ALTROMYCIN COMPOUNDS

Abstract: A mixture of novel altromycin compounds is produced by a microorganism belonging to the order Actinomycetales. The compounds exhibit cytotoxic activity against mammalian tumor cell lines and antibacterial activity against a broad spectrum of gram-positive bacteria.

Description: ALTROMYHTN COMPOUNDS This application is a continuation-in-part of copending U.S. application Ser. No. 307,555, filed Feb. 7, 1989. TECHNICAL FIELD This invention relates to a mixture of compounds, and in particular to novel altromycin compounds having cytotoxic and antimicrobial activity, as well as to a process for making the new altromycin compounds. BACKGROUND OF THE INVENTION The compounds of the present invention are related to, but distinct from, compounds of the pluramycin family which are described in U.S. 3,314,853; U.S. 3,334,016; EP 200,818; EP 274,545; Byrne et al., J. An ibiotics r 38:1040-49 (1985); Maeda et al., J. Antibiotics, Ser A, 9:75-81 (1956); and Kanda, i. Antibiotics, 24:599-606 (1971). Pluramycin compounds belong to the ant raquinone derivative class of compounds and have been found as metabolites of Streptomycetes and an Actinomadura species. Compounds of the pluramycin family exhibit potent anti- tumor activity in vivo. However, when the pluramycin compounds are administered to laboratory animals, severe adverse side reactions such as kidney toxicity result. There has, therefore, been a need for compounds that have the anti-tumor potency of the pluramycin compounds but which have a higher therapeutic index of activity, thus resulting in lower clinical toxicities. SUMMARY OF THE INVENTION The present invention comprises a fermentation isolate containing one or more altromycins which are represented by the following structural formula: in which Ri is selected from -NH2, -NHCH3, and -N(CH3)2 r and in which R2 and R3 are independently selected from hydrogen and hydroxyl, at least one of R2 and R3 being hydroxyl. The present invention also comprises the individual altromycins and their pharmaceutically acceptable salts and ester thereof. The inventive compounds are produced by a novel Nocardia- like Actinomycete microorganism which exhibits the identifying characteristics described in Tables 1-3. In one aspect of a method of the present invention, Actinomycte sp. AB 1246E-26 produces altromycins upon culturing of said microorganism in a nutrient medium. The antibacterial effects of the new compounds on specific microorganisms, together with their chemical structure and physical properties, differentiate them from other compounds having antibacterial activity. For example, the compounds of the present invention are different from the pluramycin-type compounds in both the arrangement of the substituents on the chromophore and the nature of the glycosidic substituents. The pluramycin compounds have a methyl group attached to carbon 5 of the B ring. Further, the members of the pluramycin class have an amino sugar on carbon 8 and another amino sugar on carbon 10 of the D ring of the anthraquinone-gamma-pyrone nucleus . On the other hand, the altromycins of the present invention have a neutral glycoside, a C-glycoside, attached via carbon 13 to carbon 5 of the B ring. Carbon 8 is unsubstituted in the compounds of the present invention. The altromycins have a disaccharide unit, containing a neutral O-glycoside attached to an amino sugar, on carbon 10 of the D ring. Accordingly, the altromycins of the present invention contain neutral sug/ars which are not found in the pluramycin-type compounds. Moreover, the altromycins are the metabolites of a Nocardia-like organism. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows a UV/visible spectrum of altromycin B recorded in acidic, neutral and basic methanol solutions. The altromycins A, C, D , and G exhibit essentially identical spectra. FIGURE 2 is an Infrared (IR) spectrum of altromycin B taken in deuteriochloroform solution (5%) . The altromycins A, C, and D exhibit essentially identical spectra. F FIIGGUURREE 33 iiss aa 550000 MMHHzz H NMR spectrum of altromycin A taken in deuteriochloroform (CDCI3) F FIIGGUURREE 44 iiss aa 550000 MMHHzz H NMR spectrum of altromycin B taken in deuteriochloroform (CDCI3) . DETAILED DESCRIPTION OF THE INVENTION The compounds and mixture of the compounds of the invention are made by cultivating the microorganism Actinomycete sp. AB 1246E-26 of the order Actinomycetales. The microorganism produces branched vegetative hyphae typical of Actinomycetes. Furthermore, the vegetative hyphae have a tendency to fragment into irregular, smaller, often bacillary, units, typical of Actinomycete species. The strain Actinomycete sp. AB 1246E-26 was isolated from soil collected in South Africa. A subculture of the microorganism was deposited in the permanent collection of the Agricultural Research Service at Northern Regional Research Center, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A. The accession number for the Actinomycete sp. strain AB 1246E-26 at the depository is NRRL 18371. Morpholoσy and Culture Characteristics: The microorganism of the present invention, Actinomycete sp. AB 12 6E-26, can be characterized by morphology and other culture characteristics . Further, whole-cell hydrolysates can be used to predict cell wall composition, and the combination of morphology and chemical composition can be used to classify aerobic actinomycetes into groups according to their cell wall type. Lechevalier and Lechevalier, as reported in Inter. J. Sys . Bacteriol .. 