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Publication numberUS3907779 A
Publication typeGrant
Publication dateSep 23, 1975
Filing dateMay 20, 1974
Priority dateMay 20, 1974
Also published asCA1037889A1, DE2521197A1, DE2521197C2
Publication numberUS 3907779 A, US 3907779A, US-A-3907779, US3907779 A, US3907779A
InventorsClarence Deboer, Brian Bannister
Original AssigneeUpjohn Co
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
5,6 Dihydro-5-azathymidine and derivatives
US 3907779 A
New antibiotic U-44,590 and derivatives thereof, produced by the controlled fermentation of the new microorganism Streptomyces platensis var. clarensis var nova, NRRL 8035. This antibiotic and its derivatives are active against Gram-negative bacteria. Accordingly, they can be used in various environments to eradicate or control such bacteria.
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Description  (OCR text may contain errors)

United States Patent DeBoer et al.

5,6 DlHYDRO-S-AZATHYMIDINE AND DERIVATIVES Inventors: Clarence DeBoer; Brian Bannister,

both of Kalamazoo, Mich.

Assignee: The Upjohn Company, Kalamazoo,


Filed: May 20, 1974 Appl. No: 471,322

US. Cl 260/2l1.5 R; 195/80; 424/18] lnt. Cl. C07H 19/12 Field of Search 260/21 1.5 R

References Cited UNITED STATES PATENTS l/l967 Wechter 260/2l 1.5 R

3,457,253 7/l969 Wechter 260/21 15 R Primary Examiner-Johnnie R. Brown Attorney, Agent, or Firm-Roman Saliwanchik ABSTRACT New antibiotic U-44,590 and derivatives thereof, produced by the controlled fermentation of the new microorganism .Strepwmyces platensis var. clarensis var nova, NRRL 8035. This antibiotic and its derivatives are active against Gram-negative bacteria. Accordingly, they can be used in various environments to eradicate or control such bacteria.

3 Claims, No Drawings 5,6 DIHYDRO-S-AZATHYMIDINE AND DERIVATIVES BRIEF SUMMARY OF THE INVENTION The novel antibiotic of the invention, U-44,590 is obtained by culturing Streplomyces platensis var. clarensis, NRRL 8035, in an aqueous nutrient medium under aerobic conditions. Various derivatives of U-44,590 can be made as disclosed infra. U-44,590 and its derivatives have the property of adversely affecting'the growth of Gram-negative and Gram-positive bacteria, for example, Streptococcus hemolyticus, Klebsiella pneumonia, Salmonella Sp., Serratia marcescens,.PasteureIla multocida, Hemophilus Sp., Proteus morgani and Proteus rettgeri. Accordingly, U-44,590 and its derivatives can be used alone or in combination with other antibiotic agents to prevent the growth of or reduce the number of bacteria, as disclosed above, in various environments.

U44,590 and its derivatives are also active against DNA viruses, for example, the Herpes virus and, thus, can be used to control such virus where its presence is not desired. 1

DETAILED DESCRIPTION OF THE INVENTION Chemical and Physical Properties of U-44,5 90

1516111611011 Analysis: Calcd. for cai u o c, 44.03; H, 6.17; N, 17.13. Found: c, 44.14; H, 6.08; N, 17.36.

Molecular Weight: 245 (Determined by mass spec-.

Band Frequency (Wave-Numbers) Intensity 3440 3400 3340 3190 3080 3000 2960 (N Nujol) 2930 (N) 2860 (N) 1695 sh. I683 I510 I503 I483 1463 (N) I440 I421 I407 I396 1375 (N) 1350 l3l5 I300 1280 I276 I262 I243 I230 sh. 1 I95 I I33 I093 -Continued Band Frequency (Wave Numbers) I085 I060 I01 I 985 97l 943 885 872 849 826 792 755 .735

Intensity Note: sh means a shoulder band.

Band Frequency (Wave Numbers) Intensity 1510 1482 1461 1437 1420 1406 I396 1349 1310 129s 1290 1 1275 sh. 1263 1243 1 19.5 v 1 16s I085 1060 1010 98s 97I 942 883 870 347 827 791 754 733 Note: sh means a shoulder band.

Infrared band intensities, throughout this disclosure, are indicated as S, M, and W respectively and are approximated in terms of the backgrounds in the vicinity of the bands. An 8" band is of the same order of intensity as the strongest in the spectrum; M bands are between A and as intense as the strongest band; and, W bands are less than V3 as intense as the strongest band. These estimates are made on the basis of a percent transmission scale.

The following is considered to be the structure of U- 44,590:

2 Thus, U-44,590 can be referred to by the trivial name 5,6-dihydro--azathymidine, or by its chemical name I 2-deoxy-B-D-erythro-pentofuranosyI )-5 ,o-dihydro- 5-methyl-s-triazine-2,4( lH,3H)-dione.

