PT93070A - PREPARATION PROCEDURE OF ALTROMYCINES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM - Google Patents

Description

70

Case 4654, PG.01

The present invention is a continuation in part of co-pending co-pending U.S. patent application Ser. 307,555, filed February 7, 1989.

TECHNICAL FIELD The present invention relates to a mixture of compounds and in particular to novel compounds of albomycin having cytotoxic and antimicrobial activity as well as to a process for the preparation of novel albomycin compounds.

BACKGROUND OF THE INVENTION

The compounds of the present invention are related, but are distinct, from the compounds of the pluramicin family which are described in U.S. Pat. 3,314,353; U.S. Patent No. 3,334,016; European Patent No. 200,818; European Patent No. 274-545; Byrne st., T, Antibiotics. 38: 1040-49 (1985); Maeda et al., J. Antibiotics, Sci A, 9: 75-81 (1956); ©

Kanda, J. Antibiotics. 24 = 599-606 (1971).

The compounds of the pluramicin family belong to the class of compounds derived from anthraquinone and were evidenced as the half-cells of Streptomyces and Actinomadura species.

Compounds of the pluramicin family exhibit, in vivo, potent anti-tumor activity. However. when compounds of the pluramicin family are administered to laboratory animals, they give rise to severe adverse side reactions such as toxicity to the kidneys. there is therefore a need for compounds with the anti-tumor potency of Compounds of the pluramycin family, but with a higher therapeutic index of activity, thus resulting in a decrease in clinical toxicities.

SUMMARY OF THE INVENTION The present invention relates to the process for the preparation of a lipid-containing product containing one or more albromycins, which are represented by the following structural formula:

Case 4654 = PQ = 01

in which 9 is selected from -NH2, -NHCH3 and -Ν (ΟΗ))? In which $ 233 are selected, respectively. ds between hydrogen and hydroxyl, such that at least one of the groups Î ± 2 β3 is hydroxyl. The present invention also relates to the process for the preparation of the individual albromycins and their pharmaceutically acceptable salts and esters.

The compounds of the invention are produced by an Actinomovce microorganism of the genus Nocardia which exhibits the identification characteristics described in Tables 1-3. In one aspect of a process of the present invention, strain AB1246E-26 of the species Actinomycete produces albromycins by culturing said microorganism in a nutrient medium.

The antibacterial effects of the novel compounds on specific microorganisms together with their chemical structure and their physical properties differentiate them from the other compounds having antibacterial activity.

For example. compounds of the invention of the present invention are different from the compounds pluramicin, or in the arrangement of the substituents on the glycoside substituents. The chromophore and the pluramicin compounds have a methyl group attached to the

-3Z3 O 70 619

Cas® 4654.PQ.01 -4-

of carbon 5 in ring B. In addition, members of the class of pluramicin have an amino sugar at the carbon atom 8 and the amino sugar at the carbon atom 10 of the D ring of the anthraquinone-gamma-pyrone nucleus.

In turn, the altromycins of the present invention have a neutral glycoside, a C-glycoside attached via carbon 13, to the carbon 5 of ring B. The compounds of the present invention are unsubstituted carbon atoms. The altromycins have a disaccharide unit containing a neutral 0-glycoside attached to an amino sugar at the 10-carbon of ring D. Thus, the albromicins of the present invention contain neutral sugars that are not found in compounds of the pluramicin type. In addition, albromycins are the metabolites of a Moeardia-like organism.

BRIEF DESCRIPTION OF THE DRAWINGS

UV / visible spectra of altromycin B, recorded in acidic, neutral and basic methanol solutions, are shown. Altromycins A, C, D and G exhibit essentially identical spectra. FIGURE 2 is an infrared (IV) spectrum of altromycin B in deuterochloroform solution (5¾). A, C and D altromycins exhibit essentially identical spectra. FIGURE 3 is a 500 MHz HIFI spectrum of HI1 of altromycin A in deuterochloroform (CBCI3). FIGURE 4 is a 500 MHz NMR spectrum of the altromycin S in deuterochloroform (CDCl3).

DETAILED DESCRIPTION OF THE INVENTION The compounds and mixtures of compounds of the present invention are prepared by culturing the microorganism of the species Actinomovectes AB 1246E-26 of the order Actinomycetales. The microorganism produces vegetative, branched hyphae, typical of. Acti nomycetes.

In addition, vegetative hyphae tend to fragment into small, irregular, often bacillary, snappy units typical of the species Actinomycetes. 70 619

Case 4654 =, 90.01 AS 1246E-26 strain of the species Actinomyces was isolated from soil collected in South Africa, A subculture of the microorganism was deposited in the permanent collection of "Agriculture! Research Service â € ¢ Northern Regional Research, US Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 E.U.ft., accession number for strain AB 1246E-26 of the species Actinomycephal in the depositary is NRRL 18371,

Morphology and Caractheristics of Cultures The microorganism of the present invention, of the species Actinomycete, AS 1246E-26, may be characterized by the morphology and other culture characteristics. In addition, whole cell hydrolysates may be used to provide the cell wall composition and the combination of morphology and chemical composition may be used to classify aerobic Actinomycetes into groups according to their cell wall type, Lechevalis and Lecheva, et al., Reported in Inter. J, Syst, Bacterium 1, 20? 435-443 (1970) have developed this classification scheme. The morphology of nocardioform, the chemical composition and resistance to lysozyme of strain AE 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 all cell hydrolysates. The chromatography of sugars in whole cell hydrolysates showed arabinose and galactose as diagnostic sugars. Specifically, the microorganism of the present invention has a Type IV cell wall containing msso-2,6-diaminopimelic acid and has a pattern of whole-cell type A sugars (arabinose and galactose). The chemical composition of the microorganism was also characterized by extraction and analysis of cellular menaquinones. The major menaquinone had a molecular weight of 720, as determined by mass spectrometry, indicating that menaquinone was tetrahydrogenated with 8 isoprenoid units.

In addition, the microorganism of the present invention is -6- 70 619

Case 4654.PG.01 lysozyme resistant. Lysozyme is an enzyme that breaks down the amino acid cross-linking of the amino-hexyl chains, the pseudoglycan layer of the cell walls of many, but not all, Gram-positive bacteria and lysozyme resistance constitutes a useful identifying characteristic. of the aerial growth product of strain AB 1246E-26 of the species Actinomycete has a light gray to white color, the microorganism forms a non-melanocyte diffusing pigment, several mediums growing ability to grow, with H 2 O 3 SO 4 In Table 3, the appearance and characteristics of AB 1246E-26 strain of the species Actinomycephus in various media are described in Table 3. The results are described in greater detail in Table 1. AB 1246E-26 of the species Actinomycete of Ascar ac • ------- --------- J --- --- it