20:435-443, (1970), have developed such a classification scheme. The nocardioform morphology, the chemical composition and the resistance to lysozyme of strain AB 1246E-26 are all consistent with the classification of the producing organism as a Nocardia-like organism. The meso isomer of diaminopimelic acid was found in whole cell hydrolysates. Chromatography of the sugars in the whole cell hydrolysates showed arabinose and galactose as diagnostic sugars. Specifically, the microorganism of this invention has a cell wall of Type IV, containing meso-2 f 6 f -diaminopimelic acid and having a type A whole-cell sugar pattern (arabinose and galactose) . The chemical composition of the microorganism was also characterized by extraction and analysis of cellular menaquinones . The principal menaquinone had a molecular weight of 720, as determined by mass spectrometry, indicating that the menaquinone is tetrahydrogenated with 8 isoprenoid units . Additionally, the microorganism of the present invention is resistant to lysozyme. Lysozyme is an enzyme which attacks and cleaves the amino acid cross-linking of the aminohexose cha±'hs in the peptidoglycan layer of the cell walls of many but not all Gram-positive bacteria, and resistance to lysozyme is a useful.identifying characteristic. The color of the aerial growth of Actinomycete sp. AB 1246E-26 is white to light gray. The microorganism makes a diffusible, non-melanoid pigment on several nutrient media. The appearance and cultural characteristics of Actinomycete sp. AB 1246E-26 in various media are described in greater detail in Table 1. The ability of the Actinomycete sp. AB 1246E-26 to grow on various carbon compounds in synthetic medium is shown in Table 2. Physiological characteristics are given in Table 3. Glycerol-asparagine agar G Abundant ISP 5 AM Sparse, white R Grayish reddish orange (39) ; moderate orange (53) ; grayish yellow (90) SP Light grayish reddish brown (45) Moderate White Grayish reddish orange (39) Light reddish brown (42) Abundant Yellowish white (92) Moderate reddish brown (43) Grayish reddish brown (46) Table 1 (continued) MEDIUM CULTURAL CHARACTERISTICS* Nutrient agar G Moderate AM Sparse, white R Light orange (52) SP Light grayish reddish brown (45) Czapek's agar G Moderate AM Sparse, white R Yellowish gray (93) SP Absent Calcium malate agar G: Poor AM: Sparse, white SP: Absent ATCC #172 G Moderate AM Sparse, light gray (2 4) R Grayish reddish orange (39) SP Absent G: Moderate AM: Sparse, white R: Light Gray (264) S : Absent 1.5%) Observations were made after incubation for 14 days at 28 C. * Abbreviations: G = growth; AM = aerial mycelium; R = reverse; SP soluble pigment Color names and numbers in parentheses follow the color standard in Kelly, K.L. & D.B. Judd, ISCC-NBS Color-Name Charts Illustrated with Centroid Colors, U.S. Dept. of Comm. Suppl. to Cir. 553, Washington, D.C. (1976) . Table 2 Utilization of various compounds as the sole source of carbon by Actinomycete sp. AB 1246E-26* CARBON SOURCE GROWTH Adonitol - Arabinose ++ Cellulose - Dulcitol - Fructose ++ Galactose ++ Glucose ++ Inositol ++ Inulin - Lactose - Maltose - Mannitol ++ Mannose ++ Melezitose " - Melibiose - Raffinose - Rhamnose + Ribose ++ Salicin + Sorbitol + Starch Sucrose Trehalose ++ Xylose + ++ •*= Good utilization + = Poor utilization - - Did not utilize Incubation was carried out at 28 C for 43 days * Shirling, E.B. & D. Gottlieb, Methods for characterization of Streptomyces species, Intern. J. Syst. Bacteriol. 16:313-340 (1966) . -9- Table 3 Physiological characteris ics: Actinomycete sp . AB 1246E-26 TEST REACTION * Starch hydrolysis H ? S production Melanin formation: Peptone-yeast extract-iron agar Tyrosine agar NaCl tolerance (yeast extract- malt extract agar) Growth at 4%, not at 7% Temperature range (yeast extract- malt extract agar ISP 2) Growth at 21 to 42°C, no growth at 54°C ** Litmus milk Alkaline digestion Gordon, R.E., Barnett, D.A., Handerhan, J.E., Pang, C.H-N., Int. J. Sysf- Bacteri l.. 24:54-63 (1974) . Table 3 .con nued. Smibert, R.M. and N.R. Krieg, Manual of Methods for General Bacteriology, American Socety for Microbiology, Washington D.C. pp 409-443 (1981) . Fermentation Although other culture methods are feasible, a liquid, submerged, agitated culture process is preferred. The culture is grown in a culture medium which includes a source of carbon and a source of nitrogen. Media which are useful include an assimilable source of carbon such as starch, sugar, molasses, glycerol, a combination of glucose plus molasses, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, peptone plus yeast extract or whole yeast, etc.; and other organic and inorganic ingredients which can be added to stimulate production of the compounds such as, for example, inorganic anions and cations including potassium, magnesium, calcium, ammonium, sulfate, carbonate, phosphate, chloride, etc. Further, buffers such as calcium carbonate can be added to aid in controlling the pH of the fermentation medium. Aeration can be provided by forcing sterile air through the fermentation medium. Agitation can be provided by shaking the container or by stirring the culture, for example with a mechanical stirrer. The fermentation is generally carried out in a temperature range of from about 24 C to about 35°C. The pH of the fermentation is preferably maintained between about 6 and about 9. The compounds are produced and accumulated between 3 and 9 days after inoculation of the fermentation. Isolation and P rifi cafinπ Upon completion of the fermentation, the compounds of the invention are recovered from the whole broth by repeated solvent extraction with a water-immiscible solvent, such as ethylene chloride. This is the fermentation isolate of Actinomycete sp. AB 1246E-26 which shows antibacterial activity and which " includes the compounds altromycin A, altromycin B, altromycin C, altromycin D, altromycin E, altromycin F, and altromycin G. The compounds, altromycins A through G, can be further purified by sequential