NL8401987A - DESACETYL RAVIDOMYCIN. - Google Patents

Description

I * *% * 1 VO 6403

Title: Desacetyi ravidomycin.

The present invention relates to an antibactarial and anti-tumor compound, a method of use and its synthesis. More particularly, the invention relates to an antibiotic and neoplasna-fighting agent isolated from a new species of Streptomyces, as well as their use for treating infections and preventing tumor growth.

Streptomyces, a family of the order of actinomycetai, are characterized by stable, branched filamentous growth and the formation of spore aerial nyceia. Streptomyces bacteria are abundant in the soil. Basically, Streptomvces are widespread scavengers, which break down proteins, cellulose and organic matter, such as waxes, rubber and paraffin.

Loaded up to about 40 years, only a few soil microbiologists were interested in Streptonyces. However, after isolation of acti-13 nomycin and then streptomycin, Streptomvces from soil samples around the world have been studied for antibiotic production.

Recently, a new antibiotic from a new species of Streptonyces has been successfully isolated, namely Streptomvces ravidus MRRL 20 11,300. Such an antibiotic known as ravidomycin is described in U.S. Patent 4,230,692. Ravidomycin not only has an antibacterial activity but also an anti-tumor activity.

The invention now relates to a new antihacterial and anti-tumor mid, namely desacetyl ravidomycin, to its preparation by fermentation, to methods of recovering and concentrating them from crude solutions and to methods of purification thereof. vBfnl The molecular structure of desacetyi ravidomycin is shown in% formula 1 of the formula sheet.

Ravidomycin is described in U.S. Patent 4,230,632. Its molecular structure is indicated in Figure 2 of the formula sheet.

Desacetyi ravidomycin is formed from a new strain of Streptomycas during cultivation under controlled conditions.

8401987 -2- i, named Streptonyces olivaceo-griseus. It is referred to as LL-C23201 φ / and will hereinafter be referred to as desacetyl ravidomycina.

The new antibiotic producerenda species Streptonyces olivaceo-griseus was isolated from aviation contamination at the medical research department, American Cyanamid Company, Pearl River, New York, and is kept as culture number LL-C23201 in the culture collection of the aforementioned medical research department. A viable culture of this new microorganism has been deposited at t

Agriculture Research Culture Collection, Northern Reginal Research Center, 10 U.S. Department of Agriculture, Peoria, Illinois and added to its permanent collection. The trunk has been given the designation number NRRL 15357. It will hereinafter be referred to as NRRL 15357.

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A patent application in which the said new Streotomyces species is described and for which rights have been requested has also been filed by the applicant.

NRRL 15357 culture was taxonomically characterized and identified as a new species of gray spore Stregtomyces known as Streptomyces olivaceo-griseus. The culture, physiological and morphological details of the culture were observed according to the methods outlined in Ξ.3. Shirling and D. Gottlieb, INTERNAT.

J. SYST. 3ACTERIOL., 1: 313-340 (1966). The media used in this study were selected from those recommended by T.G. Pridham et al, ANTIBIOTICS ANNUAL, biz. 947-953 (1956/57) and R.E. Gordon et al., INTERNAT J. SYST. 3ACTERIOL., 24_: 54-63 (1974) for the taxonomic study of Actinomycetes and soil bacteria, such as Streptomvces.

The chemical composition of the cell walls of the culture was determined by the method of Lechevalier, et al., ADV. APPL. MICROBIOL., 14; 47-72 (1971). Details of such observations are summarized in Tables Α-Ξ giving a general description of culture Θ30. The underlying descriptive colors are from K.L. Kelly and D.B. Judd, NAT.3UR. STANDARD, SPEC. PU3L. 440 (1976) and 1 'the associated Intar-Society Color Council, National Bureau of Standards, 1 Centroid Color Charts.