Table 1

Characteristics of cultures, species AB 1246E-26 of the species Actinosnyce I IEX FACES CULTURE CITTERS * " Agar of Gs extract Abundant yeast extract / extract: White to light gray (264) ** T-0 R: Cashew nut powder (43) ISP 2 SP: Brownish (58) Flour agar of p B Moderate white oats ISP 3 AM 2 Dispersed, white TOO T ji »V, · 1 li R · Yellowish gray (93) SP: Absent Agar of inorganic salts G: Poor nor cos-starch AM: Dispersed, white ISP 4 SP = Absent -7- 70 619

Case 4654.96 = 01 iabela 1 (continuation) MEfi CHARACTERISTICS OF CULTURE ' and glycerol-aspartic acid: Abundant paraffin AMPs dispersed, white TSP 5 Ri Color-orange-red-gray (39)? orange (53)? (90) SP = Light gray to light gray (45) a Iron and exaggerated agar: Moderate yeast tract-AM: White-peptone R: Color-white to black-white ISP (39) SP 2 Light reddish brown (42) / tyrosine fillet 6 »Abundant ISP 7 AM: Yellowish white (92) R5 Reddish brown (43) SPs Reddish brown / Nutrient agar G S Moderate AM: Dispersed5 white A: Light orange (52) SP: Lightly grayish reddish brown (45) / Czapek filigree 6: Moderate AM: Dispersed, white R: Yellowish gray (93) SP: Absent / Poor calcium AM: D i sper so * white SP: Absent 70 619

Case 4654.PG.01

Table 1 (continued) MEAT CULTURE CHARACTERISTICS * ATCC # 172 G: Moderate AM: Scattered, light gray (264) R: Red orange accented (39) SP: Absent Gause # l modified G - Moderate (KN03 0.1%, K 2 HPO 4 AM: Dispersed, white 0.05%, MgSO 4 0.05%, R: Light gray (264) NaCl 0.05%, FeSO 4 SP: Absent 0.001%, 0.1% starch, yeast 0.01%, agar 1.5)

Observations were made after incubation for 14 days at 28 ° C. * Abbreviations: G = growth; AM = aerial mycelium; R = inverse; SP = soluble pigment -X "

The names and color numbers between, parentheses, follow the color pattern of 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

Cass 4654.PG, 01 _o_

Table 2

Oio single carbon source oor

GROWTH

Use of various compounds ficfcinomycete sp. AB 1246E-26 *

SOURCE OF CARBON

Adonitol Arabinose Cellulose Dulcitol Fructose Galactose 61ucose Inositol Inulin Lactose Maltose Mannitol

Melesitosis

Melibiose

Raffinose

Ramnose

Ribose

Salicin

Sorbitol

Starch

Sucrose T r eals

Xiloss ++ = Soa use = Misuse = Hand used The incubation was

svsda s cable at 280C for 43 days

Shirlingr E.B. & D, Streptomyces species,

Sottlieb * Methods for characterization of

Intern. J, Syst, Sactsriol, 16, 313-340 (1966)

70 619

Case 4654PG. 01 -10-

Tabel 'physiological characteristics · AB 1246E-26

Actinomycete REACC30

TEST

Starch hydrolysis "

Production of H2S " '

Melanin formation / Peptide agar na-ex tr ac tg d > yeast-iron shot agar1

(4% Growth Agar, not 1% HCl, 50æl extract) was measured at 1 ° C / mperature (Growth agar at 212 ° C-422 ° C, ext. ; No yeast extract no growth at 540 ° C (60 ° F)

Decomposition of ";

In addition,

Casein +

Xanthine

Hypoxanthine *

Resistance to antibiotics (50 micrograms / ml) on nutrient agar (9 days at 28 ° C): erythrophicin gentamicin - kanamycin novobiocin - oxy - tetracycline - rifampin streptomycin vancomycin G ordon, R I n t, J. Svst. (i.e. Sarnett Bactsriol, D A *, Hía nder h a n 24: 54-63 (1974).

J pa Oj JJj >

Table 5 (continued)

Smibert, R.M. and N.R. Krieg, Manual of Methods for General Bacteriology, American Socety for Microbiology, Washington, U.S.A., pp. 409-443 (1981),

Fermentation

Although other culturing methods may be used, a stirred, liquid submerged culture process is preferred. The culture is grown in a culture medium which includes a carbon source and a source of nitrogen. Useful media include an assimilable carbon source, such as starch, sugar, molasses, glycerol, a combination of glucose and molasses, etc .; a source of assimilable nitrogen such as a protein, a protein hydrolyzate, polypeptides, amino acids, peptone plus yeast extract or whole yeast, etc. in other organic or inorganic ingredients which may be added to stimulate the production of the compounds, such as anions, and inorganic cations including potassium, magnesium, calcium, ammonium, sulfate, carbonate, phosphate, chloride, etc. In addition, buffers such as calcium carbonate may be added to assist in the control of ρ'-J of the fermentation. Aeration may be provided by forcing the passage of sterile air through the fermentation medium. Stirring may be provided by agitating the vessel itself or by shaking the culture, for example with a mechanical stirrer. The fermentation is generally carried out in a temperature range 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.

Insulation and Purification

After the fermentation is completed, the compounds of the invention are recovered from the medium by repeated extractions with a water immiscible solvent, such as methylene chloride. The fermentation product of AB 1246E-26 of the species actinomycete is thus obtained. 70 619

Case 4654 = PQ.01 exhibits anti-bacterial activity and includes the compounds altromycin A, altromycin B, altromycin C, altromycin D, altromycin E, altromycin F and altromycin 6 = The compounds, altromycins A to 6, can be further purified by sequential chromatographic techniques in countercurrent, as is well known in the art. Isolation of the altromycin compounds can be monitored by TLC sample analysis, with appropriate visualization methods, sensitive to the herein anion antron portion of the compounds. TLC can be performed using additional HPLC plates of silica from EM reagents (silica gel 60 F054 orally coated 10x10 cm, E. Merck, Darmstadt, Federal Republic of Germany). The albromycin compounds are visualized at light visible as orange-speckled, in UV light of small wavelengths as dark blue spots on a light blue background 3 to light U.V. of a large wavelength, like bright orange spots, is a moderately dark blue background. The compounds are disclosed in these plates using a Sprayer of S9 4 4 5 (in EtOH). The solvents used to develop TLC plates range from 30-90% chloroform with methanol, to make 100%, depending on the particular sample concerned. The altromycin compounds are usually within the range of Rf values of 0.2-0.6. Isolation of larger amounts of aldrich from crude extracts may also be effected by the use of 60% glycation of E.M. Reagents for layer chromatography. (E. Merck, Darmstadt, West Qermany), which may be used as a filler for large columns. The sample loads.> If they are as high as 0.8 g of sample / 10 ml of silica, assays preparations. A simple elution gradient is used. with 100% chloroform, increasing in methanol (10%) increments until a concentration of 20% in MeOH was reached.