Antibacterial Activity of U-44,590

Organism No. of lnhibition(p.glm)

Strains Staphylococcus aureus I l000 Streptococcus hemolylicusl 152 Diplococcus pneumoniae I 500. Klebsiella pneumoniae 5 2.0 1000 Salmonella sp. 4 15.6 100 Serralia marcescens 2 I25 Pseudomonas aeruginosa 5 1000 Pasteurclln mulrocida l 125 Hemophilus sp. 5 31.2 100 Proteus vulgaris 3 I000 Proteus ntirabilLr 3 1000 Proteus morgam' 3 62.5 250 Proteus rengeri 3 312 The above antibacterial spectrum was obtained by a standard agar dilution test with the following media and conditions:

Difco Brain Heart Infusion Medium was used for all test bacteria except P. multocida and Hemoph ilus species which were grown in Difco Blood Agar Base with 5% defibrinated rabbit blood. All were grown aerobically at 37 C. (except Hemophilus species, grown anaerobically) 16-18 hours. lnocula were grown ovemite (16-18 hours) at 37 C. and used to seed agar at the rate of 0.025 ml. of 10 dilution (approximately 2500 to 25,000 bacteria per drop of inoculum).

In vivo testing of U-44,590 in mice infected with selected microorganisms is as follows:-

Activity (C11,, in mg/kg) ANTlVlRAL ACTIVITY OF U-44,590

The following is an example of the antiviral activity of antibiotic U-44,590. The antibiotic is administered subcutaneouslyto mice which are inoculated intravenously with Herpes simplex virus. Treatment. is initiated two hours prior to viral infection and is followed by treatment four times daily for five consecutive days. A detailed account of the materials and methods and results are as follows:

Male mice, weighing approximately 20 gm. each, are divided into 4 groups of 20. Group I is treated with saline, Group 2 with 400 mg./kg./dose (mkd) U-44,590, Group 3 with 200 mkd U-44,590, and Group 4 with mkd U-44,590. The antibiotic is dissolved in saline and administered subcutaneously in the nape of the neck at 8 a.m., 12 noon, 4 p.m., and 8 pm. on days 0, l, 2, 3, and 4. Herpes virus at 10" dilution, 0.05 ml/mouse, equivalent to a viral dose of 40 LD s, is inoculated into the tail vein at 10 a.m. on day 0. Paralysis and death are recorded daily.

Hind leg paralysis usually preceded death by 1-2 days. All mice died that became paralyzed. Death pattern of the 4 groups, as shown in the curves which follow, illustrates the dose response obtained. Statistical analysis of the results at day 11 indicates that all 3 treated groups are significantly different from the control group (I).

Antibiotic U- 1 i,590 vs. Herpes v rus in Mice '20 mice per q oup Saline o-o 0 1 15 0 10o mg./kg./d0se Days THE MlCROORGANlSM The microorganism used for the production of U- 44,590 is Streptomyces platensis var. clarensis, NRRL 8035. A subculture of this'microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, US. Department of Agriculture, Peoria, Ill.

The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories.

A new soil isolate with hygroscopic spore masses, but with smooth, hat-shaped (crescent) or brazil-nutshaped (elliptical) spores, has been found to differ in certain characteristics from the type culture Streptomyces platensis. An outstanding difference of the new culture is the production of antibiotic U-44,590. The new isolate can be recognized as a variant of Streptomyces platensis by its cultural, microscopic, and biochemical characteristics. Therefore, it is proposed that this new isolate be designated Streptomyces platensis var. clarensis Dietz var. nova. Rule 7 of the lntemational Code of Nomenclature of Bacteria [International Code of Nomenclature of Bacteria. 1966. Edited by the Editorial Board of the Judicial Commission. of the lntemational Committee on Nomenclature of Bacteria. Int. J. Syst. Bacteriol. 16: 459-490] wasapplied in designating the variety epithet.

S treplomyces platensis var. clarensis is compared with the type species Streptomyces platensis Pittenger and Gottlieb [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces lll. Additional species descriptions from first and second studies. Int. J. Syst. Bacteriol. 18:279-392] [Tresner, H. D'., E. JpBackus, and Jean A. Hayes. 1967. Morphological spore types in the Streptomyces hygroscopicuslike complex. Appl. Microbiol. l5:637639] NRRL 2369, and two recently characterized strains: Streptomyces platensis NRRL 3593 [Evans, Ralph Henry Jr., and Samuel Owen Thomas. 1971. Antiobiotics AH272 a and AH272/3 and process for producing same. U.S. Pat. No. 3,592,925] and Streptomyces platensis NRRL 3761 [Okuda,-Tomohau, and Shigemi Awatagouchi. 1973. Antibiotics YL 704 and preparation thereof. US. Pat. No. 3,718,742];

Color characteristics: Aerial growth white to yellow to gray. Moist black hygroscopic patches on some me dia. Melanin-negative. Appearance on Ektachrome lDietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60: l 52l54] is given in Table l Reference color characteristics are given in Tables 2 and 3. The new culture may be placed in the White (W), Yellow (Y), and Gray (GY) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 1 1:335338].

Microscopic characteristics: Spore chains in tight spirals uncoiling to long open spirals. Spore chains spiral (S) in the sense of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79]. Spore hat-shaped (crescent) or Brazil-nut (elliptical) shaped. Spores are the platensis-type of Tresner and Backus [Tresner, H. D., E. J. Backus, and Jean A. Hayes. 1967. Morphological spore types in the Streptomyces hygroscopicus-like complex. Appl. Microbiol. 15:637-639]. Spore silhouette smooth by direct observation with the electron microscope. Spore surface ridged with surface markings by the carbon replication technique of Dietz and Mathews [Dietz, A. and J. Mathews. 1962. Taxonomy by carbon replication. I. An examination of Streptomyces Cultural and biochemical characteristics: See Table 4, infra.

