various lengths and short branching filaments. The following characteristics are noted: (1) the segmentation of straight hyphae, bearing long chains of cylindrical spores (0.5.times.1.0-3.0 .mu.m in size) with smooth surface, (2) constriction of hyphae to zigzag form, (3) acropetal and basipetal buddings of blastospores on repeatedly branched aerial hyphae, occasionally with single swellings terminally or laterally. These structures are formed both on the aerial and substrate mycelia. The Gram-stain is positive, and acid-fast stain is negative.
Cultural and Physiological Characteristics
The cultural and physiological characteristics and the carbohydrate utilization profile are shown in TABLES 1, 2 and 3, respectively. Strain No. G495-11 grows well and forms thick white aerial mycelium on many agar media. A yellowish, non-carotinoid, lipophilic pigment is produced in various media. Strain No. G495-11 is resistant to lysozyme, ad sensitive to NaCl at 10%.
TABLE 1______________________________________Cultural Characteristics* of Strain No. G495-11______________________________________Tryptone-yeast extract G**: No turbidity; whitish pellicle andbroth (ISP No. 1) colorless sediment D: NoneSucrose-nitrate agar G: Poor(Czapek's agar) R: Yellowish white (92) A: No or scant D: NoneGlucose-asparagine G: Moderateagar R: Yellowish white (92) to brilliant orange yellow (67) A: Moderate; white (263) D: Pale yellow (89)Glycerol-asparagine G: Abundantagar (ISP No. 5) R: Deep yellow (85) to light brown (57) A: Abundant; white (263) to light yellow (86). Formation of blastospores D: Vivid greenish yellow (97)Inorganic salts-starch G: Pooragar (ISP No. 4) R: Yellowish white (92) A: No or scant D: NoneTyrosine agar G: Abundant(ISP No. 7) R: Strong yellow (84) or dark olive brown (96) A: Abundant; white (263) to light greenish yellow (101) D: Moderate yellow (87)Nutrient agar G: Moderate R: Yellowish white (92) A: Scant; white (263) D: Pale yellow (89)Yeast extract-malt G: Abundantextract agar R: Brilliant yellow (83) to brownish(ISP No. 2) orange (54) A: Moderate; white (263) D: Dark orange yellow (72)Oatmeal agar G: Poor(ISP No. 3) R: Pale orange yellow (73) A: No or scant D: NoneBennett's agar G: Abundant R: Light yellow (86) to deep orange (51) A: Abundant; white (263) D: Dark yellow (88)Peptone-yeast extract- G: Moderateiron agar (ISP No. 6) R: Strong yellow (84) A: None D: None______________________________________ *observed during incubation at 28.degree. C. for 2 weeks. **abbreviation: G Growth; R Reverse color; A Aerial mycelium; D Diffusible pigment ***Color and number in parentheses follow the color standard in "Kelly, K L. & D. B. Judd: ISCCNBS colorname charts illustrated with Centroid Colors. US Dept. of Comm. Cir. 553, Washington, D.C., Nov., 1975".
TABLE 2__________________________________________________________________________Physiological Characteristics of Strain No. G495-11Test Response Method or medium used__________________________________________________________________________Temperature for growth Range: 12.degree. C.-40.degree. C. Bennett's agar Optimum: 25.degree. C.-32.degree. C. No growth: 10.degree. C., 42.degree. C.Gelatin liquefaction Positive (slow) 1% malt extract, 0.4% yeast extract, 0.4% glucose, 20% gelatinStarch hydrolysis Negative Starch agar plateReactions in skimmed Peptonized and not Difco skimmed milkmilk coagulatedFormation of Negative Tyrosine agar, peptone-melanoid pigment yeast extract-iron agar, and tryptone-yeast extract brothTyrosinase reaction Negative Arai's method*Nitrate reduction Negative 0.5% yeast extract, 1% glucose, 0.5% KNO.sub.3, 0.1% CaCO.sub.3.pH tolerance Growth in pH 4.5-11.0 Yeast extract-malt extract agarNaCl tolerance Growth at 7% or less Basal medium: 1% yeast extract, 2% soluble starch, 1.5% agarLysozyme tolerance Resistant to 0.01% Trypticase soy broth plus 1.5% agarYellow lipophilic pigment Non-carotinoid Pridham-Gottlieb's basal medium supplemented with xylose or galactose at 5 g/L.__________________________________________________________________________ *Arai, T. and Y. Mikami: Chromogenicity of Streptomyces, Appl. Microbiol. 23:402-406, 1972.