The Tracts sluids - »column» s scrolled s analyzed -13- 70 619

Case 4654.PG.01-as to antibacterial activity, by a diffusion test on an agar plate. A 20 microliter sample of each fraction is applied onto a paper disc. The discs are then plated onto agar plates which have previously been cultured with organisms sufficient to provide a cloudy background after incubation, typically 10 to 10w colony forming units (CFU) per ml. The formation of a clear zone around a paper disk is an indication that the test fraction contains a compound or compounds having antibacterial activity. The above described may be better understood with reference to the following examples and which are presented for illustration and not for limitation of the practice of the inventors. EXAMPLE 1 The strain AB 1246E-26 of the species Actinomycete (NRRL 13371) was maintained stored as a frozen inoculum, by freezing a portion of the original inoculum and storing at -75 ° C. The 5B7 medium (Table 4) was used for seed growth in N2B1 medium (Table 5) for fermentation,

Table 4

Sowing medium 5B7

Ingredients g / 1

Glucose monohydrate 10

R

Starch, Stable JUET '(produced by 15 A.E. Staley Manufacturing Co.,

Decatur, IL, U.S.A.)

Yeast extract (produced by 5

Difco Laboratories, Detroit, MI, USA) M2 amine type A (produced by Humko 5

Shsffield, Memphis, TM, U.S.A.)

Calcium carbonate 1

Distilled water was added to a volume of 11, the pH was adjusted to pH 7.0. 70 619Css © 4654 = PG = 01-14 Table 5. Production Medium M2ES1 - Ingredients Glucose Monohydrate Peptone F-152 (produced by Index Corp., Chicago, IL, E.ILA) Molasses (Brsr Rabbit * ', Qreen Label, produced by Del Monte Corp., San Francisco , CA, USA) Yeast extract (produced by Difeo Laboratories, Detroit, MI, USA) Yeast extract (produced by Difeo Laboratories, Detroit, MI, USA) Calcium carbonate 20 10 5 1 1 2

Distilled water was added to make up to a volume of 11 Âμl = 0 pM was not adjusted. The medium was prepared for sowing as follows: 10 ml of the sowing medium (Table 4) were placed in 25x150 mm glass seed tubes. 100 ml of the seed medium (Table 4) were placed in 500 ml Erlenmeyer flasks. The tubes were sealed with stainless steel capsules (Mortc-n closures) and the flasks were capped with "rayon" dressing rolls. The tubes and flasks were then sterilized for 35 minutes at 1212C, 103 kPa. The inoculum for the fermentation was prepared in two steps. In the first step, 5% of the frozen inoculum was inoculated into the seed tubes containing 10 ml of the 5537 seed medium. The tubes were incubated for 96 hours at 28 ° C on a rotary shaker operated at 250 rpm, with an amplitude of 5.7 cm. In a second step, 5% of the growth tube vegetative inoculum was used to inoculate the seed flasks containing 100 ml of the 5B7 seed medium. The seed flasks were 70 619, Ο '

Case 4654.PQ.Q1 -15-, ./ν'- '-; incubated for 72 hours at 28 ° C on a rotary shaker operated at 250 rpm with an amplitude of 5.7 cm.

Then, 5 percent of the seed inoculum from the second stage seed balloon was aseptically transferred to 200 500 ml Erlenmeyer flasks, each containing 100 ml of the N2B1 production medium (Table 5). The fermentation flasks were incubated for 5 days at 28 ° C on a rotary shaker. The stirrers were operated at 250 rpm with an amplitude of 5.7 cm. After completion of the incubation, the contents of the fermentation flasks were collected and combined. The volume collected was 18.0 liters. EXAMPLE 2 The fermentation broth (18 1) of Example 1 was adjusted to pH 10.0 with 6 N MCI and extracted three times with 4 L portions of methylene chloride. The methylene chloride extracts were combined and concentrated in a vertical evaporator until approximately 4 g of oil were obtained. The concentrate was partitioned into a solvent system, MeOM-S-20-CCl 4 (5: 2: 5), using a countercurrent drop device with the dense phase as the stationary phase. This device consists of 30.5 m of continuous Teflon tube, Dl (internal diameter) 0.48 cm, rolled, in a repetitive scheme 2x14 cm, around a wooden board of 2.5 cm by 15 cm. The serpentine had 96 revolutions with a total internal volume of 450 ml, approximately half of which was held as the stationary phase. Compounds exhibiting antibacterial activity were eluted at a flow rate of 5 ml / min in the numbered bouts of 26-80 (10-12 ml each, hereinafter referred to as Fraction A) which gave approximately 255 mg of a colorless oil after concentration in vacuo, and in numerated fractions of 117-121 (10-12 ml each, hereinafter referred to as Fraction B the end of which corresponds to the start of the steady state entrainment) which gave 570 mg of a red-orange oil (after concentration in vacuo),

Fraction A fraction (fractions numbered 26-80).

Fraction A (exhibiting activity -16-70 619

House 4654.PG.01 rntibactsriana) was fractionated in a countercurrent device " Coil Planet Centrifuge " (CPC), (P.C., Inc. Potomac, Maryland, U.S.A.) employing the following conditions; MeOH-HoO-CCl 4 (5: 2: 5); phase dense stationary lower end as input; flow rate 4 ml / min at SOO rpm; retention of 85-90% of the stationary phase; Collection of 10-L fractions Two bands of antibacterial activity were obtained, fractions numbered 35-41 (hereinafter referred to as Fraction Ai) and numbered fractions (hereinafter referred to as Fraction A2) 224.7 mg) was rechromatographed on the CPC device. The conditions for the chromatography of the Po fraction were as follows: 0.01 M-CCI4 aqueous MeOM-NH4 O2c (5: 2: 5); stationary dense stage; lower end as input; flow rate 4 ml / min at 800 rpm; retention of 85-90% of the stationary phase; collection of fractions

10 ml. Two bands of antibacterial activity were observed, fractions numbered 16-21 (hereinafter referred to as Fraction P00J which yielded 24.7 mg of red-orange oil after concentration in 50/110 and fractions numbered 43-80 (hereinafter referred to as Fraction & 2b) which gave 230.4 mg of pure orange after concentration in vacuo The oil obtained in the concentration of the Apa fraction was essentially the same as the material obtained from fraction A, and was identified as altromycin A. It was found It was found that the Açb fraction contained AL

Fraccionamenfco da. Fraction B (fractions numbered 57-45) The fraction B concentrate was fractionated in the CPC countercurrent device employing the following conditions: hexane-FTOOAc-MsQH-HoO3: 7: 6: 4, more dense stationary phase! lower end as input; flow rate 4 ml / min at SOO rpm;

retention of 85-90% of the stationary phase, collection of 5 ml fractions. An activity band, numbered fractions 37-45 (hereinafter referred to as fraction Bj) which yielded 29.7 mg of red-orange oil after concentration in vacuo, the red-orange oil 1 of fraction B was identified as being a mixture of altromycin C and altromycin D. Althromycin C and α-17 670 619

Case 4654, PG.01 altromycin D may be further separated in the countercurrent device described above using 0.01 ML hexane-EtOAc-MeOH-NH4 OAc. The total yield of altromycin A was 56.7 mg, total yield of the altromycin E was 230.4 mg and the combined total yield of the C and D altromycins was 29.7 mg. EXAMPLE 3