counter-current chromatographic techniques as are well known in the art. The isolation of the altromycin compounds can be monitored by TLC analysis of samples with appropriate viewing methods sensitive to the anthraquinone portion of the compouη,d!s . TLC may be performed using normal silica EM Reagents HPTLC plates (silica gel 60 F 254 precoated 10 x 10 cm, E. Merck, Darmstadt, West Germany) . The altromycin compounds are visualized in visible light as yellow-orange spots, under short wavelength UV light as dark blue spots on a light blue background and under long wavelength ' UV light as bright orange spots on a moderately dark blue background. The compounds are readily charred on these plates using a 5% cone. H 2 SO 4 (in EtOH) spray. Solvents used for development of the TLC plates range from 80-90% chloroform with methanol added to make up the remainder to 100%, depending on the particular sample to be run. The altromycin compounds are consistently found to be in the Rf range of 0.2-0.6. The isolation of larger quantities of the altromycins from crude extracts can also be accomplished by the use of EM Reagents silica gel 60 G for thin layer chromatography (E. Merck, Darmstadt, West Germany), which can be used in bulk to pack large columns. Load samples can be as high as 0.8 g of sample/ΪO mL silica on preparative runs. A simple step elution gradient is employed, starting with 100% chloroform and incrementally increasing the methanol content until a concentration of 20% MeOH is reached. Determination of Antibacterial Activity The column fractions are collected and monitored for antibacterial activity by an agar disc diffusion assay. A 20 microliter sample of each fraction is applied to a paper disc. The discs are then placed on agar plates which were previously seeded with sufficient organism to provide a turbid background after r- C incubation, typically 10 to 10 colony forming units (CFU) per mL. The formation of a clear zone surrounding a paper disc is an indication that the fraction being tested contains a compound, or compounds, with antibacterial activity. The foregoing can be better understood by reference to the following examples which are provided for the illustration, and not the limitation, of the practice of the invention. EXAMPLE 1 Actinomycete sp. AB 1246E-26 (NRRL 18371) was maintained as a frozen inoculum stock by freezing a portion of the original inoculum and storing it at -75 C. The medium 5B7 (Table 4) was used for seed growth and the medium N2B1 (Table 5) was used for the fermentation. Table 4 Seed medium 5B7 Ingredients σ/L Glucose monohydrate 10 Starch, Staclipse JUB (made by A.E. Staley Manufacturing Co., Decatur, IL) 15 Table 4 (continued) Ingredients σ/L Yeast extract (made by Difco Laboratories, Detroit, MI) NZ amine type A (made by Humko Sheffield, Memphis, TN) 5 Calcium carbonate 1 Distilled water was added to achieve a volume of 1 L. The pH was adjusted to pH 7.0. Table 5 Production medium N2B1 Ingredients Glucose monohydrate 20 F-152 peptone (made by Inolex Corp. , Chicago, IL) 10 Molasses (Brer Rabbit , Green Label, made by Del Monte Corp, San Francisco, CA) Yeast Extract (made by Difco Laboratories, Detroit, MI) Yeast Extract (made by Difco Laboratories, Detroit, MI) 1 Calcium carbonate 2 Distilled water was added to achieve a volume of 1 L. The pH was not adjusted. The medium was prepared for seeding as follows: Ten mL of the seed medium (Table 4) were dispensed into 25 X 150 mm glass seed tubes. One hundred mL of the seed medium (Table 4) were dispensed into 500 mL Erlenmeyer flasks. The tubes were covered with stainless steel caps (Morton closures) and the flasks were plugged with rayon pharmaceutical coil. Thereafter, the tubes and flasks were sterilized for 35 minutes at 121 C, 15 psi. Inoculum for the fermentation was prepared in two stages. In the first step, 5% of the frozen inoculum was inoculated into the seed tubes containing 10 mL of seed medium 5B7. The tubes were incubated for 96 hours at 28 C on a rotary shaker, operated at 250 rpm, with a stroke of 5.7 cm. In a second step, 5% vegetative inoculum from the tube growth was used to inoculate the seed flasks containing 100 mL of seed medium 5B7. The seed flasks were incubated for 72 hours at 28 C on a rotary shaker, operated at 250 rpm, with a stroke of 5.7 cm. Five percent vegetative inoculum from the second step seed flasks was then transferred aseptically to 200 x 500 mL Erlenmeyer flasks each containing 100 mL of production medium N2B1 (Table 5) . The fermentation flasks were incubated for 5 days at 28 C on a rotary shaker. The shakers were operated at 250 rpm with a stroke of 5.7 cm. After incubation was complete, the fermentation flasks were harvested and combined. The harvested volume was 18.0 liters. EXAMPLE 2 The fermentation broth (18 L) from Example 1 was adjusted to pH 10.0 with 6N HCl and extracted three times with 4 L portions of methylene chloride. The methylene chloride extracts were combined and concentrated on a vertical evaporator to approximately 4 g of oil. The concentrate was partitioned in the solvent system MeOH-H2θ-CCl4 (5:2:5) using a droplet counter- current device with the lower phase as the stationary phase. This -15- device consists of 100 feet of continuous 3/16" ID (internal diameter) teflon tubing wrapped in a 2 X 14 cm repeating pattern around a 1 inch by 6 inch wooden plank. The coil had 96 turns with a total internal volume of 450 mL, approximately one-half of this volume is retained as stationary phase. The compounds exhibiting antibacterial activity were eluted with a fløJw rate of 5 mL/min in fractions numbered 26-80 (10-12 mL each,.'hereinafter referred to as Fraction A) , which yielded approximately 255 mg of orange-red oil after concentration in vacuo r and