Isolated NRRL 15357 was compared to a suitable reference 35 strain, Streptomvces ravidus NRRL 11300, a culture that produces ravidomycin. See U.S. Patent 4,230,692. A comparison of a 14 day culture of both cultures on Hikey-Tresner agar is indicated below:

Culture Color spears mass Soluble pigments Color backside 3 ills - * - * - light gray red grayish red- 5 M33L 11300 brown - * —---— light gray no blackish green griseus h'RSL 15357

The global morphology of a colony of NHSL 15357 does not equal 10 to that of 3_. ravidus and significant physiological differences were also observed. 5.avidus nitrates are reduced and arabinose is used, but no mannitol or sucrose is used. A review in the current streptomycete literature did not reveal any described species similar to NIïRL 15357. Thus a new species was named, known as Streptonyces olivaceo-griseus.

Mi cromorphology

Sr, spores are formed in curled chains (Spira) on air track carriers of 5iSL 15357. The spores are oval-like (about 1.5-1.3 micrometers at about 2.0-2.5 micrometers), and the surface of the mature spores. is smooth when observed by scanning electron microscopy. Cell wall arrangement

Whole cell hydrolysates of cultured N3RL 15357 contain the LL isomer of dianinopineinic acid, allowing it to be placed in the type X cell wall group of Lechevalier, et al. (Supra). This is typical for all Streptomyces species.

Rate of MRRL 15357 growth

Good growth is observed in most media; moderate growth is observed on glycerol-asparagine agar; poor growth is observed on oatmeal agar; and no growth on tomata-S: 30 pasta oatmeal agar.

- Air mycelium and sooran color

Aerial mycelia is white on most media but is tinged with a rose color on inorganic salt an starch agar; trace mass are 264.

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Soluble pigments

Absent on many media; brownish tones are produced. Backside color

Greenish black to olive - blackish tones on all media.

5 Physiological reactions 3 No nitrates are reduced to nitrites within 14 days; no gelatin liquefaction takes place within 14 days; ar, no black pigment (melamine) is produced on either peptone yeast extract iron agar or tyrosine agar; strong peptonization of litmus quickly within 10 days. Carbohydrate consumption according to the method of T.G. Pridham, □. Gotlsiieb, J. 3ACTERIOL., 5 (5, 107-144 (1948), good use of glucose; moderate use of fructose and inositol; poor use of galactose, mannitol, saccharose, and xylose; no use of arabinose, raffinose, rhamose or "salicin. Different organic acids were investigated as the only carbon sources; citrate, maleate and succinate were used extensively; lactate was used only weakly; while benzoate, mucate and oxalate were not used at all. Adenine, hypo xanthine and tyrosine were hydrolyzed within 14 days, unlike guanines and xanthine.

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TABLE C

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Physiological response of Stregtomyces olivaceo-griseus NRRL 15357

Medium Incubation Quantity Physiological _________ period (days) growth___ reaction_ oepton iron _, “7 good no black xng 14 good no blacking 5 Tyrosine agar 7 good no blacking (ISP-7) 14 good greenish black pigment litmus milk 7 good low proteolysis 14 good strong proteolysis nutritional gelatin 7 low no proteolysis 10 14 low no proteolysis nitrate juice 7 good no reduction 14 good no reduction adenine agar 7 good strong hydrolysis. strong hydrolysis 15 guanine agar 7 good no hydrolysis 14 good no hydrolysis §4 0 1 3 3 7 TABLE C (Continued) -3—

Medium Incubation - Physiological acevicIneic series. response growth (days) _

Hypoxanthine 7 good no hydrolysis agar 14 good strong hydrolysis

Tyrosine- 7 good no hydrolysis agar 14 good weak hydrolysis 5 Xanthine agar 7 good no hvdr91ysis 14 good no hydrolysis

TABLE D

Carbon source consumption, from Streptomyces olivaceo-griseus NR5L 15357

10 Incubation: 14 days Temperature: about 28 ° C

Carbon source Consumption x 1-arabinose 0 fructose 2 d-galactosa 1 15 d-glucose 3 i-inositol 2 d-aannitol 1 d-raffinose 0 1-rhamnose 0 2 '> salicin 0 sucrose 1 V xylose 1% negative control 0 x 3 = good consumption 25 2 «reasonable consumption l * low consumption 8 4Q 1 9 8 7 C * yeen consumption t -10-

