CA1056822A - Acyl and phosphate derivatives of 5,6-dihydro-5-azathymidine - Google Patents
Acyl and phosphate derivatives of 5,6-dihydro-5-azathymidineInfo
- Publication number
- CA1056822A CA1056822A CA303,945A CA303945A CA1056822A CA 1056822 A CA1056822 A CA 1056822A CA 303945 A CA303945 A CA 303945A CA 1056822 A CA1056822 A CA 1056822A
- Authority
- CA
- Canada
- Prior art keywords
- inclusive
- carbon atoms
- carboxylic acid
- acyl radical
- acid acyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A new antibiotic U-44,590 known as 5,6-dihydro-5-azathymidine and its derivatives are described. These anti-biotics are active against Gram-negative bacteria and can be used in various environments to eradicate or control such bacteria. This divisional application is particularly directed to acyl and phosphate derivatives of U-44,590 having the formula wherein R and R1 are selected from the group consisting of a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; R
is hydrogen and R1 is as defined above or phosphate; or R1 is hydrogen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive, or phosphate.
A new antibiotic U-44,590 known as 5,6-dihydro-5-azathymidine and its derivatives are described. These anti-biotics are active against Gram-negative bacteria and can be used in various environments to eradicate or control such bacteria. This divisional application is particularly directed to acyl and phosphate derivatives of U-44,590 having the formula wherein R and R1 are selected from the group consisting of a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; R
is hydrogen and R1 is as defined above or phosphate; or R1 is hydrogen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive, or phosphate.
Description
~ 31~3 PJ~
~ NTfON
The no~el antibiotic of the in~ention, U-44,590 is obtained by culturing Streptomyces p.latensis ~ar. clarensis.
NRRL 80~5, in an aqueous nutrient medium under aerobic conditions. Various derivati~es of U-44,590 can be made as disclosed infra. Uw44,590 and its deri~ati~es ha~e the property of ad~ersely af~ecting the growth o~ Gram-negati~e and Gr2m-positi~e bacteria, for example, _rep~o-coccus ~ æ~L~ , Klebsiella pneumonia, Salmonella Sp., Serratia marcescens, Past~urell~ multocida7 Hemophilus Sp., Proteus ~ and Proteus rettqeri. Accordingly, U-44,590 and its deri~ati~es can be used alone or in combination --with o~her antibiotic agents to pre~ent the growth of or reduce the number of bacteria, as disclosed above, in ~arious environments.
U-44,590 and its derivati~es are also acti~e against ~ -- DNA ~iruses, for example, the Herpes ~irus and, thusJ can ~-~
be used to control such ~irus where its presence is not desired.
~
Ch~emical and Ph~cal Properties of U-44,590 ! Elemental AnalYsis.
': ~
Calcd. for CaHl5N305:
C, 4l~.o8; H, 6.17; N, 17.13.
F~ound:
C, 44.14; H, 6.oB; N, 17.36.
~L~ LI~LDh : 245 (Determined by mass spectrometry) Meltinq Point Ranqe: 141 - 142 C.
~?25 _ -5 (c, 0.9030 in H20) SolubTlit7es: Highly soluble in water, and lower aicohols, ---- .
'~
for example, methanol and ethanol; relatively insoluble in Me2CO, El:OAc, hydrocarbons, CH2Cl2 and CHCI3.
; Infrared Absorption Spectra: U-~4.590 has a characterislic infrared absorption spectrum when suspended in a mineral oil mull. Peaks are obser~ed at the following wa~e lenglh, expressed in reciprocal centimeters:
Band Frequency Intensity . 334 M
319~ M
. 3080 M
2960 (N = Nujol) S
2930 (N) S
: - 2860 (N) S
1695 sh. S
: 16~3 S
~0 1510 M
1503 M :
.
148~ M
1463 (N) S . .
~5 1421 M
1396 M :-1~7$ (N) W
~315 : -3-' '',, . ' ' , ~: ,., . .
~3 Band Frequency ,~Wa e Numbers~
130~ W
1~76 W
126~ W
1243 . S . :
1230 sh. M
: 1195 W . :
: 10 1133 W
1og~ M
1085 W ~
~0~0 S ' '.. ':
1011 S ~ : .
~72 W
8~9 W
~2~ W
7~5 W :
25 NoteJ sh means a shoulder band.
U~44~590 also has a characteristic infrared absorption spectrum when presseci in a KBr disc. Peaks are obser\~ed at the followTng wa~/e lengths expressed in reciprocal centi-meters:
3~ .
. , .
~ .
~ ~ 31~
,, Band f requ~nc I n tens i t Y
3~ W
~ NTfON
The no~el antibiotic of the in~ention, U-44,590 is obtained by culturing Streptomyces p.latensis ~ar. clarensis.
NRRL 80~5, in an aqueous nutrient medium under aerobic conditions. Various derivati~es of U-44,590 can be made as disclosed infra. Uw44,590 and its deri~ati~es ha~e the property of ad~ersely af~ecting the growth o~ Gram-negati~e and Gr2m-positi~e bacteria, for example, _rep~o-coccus ~ æ~L~ , Klebsiella pneumonia, Salmonella Sp., Serratia marcescens, Past~urell~ multocida7 Hemophilus Sp., Proteus ~ and Proteus rettqeri. Accordingly, U-44,590 and its deri~ati~es can be used alone or in combination --with o~her antibiotic agents to pre~ent the growth of or reduce the number of bacteria, as disclosed above, in ~arious environments.