The AS 1246E-26 strain of the species Actinomyces (NRRL 18371) was kept as a frozen inoculum by freezing a portion of the original inoculum and storing at -75 ° C, the seed medium for a stirred fermentation of 80 liters was prepared as follows:

10 ml of the 5B7 seed medium (Table 4) was placed into 25x150 mm sowing glass tubes. Six hundred ml of the seed medium 5B7 (Table 4) were placed in 2-Erlenmeyer flasks 1. The tubes were covered with stainless steel capsules (Morton closures) and the flasks were capped with "rayon" dressing rolls. The tubes and flasks were sterilized for 35 minutes at 12 ° C, 103 kPa. The inoculum for fermentation was prepared in two steps. In the first step, 1% of the frozen inoculum was inoculated into the seed tubes containing 10 ml of the 5B7 seed medium (Table 4). The tubes were incubated for 96 hours at 28 ° C on a rotary shaker operated at 225 rpm, with an amplitude of 5.7 cm. In a second step, 5% of the growth tube vegetative inoculum was used to inoculate the seed flasks containing 600 ml of 5B7 seed medium (Table 4). The seed flasks were incubated for 72 hours at 2820 on a rotary shaker, operated at 225 rpm, with an amplitude of 5.7 cm.

80 liters of N2S1 production medium (Table 5) were prepared in a stirred 150-liter Wew Brunswick stainless steel fermenter. The N2B1 medium was sterilized in the fermenter at 12 ° C and 103 kPa for 1 hour. The glucose was aseptically sterilized as a 50% aqueous solution at 121 ° C for 30 minutes and added to the fermenter after 70 ° C.

The antifoam agent used was XFO 371 (produced by Ivanhos Chemical Co., Mundelein, IL, U.S.A.). It was initially added in an amount of 0.01 S, becoming available if necessary. The fermenter was inoculated with 4 liters of the result of the second seed growth step. The temperature was controlled at 28Â ° C. The stirring speed was 200 rpm and the air flow was 0.7 vol / vol, minute. A pressure of 34.5 kPa was maintained. The fermentation was terminated after 120 hours. The volume collected was -SI liters. EXAMPLE 4

The fermentation broth (611) of Example 3 was charged to a 200 liter stainless steel rotary drum (Morse

Manufacturing Co. Inc., East Syracuse, M.Y., U.S.A.). The broth was shaken four times with portions of 20-24 l of methylene chloride or chloroform, with rotation, for 30-45 minutes while draining the organic solvent. The chlorinated solvent extracts were combined and concentrated on a vertical evaporator to a volume of 11. This volume was reduced to approximately 100 ml of oil on a rotary evaporator and then fractionated several times between methanol and n-heptane (1 = 2). The combined heptane phases did not reveal the presence of tremor-type 1 compounds when analyzed by TLC. The methanol phase was concentrated in vacuo and partitioned several times between water at pH 10.0 (MH 4 OH) and chloroform. centrifugation (1000 rpm) to remove a protease type interfacial precipitate, which developed during fractionation. The chloroform layer was concentrated in vacuo to give 12.09 g of a reddish-brown oily solid. The concentrate was fractionated in the MeO1 · 5 - (5: 2: 5) solvent system using a countercurrent drop preparative device (PQCC), with the dense phase as the stationary phase. The PSCC device was fabricated in situ and consisted of 60 meters of continuous tubing of Teflon. The tubing had an alternating diameter and was wound to 70619

Case 4654.PQ.01 a 3x28 cm (3x28 cm) repetitive pattern (the piping being substantially right with the excision of angled folds of the smaller diameter sections, at the top s at the base of each turn) so that a 24 cm section tubing with a DI of 4.5 mm would occupy the forming side of the serpentine droplets and a 35 cm section with a D.I. of 1.5 mm, occupied the return side of the mobile phase of the coil. The serpentine showed 100 revolutions with a steady-state retention volume of approximately 550 ml at rest. During use 90-95¾ of this stationary phase volume was retained. The serpentine had, at rest, a volume of mobile phase of approximately 70 ml. Operation in the serpentine was effected at a rate of 3-4 ml / min, collecting fractions of 10-15 ml. Approximately identical operations were performed with a 50¾ sample portion. On the basis of the TLC analyzes of the fractions, two portions of compounds of the altromycin type were obtained in each operation. The first 100 fractions, excluding the first twenty, or about twenty, which contained a significant level of impurities and few or no altromycin-like compounds, were pooled (hereinafter referred to as Fraction A4). The following 100 fractions which included stationary phase entrainment were also pooled (hereinafter referred to as Fraction 34).

Fraction A4 fractionation Fraction A4 was concentrated and re-introduced into the PGCC device in the same manner using the solvent system 1-OH-F-O-CCl (10-5 = 10). From this operation the first 120 fractions (excluding the initial 19), based on TLC analysis (hereinafter referred to as Fraction A4 <). These fractions were concentrated for further purifications using a Coil Planet Centrifuge (CPC) countercurrent device (P, C., Inc., Potomac, Maryland, E.J.A.) followed by standard column chromatography as is illustrated below in SCHEME 1. This part of the isolation scheme resulted in the isolation of altromycin E (4.7 mg) and G (6.8 mg). 70 619

Cass 4654, PS.01

Fractionation of Fraction B4 Fraction 84 was concentrated and introduced into the PQCC device (2 1/2 sample loading operations) using the MsOM-FbO-n-heptane-CCl 3 solvent system. (5 = 2: 2: 3). From each of these operations the first ~ 100 fractions (excluding the initial ones) were combined on the basis of TLC analyzes (hereinafter referred to as Fraction B4j) = This portion of the isolation scheme resulted in the isolation of altromycin F (7.8 mg), altromycin A (~ 4 mg mg), altromycin B (~1 g), altromycin C (~50 mg) and aldromycin D (~100 mg), * -01-70 619

Case 4654.PQ.01 SCHEME 1 section A4i

I

CCP

MeOH-MiiOAc 50 mM-CCl.4 (10 = 5; 10) dially stationary, bottom end as an inlet, 3-4 ml / minute. @ 10 ° C fractions of 10 ml (f) (16-23)

Silica at low pressure .. (R) (Reagents tM Lobar fa *,

SiC 60 (40-63 μ.), 2.5 x 25 cm, 34.5 kPe CHCl 3 -MeQM (9: 1), f ml (15-27) HPLC-prep. (Separation Technologies, Wakefield, RI) 2.5 x 35 cm, 30 μM MsOH-70 μM of [8% THF in 50 mM Et2 NHQAc, pH 5,0] • accommodate the active peaks in UV at f of 100-200 m]

Sprec

(3: 7 = 6: 4) Stable Densely Rase, bottom end as inlet, 4 ml / min, ε 800 rpm fractions of 10 ml

I

Altromycin E f (171-2121 -22-70 619 'ase 4654.PG.01 SCHEME 2

Fraction

CCP

MeOH-H2O-n-heptane-CCl4 (10: 4: 1: 9): dense stationary assemblies, 4 mL / minute inlet end. 0 SOO rpm, cross sections (f), 10 ml 58-81)