TABLE 3______________________________________Carbohydrate Utilization of Strain No. G495-11______________________________________ Glycerol + D(-)-Arabinose + L(+)-Arabinose + D-Xylose + D-Ribose + L-Rhamnose - D-Glucose + D-Galactose + D-Fructose + D-Mannose + L(-)-Sorbose - Sucrose - Lactose + Cellobiose + Melibiose - Trehalose + Raffinose - D(+)-Melezitose - Soluble starch - Cellulose - Dulcitol - Inositol + D-Mannitol + D-Sorbitol - Salicin -______________________________________ These results were observed after incubation at 28.degree. C. for 2 weeks Basal medium: PridhamGottlieb's inorganic medium, supplemented with yeast extract at 0.2 g/L Abbreviation: +: positive utilization, -: negative utilization.
Cellular Chemical Analyses
The following cellular components were analyzed: amino acid in purified cell wall; sugars in whole cell hydrolyzate; N-acyl type of peptidoglycan; phospholipid; and nocardomycolic acid (LCN-A). The results are shown in TABLE 4. Strain No. G495-11 has the composition of cell wall Type IV-A and phospholipid Type IV. The N-acyl type of peptidoglycan is acetyl. Nocardo-mycolic acid is not contained.
TABLE 4______________________________________Cell Chemistry of Strain No. G495-11 Diagnostic Component______________________________________Purified cell wall Meso-diaminopimelic acidWhole cell hydrolyzate Arabinose and galactoseN--Acyl type of peptido- Acetylglycan (glycolate test)Phospholipid Phosphatidylethanolamine (PE), phosphatidylglucosamine (Glu NU), and phosphatidylcholine (PC)-traceNocardo-mycolic acid Absent______________________________________
Occurrence of Variants
The origninal culture of strain No. G495-11, which was maintained at -20.degree. C. for 8 years, occurred in the following three tentative morphotypes:
Type 1*: Formation of long and well-branched stable aerial mycelium
Type 2*: Formation of short rudimentary aerial mycelium
Type 3*: Lacking the ability to form aerial mycelium
* Type 1 includes a purified culture, strain No. G495-11.
A variant No. 028 of Type 3 develops true vegetative mycelium which fragments into short filaments and rods. The surface of growth is wrinkled, granular or butyrous on the descriptive agar media. The physiological characteristics, such as formation of yellow pigment, profile of carbohydrate utilization and productivity of antibiotic boxazomycin are well consistent with the aerial mycelium-forming strain No. G495-11. A difference from strain No. G495-11 is observed in positive nitrate reduction of variant No. 028.
Toxonomic Position
Based on the major characteristics mentioned above, strain No. G495-11 was compared with genera Pseudonocardia, Micropolyspora, Saccharomonospora, Actinopolyspora, Saccharopolyspora and two atypical species of genus Nocardia, N. autotrophica and N. orientalis. Morphologically, strain No. G495-11 forms segmented straight long hyphae with total sporulation, blastospore-like segmented short hyphae with apical swelling, and zigzag constricting hyphae. Although the two know species of genus Pseudonocardia are reported to have Type P-III phospholipid including phosphatidylcholine, the other major characteristics indicated that strain No. G495-11 is a species of the genus Pseudonocardia.
The following example illustrates the best mode contemplated for carrying out the invention.
EXAMPLE
Antibiotic Production
The stock culture of strain No. G495-11 was maintained on an agar slant having a medium composition of 1% malt extract, 0.4% glucose, 0.4% yeast extract, 0.05% CaCO.sub.3 and 1.6% agar (pH 7.0). The culture was incubated at 28.degree. C. for two weeks. A well grown agar slant was used to inoculate a vegetative medium consisting of 3% glucose, 3% soybean meal, 0.5% NaCl and 0.1% CaCO.sub.3. The pH of the medium was adjusted to 7.0 before sterilization. After incubation at 28.degree. C. for 2 days on a rotary shaker (250 rpm), 5 mL of the growth was transferred into a 500 mL Erlenmeyer flask containing 100 mL of a production medium having the same composition as the vegetative medium. The antibiotic activity in the fermentation broth was monitored by the paper disc-agar diffusion method using Bacillus subtilis PCI 219 as the test organism. The fermentation was continued for 4 to 5 days at 28.degree. C. on a rotary shaker and the production of boxazomycin reached a maximum of 50 mcg/mL.