U-44,590 and its derivati~es are also acti~e against ~ -- DNA ~iruses, for example, the Herpes ~irus and, thusJ can ~-~
be used to control such ~irus where its presence is not desired.
~
Ch~emical and Ph~cal Properties of U-44,590 ! Elemental AnalYsis.
': ~
Calcd. for CaHl5N305:
C, 4l~.o8; H, 6.17; N, 17.13.
F~ound:
C, 44.14; H, 6.oB; N, 17.36.
~L~ LI~LDh : 245 (Determined by mass spectrometry) Meltinq Point Ranqe: 141 - 142 C.
~?25 _ -5 (c, 0.9030 in H20) SolubTlit7es: Highly soluble in water, and lower aicohols, ---- .
'~
for example, methanol and ethanol; relatively insoluble in Me2CO, El:OAc, hydrocarbons, CH2Cl2 and CHCI3.
; Infrared Absorption Spectra: U-~4.590 has a characterislic infrared absorption spectrum when suspended in a mineral oil mull. Peaks are obser~ed at the following wa~e lenglh, expressed in reciprocal centimeters:
Band Frequency Intensity . 334 M
319~ M
. 3080 M
2960 (N = Nujol) S
2930 (N) S
: - 2860 (N) S
1695 sh. S
: 16~3 S
~0 1510 M
1503 M :
.
148~ M
1463 (N) S . .
~5 1421 M
1396 M :-1~7$ (N) W
~315 : -3-' '',, . ' ' , ~: ,., . .
~3 Band Frequency ,~Wa e Numbers~
130~ W
1~76 W
126~ W
1243 . S . :
1230 sh. M
: 1195 W . :
: 10 1133 W
1og~ M
1085 W ~
~0~0 S ' '.. ':
1011 S ~ : .
~72 W
8~9 W
~2~ W
7~5 W :
25 NoteJ sh means a shoulder band.
U~44~590 also has a characteristic infrared absorption spectrum when presseci in a KBr disc. Peaks are obser\~ed at the followTng wa~/e lengths expressed in reciprocal centi-meters:
3~ .
. , .
~ .
~ ~ 31~
,, Band f requ~nc I n tens i t Y
3~ W
2~37~ W
2~60 W
2~70 W
1697 sh~ S
16~5 S
146~ S
14~7 M
134g W
: ~310 W
1~75 sh. M
2 5 1~!63 M
1~95 W
-~
~
' .
0and Fr~qucnc : 1013 5 M
9~5 W
88 ~5 W
87~ W
. 10 ~47 W
`, 827 W
7gl M
`: . .754 W
.. . .
, 7~ W
.
Note: sh means a shoulder band.
3nfrared band intensities~ throughou~ this disclosure, are indicated as ~t5~1~ "M", and "W" respectively and are appro~imated in terms of the backgrounds in the vicinity : of the bands. An "S" band is of the same order of inten-2~ sity as the strongest in the spectrum; "M" bands are be~
~ween 1/3 and 2/3 as intense as the strongest band; and, "W" bands are less than 1/3 as intense as the strongest band. These estimates are made on the basis of a percent transm i ss ion sc~ l e .
' 25 The fol lowTng is cons idered to be the structure of ~
~J-44,59: :
, .. :
' . - , , 31 1~3 ~ 3~
. H~N~ s N -CH 3 ~NJ
:' 5' HOH~ ~ ~
. ~ ' ; I H
ThusJ U-44,590 can be referred to by the ~r i~ ial name . . ~ .
5,6-dihydro-5-a2athymidine, or by its chemical name 1- : -~2-deoxy-,B-D-~-pentofuranosyl ) -5,6-d i hydro-5-methy 1-.~ .
, : s-tr i3z ine-2,4(1Hg3H)-dior,e.
hnt_ibacterl a! ~Acti~ity of U-~4~590 ~:-Orqan i sm No . ID~ I n h; b i t ! on i,u~m l ) - i .Stra ins ~ ~ Staphylococcus aureus 1 ~1000 . ~ -: ~ ~.@e~ hemolYticus 1 15.2 . 20 Dip?oc~us ~Ym~ 1 500.
i . . .
~ : ~ ~m~ 5 2 .0 - ~looo ; : ~ sp....................... 4 15.6 - ~looo :
~ ~ 2 125 :-E5~ aer_~i osa 5 ~1000 :~:
~L~ mul toc ida 1 125 5p. 5 31.2 - ~1000 . uiqaris 3 >1000 .
~!~ 3 ~1000 ~ m rgani :S . 62 .5 - 250 ~: .
3l:~ ~Q~ ~g~ 3 : 31.2 - >1000 ~ ' ~
~7- . .
. ,. . ~
314~
The ab~e 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 Hemoehilus species which were grown in Difco Blood Agar Base with 5% de-fibrinated rabbit blood. All were grown aerobically at 37 C. (except ~ e~ species, grown anaerobically) 16-18 hours. Inocula were grown o~ernite (16-18 hours) o at 37 C. and used to seed agar at the rate of 0.025 ml.
of 10 3 dilution (approximately 2500 to 25,000 bacteria per drop of inoculum).