Carbon utilization: Growth on carbon compounds was determined in the synthetic medium of Pridham and Gottlieb [Pridham, T. G and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:107-114], Table 5 and in the synthetic medium of Shirling and Gottlieb [Shirling, E. B. and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340], Table 6.

Temperature: The cultures grew well at 18 37 C. on Bennetts, Czapeks sucrose, maltose-tryptone, and Hickey-Tresner agars. Optimum growth was at 24 37 C. The new culture and the type culture did not grow at 45 C. The cultures designated NRRL 3593 and NRRL 3761 grewat 45 C. but not at 55 C..

Antibiotic-producing properties: See Table 7, infra.

Source: Soil Type culture: Streptomyces platensis Pittenger and Gottlieb NRRL 2364.

Type variety: Streptomyces platensis var. platensis NRRL 2364.

Variety: Streptomyces platensis var. clarensis Dietz var. nova.

Table 1 Appearance of .Sm'pmm n'a plulumii ('ulturcs mi Fklachrumc l)clcrplrm'mix \ar. S. plulr'mix S. plulvm'iw Agar medium minalion r/urrmix NRRl HU35 NRRL 2364 NRRl. 3593 Bennett's S lammlcrgra with Lawnder-gra Lavender-gray black patches R (roam-yclluw-pink Pink-tan Ycllu (/alpck's sucrose S l.a\cmlcr-gra -\\hilc Lavender-gray l.a\ cmlcr-gra -\\'hitc R ('rcam-yullmx lalc pink-gm Bright \'clln\\ Klallusc-tryptunu S l awndcr-gra \\ith l.a\cnv.lcr-gra lavender-gm hlack patches R ('n -am-ycllu\\-pink Pink-tan Ycllou-tan Pcptunwirun S l race graywhitc Trace lawmlcngra) 'lracu gray R (ream )clln Ycllu Yclltm-tan (l. I" \l'll\lllk 5 Trace gm with lalc Lawndurgray 'l'racc gray hlack patches R Pale ulluw Pink-tan (nlnrlcss ('ascin starch S Lavender-gm Lawmlcngra LaYcndcr-gra R lalc cream lalc grayycllow ('ruum 5' Surlauc R Rr-wrsu Table 3 Continued Color Name lelg (iolden oli\e lll7g Moderate olive Iha Pearl shell tint )Zgm Yellowish white Ica Light ivory. eggshell XJgm Light yellow Ztlc Natural. string )3gm Yellow'is'h gray Ice Biscuit. ccru. oatmeal. sand )Ugni (irayish yellow- 21h Bamhom huff straw wheat 7 947g Moderate yellow 89m Pale yellow Zfe Sih er gray 94g Light olive hrown l lZgm Light olive gray Zgc liamhoo. ehamois )Ugm (irayish yellow- Ige ('oyert tan. griegc Jtlgm (ira ish yellow Zic Light mustard tan )lgm Dark grayish yellow 94g Light olive brown 106g Light olive Zih Dark cmert gray l lZm Light olive gray l l3g Olive gray Bca Pearl pinl\ shell 73gm Pale orange yellow 3n- Siher gray (\Sgm Light brownish gray 3gc Light tan 7(wgm Light yellowish hrown 3ge Beige. camel 79m Light grayish yellowish brown J-lrn Light olive hrow'n 3ie ('amel. maple sugar. tan 76m Light yellowish hrow'n 77g Moderate yellowish brown Rig Beige hrown. mist brown Xtlm (irayish yellowish hrown )Sg Moderate olive brown 3li l5ea\ er Xllm (irayish yellowish brown 95g Moderate olive brown 'Jacohson. l-. I t. (iramille. and L. loss, lI-tts'. (olor harmony manuaL 3rd ed. (ontaiuer Corporation otAmeriea.