Isolation and Purification
The harvested broth (128 L, pH 7.8) was separated to mycelial cake and supernatant in a Sharpless centrifuge. The mycelial cake was stirred for 30 minutes with methanol (30 L) and the methanolic extract evaporated in vacuo to yield an aqueous concentrate. This was combined with the supernatant obtained above, adjusted to pH 2.0 and extracted twice with ethyl acetate (75 L each). After washing with water (50 L), the ethyl acetate extract was shaken with dilute NaOH (35 L, pH 8.6) to transfer the activity into the aqueous solution. Upon acidification to pH 2.0 with 6N hydrochloric acid, a crude yellow solid of boxazomycin complex precipitated from the aqueous solution (24.1 g). Part of this solid (22 g) was dissolved in alkaline water (350 mL, pH 10.0) and the insoluble material was removed by filtration. Acidification of the filtrate to pH 5.0 deposited a partially purified solid complex (12.2 g) which consisted of boxazomycin A (the major component) and B (the minor component), as revealed by thin layer chromatography (TLC) (system BT-103: CHCl.sub.3 --MeOH--AcOH=80:20:4 in volume). The complex solid (12.2 g) was dissolved in a small volume of CHCl.sub.3 --MeOH--AcOH (80:20:4) mixture and applied on a silica gel column (6.7.times.50 cm) which had been equilibrated with the same solvent. The column was eluted with the above-mentioned solvent mixture, and the eluate was collected in fractions (20 mL) and monitored by bioassay against B. subtilis PCI-219 and TLC (BT-103). Fractions containing boxazomycin B (Fr. Nos. 23-27) were combined, concentrated in vacuo and lyophilized to give a pale yellow, homogeneous solid (0.19 g). Fraction Nos. 29-60 were similarly worked up to afford pure boxazomycin A solid (2.37 g). For crystallization, boxazomycin A (160 mg) was dissolved in 250 mL of hot methanol, filtered, and the filtrate was allowed to stand at room temperature to deposit pale yellow, fine needles of boxazomycin A free acid (85 mg). One gram of the above solid sample was dissolved in methanolic 0.2N NaOH (40 mL) at 70.degree. C. and the insolubles were removed by filtration. Upon standing at room temperature, di-sodium salt of boxazomycin A crystallized as orange columns (500 mg) which were recrystallized from aqueous methanol (MeOH--H.sub.2 O=10:3). The yield was 305 mg.
Physico-chemical properties
Boxazomycin A and B are yellow, weakly acidic substances showing similar physico-chemical properties. The two components are differentiated by two TLC systems (TABLES 5 and 6). Boxazomycin A was isolated as crystals both in free acid and di-sodium salt forms, while boxazomycin B has been obtained as an amorphous powder. Boxazomycin A and B free acids are readily soluble in alkaline water and dimethylformamide, slightly soluble in water, methanol, ethanol and acetone, but practically insoluble in ethyl acetate, chloroform, benzene and n-hexane. Boxazomycin A di-sodium salt is soluble in water. They gave a positive response to ferric chloride, concentrated H.sub.2 SO.sub.4 and iodine reagents but were negative in ninhydrin and anthrone tests. Molecular formulae of C.sub.14 H.sub.12 N.sub.4 O.sub.5 and C.sub.14 H.sub.12 N.sub.4 O.sub.4 were assigned to boxazomycin A and B, respectively, from their mass spectral and microanalytical data. Both components exhibited a similar UV spectrum in neutral and alkaline solutions. The physico-chemical data of boxazomycin A and B are summarized in TABLES 5 and 6, respectively. Their IR spectra (FIGS. 3 and 4) exhibited primary amide bands at 1650 and 1630 cm.sup.-1, and OH and/or NH absorptions at 3350 and 3450 cm.sup.-1. The .sup.1 H-NMR spectrum of boxazomycin A (FIG. 2) showed the presence of one methyl (.delta.2.25 ppm, s), one methylene (.delta.4.40 ppm, s) and two aromatic protons (.delta.6.35 ppm, s and 7.85 ppm, s). The .sup.1 H-NMR spectrum of boxazomycin B differed from that of boxazomycin A only in the presence of an additional methyl group (.delta.2.20 ppm, s) instead of the methylene group of the latter. The .sup.13 C-NMR spectrum of boxazomycin A (FIG. 1) displayed 12 carbon signals, with two of them showing double intensity. Upon acetylation in pyridine, boxazomycin A afforded crystalline tetra-N-acetate (MS: molecular ion at m/z 484).