- In ~i~o testing of U-44,590 in mice infected with selected microorganisms is as follcws: -Acti~ity (CD50 in mg/kg) Miceg iP ~ ~~ ~~~
Challenge Subcutaneous Oral g~ Q____ Rou~te Route Salmonella flexneri 40 38 (25-57) 62.5 .
` Escherichia coli 79 141 (116-172) 218 (i54-~07) Prot,eus mirabilis1259 152 (96-240) 101 (66-156) Proteus _ ~ 79 100 (66-152) <62.5 1~0 ~30 s hemo-YtiCUs, 100 - ~60 ANT!VIRAL ACTIVIT~ O~ J590 The following is an example o-f the anti~iral acti~ity of antTbiotic U-44,590. The antibiotic is administered sub-cutaneously to mice which are inoculated intra~enously with !~LL~ ~irus. Treatment is inttiated two hours prior ~0 to ~iral infection and is followed by treatment four times ' 31 l! 3 daily for fi~e consecuti~e days. A detailed account of the materials and methods and results are as follows:
Male mice, weighing approximately 20 gm. each, are di~ided into 4 groups of 20. Grsup 1 is treated with saline, Group 2 with 400 mg./kg./dose (mkd) U~47590, Group 3 with 200 mkd U-44,590, and Group 4 with 100 mkd U-44,590. The antibiotic is dissol~ed in saline and administered subcutaneously in the nape of the neck at 8 a.m., 12 noon, ~ p.m., and 8 p.m. on days 0~ 1, 2, 3, and 4. Herpes virus at-10~1 5 dilution, 0.05 ml/mouse, .
equi~alent to a ~iral dose of 40 LD50sg is inoculated into the tail ~ein at 10 a.m. on day 0. Paralysis and death are recorded dai1y.
Hind leg paralysis usually preceded death by 1-2 days. All mice died that became paralyzed. Death pattern of the ~ groups, as shown in the cur~es which follow, il1ustrates the dose response obtained. Statis~ical analysis of the resu7ts at day 11 indicates that all 3 tre~ted groups are significant1y different from the con-trol group (1).
.:
' , ~
311~3 o ... . . ~
~' ' 1 ~n ~
~ ~ _. :t, ~ D ~
1 , ~
~ Z
' ~
~JlO 0 3 0 z u~ o C~ ~D ~ .
~D -~10- .
THE MiCROORGANISM
The microorganism used for the production of U-44,590 is streptomYces platensis ~ar. clarensis! NRRL 8035. A
subculture of this micronrganism can be obtained from the permanent collection of the Northern Regional Research ; Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S .~ .
The microorganism of this in~ention was studied and characterized by Alma Dietz of the Upjohn Research Labora-~ories.
A new soil isolate with hygroscopic spore masses, butwith smooth, hat~shaped (crescent) or brazil-nut-shaped (elliptical) spores, has been found t~ differ in certain - characteristics from thP type culture ~ æ~Y~ platensis.
An outstanding difference of the new culture is the produc-; tion of antlbiotic U-44,590. The new isolate can be recog- -ntzed as a ~ariant of Streptom~ces platensis by its cultural, microscopic, and biochemical characteristics~ Therefore, it is proposed ~hat this new isolate be designated strePtom~es ~ ar. clarensis Dietz ~ar. no~a. Rule 7 of the International Code of Nomenclature of Bacteria L Internation-al Code of Nomenclature of Bacteria. 1966. Edited by the Editorial Board of the Judicial Commission of the lnterna- ~
tion~l Committee on Nomenclature of Bacteria. Int. J. : ~ :
Syst. Bacteriol . 16: 459-490] was appl ied in des ignating the Yarlety epithet.
~ es~mY~ p,latens_is ~ar. clarensis i5 cornpared with ; th~ type spec7es S e~ platensis Pi~tenger and Gott-lteb ~Shirling, E.B,, and D. Gottlieb. 1968. Cooperati~e description of type cultures of ~treptomyces lll. Addi-tional species descriptions from firsL and second studis~s.
Int. J. Syst. Bacteriol. 18:279 392] [Tresner, H.D., E.J.
Backus, and Jean A. Hayes. 1967. Morphological spore types in the Streptomyces hyqroscopicus-like cornplex .
Appl. Microbiol. 1~:637~539] NRRL 2369, and two recently s~haracterized strains: StrePtomyces platensis NRRL 3593 [E~ans, Ralph Henry Jr.; and Samuel Owen Thomas. 1971.
Antibiotics AH272~2 and AH272~z and process for producing same~ U.S. Patent 3,592,925~ and Streptomyces platensis IIRRL :~761 [ûkuda, Tomohau, and Shigemi Awatagouchi. 197~.
Antibiotics YL 704 and preparation thereof. U.S. Patent ~5,718,742]. "
Color~characteristics: Aerial growth white to yell~w to gray. Moist black hygroscopic patches on some media.
Melanin-negative. Appearance on Ektachrome ~Dietz, A.