I

CCP CMC13-CC14-HeOH (50 mM Et2 NHOAc pH 5.0 (15: 135: 150: 60) less dense stationary phase, 4 mL / min at the end of the reaction,

I

CCP CCI4-M3OH (Et) 3WH0Ac 50 mil pH 5.0 (15: 15: 6) less dense stationary phase, 4 mL / minute @ 800 rpm, 159) (recovered stationary phase)

CCP CHC13-CC14-MeOH-H20 (1 = 4: 5: 2) less dense stationary phase, inlet end 4 ml / min 0 800 rpm, 10 ml v

CCP hexane-EtOAc-MeOH-N2 OAc 10 mH (3: 7: 6: 4) less dense stationary phase, inlet end 4 ml / minute ® 800 rpm,

Altromycin B (22-80)

Altromycin A f (96-125) (recovered stationary phase) • Altromycin D f (72-104)

Altromycin C (107-118) Altromycin F (120-128) 70 619

Case 4654, PG. EXAMPLE 5

Characterization of the A, B, C, D, E, F and 8

The altromycins of the present invention may be dried to the glass and are red-orange compounds which are readily soluble in methanol (MeOH), chloroform (CHCl3), carbon tetrachloride (CCl4), dimethylsulfoxide (DMSO), ethyl (EtOAc), benzene or dilute acid; soluble moderates in 0.01 M phosphate buffer (pH 7); and slightly soluble in n-hexane or water. When chromatographed on a reverse phase HPLC column (4.6 mm x 25 cm) using MeQH-NMUiOAc · 0.05 M (60-40) at 2 ml / min, the altromycin A showed a retention time of 10.8 minutes 3 to altromycin S a retention time of 12.8 minutes. Fast atom bombardment mass spectrometry (FAB), high resolution , in the JL ion mode. positive reaction gave a mass (M + H) " of 912.3645 (calculated value, 912.3654) for altromycin A, a molecular formula corresponding to C45H57NO1Q. A mass of 926.3828 (calculated value, 926.3810) corresponding to the molecular formula C47 H59 NO3 was obtained with the β-altromycin. The mass (MsH) of altromycin C was 896.3686 (calculated value, 896.3705), corresponding to a molecular formula C46M57MO17 and the mass () for aldromycin D was 910.3868 (calculated value 910.3861), corresponding to a C47H59MQ17 molecular formula. The mass (M + H) of the altromycin E was 896.3722 (calculated value, 896.3705) corresponding to a molecular formula C46H57W7.7. Altromycin F gave a mass of 910.3832 (calculated value, 910.3867), corresponding to a molecular formula 247H59NO17 and the (MsH) 'mass of altromycin 8 was 898.3497 (calculated value, 898.3497) corresponding to C46 H55 NO3.

The ultraviolet / visible (UV) spectroscopic results (Figure 1) were essentially identical for slmromycins A, Ξ, C, D and 8 s and placed in the class of compounds derived from anthraquinone. The maximum UV absorption in 70 619 * τ..Γ! 4654.PS.01 & ΰ, 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. Absorption maxima in the JV, acid solution, are at 424 nm, 276 nm, 243 nm at 215 nm. The UV spectra of the E and F altromycins are essentially identical to each other, but differ from those of the altromycins A to D and Q in the shifted spectra in basic acid solution. The summary of the UV results for the altromycin compounds is given below in Table 6, The infrared (IV) spectrum of the altromycin B is shown in Figure 2.

Table 6

UV Absorption Maximums for Altromycins A to G

Lmax NaOH 490 nm. 368 nm, 304 nm and -MeOH 252 nm Altromycins A-D 424 nm. 242 nm and 212 nm MCI- 424 nm, 276 nm, 243 nm and -MeOH 215 nm NaOH- 545 nm, 338 nm s 250 nm -MeOH Altromycins E s F L (ia> 440 nm, 241 nm s 209 nm Lffiax-d-1 M HCl-MeOH 424 rim, 241 nm and 207 nm --niax-f® w NaOH- 488 nm 366 nm, 302 nm s -MeOH 250 nm Altromycin Q LfiiaxMs0H 424 nm 242 nm s 209 nm -max ^ s ® ^ HCl-MeOH 423 at, 273 nm, 242 nm and 209 nm = -ma; - Lambdamax = wavelength at the maximum absorption.

The spectroscopic results of RHN (Tables 7 and S are FI6U3 in AS 3 and 4) establish that the altromycins A, B, C, D, E, F constitute a new group of compounds of the class of compounds 70 619 Case 4654. PS, 01 --Τ-1 /

Anthraquinone derivatives. Altromycins E and F are similar to altromycins A and B, respactivaments, with the proviso that the altromycins E and F have a proton on the carbon atom 13, replacing the hydroxyl functional group γ-altromycin 8 differs from the altromycins A and E because carbon 4 " of the amino sugar provides a primary amine as opposed to a mono or dimethyl amine. The NMR chemical shift results of NMR and the NMR / chemical shift / coupling results are shown in Tables 7 and 8, respectively.

Table 7 1T ΐ.

Chemical shifts of RMM-β- to Altromycins Aβ-

Carbon N2. ft B C D rr e_ F G DEPf ° Ã *. 167.5 167.4 167.0 167.0 165.7 165.7 167.5 0 3 111.1 110.9 110.9 110.8 111.3 111.3 111.1 Cl ·! 4 180.2 180.0 179.4 179.4 178.5 178.6 180.2 Θ 4 to 126.6 126.4 126.5 126.4 126.3 126.3 126.3 126.6 Θ 5 149, 2 149.1 149.4 149.2 148.2 148.2 149.3 0 6 122.5 122.4 122.9 122.8 24.1 124.0 122.5 Cl ·! 6a 137.2 137.1 137.0 136.9 136.9 136.9 137.2 B 7 131.2 181.0 181.2 181.2 131.5 181.4 181.2 to 7a 130.3 130.2 130.4 130.3 130.5 130.5 130.5 q OO 119.8 119.7 119.7 119.7 119.5 119.5 119.8 CM 9 133.6 133.7 133 , 4 133.6 133.3 133.5 133.7 CM 10 141.3 141.1 141.0 140.9 141.0 140.9 140.7 0 11 159.1 159.2 159.3 158, 9 159.3 159.3 159.4 to 11a 115.8 115.6 115.9 115.7 115.9 115.9 115.9 to 12 186.9 186.9 187.1 186.9 187.5 187.4 186.9 θ 12a 121.8 121.6 121.6 121.6 120.8 120.8 121.8 to 12b 156.8 156.7 156.6 156.5 156.1 156.1 156 , 8 to 13 80.9 80.8 79.0 78.9 - - 80.9 to 13 - - - - 48.3 48.2 - CH 14 59.8 59.7 59.8 59.7 59, 8 59.7 59.8 a