Table 4 Cultural and Biochemical Characte Deterristics of .S'lrcpluniycus plulmrris Cultures Medium mination NRRL X035 NRRL 2364 NRRL 3593 NRRL 3761 Agar Peptone-iron S Pale gray Pale gray Pale graypink R Pale oli\ e-tan Tan Yellow-tan Yellow P Yellow O Melanin-negative Melanin-negative Melanin-negative Melanin-negative (alcium malate S Pale gray Pale tan Pale gray Pale gray'white R (iray Pale tan (iray Gray P Malate not Malate not Malate not Malate slightly soluhilized soluhilized soluhilized solubilized Glucose asparagine Trace pale gray Pale pink-tan Pale graypink Gray-white R Pale olive-tan Pink-tan Pale yellow-tan Pale salmon P Pale \ellow Skim milk S Pale gray-pink Pale pink on Pale gray on Traee white on edge edge edge R Orange-tan lalc orange Pale orange Yellow l Orange-tan Pale orange Yellow=orange Yellow Casein not ('asein not Casein not Casein not soluhilized soluhilized soluhilized soluhilized 'lyrosine S Pale gray-pink White Pale gray-cream Pale gray-pink R Yellow Yellow Yellow Orange-tan P Yellow Yellow Yellow Orange-tan O Tyrosine Tyrosine Tyrosine Tyrosine soluhilized soluhilized soluhilized soluhilized Xanthine S lale gray pirlk White Pale gray Pale gray-pink R Yellow Pale yellow Pale yellow Yellow l Pale yellow Xanthine slightly Xanthine slightly Xanthine not i Xanthine solusoluhilized soluhili/ed soluhilized hilized under under growth under growth growth Nutrient starch S Pale gray-pink Pale pink Pale gray pink Pale gray-pink R Yellow Yellow Lemon-yellow Pale yellow l Yellow Starch hydrolyzed Starch hydrolyzed Starch liydrolyzed Starch hydrolyzed Yeast e\tract- S (iray with Pale pink tan White 4 Pale gray-pink nialt c\tract hlack patches R Yellow -tan Red-tan Deep yellow Yellow 1 Yellow Yellow Yellow Bennett's S l a\elider-gray La cnder gray Pale hnentler- Heavy gray with black with black gray patches patches R ()Ii\egra Red-tan Light oli\'c (ream-tan l Pale yellow- Pink-tan Light oliyc olive Table 4 Continued Cultural and Biochemical Characteristics of S/repmmy-t'ar pluu'nxiw' Cultures Detert'lmz'nxix S. plulcnxis S. p/ulcm'ix S. plulz'm'ix Medium mination 'NRRl. 8035 NRRl. 2364 NRRL 3593 NRRL 37M (Lapeks sucrose S Gray-white Gray-black in White Sparse graycentcr, light white gray on edge R Yellow Gray-green Yellow (ream P Pale yellow Yellow Maltose-tryptone S Lavender-gray Pale gray Gray-white Gray-white R Olive-green Creanryellow- Olive Pale olive-cream pink P Oli\'eyello\\' Pale yellow Pale olive Pale tan Hiekey-Tresner S Gray with black Black with gray Deep gray-white Gray with black (modified) center edge patches R Olive Olive-tan Light olive Pale olive-cream P Pale olive Light olive Peptone-yeast S Pale gray Trace white extract-iron I R Yellow Pale yellow Yellow Yellow llSP-o O Melanin-negative Melanin-negative Melanin-negative Melanin-negative Tyrosine (ISPJ) S Gray with black Gray with black Pale gray-white Pale salmon patches patches R Pale olive Pale cream-pink Pale olive Pale red-tan O Melanin-negative Melanin-negative Melanin-negative Melanin-negative Gelatin Plain S White Colorless \ege- Colorless \'ege- Colorless \'egetative growth tative growth tative growth P Yellow Trace tan Trace brown 0 Trace liquefaction No liquefaction No liquefaction No liquefaction Nutrient S Trace vegetative Colorless \'ege- White White growth tative growth P Trace yellow 0 Trace liquefac Trace to no No liquefaction No liquefaction tion liquefaction Broth Synthetic nitrate 0 Trace bottom Colorless sur- Trace bottom Trace bottom growth face pellicle growth growth Nitrate not re and bottom Nitrate not re- Nitrate reduced duced to nitrite growth dueed to nitrite to nitrite Nitrate not reduced to nitrite Nutrient nitrate 0 Trace white Trace white Trace bottom Trace bottom surface ring surface ring growth growth Floeculent Flocculent Nitrate not Nitrate reduced bottom growth bottom growth reduced to to nitrite Nitrate not re- Nitrate not rcnitrite duced to nitrite duccd to nitrite Litmus milk 0 Surface pellicle Surface pellicle Surface pellicle Surface pellicle Litmus reduced Partial peptonization coagula- No change pH 6.8

Partial peptonization pH 7.0

Partial peptoni- Zation pH 6.8

Utilization of Carbon Compounds by SII'IIIUI)I \'('('.S pluu'm'ix Cultures in the Synthetic Medium of Pridham and Gottlieb* S. platens-is w, ('Iurcnxix S. platens-ix S. plulensis S. plulunsix NRRl. 8035 NRRl. 2364 NRRL 3593 NRRl. 3761 CONTROL (no carbon compound added) I. O-Xylose 2. l.-Arabinosc 3. Rhamnose 4. ()-Fructose 5. O-Galactose o. D-(ilucose 7. ()-Mannose 8. Maltose 9. Sucrose l(). Lactose l l. ('ellobiose l2. Raffinose l3. Dextrin 14. lnulin ("l 15. Soluble starch lo. (ilycerol I7. Dulcitol l K. D-Mannitol l). D-Sorhitol It). lnositol 2 l. Saliein l Phenol Table 5' Continued lftilimtion ofCurhon Compounds h .S'lruplunl u't'.\ plulvmim (ulturcs in the Synthetic Medium of Pridhum and (iottlieh v (lUULl lltllilutinlt (*1 Poor utili/ntion I I Doubtful utllllutiou No utili/ntion "l'ritlluml. I 1 (5., and l). (iottlieb. 19-15. The utilimtion olcnrhon compounds l some Aetinontwetules its on aid for weeks determination. .l. Bucteriol. 5l illl7-l l-l Table 6 Utilization of Carbon Compounds by .S'lrepmnrvcuv plulr'nxis Cultures in the Synthetic Medium of Shirling and Gottlieb S. plzlleluis' NR RI Z 3 64 S. plulmm'ix NR R L 3 5 9 3 CONTROLS negativehusal medium Positive-basal medium plus D-glucose CARBON COMPOUNDS l.-Arahinose Sucrose D-Xylosc lnositol D-Mannitol D-Fructosc Rhumnose Ruffinosc Cellulose Slight grmflh Slight growth Good growth iood growth i +iii+li i iii+ii 'i iii ii Slight growth Good growth Slight growth Good growth Strong utili/ntion Positive utiIimtii-n l'tili/ntion tloubtl'ul l'tili/ution negzltite 'Shirling. l1. l3.. ilI'Kl l). (iottlieh. who, Methods for chorncteri/otion of .\!!'4'/Hulll \(r'.\

Buuteriol. Hull 340 species. Int. J. Syst.