;~ 1954. Ektachrome transparencies as aids in actinomycete classlficakion~ Ann. N.Y. Acad. Sci. 60:152-154~ Is ~T~en in Table 1. Reference color characteristics are g7~en in Tables 2 and ~. The new culture may be placed in the White (W), Yellow (~), and Gray (GY) color series ;
of Tresner and Backus [Tresner, H.D., and E.J. Backus.
1963. System of color wheels for streptomycete ta>~c~nomy.
Appl. Microbiol. 11 :335-338] .
-~h~_: Spore chains in tight spirals 25 uncoiling to long open spirals. Spore chains spiral (S) in ~he sense of Pridham et al. ~Pridham, T.G., C.W. Hesseltlne, and R.G. Benedict. 1958. A guide for the classification of s~reptomycetes according to selected groups. Placement of 5tr~ins in morphological sections. Appl. Microbiol. 6:52-79].
:~50 Spor~ hat-shaped (crescent) or Brazil-nut (elliptical) shap~d.
, _ ' 3143 Spores are the latens~is-type of Tresner and Back~s [Tresner, H.D.I E.J. Backus, and Jean A. Hayes. 1967. Morphological spore types in the Streptomyces hyqros ~ -like complex.
Appl. Microbiol. 15:637-639]. Spore silhouette smooth by direct obser~ation with the electron microscope. Spore surface rldged with surface markings by the carbon replica-~ion technique of Dietz and Mathews [Dietz, A. and J. Mathews.
1962. Taxonomy by carbon repl ication. I . An examination of Strepto~ hvqroscopicus. Appl. Microbtol. 10:258-263].
Cultural and biochemical characteristics: See Table 4, infra.
Garbon utllization: Growth on carbon eompounds was deter- -m;ned in the synthetic medium of Pridham and Gottlieb [Prid-ham~ T.G., and D. Gottlieb. 1948. The utilization of car- -~
bon campounds by some Actinomycel:ales as an aid for species determination. J. ~acteriol. ~):107-114~, Table 5 and in the synthetic medium of Shirling and Gottlieb [Sh;rling, E.B.
and D~ Gottlieb. 1966. Methods for characterization o~
StrePtomyces species. Int~ J. Syst. Bacter;ol. 16:313 3~0], ~able 6.
I~E~ : The cultures grew well at 18 - 37 C. on ~en-nett's, Czapek's sucrose, maltose-tryptone, and Hickey-Tres-ner agars. Optimum growth was at 24 - ~7 C. The new culture and the type culture did not grow at 45 - 55 C. The cultures desTgnated NRRL 3593 and NRRL ~761 grew at 45 C. but not at ~5 55 ~-I~L~L~a5~9:eI~e~5~L~LPrEerties See Table 7, infra.
Soil T ~ ~ t~ latensis Pittenger and Gottl;eb NRRL 236~.
30 I~ s~ ar. platensis NRRL 2364. -..
~143 ~: Streptomyces ~ ~ar. clarensis Dietz ~ar. no~a.
.. . . . .
~,,: . - , s ~ ~ . - , ' , ,~ ~'' ' ' ~ , ', : ' ' ', , : ... ~, ,.,, . : .
,. . .
, ' ' ' . ' '; ' , ' .
, . , -14 , ' . ... .. . . . .
. . , ~4~
,~ :
~ 5~ ~
:
~:
~ .
V~ ~ ;~
._ L
~: o~ 1~ c -If~ I I Q~
JJ ~ ~ S_ :~ L ~ V
* ~ ~ a~
a~ _ J ~ 3 ~ V ~ 3 E CL cc C ~ c ~ C O ~ ~ :
z ,~ _ > ---> ~
J ~ ~ J ~ t_ ~ :.
~V . , .
~C ~ ' ILI ".' ~: ' : O ~ ' ' "' ': ', ~, ut ~ ~ n~ ~ Ql .
rD n~.Rl ~ ''
2~60 W
2~70 W
1697 sh~ S
16~5 S
146~ S
14~7 M
134g W
: ~310 W
1~75 sh. M
2 5 1~!63 M
1~95 W
-~
~
' .
0and Fr~qucnc : 1013 5 M
9~5 W
88 ~5 W
87~ W
. 10 ~47 W
`, 827 W
7gl M
`: . .754 W
.. . .
, 7~ W
.
Note: sh means a shoulder band.
3nfrared band intensities~ throughou~ this disclosure, are indicated as ~t5~1~ "M", and "W" respectively and are appro~imated in terms of the backgrounds in the vicinity : of the bands. An "S" band is of the same order of inten-2~ sity as the strongest in the spectrum; "M" bands are be~
~ween 1/3 and 2/3 as intense as the strongest band; and, "W" bands are less than 1/3 as intense as the strongest band. These estimates are made on the basis of a percent transm i ss ion sc~ l e .
' 25 The fol lowTng is cons idered to be the structure of ~
~J-44,59: :
, .. :
' . - , , 31 1~3 ~ 3~
. H~N~ s N -CH 3 ~NJ
:' 5' HOH~ ~ ~
. ~ ' ; I H
ThusJ U-44,590 can be referred to by the ~r i~ ial name . . ~ .
5,6-dihydro-5-a2athymidine, or by its chemical name 1- : -~2-deoxy-,B-D-~-pentofuranosyl ) -5,6-d i hydro-5-methy 1-.~ .