Table 7 (continued) -¾c 19 &quot; 7 19 6 "* 7 ^ 19.8 19.7 20.0 199 19.6 Go 16 62.7 / * r- 5 C3 62 and b 62, b * 7 E, 62.5 62.7 CH 13.4 13.3 13.4 13.3 13.5 13.4 1 &quot; 7? 18 170.5 170.4 170.9 170.9 170.4 1 7fi] 4 JL tf -W and 170.5 g 19 b2.6 b2, b 52.4 52.3 H7 3 * · co 75T 3d y . C (O) -O-C (O) -O-C (O) -O- CD &gt; 68 3 2 0 0 0 0 0 0 0 0 0 0 0 0 69.0% PJI A 7 80.2 80.1 74.8 74.9 Oli c ϋΐ jj 81.4 80.2 Ui t T i 67.9 67.9 - - <&lt;? &gt; 0 UU JV 67.9 68.0 Λυ ϊ_. Ϊ 2 C 7 - - 26.1 26.0 - - - ru-Uj 29 .S 7 * T * 7 TJ · j / 70 7 70.2 * 7 -? 74.0 73 7 Go to • • • • • • • • • • • • • • • • • • 14.1 14 0 14.7 14.7 14 7 14.6 14.0 Lj. - 57.9 ζ7 or D / and lU bb ^ 6 ΓΓ ir1 bb b 7 57.1 57.2 Ci-s3 * 3 &quot; 70.3 70.7 70.2 70.8 70.3 70.8 70.0 CM Τ 'n 77 7 * * 5 * θ' * * s XJ ^ L and C? 77.8, 77.7, and 77.7. EXAMPLE 76.0 (b) and (b). · · And - * r * 0 to 30 and 4, 51,7 θ C ti 40.3 dtJ. 7 * - 7 s 40,4 44,7 40 4 and - 44,3 / i £ t 9 * '' 7 11 62,1 SO 62,3 69 '' 7 62,3 ^ Th τ 1, 2, 3, 4, 5, 7, 11, 14, 13, 14, 13, 14, 13, or 14 1 7 'CH3 λ 11 m * ~ 0' &gt; 3 94 1 14.0 23.8 13.9 10.3 14.1 ** * and * 32.6 CM ** 27.9 40.3 27.8 40.3 28.1 40.4 Cr z z N (CM3) n η = 1 r- | 7¾ η = 1 n = 2 η = 1 n = 2 n = 0 1 11 7 97 &lt; $. 9. and 94.5 93.3 CH? 30, S 31.1 30.9 31.1 30.9 71 1 - and 30.8 CM2 * 711? 74.8 74.9 74.8 74.9 74.9 75.0 74.8 CM 72.1 72.1 72.1 72.0 72 _ 2 * * - 7 ^ ΎΟ O &quot; 1 H-NMR (DMSO-d6):? 7 65.4 65.0 f c * 7 QO and 65.0 65.4 65.1. 7,77 17,6 17,7 17,6 * 9 r 17,7 17,8 V-8 6 ^ T! ? A 99 9 w? - 96 1 56.1 56.1 (c): 9A (5) p, 7K ... ν ϋ Τ Τ Τ Λ Λ Λ 3 3 3 3 3 MHz in CDCl3, Οϊ * Ξ553 V? X-03. Warm-up to TMS.

13 C-NMR Dfc.EN results for the alleles TABLE 8 - Chemical shifts and 1H NMR coupling to; (1) Altromycin B (2) Altromycin C (3) Altromycin D (4) Altromycin E (5) Altromycin F (6) Altromycin S (7) Althromycin A (6)

multiplicity -28- 70 619

Case 4654.PQ.01 EXAMPLE 6

Antibacterial Affectivity The antibacterial activity of the albomycin compounds of the present invention against various bacteria is illustrated in Table 9. Minimum inhibitory concentrations (CIFi) are determined by the agar dilution method, which is described hereinbelow, using agar &quot; Brain Heart Infusion &quot; (EHI).

Twelve petri dishes each containing 1: 2 successive aqueous dilutions of the test compounds were mixed with 10 ml sterile SHI agar. Each plate was inoculated with 1: 100 dilutions (or 1:10 for growth strains Micrococcus and Streptocoocus) cultures from overnight cultures of up to 32 different microorganisms using a Steers t-block. calibrated to release about 10 &quot; colony forming units (CFUs). In addition, a control plate was prepared using BHI agar, containing no test compound, ® which was inoculated at the beginning and at the end of each test. The inoculated plates were incubated at a temperature from about 35 ° C to about 37 ° C for approximately 20-24 hours. Cyprofloxacin was used as a reference antibacterial agent. After incubation, each of the agar plates was observed reliably on the presence or absence of growth of microorganisms. MIC was defined as the lowest concentration of test compound with no growth. The small halo, caused by the inoculum patch, was weighed against growth on a control plate containing no test compound,

The results are represented by the data given in Table 9 below. 70 619

Case 4654.PQ.01

Table 9 CXM ('gram / ml) for CT8SC1 bacteria. ascorbic acid ORGANISM ALTROMICINE A B esp Acochinetobacter sp. 100> 100 100 Enterococcus faecalis ATCC 8043 6.2 3.12 3.1 Eschsrichia coli DC-2> 100> 100 100 Enterobacter aerogenes ATCC 13048> 100> 100 Enterococcus faecalis ATCC 8043 6.2 3.12 3.1 Eschsrichia coli DC-2> 100 100 Escherichia coli H560 &gt; 100> 100 100 Eschsrichia coli JUHL> 100> 100 Escherichia coli KNK 437 25 25 25 Eschsrichia coli SS 0.39 0.20 <9> Klebsiella pneumonia ATCC 8045> 100> 100 100 Micrococcus luteus ATCC 4698 1.56 0.39 1.56 Micrococcus lutsus ATCC 9341 0.2 0.10 &lt; 0.39 Provided for CMX 640> 100> 100> 100 Pseudomonas aeruginosa A5007 100> 100 50 Pseudomonas asruqinosa BMH10 50 50 100 Pseudomonas aeruginosa K799 / 61 0.39 0.10 0.7 S Pseudomonas aeruginosa K799 / MT 3.1 3.12 3.1 Pseudomonas cepacia 2961. &gt; 100> 100 50 Staphylocoecus aureus 45 1.56 0.39 1.56 Staphylocoecus aureus 45 RAR2 6.2 3.12 6.2 Staphylococcus aureus A5177 1.56 1.56 1.56 Staphylocoecus aureus ATCC 6538P 0.78 0.39 0.73 Staphylococcus aureus CMX 503 - 1.56 - Staphylocoecus aureus CMX 553 3.1 3.12 3.1 staphylocoecus aureus 642A 3.1 - 3 1 ^> - Staphylocoecus aureus CMX 686B - jL "- Staphylocoecus aureus 3519 6.2 - 6.2 Staphylocoecus aureus NCTC 10649 3.1 - 3.1 Staphylocoecus epidermidis 3519 6.2 1.56 6.2 Strsptococcus agalactiae CMX 508 0.39 0 , 39 &lt; 0.39 Strsptococcus bovis A5169 0.39 3.12 1.56 Streptococcus pyogenes 930 0.39 0.20 &lt; 0.39 Streptococcus pyogenes 2548 0.78 0.39 0.78 Strsptococcus pyogenes EES61 0, 39 0.39 &lt; 0.39 -30- 70 619

Case 4654 = 96.01 EXAMPLE 7

Cytoxicity

The compounds of the present invention exhibit potent in vitro activity against both types of human cells in d-3 'urine. The toxicity of the A, B and C allemoses against HeLa cells shown in Table 10 shows the toxicity of the A, E and D altromycins against human and murine tumor cell lines shown in Table 11. Inhibitory concentrations capable of killing 50% of the cells (IC 50) were determined in an immersion color test for determination of cytotoxic activity against culture cells according to the following protocol - a three-day microtiter assay was used to measure the metabolic activity of cells of culture exposed to a range of drug concentrations. The metabolic activity was measured by the ability of the cells to reduce the tetrazolium dye, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), to a formazan, quantifiable.