The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions.

'lt is to be understood, also,that for the preparation of dextrin, molasses, and the like. Preferred nitrogen sources include comsteep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the effected at any temperature conductive to satisfactory growth of the microorganism, for example, between about 1 8 and 40 C., and preferably between about 20 and 32 C. Ordinarily, optimum production of the compound is obtained in about to 'days. The medium normally remains neutral during the fermentation. The final pH is dependent,'in part, on the buffers present,

' if any and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks,

I it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compound, so long as a good growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation and purification of the compound of the subject invention, for example, solvent extraction, partition chromatography, silica gel chromatography, liquidliquid distribution in a Craig apparatus, absorption on resins, and crystallization from solvents.

In a preferred recovery process the compound of the subject invention is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is recovered from the filtered or centrifuged broth by adsorption on activated carbon. The'activated carbon is then washed with water to remove some impurities. This is followed by elutions with acetone: water solutions which remove the antibiotic from the activated carbon. The acetone in the eluates is removed, advantageously by evaporation, and the remaining aqueous residue is lyophilized to afford a crude preparation of antibiotic U-44,590.

A preferred purification procedure is to subject a crude preparation of U-44,590, as described above, to chromatography on silica gel from which U-44,590 is eluted. Fractions which show activity against the bacterium Klebsiella pneumoniae on a standard agar plate test, are pooled and taken to dryness to yield a relatively pure preparation of U-44,590. Further purification is achieved by acetylation to a crystalline diacetate derivative of U-44,590. Zemplen [G. Zemplen and E. Pacsu, Ber., 62, 1613 (1929)] de-esteriflcation (transesterification) with sodium methoxide in methanol, and neutralization of the catalytic amount of base withcarbon dioxide gives the free antibiotic U-44,590 which crystallizes readily from methanol-ethyl acetate to give a pure preparation of U-44,590.

Antibiotic U-44,59O is active against Streptococcus hemolyticus and, thus, can be used to disinfect instruments, utensils or surfaces when contaminated with this microorganism, where the inactivation of this microorganism is desirable. Also, U-44,590 is active against Escherichia coli and can be used to reduce, arrest,,and-

- /or eradicate slime production in papermill systems because of its antibacterial action against this bacterium. Antibiotic U-44,590 can also be used to prolong the life of cultures of Trichomonas foetus, Trichomonas hominis, and Trichomonas vaginalis by freeing them of Escherichia coli contamination. Further, U-44,59O can 'be used to inhibit the growth of E. coli in hospital flower vases where it has been reported to exist and present a hazard to hospital patients. See Clinical Medicine, February, 1974, Page 9.

Novel acyl derivatives of U-44,590, as disclosed herein, can be used for the same antibiotic purposes as U-44,59O in environments possessing means to deacylate the compound to U-44,590. Thus, the acyl derivatives of U-44,590 can be used to treat laboratory mice infected with a Gram-negative bacteria, for example E. coli, as disclosed herein. Further, acyl derivatives of U- 44,59O can be used, advantageously, to upgrade U- 44,590. This is accomplished by acylating U-44,590, recovering the acylated compound relatively free of impurities, then deacylating the acylated U-44,590 to give U-44,590 in a more purified form.

The following examples are illustrative of the process and productsof the present invention but are not to be construed as limiting. All percentages are by weight and solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE I Part A Fermentation A soil stock of Streptomyces platensis var. clarensis, NRRL 8035 is used to inoculate a series of 500-ml. Er-

lenmeyer flasks, each containing ml. of sterile seed v medium consisting of the following ingredients:

Glucose monohydrate IOGm/l Bacto Peptone (Difco) l0 Gmll Bacto Yeast Extract (Difco) 2.5 Gm/l Deionized water Balance Brer Rabbit Molasses (RJR Foods Inc., N.Y., N.Y. l00l7) 20 ml Yeast Extract (Difco) Detroit,

Michigan I Gmll Glucose monohydrate l0 Gmll Dextrin (Corn Products Co. International Inc., International Plaza,

Englewood Cliffs, New Jersey 07632) 10 Gmll Proteose Peptone No. 3 (Difco) l0 Gmll Tap water q.s. Balance The presterilization pH is 7.0. The inoculated fermentation flasks are incubated at a temperature of 28 C. on a Gump rotary shaker operating at 250 rpm. with a 2% inch stroke. Ucon antifoam (a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.) is used if needed. Harvest is usually after 5 to 12 days of fermentation.

The antibiotic titer of the fermentation beer can be monitored by an agar plate disc assay using the bacterium Klebsiella pneumoniae. This bacterium is inoculated into the assay agar (Streptomycin Assay Agar, BBL, Cockeysville, Md., 21030) of the following composition:

Deionized water 1 adjust 'pH to 7.9 with Sterilize at 121 C. lbs. steam pressure) for 15 minutes.