, : s-tr i3z ine-2,4(1Hg3H)-dior,e.
hnt_ibacterl a! ~Acti~ity of U-~4~590 ~:-Orqan i sm No . ID~ I n h; b i t ! on i,u~m l ) - i .Stra ins ~ ~ Staphylococcus aureus 1 ~1000 . ~ -: ~ ~.@e~ hemolYticus 1 15.2 . 20 Dip?oc~us ~Ym~ 1 500.
i . . .
~ : ~ ~m~ 5 2 .0 - ~looo ; : ~ sp....................... 4 15.6 - ~looo :
~ ~ 2 125 :-E5~ aer_~i osa 5 ~1000 :~:
~L~ mul toc ida 1 125 5p. 5 31.2 - ~1000 . uiqaris 3 >1000 .
~!~ 3 ~1000 ~ m rgani :S . 62 .5 - 250 ~: .
3l:~ ~Q~ ~g~ 3 : 31.2 - >1000 ~ ' ~
~7- . .
. ,. . ~
314~
The ab~e 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 Hemoehilus species which were grown in Difco Blood Agar Base with 5% de-fibrinated rabbit blood. All were grown aerobically at 37 C. (except ~ e~ species, grown anaerobically) 16-18 hours. Inocula were grown o~ernite (16-18 hours) o at 37 C. and used to seed agar at the rate of 0.025 ml.
of 10 3 dilution (approximately 2500 to 25,000 bacteria per drop of inoculum).
- In ~i~o testing of U-44,590 in mice infected with selected microorganisms is as follcws: -Acti~ity (CD50 in mg/kg) Miceg iP ~ ~~ ~~~
Challenge Subcutaneous Oral g~ Q____ Rou~te Route Salmonella flexneri 40 38 (25-57) 62.5 .
` Escherichia coli 79 141 (116-172) 218 (i54-~07) Prot,eus mirabilis1259 152 (96-240) 101 (66-156) Proteus _ ~ 79 100 (66-152) <62.5 1~0 ~30 s hemo-YtiCUs, 100 - ~60 ANT!VIRAL ACTIVIT~ O~ J590 The following is an example o-f the anti~iral acti~ity of antTbiotic U-44,590. The antibiotic is administered sub-cutaneously to mice which are inoculated intra~enously with !~LL~ ~irus. Treatment is inttiated two hours prior ~0 to ~iral infection and is followed by treatment four times ' 31 l! 3 daily for fi~e consecuti~e days. A detailed account of the materials and methods and results are as follows:
Male mice, weighing approximately 20 gm. each, are di~ided into 4 groups of 20. Grsup 1 is treated with saline, Group 2 with 400 mg./kg./dose (mkd) U~47590, Group 3 with 200 mkd U-44,590, and Group 4 with 100 mkd U-44,590. The antibiotic is dissol~ed in saline and administered subcutaneously in the nape of the neck at 8 a.m., 12 noon, ~ p.m., and 8 p.m. on days 0~ 1, 2, 3, and 4. Herpes virus at-10~1 5 dilution, 0.05 ml/mouse, .
equi~alent to a ~iral dose of 40 LD50sg is inoculated into the tail ~ein at 10 a.m. on day 0. Paralysis and death are recorded dai1y.
Hind leg paralysis usually preceded death by 1-2 days. All mice died that became paralyzed. Death pattern of the ~ groups, as shown in the cur~es which follow, il1ustrates the dose response obtained. Statis~ical analysis of the resu7ts at day 11 indicates that all 3 tre~ted groups are significant1y different from the con-trol group (1).
.:
' , ~
311~3 o ... . . ~
~' ' 1 ~n ~
~ ~ _. :t, ~ D ~
1 , ~
~ Z
' ~
~JlO 0 3 0 z u~ o C~ ~D ~ .
~D -~10- .
THE MiCROORGANISM
The microorganism used for the production of U-44,590 is streptomYces platensis ~ar. clarensis! NRRL 8035. A
subculture of this micronrganism can be obtained from the permanent collection of the Northern Regional Research ; Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S .~ .
The microorganism of this in~ention was studied and characterized by Alma Dietz of the Upjohn Research Labora-~ories.
A new soil isolate with hygroscopic spore masses, butwith smooth, hat~shaped (crescent) or brazil-nut-shaped (elliptical) spores, has been found t~ differ in certain - characteristics from thP type culture ~ æ~Y~ platensis.
An outstanding difference of the new culture is the produc-; tion of antlbiotic U-44,590. The new isolate can be recog- -ntzed as a ~ariant of Streptom~ces platensis by its cultural, microscopic, and biochemical characteristics~ Therefore, it is proposed ~hat this new isolate be designated strePtom~es ~ ar. clarensis Dietz ~ar. no~a. Rule 7 of the International Code of Nomenclature of Bacteria L Internation-al Code of Nomenclature of Bacteria. 1966. Edited by the Editorial Board of the Judicial Commission of the lnterna- ~
tion~l Committee on Nomenclature of Bacteria. Int. J. : ~ :
Syst. Bacteriol . 16: 459-490] was appl ied in des ignating the Yarlety epithet.
~ es~mY~ p,latens_is ~ar. clarensis i5 cornpared with ; th~ type spec7es S e~ platensis Pi~tenger and Gott-lteb ~Shirling, E.B,, and D. Gottlieb. 1968. Cooperati~e description of type cultures of ~treptomyces lll. Addi-tional species descriptions from firsL and second studis~s.