fractions numbered 117-121 (10-12 mL each, hereinafter referred to as Fraction B the end of which corresponded to the beginning of the blow-out of the stationary phase) which yielded 570 mg of orange- red oil (after concentration in vacuo.. Partitioning of Fraction A (fractions numbered 26-80) Fraction A concentrate (displaying antibacterial activity) was partitioned on a Coil Planet Centrifuge (CPC) counter-current device (P.C. Inc, Potomac, Maryland) employing the following conditions: MeOH-H2θ-CCl (5:2:5); lower phase stationary; tail as inlet; 4 mL/min flow rate at 800 rpm; 85-90% stationary phase retention; 10 L fractions were collected. Two bands of antibacterial activity, fractions numbered 35-41 (hereinafter referred to as Fraction A**_) and fractions numbered 71-105 (herein referred to as fraction A2) were obtained. The concentrate from fraction A2 (224.7 mg) was rechromatographed on the CPC. The conditions for rechromatography of fraction A2 were as follows : MeOH-0.01 M aqueous NH4OAC-CCI4 (5:2:5); lower phase stationary; tail as inlet; 4 mL/min flow rate at 800 rpm; 85-90% stationary phase retention; 10 mL fractions were collected. Two bands of antibacterial activity were observed, fractions numbered 16-21 (hereinafter referred to as Fraction A 2a ) which yielded 24.7 mg of orange-red oil after concentration in vacuo and fractions humbered 48-80 (hereinafter referred to as Fraction A2 13 ) which yielded 230.4 mg of orange-red oil after concentration in vacuo. The oil obtained from concentrating the fraction Rza . wa s essentially identical to the material obtained from the fraction i and was identified as altromycin A. The fraction A 2b was found to contain altromycin B. Partitioning of Fraction B .fractions numbered 37-45. The concentrate from fraction B was partitioned on the CPC counter-current device employing the following conditions: hexane-EtOAc-MeOH-H 2 θ (3:7:6:4 lower phase stationary); tail as inlet, flow rate 4 mL/min at 800 rpm; 85-90% stationary phase retention; 8 mL fractions were collected. One band of activity, fractions numbered 37-45 (hereinafter referred to as fractions Bi) was found which yielded 29.7 mg of orange-red oil after concentration in vacuo. The orange-red oil from fraction Bi was identified as a mixture of altromycin C and altromycin D. Altromycin C and altromycin D can be further separated on the counter-current device described above, using hexane-EtOAc-MeOH- 0.01 M NH 4 OAc. Total yield of altromycin A was 56.7 mg, the total yield of altromycin B was 230.4 mg, and the combined total yield of altromycins C and D was 29.7 mg. EXAMPLE 3 Actinomycete sp. AB 1246E-26 (NRRL 18371) was maintained as a frozen inoculum stock by freezing a portion of the original inoculum and storing it at -75 C. Seed medium for an 80-liter stirred fermentation was prepared as follows : 10 mL of the seed medium 5B7 (Table 4) were dispensed into 25 x 150 mm glass seed tubes. Six hundred mL of the seed medium 5B7 (Table 4) were dispensed into 2 L Erlenmeyer flasks. The tubes were covered with stainless steel caps (Morton closures) and the flasks were plugged with rayon pharmaceutical coil. The tubes and flasks were sterilized for 35 minutes at 121°C, 15 psi. Inoculum for the fermentation was prepared in two stages . In the first step, 1% of the frozen inoculum was inoculated into the seed tubes containing 10 mL of seed medium 5B7 (Table 4) . The tubes were incubated for 96 hours at 28°C on a rotary shaker, operated at 225 rpm, with a stroke of 5.7 cm. In a second step, 5% vegetative inoculum from the tube growth was used to inoculate the seed flasks containing 600 mL of seed medium 5B7 (Table 4) . The seed flasks were incubated for 72 hours at 28°C on a rotary shaker, operated at 225 rpm, with a stroke of 5.7 cm. Eighty liters of production medium N2B1 (Table 5) were prepared in a 150-liter New Brunswick stainless steel stirred fermentor. The N2B1 medium was sterilized in the fermentor at 121°C and 15 psi for 1 hour. The glucose was sterilized separately as a 50% aqueous solution at 121°C for 30 minutes and added to the fermentor after sterilization. Antifoam was XFO 371 (made by Ivanhoe Chemical Co., Mundelein, IL) . It was added initially at 0.01%, then was available on demand. The fermentor was inoculated with 4 liters of the 2nd step seed growth. The temperature was controlled at 28°C. The agitation rate was ' 200 rpm and the air flow was 0.7 vol/vol/min. A head pressure of 5 psi was maintained. The fermentation was terminated at 120 hours. The harvest volume was 61 liters . EXAMPLE 4 The fermentation broth (61 L) from Example 3 was charged into a 200 liter stainless steel roller barrel (Morse Manufacturing Co. Inc., East Syracuse, N. Y.) . The broth was extracted four times with 20-24 L portions of methylene chloride or chloroform, rolling for periods of 30-45 minutes and draining off the organic solvent. The chlorinated solvent extracts were combined and concentrated on a vertical evaporator to a volume of 1 L. This volume was reduced to approximately 100 ml of oil on a roto-evaporator and then partitioned several times between methanol and n-heptane (1:2) . The combined heptane layers did not contain altromycin-type compounds when analyzed by TLC. The methanolic layer was concentrated in vacuo and and partitioned several times between pH 10.0 (NH4OH) water and chloroform. Centrifugation (1000 rpm) was required to remove a proteose-like interfacial precipitate that developed during partitioning. The chloroform layer was concentrated in vacuo to yield 12.09 g of an oily red-brown solid. The concentrate was partitioned in the solvent system MeOH- H2O-CCI4 (5:2:5) using a preparative droplet counter-current (PDCC) device with the lower phase as the stationary phase. The PDCC device was made in-house and consisted of 60 meters of continuous teflon tubing. The tubing alternated in diameter and was coiled in a 3 x 28 cm repeating pattern (the tubing being essentially straight except for sharp bends of the smaller diameter sections at the top and bottom of each loop) such that a 24 cm section of 4.5 mm ID tubing occupied the droplet formation side of the coil and a 35 cm section of 1.5 mm ID tubing occupied the mobile phase return side of the coil. The coil had 100 loops with an approximate stationary phase retention volume of 450 ml on standing. 