TABLE E

Consumption of Organic Acids by S trentomyces olivaceo-griseus NRKL 15357 on Gordon's Modification 5 of Koser's Basal Agar (Koser's Citrate Agar)

Incubation: 14 days Temperature: about 28 ° C

Carbon source Consumption

Benzoate 10 Citrate +

Lactate +

Maleate +

Mucic acid

Oxalate 15 Succinate + no consumption +: strict consumption +: some consumption

It is clear that the present invention is not limited to the production of this antibacterial antitumor agent1 until the particular Streptomyces organism designated as "3®Rirtt5T5T7" or non-organisms - which fully conforms to the above growth and microscopic properties, which are illustrative only. purposes are given.

Also, for the production of desacetyl ravidomycin, the use of natural mutants of the organism referred to as NSHL 25 15357 and mutants generated therefrom by various methods is contemplated. such as X-ray exposure, ultraviolet radiation, nitrogen mustard, actinophages, etc.

The antibacterial and anti-tumor agent desacetyl ravidomycin is% in vgtro active against a plurality of gran-positive bacteria and neoplasms.

The in vitro antibacterial spectrum of desacetyl ravidomycin was determined by the agar plate thinning method with ilueller-Hinton agar and an inoculum of each test organ of approximately 10 8401987 • m -11 koionic units delivered by a Steers replication device.

The minimum inhibitory concentration was defined as the lowest concentration of desacetyi ravidcmycin, which inhibited visible growth after approximately IS hours of incubation at about 33 ° C. The 5 results are shown in Table F.

TA3EL F

Antibacterial spectrum of desacetyi ravidcmycin

Gram-positive bacteria Minimum inhibitory concentration (ajcg / sil)

Staphylococcus aureus, Smith £ 0.25 10 "" LL No. 14 40.25 "* LL No. 27 0.5" "LL No. 45 0.5 "" ATCC 25923 0.5

Staphylococcus eoidermidis, CKC-33-56 1 15 "" ATCC 12223 0.5

Strestorayces faecalis, ATCC 29212 £ 0.25

Streptococcus nutans ATCC 27352 1

Streptococcus sanguis GOBCa! £ 0.25 -, creptococcus (enterococcus) s? OSU-75-1 <0.25 20 - i! . 5M-77-15 <0.25

Micrococcus lutaus, PCX 1001 <0.25 3acillus snht -.- H-is. ATCC 6633 £ 0.25 3acillus cereus, PCI 213 0.5 3 in vivo research systems and schemes have been developed by the National Cancer Institute for the investigation of bonds for their use as neoladeic agents. These are listed in "Cancer Chemotherapy Reports", Part III, 3and 3, no. 2 (1972), Λ Deran, st al. These schedules have led to standardized sequence tests generally followed in the wet field of studies of 30 antituner agents. Of these systems, lynphytic P338 and nelanotic melanoma 8401987-12 chemistry are particularly significant for the present invention. Such neoplasms are found in mice. In general, good anti-tumor activity is shown as a percentage in these schemes. increase in mean survival time of the treated animals (T) relative to the control animals (C) predicting similar results in human leukemia. ld.

The compound desacetyl ravidomycins has the property of inhibiting growth of transplanted mouse tumors as shown in the following experiments.

Lynphocytic leukemia P383 test.

The animals used were 3DF1 mice, of a sex, with a minimum weight of about 17 g with a deviation of no more than about 3 g.

There were 5 or 6 mice per group to be tested. The tumor graft 15 was followed by intraperitoneal injection of about 0.5 already diluted 6 ascitic fluid containing about 10 cells of lymphocytic leukemia P338. The oroo compounds were administered intraperitoneally in an amount of about 0.5 ml in about 0.2% Kiucsl in normal brine on days 1.5 and 9 (relative to the tumor inoculation) at the indicated doses. The mice were weighed and the surviving specimens recorded regularly for 30 days. The results of this test are shown in Table G.