Int. J. Syst. Bacteriol. 18:279 392] [Tresner, H.D., E.J.
Backus, and Jean A. Hayes. 1967. Morphological spore types in the Streptomyces hyqroscopicus-like cornplex .
Appl. Microbiol. 1~:637~539] NRRL 2369, and two recently s~haracterized strains: StrePtomyces platensis NRRL 3593 [E~ans, Ralph Henry Jr.; and Samuel Owen Thomas. 1971.
Antibiotics AH272~2 and AH272~z and process for producing same~ U.S. Patent 3,592,925~ and Streptomyces platensis IIRRL :~761 [ûkuda, Tomohau, and Shigemi Awatagouchi. 197~.
Antibiotics YL 704 and preparation thereof. U.S. Patent ~5,718,742]. "
Color~characteristics: Aerial growth white to yell~w to gray. Moist black hygroscopic patches on some media.
Melanin-negative. Appearance on Ektachrome ~Dietz, A.
;~ 1954. Ektachrome transparencies as aids in actinomycete classlficakion~ Ann. N.Y. Acad. Sci. 60:152-154~ Is ~T~en in Table 1. Reference color characteristics are g7~en in Tables 2 and ~. The new culture may be placed in the White (W), Yellow (~), and Gray (GY) color series ;
of Tresner and Backus [Tresner, H.D., and E.J. Backus.
1963. System of color wheels for streptomycete ta>~c~nomy.
Appl. Microbiol. 11 :335-338] .
-~h~_: Spore chains in tight spirals 25 uncoiling to long open spirals. Spore chains spiral (S) in ~he sense of Pridham et al. ~Pridham, T.G., C.W. Hesseltlne, and R.G. Benedict. 1958. A guide for the classification of s~reptomycetes according to selected groups. Placement of 5tr~ins in morphological sections. Appl. Microbiol. 6:52-79].
:~50 Spor~ hat-shaped (crescent) or Brazil-nut (elliptical) shap~d.
, _ ' 3143 Spores are the latens~is-type of Tresner and Back~s [Tresner, H.D.I E.J. Backus, and Jean A. Hayes. 1967. Morphological spore types in the Streptomyces hyqros ~ -like complex.
Appl. Microbiol. 15:637-639]. Spore silhouette smooth by direct obser~ation with the electron microscope. Spore surface rldged with surface markings by the carbon replica-~ion technique of Dietz and Mathews [Dietz, A. and J. Mathews.
1962. Taxonomy by carbon repl ication. I . An examination of Strepto~ hvqroscopicus. Appl. Microbtol. 10:258-263].
Cultural and biochemical characteristics: See Table 4, infra.
Garbon utllization: Growth on carbon eompounds was deter- -m;ned in the synthetic medium of Pridham and Gottlieb [Prid-ham~ T.G., and D. Gottlieb. 1948. The utilization of car- -~
bon campounds by some Actinomycel:ales as an aid for species determination. J. ~acteriol. ~):107-114~, Table 5 and in the synthetic medium of Shirling and Gottlieb [Sh;rling, E.B.
and D~ Gottlieb. 1966. Methods for characterization o~
StrePtomyces species. Int~ J. Syst. Bacter;ol. 16:313 3~0], ~able 6.
I~E~ : The cultures grew well at 18 - 37 C. on ~en-nett's, Czapek's sucrose, maltose-tryptone, and Hickey-Tres-ner agars. Optimum growth was at 24 - ~7 C. The new culture and the type culture did not grow at 45 - 55 C. The cultures desTgnated NRRL 3593 and NRRL ~761 grew at 45 C. but not at ~5 55 ~-I~L~L~a5~9:eI~e~5~L~LPrEerties See Table 7, infra.
Soil T ~ ~ t~ latensis Pittenger and Gottl;eb NRRL 236~.
30 I~ s~ ar. platensis NRRL 2364. -..
~143 ~: Streptomyces ~ ~ar. clarensis Dietz ~ar. no~a.
.. . . . .
~,,: . - , s ~ ~ . - , ' , ,~ ~'' ' ' ~ , ', : ' ' ', , : ... ~, ,.,, . : .
,. . .
, ' ' ' . ' '; ' , ' .
, . , -14 , ' . ... .. . . . .
. . , ~4~
,~ :
~ 5~ ~
:
~:
~ .
V~ ~ ;~
._ L
~: o~ 1~ c -If~ I I Q~
JJ ~ ~ S_ :~ L ~ V
* ~ ~ a~
a~ _ J ~ 3 ~ V ~ 3 E CL cc C ~ c ~ C O ~ ~ :
z ,~ _ > ---> ~
J ~ ~ J ~ t_ ~ :.
~V . , .
~C ~ ' ILI ".' ~: ' : O ~ ' ' "' ': ', ~, ut ~ ~ n~ ~ Ql .
rD n~.Rl ~ ''
3 ~ ~ ~Jl a~ . :-.
1~ I i ,~
V C~J ' C L. ~~_ C
~ 0 ~ 1~
_ _ J ~ ~ ~ CL~:1 V
U~ Q tl~ C I C C I 0 ~5 -C ce: ~ ) v 0 . Z ~ ~ > ~
l J Q J CLJ t~
. ' ..