The test compounds were dissolved in dimethylsulfoxide (DM30) and diluted, first with Earle's Balanced Salt Solution and then with culture medium, up to twice the highest concentration of test compound. From this concentrated solution, serial dilutions of 1: 2 were prepared in 96 well microtiter plates each containing twice the desired final concentration of the compound. Each concentration was tested was triplicate and compared to triplicate, drug-free controls.

The cells were cultured in the same medium as that used to dilute the compounds after harvesting by trypsinization, as described in Bird, S.R. &amp; F.T. Forrester, Basic Lab-ratory Tschniques in Cell Culture, U.8. Dspt. of Health &amp; Human Services, Virology Training Branch, Atlanta Seorgia, ρρ S4-S5, 104-105, 111 (19S1), the viable cell counts were adjusted to cell density at 25,000 cells / ml. The inoculum (0.1 ml) was then added to each well, to a 70 619 Casa 4654, PQ.01

Addition of the inoculum was used to dilute the test compounds to the desired final concentration.

The title plates were incubated for three days at 36Â ° C in a humidified atmosphere containing 5Âμ of carbon dioxide,

At the end of the three days 20 microlifers of 5 mg / ml MTT in phosphate buffered saline were added to each well. microtiter plate. The microtiter plates then returned to the incubator for two to four hours to allow the surviving cells to reduce the dye. After this incubation, the medium was removed by aspiration. the color is not reduced. DMSO was added to each well to dissolve the final, water-insoluble final product resulting from dye reduction so that it could be measured, spectrophotometrically, at 570 nm.

Table 10 CXgn of the altromycins A, Be C-j-D sweating used against cell lines of culture

Altromycin Cell line Clcn A Hr-Mela <0.007 E Mela <0.015 C -: D Mela <0.006 micrograms per milliliter.

Hela cells were purchased from the ATCC, Catalog CCL2 = The medium used was the Minimum Essential Medium (MEM), with 10¾ fetal calf serum and 1¾ d non-essential amino acids. 70 619 .....

Case 4654 = 79.01 -32-

Tabsla 11 ICc, n of the altromycins A.B and D when used against mouse and human tumor cell lines 0549 HCT-S MT-29 P388 (human lung) human colon (leukemia Compound human)) mouse 01 thromycin A 0.00844 0.00322 0.0235 0.0001292 ftlromynein 1 0.00357 0.00186 0.0013 0.0000867 01 thromycin D 0.00515 0.00131 0.00117 Adriamycium 0.0513 0 7 0.11174 0.0150 Etoposide 1.080 1.033 0.0618

The in vivo anti-tumor characteristics of the α, β-3-alpha-3-aminocinins were determined using the asiatic leukemia tumor line 7388 = Before the test, P388 was propagated intraperitoneally in DBA / 2 female mice. Ascites tumors were injected intraperitoneally into female S5D2FI mice at a concentration of 10-cells per mouse. Mice were treated with anti-tumor agents administered once daily for five consecutive days using fluorouracil as a positive control.

All drugs were administered intraperitoneally in 5% aqueous solution or in another appropriate diluent. The drug efficacy was determined by prolonged survival. The survival time was calculated as% T / C, the mean value or the intermediate value of survival time of the treated mice expressed as a percentage of the mean value or intermediate value of the survival time of the control mice. Compounds having% T / C values greater than 125 were considered to have significant activity. Treatment groups contained 10 mice and the control groups contained 20 mice. The toxicity of the anti-tumor agents was evaluated in these tests by comparing the weight of the mice before treatment and 5 days post-treatment. The LD 50 values for ura® alone administered intraperitoneally were of 0.3, 0.2 3 0.1 mg / kg for the altromycins A, 2 and D, respectively = 0s-33-

/, J 70619

Case 4654, PG.01 alleromycins demonstrated reproducible activity in the systemic leukemia model of P33S, as shown in Table 12.

Table 12 in vivo fictivity of the altromycins. S and D versus systemic P3S8 (T / C-100) (%) Compound dose (mg / kg) Scheme Mean Intermediate value Curing rate Altromycin A 0.23 QD 1-5 69> 88 5/10 0.14 0D 1-5 77 90 3/10 0.08 QD 1-5 59 64 0.05 GD 1-5 51 46 0.25 Dl, D5 57 77 4/10 0.15 D1, D5 61 55 0.09 Dl, D5 38 32 0.05 Dl, D5 33 32 Altromycin B 0.05 QD 1-5 53 50 0.03 QD 1-5 51 46 0.018 QD 1-5 43 46 0.011 QD 1-5 33 27 0.014 Dl, D 5 64 73 0.08 Dl, D5 47 46 1/10 0.05 Dl, D5 55 50 0.03 Dl, D5 38 36 5-Fluoruracil 10 QD 1-5 65 64 Altromycin D 0.150 QD 1-5 Toxic Toxic 0.075 QD 1 -5 -10 24 0.037 QD 1-5 58 68 0.018 QD 1-5 50 59 5-Fluoruracil 10 QD 1-5 98 95 1/10 QD1-5 = Test compounds administered daily for 5 consecutive days.