λ 8401987 -13-

ΤΑ3ΞΙ »G

Desacstyl ravidomycin and ravidomycin activity against 3SS leukemia in whizzing

Compound Dose Mean T / CxIGC

(mg / kg) survival% duration (days) _

Desacetyi Ravidomycin 25 20.5 174 (Test 2) 12 19.5 165 6 17.5 143 3 15.5 131

Control - 11, S

1,4-dihydroxy-5,8-bis 2- 1,6 13,2} 154 (2-hydroxye thylacino) ethyl, aiiiinoj anthraquinone, dihydrochioride (hereinafter referred to as positive control)

Desacetyl Ravidomycin 25 16.5 133 (Test No. 2) 6 14 156 1.6 12.5 139 0.4 10.5 117 Control - 9

Positive control 0.4 14 156

Ravidomycin hydrochloride 50 21 173 25 20.5 174 12 17 144 6 12 102 3 12 102

Control - 11.3

Positive control 1.6 13.2 y 154 \ - 8401937 TABLE G (continued) -14-

compound 'Dose mean T / Cx100

survival daur * ______ (days)

Ravidomycin 400 16 173 100 17.5- 194 25 14 156 6 10 111 5 1.6 10 111

Control - 9

Positive control 0.4 14 156

Melanotic melanoma 316 groove

The animals used were BDF1 mice, all of one sex 10 weighing at least about 17 g within a deviation of about 3 g.

There were normally 6 animals per test group. A portion of about 1 g of melanotic melanoma 316 tumor was homogenized in about 10 ml of a cold balanced saline and approximately 0.5 ml aliquot of the homogenate was implanted intraperitoneally in each of the 15 mice. The test compounds were administered intraperitoneally on days 1-9 (relative to the tumor inoculation) in different dosages. The mice were weighed and the survivors recorded regularly for 60 days. The mean survival time and the survival time ratio for the treated (T) / 20 control (C) mice was calculated. The positive control compound was the same as that used in the P38S test. The results are reported in Table H compared to ravidomycin.

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8401987 -15- TA5EL3

Desacetvl ravidomycin an ravidomycin activity against 315 melanoma in mice

Dose Average Ί1 / CxlGo ng / kg survival% duration ('days)

Dasacetul ravidomycin 12 33 184 3 32 155 0.3. 29.5 143 0.2 26 126 5 Control ~ 20.7

Positive control 0/3> 39> 190

Ravidomycin hydrochloride 12 39 138 3 32 155 0.3 27.5 133 10 0.2 23.5 114

Control "20.7 0.8> 39> 190

Positive control

General fermentation conditions

The cultivation of S trap tonyces olivaceo-grissus NREL 15357 can be performed in a large number of liquid culture media. Media useful for the production of desacetyl ravidomyclene include an assimilable carbonone such as starch, sugar, molasses, glycerol, etc .; an assimilable nitrogen bar such as proteins, protein hydrolysatan, polypeptides, amino acids, corn juice, etc .; as well as inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, rosrate, chloride, etc. Trace elements such as boron, molydene, copper, etc. are introduced as impurities of other media components. Aeration in tanks and bottles is accomplished by pressing sterile luent down onto the surface of the fermenting medium. A further movement in tanks is provided with the aid of 23 è 4 0 1 9 8 7 «'-16- \ of mechanical stirrers.

If necessary, an anti-foaming agent such as giant oil can be added.

General procedure for the isolation of desacetyl ravidomycin

Desacetyl ravidomycin is recovered from the fermentation mass by extraction with an organic solvent such as dichloomethane, followed by concentration of the extract and chromatography of this extract on a silica gel column eluting with a suitable solvent system.

The invention will now be explained in more detail by the following non-limiting examples.