¦_ . . . ..U~ L~ ., r~ ' ' (~ ,. .
.) .. o r J- C
, CO .~ !~ .-- ~ V
E ~t~ ~- C C .-- c O > J 3 ~-- ~ 2 .--ul Q
r ._ ~ ~ 3 j~
U~ ~ V_ >,_ I ~ ._ ~ v) S~ Q O ~ ~J ~. . - .
1~ ITl C Q)>~
,, O ' q~ ~ v I ~ ~ ~ ~ I
E 1:: E C O E O E
î~ Q~ ~ ~D
_ ~ îT) D
lU I
I C
Q) ~ '-- 0 Cl E c~-~E
, ., ~ ~ -. '"
~ ~ o .~ . Ul ~_ ,_ U~
~I) v E u x ", c aJ Q~ O
c a. ~
C 9 ~ o, a~ ~ D aJ
~: ~ ~ S: ~
.. . .
D J
'1~5~
., ' ..
, V) >.
. ~ .
C ~ rD U~ ~ o .~ ~ ~ ~ U~ .. ._ v U~ ~ C~ a _ ~ ", , _ ~ . C
J ~ O C~ ~ _ t~ ~_ O ~D ~ V~ '' In , . ..
~ .
C~
>~
~ C~
~ , ~
* -- E : .
', Q~ .UI O ~ C~ C
C ~n- v C) I ~
~O ~ C L_ ro ~1 C\l ~ ~ ~ ~7 C
V =_ l C ._ .
J C~ Y O C) ~: ~
:~, c ~nlz ~ Q I n.~ ~
O D
Ul "~ . .
0 V\
, J ~ ~ Q) > J ~ U
x: Y ~ c ,, I . 1~ - c~
:'. l ~ ,~v ~ ~ E Q
I ~ V~ ~ ~ C, ;~
I ~ ~ C~ O ~ I'D Ln I _ C~ V ~
C ~ C) ~ C J.J I
~ u ~
_ r~---- >-- ~L~
,,:O ~1 o ~ o ~
5: ~O
~ a) c)~ o .-C:~ ~ E c:~- ~ ~ y ,~ :
~1_ .
,,: ~U~ ~
a~ u E ~ a,~
8 ~ ~ 1" ID v u~ . .
. O J~ rD ~ Ct t ~ , ,, ~ ~,. .
C~ ~ ~ ~ .. Z
~ C ~ ~ y _ ~, ~ ~ a) ~ c S~ Q 10 ,~ v~ 11 11 -- C .
ro Q Cl . t~J O .4.
~- ~s a o .
-~ 31 ~q~5~
C ~
V ~ _,_ ~ _.._~
O J F ~J) ~ E E E E E E
a~ ~ to u ~.) (l\ u tJ~¦Z N N 9 D N I N tU I ~N I ~ N I
V ~S ,: :
(~.VJ D
. ~n _~ ~ ~ ~` ~
~_ ~a~ cr~ O ~--~
' V~ ~V Lt'~
. ~ 11)~ ~ ~ 'J' ^-- ~ ~ --` cn ~_, ..
Q~ ~13_ J E~ E~ E~ E~ E~
~ .C~
r_ ~ C~
O. ~IZN ~1 I N -1 I N ~t I N ~1 I C\l ~1 . ~ _ ' : .
,u~ . E , . -v >~~n - . Q cc ~t O
, Q) O Q~ ~D
~, ~ Ev 1 CUv ~~11 N
~n ~_ J E ~:n E ~ E E E E E
~ c~: a~ u ~ a~ a) u ~ u a~ a - 1~ 0~lZ c~
1~ . .
~,~ . .u o ,_, ' ~ ~ ~ E cn ._ ~ ~ Q~ q ,, ~ u~ u) ~
al ._ ,_ ~) Il~ 1~ N
v c~ U~ O N ~ O O
Q~ <1~ 1~ .~ v ~ ' D '~a ~ :o _- v_~ ~ ~ ~"~
Q U J ~
n~ ~:n a~ n u O ~¦ > Z C~J N I tl~ "~
O ' . , .
U Q~ O
Q ~ E ~ ~ ~ n ~ ~ tL L~ L ~ ~ Q ~
Yj Q~ . ' ', .' .
~ v ,,:
E u u .-._ v~ a~ v ~-~ ~ c ~ )~ X--~
Q~ ~ _ ~ Q~ O I aJ a~
:~: vv o u~ ~ c Q) a~ ~- o Q q) V~ v v o,, ~ cQ U V ~ v ': 0 C~ ~ ~
~ n~ v -'~ a~ v -17- :
:, . . . .... . . ... . . ..
,u~ .' ~ ~o~ ~~
c _l ~ ~J) E Cl) E
~ u .' ~ o~ -- u ~ a) ~ u a ~Iz ., *
~, oo V\ .~ -: o~ ._ _ , ,1 ~ t~
O L~ C
ILI ~ ~ ~
.~ _ E ~ E ~ 1~ E
~ ~ J ~ ~ ~ ~
u~ ~: a~ u u u ~ ~ c 0 ~ ~ . ~ ~ -CJ O) D ~ O . :~
, ~ ~nlz ~C~I I C`J CU I CU C~l I E
_ ~ ,_ ~a ~ ~ .~ O
,u~ r c ~ 0 0 c c . O a) ~D J U
a~ E ~ 1~\ lu .