Dl, D5 = Test compounds administered daily, only on days 1 and 5. 70 619, .-

Case 4654.PG.01 -34- -

EXAMPLE S

A, E and D altromycins were also tested against two solid tumors, Lewis lung carcinoma and ovarian sarcoma 5076. Prior to the test, these tumors were propagated subcutaneously in C57B1 / 6 female mice. Lung (solid) tumors of Lewis lung or M5076 were implanted subcutaneously as a 1:10 homogenate in B5D2FI female 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 a positive control drug. For mice implanted with M5076 (solid) tumors, test compounds were administered 4 times daily for 11 consecutive days. Cis-platinum was used as a positive control. All drugs were given intraperitoneally in 5% aqueous dextrose. Drug efficacy was determined by reduction of tumor weight. Inhibition of tumor weight was expressed as a T / C function, ratio of treated tumor tumor weight to control mouse tumor weight, and the results are shown in Tables 13 and 14, below. Altromycin D produced approximately 90¾ inhibition of tumor weight relative to the tumors of the untreated controls; altromycin B produced a maximum of 60% inhibition. Against ovary M5076 tumor growth as subcutaneous implantation, altromycin A was shown to be slightly more active than aldromycin D (Table 14). 70

Case 4654, PG.01

Table 13

In vivo activity of the A, B and D altromycins against subcutaneous implantations of Lewis lung carcinoma

Dose * Inhibition of tumor weight Compound mg / kg) Mean Intermediate value Altromycin A 0.30 0 0 0.15 27 15 0.075 0 0 0.037 0 0 Altromycin B 0.075 toxic toxic 0.037 31 60 0.018 13 20 0.009 0.03 16 Altromycin D 0.075 toxic toxic 0.037 86 89 0.018 28 61 0.009 0 0 Cyclophosphamide 100 99 50 90 90

A, B and D altromycins were administered daily for 9 consecutive days? cyclophosphamide was administered only on day one. Inhibition of tumor weight was calculated as (1-T / C) x 100. 70,619;

Case 4654.PG.01 -36-

Table 14

In vivo activity of the A, B and D altromycins against subcutaneous implantations of ovary sarcoma M5076 Dose * Inhibition of tumor weight Compound (mg / kg) Mean Intermediate value Altromycin A 0.20 69 72 0.10 60 47 0.05 51 45 0.025 29 21 0.0125 30 4 Altromycin B 0.15 toxic toxic 0.075 toxic toxic 0.038 toxic toxicant Altromycin D 0.075 toxic toxic 0.038 45 37 0.019 54 47 Cyclophosphamide 100 100 100 50 94 93 fischromycins were administered twice daily for 6 days. Cyclophosphamide was administered once on day 1. EXAMPLE 9

The B and D altromycins were tested against a human tumor xenograft in the subrenal capsule assay (ECSR) as described in Bogden et al., Cancer 48: 10-20 (1981). The human colon tumor line, LS174T was maintained by serial propagation in BALB / C athymic peeled mice. The tumors were removed from the peeled mice after reaching 0.2-0.3 grams and were fragmented into 1 mm pieces. The tumor fragments were then implanted into the renal capsule of CDF1 mice. Immediately after implantation, the tumor mass was measured with a stereomicroscopic dissecting microscope, equipped with a 70 619 micrometer

In the case of ocular units, calibrated in ocular units (OMU). The mean tumor mass was calculated in SMO. Three days after tumor implantation, the mice received a single intravenous injection of altromycin B or D or 5-fluorouracil, as reference. The results are shown in Table 15 below.

Table 15

In vivo activity of Aβ B altromycins against human colon tumor LS174T. evaluated by the subrenal capsule assay

Dose Weight of Compound (mg / kg) tumor (mg) N Comment Altromycin B 0.5 2.47 9 Active 0.025 3.21 10 Active Altromycin D 0.5 2.92 7 Active 0.25 3.64 8 Inactive 5 -Fluorouracil 50 3.15 8 Active 5% Dextrose 4.26 7 Control

Formulation

The compounds of the present invention may be administered orally, nasally, ophthalmically, parenterally, vaginally, rectally, topically or aerosolly, in conventional dosage unit formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, adjuvants and carriers as desired. The term &quot; parenteral &quot; 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 non-toxic, pharmaceutically acceptable diluent or solvent, for example a 1,3-butanediol solution. Among the acceptable carriers and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Additionally, fixed sterile oil, including synthetic mono- or diglycerides, may be used. It is also possible to use fatty acids, such as oleic acid, in the preparation of injectable formulations.

Suppositories for rectal or vaginal administration of the active ingredient may be prepared by mixing the active ingredient with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols which are solid at room temperature but liquid at the rectal / vaginal temperatures and which will melt therefore in the rectum or vagina releasing the active ingredient.

Solid dosage forms, for oral administration, may include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also include, as is customary practice, additional substances in addition to inert diluents, for example, 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 may additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs, containing inert diluents, commonly used in the art, such as water. Such compositions may also comprise adjuvants such as wetting, emulsifying and suspending agents and sweetening, flavoring and perfuming agents.

The compounds of the invention may be used for the therapeutic treatment of a mammalian host affected by a microbial infection or an experimental host animal affected by a malignant tumor. The compounds are useful for 70

Case 4654.PG.01 L-39- topical treatment of infectious diseases. Additionally the compounds may be used in disinfecting and sterilizing solutions or as preservatives in plastics, paints or fabrics.

The total daily doses given to a patient in single or divided doses may be, for example, 0.01 to 50 mg and more usually 0.01 to 10.0 mg. The dosage unit compositions may contain submultiples of these values to make up the daily dose. The amount of active ingredient that can be combined with the carrier materials to produce a unit dosage form will vary according to the patient being treated and the particular mode of administration.

However, it should be understood that the dosage level, specific for any particular patient, will depend upon several factors such as 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; the drugs used in combination, and the severity of the particular disease being treated.

Although the present invention encompasses the preparation of the albromycin compounds, it is not limited to the use of strain AB 1246E-26 of the species Actinomycete or other strains corresponding to the same description, but includes, on the contrary, the use of variants of such organisms such as those obtained, for example, by selection or mutation, under the action of ultraviolet or X-ray, or by bombardments of nitrogen. The foregoing description is merely illustrative of the invention and is not intended in any way to limit it to the said compounds. Variations and modifications, which will be obvious to those skilled in the art, are understood to fall within the scope and nature of the invention, which are defined in the appended claims.

Claims (7)

70 619 Case 4S4 &gt; PG &gt; INDICATIONS 1. A process for the production of compounds of formula in which R1 is selected from -NH2 * NHCH3 and -M (01-13) 2 and R3 are independently selected from hydrogen and hydroxy, such that at least one of 1 → R 3 is hydroxyl, which comprises culturing a microorganism belonging to the order of the Actinomyces in a nutrient medium. A method according to claim 1, characterized in that the microorganism has a Type IV cell wall and is resistant to lysozyme. 3. A process according to claim 2, wherein the microorganism exhibits, on agar with malt extract-extract, abundant growth characteristics with light gray to white outcrops, a moderate, when viewed from the reverse side to a mild brown soluble pigment. A method according to claim 1, characterized in that the microorganism exhibits the distinguishable characteristics of the species of Actinomycete AB 1246E-26, deposited under the accession number NRRL 18371. -41-70619 Dase 4654, PS, 01 A method according to claim 1, characterized in that the microorganism is AB 1246E-26 of the species Actinomycete. deposited under accession number NRRL 18371 = 6. A process according to claim 1, further comprising the step of extracting from the culture medium; a fermentation isolate containing the compounds. A process according to claim 1, further comprising the step of purifying the compounds by chromatographic partitioning of the fermentation isolate. A process for the preparation of a pharmaceutical composition having anti-bacterial or cytotoxic activity, characterized in that it comprises combining a pharmaceutical carrier and a pharmaceutically effective amount of a compound prepared according to the preceding claims, Lys. -6. FEV. 1950 By ABBOTT LABORATORIES