Phosphate buffer (0.1N pH 6.0) is used as the diluent. The agar plates are incubated at 37 C.-for 16-18 hours. Presence of antibiotic U-44,590 is evidenced by the zone of inhibition around a paper disc to which a fermentation sample was previously applied. The diameter of the zone of inhibition reflects the potency of the 20 antibiotic sample. Thus, a 20 mm. zone of inhibition using a 12.7 mm. paper disc to which 0.08 ml. of antibiotic sample has been applied is expressed as one bio unit per m1. (1 BU/ml.).

Part B Recovery Whole fermentation beer (ca 1600 ml. assaying 5 BU/ml. against K. pneumoniae), obtained as described above, is filtered using diatomaceous earth as a filter aid. The filter cake is washed with water. The clear beer and wash (1800 m1.) is then passed through an ac- 30 tivated carbon column. The column measures 2.8 X 44 cm. and contains 126 grams of activated carbon. The carbon column is washed with 1750 ml. water and the wash is discarded. The column is then washed with 1 liter each of a 1%, 2% and 5% acetone:water concentration. These eluates are also discarded. The column is then eluted with 1 liter each of a 10%, and 50% acetone:water concentration. These eluates, which contain antibiotic U-44,590, are pooled and the acetone is removed on a rotatory evaporator at C./ 15 mm. Hg. The resulting acetone-free preparation is shell-frozen to an aqueous residue and then lyophilized, yield, 3.55 grams assaying 2 BU/mg. of U-44,590 against K. pneumoniae. This preparation, labeled for convenience as Solid A, is then subjected to further recovery procedures as follows.

A silica gel (Merck-Darmstadt Cat. 7734) column is prepared from 420 grams of silica gel packed in methanol: chloroform (1:1 v/v). The column measures 3.8 X 88 mm. Solid A, obtained as described above, is added on the top of the column and the column is then eluted with methanol: chloroform (1:1 v/v). Active fractions, as determined by the abovedescribed K. pneumoniae assay, are pooled and the solvent is removed from said pooled fractions by use of a rotary evaporator at 30 C./l5 mm. Hg; yield, 830 mg. assaying 7.5 BU/mg. of antibiotic U-44,590.

Part C Purification No. 1

A preparation of antibiotic U 44,590, obtained as described above in Part B., is subjected to chromatography on silica gel using the solvent system ethyl acetate: methanol (6:1 v/v) to give a purer preparation containing U-44,590. The procedure for this purification step is as follows: 65

A column of silica gel (Merck-Darmstadt, 1 15 grams/gram of the U-44,590 preparation being chromatographed) in ethyl acetate: methanol (6:1 v/v) is prepared by pouring a slurry of silica in the solvent into the column to give a height-diameter ratio of 10:1 after being packed. The U-44,590 preparation, obtained as described above in Part B, is dissolved in methanol, silica is added (three times the weight of the U-44,590 preparation used), and this is then taken down to a dry powder on a rotatory evaporator at 40/15 mm. Hg. The resulting dry solid is added to the top of the silica column through a small head of the solvent ethyl acetatezmethanol (6:1 v/v). After a forerun of4 liters, ml. fractions are collected and assayed for activity against K. pneumoniae. Active fractions are also tested for solids content. Fractions greater than 50, BU/mg are pooled and then taken to dryness in a rotatory evaporator at 40 C./7mm. Hg. to yield a syrup. Fractions and their K. pneumoniae (K.p.) activity and solids from a usual run are as follows:

Fraction Zone of Wt. 'of solid Number 1 Inhibition in Fraction (using 12.7 (rnqm) mm. discs) Fractions 120-180, incl. are pooled and taken to dryness on a rotatory evaporator at 40/7 mm. Hg. to give a syrup, wt. 2.66g, assaying 54 K.p. BU/mg (Fractions 181-240, incl. give a syrup, 830 mg; 32 BU/mg., and fractions 241-300, incl. give a syrup, wt. 710 mg., assaying 11 BU/mg.). The standard assayed 4 BU/mg. against the usual assay for this standard of 6 BU/mg.

Part D Purification No. 2

The preparations of U-44,590 obtained as described in Part C., can be further purified to a preparation of essentially pure U-44,590 by passage over another silica gel column using this time the solvent system methanol: methylene chloride (1:8 v/v). The procedure is as follows:

A U-44,590 preparation, as obtained in Part C., (2.28 grams) is dissolved in methanol and 7 grams of silica gel, as described in Part C., is added. The solvent from this mixture is removed on a rotary evaporator at 40 C. /7 mm. Hg. The resulting solid is added to a column of silica gel [750 g., 4.8 X 96 cm, hold-up volume 1500 ml., made up in MeOH-CH CI (1:8 v/v)]. A forerun (1100 ml.) is collected, followed by 50 ml. fractions. Fractions 141-200, inclusive, weigh 390 mg. when taken to'dryness in the form of a syrup. This material is shown to be almost pure U-44,590 by thin layer chromatography (tlc).

. The tlc is conducted on silica gel plates using the solvent system MeOH-CI-I Cl (1:9 v/v). Zones of the antibiotic are detected by spraying the plates with 10f/M- n spray, and with 50% aq. H 80 followed by heating at 1 C. for ca 10 min. The Rf of the active material in this solvent system is 0.1 1.