90-95% of this volume of stationary phase was retained during use. The coil had a mobile phase volume of approximately 70 ml on standing. The coil was run at a rate of 3-4 ml/min, collecting 10-15 ml fractions. Two nearly identical runs were made on a 50% portion of sample. Based on TLC analysis of the fractions, two pools of altromycin-type compounds were obtained from each run. The first 100 fractions, excluding the initial twenty or so which contained significant impurities and little if any altromycin-types, were pooled (hereinafter referred to as Fraction A4) . The second ~100 fractions, which included the stationary blow-out, also were pooled (hereinafter referred to here as Fraction B4) . Partitioning of Fraction A4 Fraction A4 was concentrated and again run on the PDCC device in the same manner using the solvent system MeOH-H2θ-CCl4 (10:5:10) . From this run the first 120 fractions (excluding the initial 19) were combined on the basis of TLC analysis, and are hereinafter referred to as Fraction 4i) . These fractions were concentrated for further purifications using a Coil Planet Centrifuge (CPC) counter-current device (P.C. Inc., Potomac, Maryland) followed by standard column chromatographies as outlined in SCHEME 1, below. This portion of the isolation scheme resulted in the isolation of altromycin E (4.7 mg) and G (6.8 mg) . Partitioning of Fraction B4 Fraction B4 was concentrated and run on the PDCC device (2 runs with 1/2 sample load) using the solvent system MeOfi-H2θ-n- heptane-CCl4 (5:2:2:3) . From each of these runs the first ~100 fractions (excluding the initial ~6) , hereinafter referred to as Fraction B4ι f were combined on the basis of TLC analysis. These fractions were concentrated for further purification using a CPC as outlined in SCHEME 2, below. This portion of the isolation scheme resulted in the isolation of altromycin F (7.8 mg) , altromycin A (~400 mg) , altromycin B (~1 g) , altromycin C (~50 mg) and altromycin D (-100 mg) . SCHEME 1 Fraction A4χ . PCC MeOH-50 mM NH 4 OAc-CCl 4 (10:5:10) lower phase stationary, tail as inlet, 3-4 ml/min. @800 rpm, 10 ml fractions (f) f(16-23) Low Pressure Silica EM Reagents Lobar® LiCroprep® Si 60 (40-63μm), 2.5 x 25 cm, 5 psi, CHCl 3 -MeOH (9:1), 5 ml/f f(15-27) Prep-HPLC SepTech® Macrobore® C 18 (10 μ ) (Separation Technologies, Wakefield, R. I.) 2.5 x 35 cm, 30% MeOH- 70% [8% THF in 50 mM (Et) 3 NHOAc pH 5.0] collected UV active peaks in 100-200 ml/f Altromycin f(18-20) . PCC hexane-EtOAc-MeOH-10 mM NH 4 OAc (3:7:6:4) lower phase stationary, tail as inlet, 4 ml/min. Θ800 rpm, 10 ml fractions Altrom Iycin E f(171-212) SCHEME 2 Fraction B4χ PCC MeOH-H 2 0-n-heptane-CCl 4 (10:4:1:9) lower phase stationary, tail as inlet, 4 ml/min. @800 r P m r iO ml fractions (f) f (38-81) f (82-113) PCC PCC CHCl 3 -CCl 4 -MeOH- CCl 4 -MeOH-50mM (Et) 3 NHOAc 50 mM (Et) 3 NHOAc pH 5.0 pH 5.0 (15:135:150:60) (15:15:6) upper phase stationary, tail upper phase stationary, as inlet, tail as inlet, 4 ml/min. 6800 rpm, 10 ml/f 4 ml/min. @800 rpm, 10 ml/f f(68-124) f (148-159) (recovered stationary) PCC hexane-EtOAc-MeOH-10 mM PCC NH 4 OAc CHCl 3 -CCl 4 -MeOH-H2θ (3:7:6:4) (1:4:5:2) upper phase stationary, upper phase stationary, tail tail as inlet, as inlet, ml/min. @800 rpm, 10 ml/f 4 ml/min. @800 rpm, 10 ml/f Altromycin D Altromycin B f(72-104) f(22-80) Altromycin I Altromycin C f(96-125) f(107-118) (recovered Altromycin F stationary) f(120-128) EXAMPLE 5 Characterization of altromycins A. B. C .D. E. F. and G The altromycins of this invention can be dried to a glass and are orange-red compounds which are readily soluble in methanol (MeOH), chloroform (CHC1 3 ) , carbon tetrachloride (CCI 4 ), dimethyl sulfoxide (DMSO) , ethyl acetate (EtOAc) , benzene or dilute acid; moderately soluble in 0.01M phosphate buffer (pH 7); and slightly soluble in n-hexane or water. When chromatographed on a reverse phase Ciβ High Performance Liquid Chromatography (HPLC) column (4.6 mm X 25 cm) using MeOH-0.05 M NH 4 OAC (60:40) at 2 mL/min, altromycin A had a retention time of 10.8 minutes and altromycin B a retention time of 12.8 minutes . High resolution Fast Atom Bombardment (FAB) mass spectrometry m the positive ion mode gave an (M+H) + parent mass of 912.3645 (calculated 912.3654) for altromycin A, corresponding to a molecular formula of C 46 H 57 NOi8- Altromycin B gave an (M+H)+ parent mass of 926.3828 (calculated 926.3810), corresponding to a molecular formula of C 47 H 5 gNOιg. The (M+H)+ parent mass of altromycin C was 896.3686 (calculated 896.3705), corresponding to a molecular formula of C.gH^NO-..,, and the (M+H) for altromycin D was 910.3868 (calculated 910.3861), corresponding to a molecular formula C 4 7H59NO 1 7. The (M+H)+ parent mass of altromycin E was 896.3722 (calculated 896.3705) corresponding to a molecular formula of C gH57NOχ7. Altromycin F gave an (M+H) + parent mass of 910.3832 (calculated 910.3867) corresponding to a molecular formula of C47H59NO17 and the (M+H) + parent mass of altromycin G was 898.3497 (calculated 898.3497) corresponding to C46H55 O18• Ultraviolet/visible (UV) spectroscopic data (Figure 1) were essentially identical for altromycins A, B, C ,D and G and place them in the