TABLE 5__________________________________________________________________________Physico-Chemical Properties of Boxazomycin A Boxazomycin A Free acid Di--Na salt__________________________________________________________________________Nature Pale yellow needles Orange columnsM.P. >275.degree. C. (dec.) >270.degree. C. (dec.)Microanalysis Calcd for C.sub.14 H.sub.12 N.sub.4 O.sub.5 1/2CH.sub.3 OH C.sub.14 H.sub.10 N.sub.4 O.sub.5 Na.sub.2 --2H.sub.2 O C 52.41; H 4.22; C 42.43; H 3.56; N 16.87 N 14.41; Na 11.60Found C 52.80; H 4.21; N 16.59 C 41.99; H 3.24; N 14.42; Na 12.21EI-MS 316 (M), 299, 215UV .lambda..sub.max nm(.epsilon.) 364 (13,500)in EtOH--DMF 379.sup.sh (12,600)(95:5) 420.sup.sh (6,100)in 0.1N NaOH 219 (28,700) 230.sup.sh (24,800) 259.sup.sh (13,100) 403 (20,200)TLC (SiO.sub.2): RF 0.14n BuOH--AcOH--H.sub.2 O (3:1:1)CHCl.sub.3 --MeOH--AcOH (80:20:4) 0.22__________________________________________________________________________
TABLE 6______________________________________Physico-Chemical Properties of Boxazomycin B Boxazomycin B Free acid______________________________________Nature Pale yellow powderM.P. >270.degree. C. (dec.)Microanalysis C.sub.14 H.sub.12 N.sub.4 O.sub.4Calcd for C 56.00; H 4.03; N 18.66Found C 55.63; H 3.82; N 18.34EI-MS 300 (M), 283, 215UV .lambda..sub.max nm(.epsilon.) 364 (14,800)in EtOH--DMF 379.sup.sh (12,800)(95:5) 420.sup.sh (2,200)in 0.1N NaOH 219 (28,700) 230.sup.sh (24,100) 259.sup.sh (11,600) 403 (21,100)TLC (SiO.sub.2): RF 0.44n BuOH--AcOH--H.sub.2 O(3:1:1)CHCl.sub.3 --MeOH--AcOH 0.54(80:20:4)______________________________________
Antimicrobial Activity and Toxicity
The minimum inhibitory concentrations (MICs) of boxazomycin A and B were determined by the serial agar dilution method. Nutrient agar (Eiken) was used for aerobic bacteria, GC medium (Eiken) for fastidiuous organisms and GAM agar medium (Nissui) for anaerobic bacteria. TABLE 7 shows the in vitro antibacterial activity of boxazomycin A and B in comparison with kanamycin. The two components of boxazomycin showed similar antibacterial spectra, inhibiting Gram-positive aerobic and Gram-positive/Gram-negative anaerobic organisms, while kanamycin was inactive against anaerobes. The intrinsic activity of boxazomycin A was comparable to or slightly higher than that of boxazomycin B. As shown in TABLE 8, boxazomycin A and B showed enhanced activity in acidic media, especially against anaerobic bacteria.
The in vivo efficacy of boxazomycin A was assessed in an experimental model of mice infected by strains of Staphylococcus aureus and Clostridium perfringens. Mice were challenged with a 100.times.LD.sub.50 dose of the pathogens in a 5% suspension of hog gastric mucin (American Laboratory, Omaha, Nebr.). Boxazomycin A was dissolved in saline and administered to mice intramuscularly just before the bacterial challenge. As shown in TABLE 9, boxazomycin A showed protective effect in mice against both infections, but its in vivo efficacy was lower than that expected from its in vitro activity. Boxazomycin A did not show any toxic symptoms up to a dose of 400 mg/kg (i.m.).