Part E Purification No. 3

The preparation of antibiotic U-44,590 obtained in Part D can be further purified by acetylation of the preparation followed by deacetylation and crystallization. The procedure for acetylation is as follows:

A sample (ca. 22 g.) of U-44,590 preparation, prepared as described in Part D and assaying 160 BU/mg, is dissolved in pyridine (300 ml), and to this solution, stirred magnetically, is added acetic anhydride (150 ml) over the course of 45 min. After standing overnight at room temperature, volatile materials are removed as completely as possible on a rotatory evaporator at 40/15 mm. Hg., and finally under high vacuum, to give a tan syrup. I

This syrup is stirred with CH CI (200 ml), and a colorless, flocculent precipitate is removed by filtration and washed with CH Cl until the washings are colorless. The precipitate is discarded. The combined filtrate and washings are washed with aqueous HCl (N/20, 100 ml) twice, the aqueous layer being acidic after the second wash. The aqueous layers are discarded. The organic phase is then washed with water (100 ml), saturated aqueous NaHCO (100 ml), again with water (100 ml), and dried (Na SO The aqueous layers are discarded.

Removal of solvent on a rotatory evaporator at 40 and mm. Hg. gives a dark syrup (21.10 g), which is dissolved in EtOAc (50 ml) by warming on a steambath. On cooling, crystallization occurs; the solid is removed by filtration, washed with EtOAc, and dried at 60/ 15 mm. Hg., to give essentially pure 3,5'-di-O- acetylated U-44,590 (12.01 g., m.p. l23124.5 C.).

Recrystallization from the same solvent gives U-44,590 diacetate, having a melting point 124-125 C. This compound is then labeled U-44,474.

U-44,474 is deacetylated to afford essentially pure U-44,590 by the Zemplen procedure which is as follows.

The crystalline diacetate U-44,474 (24.90 g) is stirred magnetically in methanol (400 ml), and methanolic sodium methoxide (Stauffer Chem. Co. 25%, 5 drops) is added. Stirring is continued till the solid has dissolved (Drierite tube), and the solution allowed to stand at room temperature for about 2 hours. Solid carbon dioxide, in small pieces, is then added cautiously, with stirring, to neutralize the methoxide, and the solvent is removed on a rotatory evaporator at 40 and 15 mm. Hg., giving a colorless oil.

The residue is dissolved in methanol (50 ml) by warming on a steambath and diluted with ethyl acetate (50 ml). Crystallization occurs on cooling. The solid (12.39 g) is collected on a sintered filter at the pump, washed with methanol, and dried in a vacuum oven at 60/15 mm. Hg. Antibiotic U-44,590 crystallizes in colorless prismatic needles, l41- 142. Removal of solvent from the filtrate plus washings on the evaporator and crystallization from methanol-ethyl acetate gives additional material (1.91 g, m.p. 140.514l.5).

EXAMPLE 2 The acylating procedure described in Example 1,

Part E can be substituted by acylating U-44,590 with any readily-available acylating agent to give acylated U-44,590. This acylated U-44,590 product can then be deacylated by methods well known in the art to yield a purified preparationof U-44,590. Readily-available I EXAMPLE 3 As disclosed in Example 2, various acylates of U- 44,950 can be made, and these acylates areuseful to upgrade U 44,590. By following the procedure of Example 1, Part E, the 3,5'-di-esters of U-44,590 are formed.

The 5'-mono-esters can be formed by standard procedures using a minimum amount of acylating agent.

The 3-mono-esters and phosphate can be formed by tritylating U-44,590 to give the 5-'-trity1 derivative, acylating this compound with the desired acylating agent, selected from those disclosed above, to give the 3'- mono-ester 5 '-trityl derivative, which then can be converted to the 3'-mono-ester by removal of the trityl group. The tritylationprocedure disclosed in U.S. Pat. No. 3,426,012, Columns 4 and 5, or other standard tritylation procedures can be employed. The trityl group can be removed by using the procedure disclosed in U.S. Pat. No. 3,426,012, Column 6.

EXAMPLE 4 The 5-phosphate of U-44,590 can be prepared by procedures as disclosed in the work of D. Mitsunobu, K. Kato, and J. Kimura [J. Amer. Chem. Soc., 91, 6510 (1969)]. This compound can be used for the same purposes as U-44,590.

The compounds, described above, being the derivatives of U-44,590 which are within the scope of the subject invention, can be shown by the following structural formula wherein R and R are Selected from the group consist- Band Frequency ing of a carboxylic acid acyl radical of from 2 to 18 car- (wave Numb) bon atoms, inclusive; or a halo-, nitro-, hydroxy-, 328 W amino-, cyano-, thiocyano-, and lower alkoxy- 2930 a substituted hydrocarbon carboxylic acid acyl radical of 5 2880 w from 2 to 18 carbon atoms, inclusive; R is hydrogen gig and R is as defined above or phosphate; or R is hydro- 1732 5 gen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a'halo nitro-, hydroxy-, 14 3 s aminocyano-, thiocyano-, and lower alkoxy- Egg M substituted hydrocarbon carboxylic acid acyl radical of 1370 m from 2 to 18 carbon atoms, inclusive, or phosphate. 1248 5 Additional characterization date for U-44,474, pre- R5; 5 M pared as d1sclosed 1n Example 1, Part E, 1s as follows: 1145 w Elemental Analysis: Calcd. for C H N O Found: :82; C, 47.41; H, 5.82; N, 12.76; 0, 34.01. 1030 M Molecular Weight: 329 (Determined by mass specm trometry) 986 M Infrared Absorption Spectra: U-44,474 has a characas; M teristic infrared absorption spectrum when suspended 932 a, in a mineral oil mull. Peaks are observed at the follow- 890 M ing wave lengths expressed in reciprocal centimeters: Egg 790 M 773 M Band Frequency 754 M (Wave Numbers) Intensity 739 W 3210 M 3080 M 2960 (oil) S 2930 (011) s We claim. 2860 (oil) 5 l. Antibiotic U-44,590, havmg the structure 1750 s 1732 S 0 1702' 5 I520 S I468 (oil) 8 1411 M 1379 (oil) 5 H CH3 1358 w 1327 w 1318 w 1310 w 1298 w 1280 M I 1250 s 40 1227 s H88 M 1148 w 1113 w 1097 s 1057 M 1030 S 1011 M 1000 M 987 M OH 964 M 957 M 322 $3 2. Compounds of the formula 392 M 0 863 M 828 M 791 M 775 M A 755 M H -N N c113 740 w 721 (oil) w 675 w 668 w 0 N U-44,474 also has a characteristic infrared absorption spectrum when pressed in a KBr disc. Peaks are observed at the following wave lengths expressed in reciprocal centimeters:

Band Frequency 5 (Wave Numbers) Intensity 3420 (water) W H 210 M 3 OR 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-,

amino-, cyano-, thiocyano-, and lower alkoxysubstituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive, or phosphate.

3. A compound, U-44,474, having the formula in claim 2 wherein'R and R are acetyl.

UNITED STATES PATENT AND TRADEMARK OFFICE EEHFICATE OF (IQRRECTION PATENT N0. 3,907,779 9 DATED September 23, 1975 INVENTOR(S) I Clarence DeBoer and Brian Bannister it is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

a Column 5 iine 25, for "Inhibition (pg/m)" read Inhibition (pig/mi i ine 50, for "15.6- 100" read 15.6- 1000 i ine E i, for "31 .2- 100 read 31.2 1000; i ine 38, for "Eli- 10" read 31,2- lOOO Coi umn 4, i ine 7, for "Activity (CH read Activity (CD Column 11, Table 5:

for "1.. O-Xyiose read D-Xyiose for O-Fructose" Q read D-Fructose for "5. O-Gaiactose" read D-Gaiactose for "7. O-Mannose" read D-Mannose Column 18, line 21 for (mqm)" read (mgm) Column 21, line 15, for "date' read data Q Signed and Scaled this eighth Day of Junel976 [SEAL] Arrest:

RUTH C. MASON C. MARSHALL DANN Anem'ng 0mm (:mmlssioner ufParems and Trademark: Q

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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3988314 *Oct 10, 1975Oct 26, 1976The Upjohn CompanyViricides; 1-methyl-pseudouridine; fermentation of streptomyces
US4022889 *Jun 12, 1975May 10, 1977The Upjohn CompanyTherapeutic compositions of antibiotic U-44,590 and methods for using the same
US4140850 *Aug 29, 1977Feb 20, 1979The Upjohn CompanyImmunosuppressive, viricides, silylation, cyclization, desilylation
US4171431 *Jun 1, 1977Oct 16, 1979The Upjohn CompanyNucleosides and process
US4239753 *Dec 12, 1978Dec 16, 1980The Upjohn CompanyComposition of matter and process
US4423212 *Dec 18, 1980Dec 27, 1983The Upjohn Company1-(2-deoxy-b-d-pentofuranosyl)-5,6-dihydro-s-triazines
US4477442 *Nov 5, 1980Oct 16, 1984The Upjohn CompanyComposition of matter and process
US5443972 *May 6, 1994Aug 22, 1995Sankyo Company, LimitedProcess to prepare leustroducsins
US5647957 *Jun 7, 1995Jul 15, 1997Ranpak CorporationRecycling
US5686262 *Jun 7, 1995Nov 11, 1997Ranpak CorporationRecycle process for the production of low-cost soluble collagen
US5700353 *Jun 7, 1995Dec 23, 1997Ranpak CorporationPaper strengthened with solubilized collagen and method
US5700354 *Jun 7, 1995Dec 23, 1997Ranpak Corp.High strength, tensile strength
US5707491 *Jun 7, 1995Jan 13, 1998Ranpak CorporationPaper strengthened with solubilized collagen and method
US5711853 *Jun 7, 1995Jan 27, 1998Ranpak Corp.Pulp from newsprint or eucalyptus fibers
US5714042 *Jun 7, 1995Feb 3, 1998Ranpak CorporationA paper sheet or board prepared from a mixture of solubilized collagen and cellulosic pulp (virgin, broke or reclaimed) with good tensile, burst and tear strength
US5736010 *Jun 7, 1995Apr 7, 1998Ranpak CorporationForming collagen and cellulose mixture into sheets
US5810970 *Jun 7, 1995Sep 22, 1998Ranpak CorporationMixing cellulosic material, diluting and adding an aluminum rosin forming a pulp slurry
DE2735378A1 *Aug 5, 1977Feb 23, 1978Upjohn Co5,6-dihydro-syn(s)-triazinnukleoside und -nukleotide sowie verfahren zu ihrer herstellung
U.S. Classification536/28.3, 435/87, 435/903
International ClassificationA61P31/04, A61K31/7042, A61K31/706, C12R1/585, C12N1/20, A61K31/70, A61K31/7052, C12P1/06, C12P19/38, C07H19/12
Cooperative ClassificationY10S435/903, C07H19/12
European ClassificationC07H19/12