anthraquinone derivative class of compounds . The UV absorption maxima in basic solution are at approximately 490 nm, 368 nm, -304 nm and 252 nm. The UV absorption maxima in neutral solution are at 424 nm, 242 nm, and 212 nm . The UV absorption maxima in acidic solution are at 424 nm, 276 nm, 243 nm, and 215 nm . The UV spectra of altromycins E and F are es sentially identical to each other, but differ from altromycins A thorough D and G in the acid and base shifted spectra . A tabulation of the UV data for altromycin compounds is given in Table 6 below . The infrared ( IR) spectrum of altromycin B is shown in Figure 2 . Table 6 UV Absorbtion Maxima for Altromycins A through G . Irøaxl . O N NaOH- 490nm, 368nm, 304nm and 252 nm MeOH Altromycins A-D LmaxMeOH 424 nm, 242 nm and 212 nm Lmaxl . O N HCl-MeOH 424 nm, 276 nm, 243 nm and 215 nm Lχnaχl .0 N NaOH- 545 nm, 338 nm and 250 nm MeOH Altromycins E and F 1-maχMeOH 424 nm, 241 nm and 209 nm Lmaχl .0 N HCl-MeOH 424 nm, 241 nm and 207 nm Lma l .0 N NaOH- 488 nm, 366 nm, 302 nm and 250 nm MeOH Altromycin G LmaxMeOH 424 nm, 242 nm and 209 nm Lmaχl - 0 N HCl-MeOH 423 nm, 273 nm, 242 nm and 209 nm L max = Lambda m x = wavelength of absorbtion maxima . NMR spectroscopic data (Tables 7 and 8 and FIGURES 3 and 4 ) establish that altromycins A, B, C, D, E, F, and G are a new group of compounds in the anthraquinone derivative class of compounds . Altromycins E and F are like altromycins A and B respectively, with the exception that altromycins E and F are each found to have a proton on carbon 13 replacing the hydroxyl functionality . Altromycin G differs from altromycins A and B in that carbon 4" of the amino sugar bears a primary amine as opposed to a mono- or dimethyl amine. 1 ^C NMR chemical shift data and ---H NMR chemical shift/coupling data are listed in Tables 7 and 8, respectively. Table 7 13 C-NMR Chemical Shifts for Altromycins A-G a Carbon DEPT r No. a Measured at 75 or 125 MHz in CDC1 3 ; chemical shifts in ppm from TMS. • ° 13 C NMR DEPT data for altromycins. Table 8 Η NMR Chemical Shift Assignments and Coupling Data for Altromvcins A-G in CDCh .ppm from TMS. a m: multiplicity b n: (1), 1 ; (2). 2; (3 ). 1 ; (4), 2 * . (5), 1; (6). 2; (7), 0. EXAMPLE 6 Antibacterial Activity The antibacterial activity of the altromycin compounds of the present invention against a variety of bacteria is,illustrated in Table 9. Minimum inhibitory concentrations (MICs) were determined by the agar dilution method, described hereinbelow, using Brain Heart Infusion (BHI) agar. Twelve petri dishes were prepared, each containing successive aqueous 2-fold dilutions of the test compouncLs mixed with 10 mL of sterilized BHI agar. Each plate was inoculated with 1:100 (or 1:10 for slow growing strains, primarily Micro"coccus and Streptococcus) dilutions of overnight cultures ^ of up to 32 different microorganisms, using a Steers replicator block calibrated to deliver approximately 10 colony forming units (CFU) . In addition, a control plate using BHI agar containing no test compound was prepared and inoculated at the beginning, and at the end, of each test. The inoculated plates were incubated at from about 35 C to about 37 C for approximately 20-24 ho ' urs. Ciprofloxacin was used as a reference antibacterial -agent. After incubation, each agar plate was observed for the presence or absence of microorganism growth. The MIC was defined as the lowest concentration of test compound yielding no growth. A slight haze caused by the inoculum spot as compared to growth on a control plate containing no test compound was disregarded. The results are represented by the data in the following Table 9. Table 9 MICs (microgram/mL. for aerobically grown bacteria ORGANISM Actinetobacter sp. CMX 669 Enterobacter aeroσenes ATCC 13048 Enterococcus faecium ATCC 8043 Escherichia coli DC-2 Escherichia coli H560 Escherichia C_Q1± JUHL Escherichi £ £ li KNK 437 Escherichia ∞ϋ ss Klebsiella pneumoniae ATCC 8045 Micrococcus luteus ATCC 4698 Micrococcus luteus ATCC 9341 Providencia stuartii CMX 640 Pseudomonas aeruginosa A5007 Pseudom n s aeru inosa BMH10 Pseudo onas aeruginosa K799/61 Pseudomonas aeruσinosa K799/ T Pseudomonas cepacia 2961 Staphylococcus aureus 45 Staphylococcus aureus 45 RAR2 Staphylococcus aureus A5177 Staphylococcus aureus ATCC 6538P Staphylococcus aureus CMX 503 Staphylococcus aureus CMX 553 StaphylQCgCCHS aureus 642A Staphylococcus aureus CMX 686B Staphylococcus aureus 3519 Staphylococcus aureus NCTC 10649 Staphylococcus eoidermidis 3519 Streptococcus agalactiae CMX 508 Streptococcus bovis A5169 Streptococcus pyoσenes 930 Streptococcus pyogenes 2548 Streptococcus pyogenes EES61 EXAMPLF. 7 Cytotoxicity The compounds of the present invention exhibitied potent in vitro activity against both human and murine cell types . The cytotoxity of altromycins A, B, and C+D is against HeLa cells ' is illustrated in Table 10 and the cytotoxicity of the altromycins A, B, and D against human and murine tumor cell lines is illustrated in Table 11. The inhibitory concentrations capable of killing 50% of the cells (IC 5 0S) were determined in a colorimetric assay for cytotoxic activity against cultured cells according to the following protocol: A three day microtiter assay was used to measure the metabolic activity of cultured cells exposed to a range of drug concentrations. Metabolic activity was measured by the cell's ability to reduce the tetrazolium dye, 3-(4,5-dimethylthiazol- 2-yl) -2, 5-diphenyltetrazolium bromide (MTT) , to a quantifiable colored formasan derivative. Test compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted, first with Earle's Balanced Salt Solution, followed by culture medium, to twice the highest concentration of the compound to be tested. From this concentrated stock, two-fold serial dilutions were prepared in 96-well microtiter trays, each well containing twice the desired final concentration of the compound. Each concentration was tested in triplicate and compared to triplicate drug-free controls. The cells were grown in the same medium used for __.