Example I

Inoculla preparation

An inoculum medium according to the following composition was prepared:

Meat extract approximately 0.3% 15 Tryptose approximately 0.5%

Dextrose approximately 1.0%

Yeast extract approximately 0.5% water to 100%

This medium was adjusted to a pH of about 6.3 and then sterilized. Approximately 50 ml of this medium was placed in a 250 ml flask and inoculated with mycelial scraps from an oblique agar culture of Streptomyces olivaceo-griseus (NRRL 15357). The flask was incubated at about 32 ° C on a rotary shaker rotating at 130-200 rpm for about 48-72 hours.

Example II Fermentation

The following termentation medium was prepared:

Glucose approximately 1.5%

Glycerol approximately 1.5% 30 Soybean meal approximately 1.5% 0 Calcium carbonate approximately 0.1% a Sodium chloride approximately 0.3% ^ Water to 100% 35 The medium set for sterilization thereof to a pH of about 6.7 was divided into about 100 ml aliquots in 500 µl Srlen-8401987 -x /% m ayer flasks. The flasks were then sterilized and inoculated with about 5.0 µl of the inoculun prepared according to Example I. The fermentation was performed at about 23 ° C for G days after which time the mass was harvested. This nassa contained about 10 mcg / ml desacetyi ravidonycin.

Example III

ramen tat is wet defined medium

The following cementation was prepared:

Sodium nitrate approximately 0.1% 10 Ferrous sulfate heptahydrate approximately 0.31%

Magnesia sulfate heptahydrate approximately 0.02%

Calcium carbonate approximately 0.5%

Dextrose approximately 1.5%

Sodium acetate approximately 0.25% 15 More to 100%

The medium adjusted to a pH of about 7.2 for sterilization was divided into about 100 nl portions into 500 nl Erlenny flasks. The flasks were sterilized and inoculated with approximately 50 µl of the inoculun described in Example I. The fermentation was carried out at about 23 ° C for 6 days after which time the post-harvest was harvested. . This nassa contained about 5 ng / ml desacetyi ravidonycin.

Example IV

Isolation of desacetyi ravidomycin

The 1 liter harvested post from Example II was extracted twice with approximately 5Gg nl portions of dichloomethane. The dichloroethane extracts were combined, centrifuged, dried over anhydrous sodium sulfate and then evaporated under vacuum at about 35 ° C. The residue was dissolved in about 2 ml of ethanol: dichloromethane (1: 1) and applied to a 15x400 am column containing 40 g of silica gel packed with the same solvent mixture. The column was developed with the same solvent mixture at an amount of about 2-3 ml per minute, fractions being collected at approximately 12 minute intervals.

Fractions 15-26 were combined and evaporated to give about 3.5 ng desacetyi ravidonycin.

33 Desacetyi ravidomycin was just honored on its own or in combination.

8401987

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* * -18- / pharmaceutically acceptable carrier administered or used. Of course, any material to be used as a carrier should be pure and virtually non-toxic in the amounts used and should not interfere with the biological activity of desacetyl ravidoraycine.

o 8401987

Claims (6)

1- Connection according to stove 1 of the formula sheet. 2. Method for running the growth of tumors in warm blooded animals, characterized in that an effective amount of a compound of formula 1 of the formula sheet is administered to gancerad animal. A method for treating bacterial infections in warm-blooded animals, characterized in that said animal is administered an effective amount of a compound according to formula 1 of the formula sheet. 4. A method for producing desacetyl ravidomycin, with the knowledge that Streptonyces olivaceo-griseus NERL 15357 is grown in a liquid medium containing assimilable columns of carbon, nitrogen and inorganic salts until significant amounts of desacetyl ravidomycin are present in such medium. . Method according to claim 4, characterized in that the cultivation is carried out at about 24-32 ° C for 4-6 days. A pharmaceutical composition comprising a compound of formula 1 of the formula sheet, and a pharmaceutically acceptable carrier therefor. Q \ 8401987 τ QH OCH3 ΛΛ och3 Η-Ο h— ^ II CH3 / / Η Qvri ^ -J / 0 Sth3 · 2 OH OCH3 ^ I och3 H-0 CH3. ff V \ A = ch3c / ^ <- ^ y o ch3 ΓΆ S £ l American Cyanaraid Company '84 0 1 9 8 7