TABLE 7__________________________________________________________________________Antibacterial Activity of Boxazomycin A and B MIC (mcg/mL) BoxazomycinTest organisms Test medium A B Kanamycin__________________________________________________________________________Staphylococcus aureus 209P A 0.4 0.4 0.4S. aureus Smith A 0.4 0.4 0.8S. aureus BX-1633.sup.b A 0.8 0.8 0.8S. aureus A20239.sup.c A 0.4 0.8 >100S. aureus A22421.sup.d A 0.8 0.8 0.8S. epidermidis A22547.sup.c A 0.4 0.2 25Streptococcus pyogenes S-23 B 3.1 12.5 25S. pneumoniae IID B 3.1 25 25S. faecalis A9612 A 0.8 3.1 25Micrococcus luteus PCI1001 A 0.4 0.8 3.1Bacillus subtilis PCI 219 A 3.1 1.6 0.2Mycobacterium smegmatis 607 A 1.6 3.1 0.8M. phlei D88 A 0.8 1.6 1.6M. smegmatis 607 D46.sup.c A 1.6 3.1 >100Escherichia coli NIHJ A >100 >100 1.6Klebsiella pneumoniae D-11 A >100 >100 0.4Proteus mirabilis A9554 A >100 >100 3.1Pseudomonas aeruginosa D-113 A >100 >100 25Neisseria gonorrhoeae A15112 B >100 >100 0.4Haemophilus influenzae A9729 B >100 >100 0.4Clostridium difficile A21675.sup.e C 3.1 6.3 >50C. perfringens A9635 C 3.1 12.5 >50Propionibacterium acnes C 3.1 6.3 >50A21933Peptostreptococcus C 3.1 6.3 >50anaerobius A21905B. fragilis A22534.sup.b C 3.1 12.5 50Bacteroides fragilis A22693 C 6.3 12.5 50__________________________________________________________________________ .sup.a A: Nutrient agar (Eiken) B: GC medium (Eiken) C: GA agar (Nissui) .sup.b Lactamase producer .sup.c Kanamycinresistant .sup.d Methicillinresistant .sup.e Clindamycinresistant
TABLE 8__________________________________________________________________________Effect of pH on Antimicrobial Activity MIC (mcg/mL) Boxazomycin A Boxazomycin B pH 6 pH 7 pH 6 pH 7__________________________________________________________________________B. subtilis PCI 219 0.8 6.3 0.2 1.6M. luteus PCI 1001 0.1 0.4 0.2 0.8S. aureus 209P 0.4 0.8 0.2 0.8E. coli NIHJ >100 >100 >100 >100K. pneumoniae D11 >100 >100 >100 >100C. perfringens A9635 0.2 3.1 0.4 6.3P. acnes A21933 0.4 6.3 0.2 6.3B. fragilis A20926 0.05 3.1 0.1 3.1S. necrophorus A15202 0.4 3.1 0.4 6.3__________________________________________________________________________
TABLE 9______________________________________In Vivo Activity of Boxazomycin APD.sub.50 (mg/kg, i.m.)Test organisms Boxazomycin A Kanamycin______________________________________Staphylococcus aureus Smith 400 1.4Clostridium perfringens A9635 220 --______________________________________
As shown above, boxazomycin A and B possess antimicrobial activity and are thus useful in the therapeutic treatment of mammals and other animals for infectious diseases caused by such microorganisms. Additionally, the compounds may be utilized for other conventional applications of antimicrobial agents such as disinfecting medical and dental equipment.
The present invention, therefore, provides a method for therapeutically treating an animal host affected by a microbial infection, which method comprises administering to said host an effective antimicrobial dose of boxazomycin A or B, or a pharmaceutical composition thereof.
In another aspect, the present invention provides a pharmaceutical composition which comprises an effective antimicrobial amount of boxazomycin A or B in combination with an inert pharmaceutically acceptable carrier or diluent. These compositions may be made up in any pharmaceutical form appropriate for the desired use, e.g., oral or parenteral administration.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use. Preparations for oral administration include tablets, capsules, pills, powders, granules and liquid forms such as solutions, emulsions, suspensions, syrups and elixirs. Some of the liquid forms may also be appropriate for topical application.
This invention also contemplates use of boxazomycin A and B in the form of nontoxic acid addition salts with a variety of organic and inorganic salt-forming reagents. Such nontoxic salts are well known to those skilled in the art, and include acid addition salts formed with such pharmaceutically acceptable organic or inorganic acids as sulfuric, phosphoric, hydrochloric, acetic, propionic, oleic, palmitic, citric, succinic, tartaric, glutamic, pantothenic, et. These salts are formed, isolated, purified and formulated by the methods generally employed in salt formation for antibiotics, and are employed in the same manner as the boxazomycin A and B compounds per se.
It will be appreciated that the actual preferred amounts of boxazomycin A and B antibiotics used will vary according to the particular component, the particular compositions formulated, the mode of application, and the particular situs, host and disease being treated. Many factors that modify the action of the drug will be taken into account by those skilled in the art, for example, age, body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities, and severity of the disease. Administrations can be carried out continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines.

Boxazomycin A and B, new antibiotics containing benzoxazole nucleus

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