__.luting the compounds. After harvesting by trypsinization as described in Bird, B.R. & F.T. Forrester, Basic Laboratory Techniques in Cell Culture, U.S Dept. of Health & Human Services, Virology Training Branch, Atlanta Georgia, pp 84-85, 104-105, 111 (1981), viable cell counts were determined and cell density was adjusted to 25,000 cells/mL. Inoculum (0.1 mL) was then added to each well for a final concentration of 2,500 cells per well» Addition of the inoculum was used to dilute the test compounds to the desired final concentration. Microtiter trays were incubated for three days at 36 C in a humidified atmosphere containing 5% carbon dioxide. After three days, 20 microliters of 5 mg/mL MTT in phosphate-buffered saline solution was added to each well of the microtiter trays. The microtiter trays were then returned to the incubator for two to four hours to allow the surviving cells to reduce the dye. After this incubation, both the medium and the unreduced dye were removed by aspiration. DMSO was added to each well to dissolve the water-insoluble, colored end product of the dye reduction so that it could be measured spectrophotometrically at 570 nm. Table 10 IC50S of Altromycins A, B and C+D When Used Aσainst Cultured Cell Lines <*. Altromycin Cell line A Hela* B Hela C+D Hela * micrograms per milliliter. ** Hela cells were acquired from ATCC, Catalog # CCL2. Medium used was Minimum Essential Medium (MEM) , 10% Fetal Calf Serum and 1% non-essential amino acids. and D were determined using the ascitic leukemia tumor line P388 . Prior to testing, P388 was propagated intraperitoneally in female DBA/2 mice. The ascitic tumors were intraperitoneally injected in female B6D2FI mice at a concentration of 10^ cells per mouse. The mice were treated with antitumor agents administered once daily for five consecutive days, with fluorouracil being used as a positive control. All drugs were administered intraperitoneally in 5% aqueous dextrose or other suitable diluent. Drug efficacy was determined by prolonged survival. Survival time was calculated as %T/C, the mean or median survival time of treated mice expressed as a percentage of the mean or median survival time of control mice. Compounds having %T/C values greater than 125 were considered to have significant activity. Treatment groups contained 10 mice and control groups contained 20 mice each. The toxicity of antitumor agents in these tests were evaluated by comparing the weight of mice prior to treatment and 5 days after treatment. LD 5 0 values for single intraperitoneal administration were 0.3, 0.2 and 0.1 mg/kg for altromycins A, B and D, respectively. The altromycins demonstrated reproducible activity in the P388 systemic leukemia model as illustrated in Table 12. Table 12 (continued. QD1-5 = Test compounds administered daily for 5 consecutive days. D1,D5 = Test compound administered daily at day 1, day 5 only. EXAMPLE 8 Altromycins A, B, and D were also tested against two solid tumors, namely, Lewis lung carcinoma and M5076 Ovarian sarcoma. Prior to testing these tumors were propagated subcutaneously in female C57B1/6 mice. Lewis lung carcinoma or M5076 (solid) tumors were implanted subcutaneously as a 1:10 brei in female B6D2 1 mice weighing 16-20 grams. For mice implanted with Lewis lung carcinoma tumors, test compounds were administered daily for 9 consecutive days. Cyclophosphamide was used as the positive control drug. For mice implanted with M5076 (solid) tumors, test compounds were administered 4 times daily for 11 consecutive days . Cisplatinum was used as a positive control. All drugs were administered intraperitoneally in 5% aqueous dextrose, Drug efficacy was determined by reduction in tumor weight. Tumor weight inhibition was expressed as a function of T/C, the ratio of tumor weight of the treated mice to the tumor weight of control mice, and is illustrated in Tables 13 and 14, below. Altromycin D produced nearly 90% tumor weight inhibition as compared to untreated control tumors; altromycin B produced a maximum of 60% inhibition. Against the M5076 ovarian tumor grown as a subcutaneous implant, altromycin A was slightly more active than altromycin D (Table 14) . 50 90 90 * Altromycins A, B, and D were administered daily for 9 con¬ secutive days; cyclophosphamide administered on day one only, ** Tumor weight inhibition calculated as (1-T/C)xl00. 50 94 93 * Altromycins were administered twice daily for 6 days, Cyclophosphamide was administered once on day 1. EXAMPLE 9 Altromycins B and D were tested against a human tumor xenograft in the subrenal capsule assay (SRCA) as des-cribed by Bogden et al., Cancer 48:10-20 (1981) . Human colon tumor line, LS174T, was maintained by serial propagation in nude, athymic BALB/C mice. Tumors were removed from the nude mice after reaching 0.2-0.3 grams and were minced into 1 m ^ pieces. The tumor fragments were then implanted into the renal capsule of CDFl mice. Immediately after implantation the tumor mass was measured with a steroscopic dissecting microscope fitted with an ocular micrometer calibrated in ocular units (OMU) . The mean tumor mass in OMU was calculated. Three days after tumor implantation mice received a single, intravenouse injection of altromycin B or D or 5-fluorouracil as reference. The results are illustrated in Table 15 below. Formulation The compounds of the present invention may be administered orally, nasally, opthalmically, parenterally, vaginally, rectally, topically or in an aerosol, in dose unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants and vehicles, as desired. The term "parenteral" as used herein includes subcutaneous, intravenous, or infusion techniques. Injectable formulations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic pharmaceutically acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oil may be employed including synthetic mono- or diglycerides . In addition, fatty acids such as oleic acid find use in the preparation of injectables. Suppositories for rectal or vaginal administration of the active ingredient may be prepared by mixing the active ingredient with a suitable, nonirritating excipient such as cocoa butter and polyethylene glycols which are a solid at room temperatures but liquid at the rectal/vaginal temperatures and will therefore melt in the rectum or vagina and release the active ingredient. Solid dosage forms for the oral administration may include capsules, tablets, pills, powders and granules. In such 'solid dosage forms, the active ingredient may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixers containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents and sweetening, flavoring and perfuming agents. The compounds of the invention can be used for therapeutically treating a mammalian host affected by a microbial infection or an experimental animal host affected by a malignant tumor. They are useful for the topical treatment of infectious diseases. Additionally, they can be used in disinfectant and sterilizing solutions or as preservatives in plastics, paints or fabrics. Total daily doses administered to a patient in single or divided doses can be in amounts, for example, 0.01 to 50 mg, and more usually 0.01 to 10.0 mg. Dosage unit compositions may contain submultiples thereof to make up the daily dose. The amount of the active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the compound; drugs used in combination and the severity of the particular disease undergoing therapy. Insofar as the present invention includes the manufacture of the altromycin compounds it is not limited to the use of Actinomycete sp. AB 1246E-26 or other strains corresponding to the same description, but includes the use of variants of these organisms such as are obtained, for example, by selection or mutation such as under the action of ultraviolet rays or X-rays or nitrogen mustards. The foregoing is merely illustrative of the invention and is not intended to limit the invention to the disclosed compounds . Variations and changes which are obvious to one skilled in the art are intended to be within the scope and nature of the invention which are defined in the appended claims.

Claims: £L M£ What is claimed is : A fermentation isolate of an Actinomycete,species comprising a compound of the formula: wherein Ri is selected from -NH2, -NHCH3 and -N(CH3)2,- and R2 and R3 are independently selected from hydrogen and hydroxyl such that at least one of R2 and R3 is hydroxyl. 2. A compound selected from the formula: wherein Ri is selected from -NH 2 , -NHCH 3 and -N(CH3) 2 , and 2 and R 3 are independently selected from hydrogen and hydroxyl such that at least one of R 2 and R 3 is hydroxyl, or a pharmaceutically acceptable salt or ester thereof. 3. - A process for producing the compounds altromycins A through G comprising culturing a microorganism belonging to the order Actinomycetales in a nutrient medium. 4. The process according to Claim 3 wherein said microorganism is Actinomycete sp. AB 1246E-26. 5. A substantially biologically pure culture of the microorganism Actinomycete sp. AB 1246E-26, said culture producing altromycins A through G upon culturing of said microorganism in a nutrient medium. -41- 6. A substantially biologically pure culture of the microorganism Actinomycete sp. AB 1246E-26, said culture characterized by: a. an appearance in media as described in Table 1; b. an ability to grow on carbon sources in synthetic medium as described in Table 2; and c. physiological characteristics as describ€d in Table 3. 7. A compound essentially characterized by: a. a nuclear magnetic resonance spectrum as shown in FIG. 3 of the accompanying drawings; b. an infrared absorption spectrum as shown in FIG. 2 of the accompanying drawings; and c. an ultraviolet/visible spectrum as shown in FIG. 1 of the accompanying drawings. 8. A compound essentially characterized by: a. a nuclear magnetic resonance spectrum as shown in FIG. 4 of the accompanying drawings; b. an infrared absorption spectrum as shown in FIG. 2 of the accompanying drawings; and c. an ultraviolet/visible spectrum as shown in FIG. 1 of the accompanying drawings. 9. A compound selected from the compounds essentially characterized by 13 C NMR data as shown in Table 7. 10. A pharmaceutical composition having antibacterial activity comprising a pharmaceutical carrier and a therapeutically effective amount of a compound chosen from the compounds of Claim 2. 11. A method of treating a bacterial infection comprising administering to a patient a therapeutically effective amount of a composition chosen from the compositions of Claim 10. 12. A pharmaceutical composition having cytotoxic activity comprising a pharmaceutical carrier and a therapeutically effective amount of a compound chosen from the compounds of Claim 2. 13. A method of treating tumors comprising administering to a patient a therapeutically effective amount of a composition chosen from the compositions of Claim 12.

Inventors: BRILL GREGORY M, JACKSON MARIANNA, KARWOWSKI JAMES P, MCALPINE JAMES B, THERIAULT ROBERT J, BRILL, GREGORY, M., JACKSON, MARIANNA, KARWOWSKI, JAMES, P., MCALPINE, JAMES, B., THERIAULT, ROBERT, J.
Applicants: ABBOTT LAB, ABBOTT LABORATORIES
Application year: 1990
Main classification: C07D311
IPC codes: C07H17, C12P19