V ~ (~ C~l ~~
11~ X . _~ . E V) E E ~ ~ E ' C o a:: ~ c In O ~nlz ,_ ._ _ U -~
_ E ~ :
& U7 ,v) ~0 ~ ~ i~ V~
~' ~ ~ ~ ~0 ,~
- O _ _ E cn E ~ ~c~ c I:L ~J J ~
.' tn Ix: ~:n u-- ~ u ~ u ~--~ . ~ ~ ~ ~`~ ~ .. L
r~1 a z ~ w ,' ,,~ ~ :
,,, o C o ~Q ~ E C ~ ~,0 :~
~::
., ~ -:
a l_ Q : .
O .~ ~ , , V-- c ~ O
S: 3 8 " ~ a~ .
w Q ~ V ~~ ._ c ~, -- V~ O C
C Q1 ~ I ~ 0 0 U~
~) ~ ~ D tO
Q~ ~ ~ ~ ~ v Q ~ to ~:L O V
v-- o ~ ~ ~ O
:.. ,".o -- v -18 ~
. . ; .' . ' ': .
~ ~:
~, ~ E
a ~D c~l .
0~ E E
~ ~ .~ ;J
_ _l E~ E ~ E E E ` E ~ E
a~ ~ ~1~ co cl) coE ~ E
~ ~ ~ ~ C~ar,o ~o : ~ , ~IZ C~ I N 00 1 C1~ C~ I ~ C~ I ~ ~ I .
. . ....... ...
V~ .,.
.' * ~ E E cn E
*~ a~ 1~
:~ Ll~v L~ O O O N
n~ ~\ . ~
c~_ ~ E ~E E E ~E cn E
~. Ct~ ~0C~ ~ 1~ 0t~O ~
U~
, _ ~ . ' ~.
._ u :~ E
~V ._ v cu E E a~o cn E tn ~ (~ N ~ ::r ~ O N u~
.. tD ~ _ ~It~ Ci~ ~ ` Ci~ O~
_ ~Cl _I " E ~ F E ~ E
zc~: ~ ~n E E E ~ ~ E ~ E E
Q~ ~ ~ ~ cr s ~ O O O
- .~nlz c~
,"~ . . ' .:
- v E
., . ., ,u) v) ,' . ~ L~ l:ln 1:~1 ~) ~ .. . .
. ~ Q~ ~ ~ ~ C~ C`l cn c~l , .
.1 ~ ~ ~ O ,I E C~ E cnc~
~. ~ ~ ~ oO ~ ~.~ o .; O ~ _ ~
~ V ~ ~Q ~J J N ~ O N ~ O o~
~-- :u~¦ ~ Z -i 00 ~ N ) ~ ~ a ) I ~ ~ a~ I C~ ~ ,, / .~
. , , O ~ E c ~
~ : . .
: ~: . ', ' .
~J
i o . I : , :
:~ w_ . ~,, ~, c S~ ~ v : :
0 ~ :~
v~ ~ v ~ . .
~ _ t~ X X--CU ~ E ~ y O
Q~ ~ ' . ~ v w Q
c n s~ V ~
n -~ ~ C. ~ u 11 n~ N U~ J E---I~ 'Clt a~
~' .
-19 ~
; .
/
~ ' , ' , ,; , , , ' ' :
., ~ , , 31 1~;~
:
v) .-c ~l ~ ~ ~ o _ ~ ` E ` EE E '1:1 ~ E ~ E ~7~ 1: C
~ O cr. ~ O C'J ~
nlz aoa~
~, ~ .
,u~ O
: * C I~ C
If~ ~ lS~ E E --~u ~ ~ cr~
U~ _ J ~` E E E E ` C
u~ E 1~7 ~) CJ)~Jl ~n q3 ~ ~: ~r, o ~ o c~
' ,~ tl~ ~ ' 3 . ,,_ .
~ ~ L. O
~ ~ 1~
.~ ~ U ~ ~ , ~0 ~ :
E ~ u~
. . a~ .. _ ~ ~ ~ cn ~o cn E Q~
C~.l Ln ~ ~ J E
1~ _ ~ t~
_ ~ ~I _l ~ ` E ~` ~ E v~ `
:, CL a~ t:~ E E E c~i E E ~n ~D.--Z . ~ . ~ O Crl,~D ~ 0 u~ ~12 O
O v E
~~ QJ .U~
~:: ~ L~ cn Q~ ~1 ~ N ~
v ~ O ~nCU ~ E ~D 1S~
11~ t~ 00 L~ ~ Ir~ a`~ cu u~ .
O _ _ c~
. ~ : Q ~ J q E ~ E E E .~
u~ ot: E ~I E cn ~)~ ~, V ~ ~J CU O t--O ~D O
._ V~ Z aD ~1 l CO oo ~ (u C5~ ~ ` ~ :
V) ._ . g a~ :
Q~ C -~ E ~ ~, I O C
r~o ~ E C J~ . ~ , ~ .
t:~ O
a ~3 0 U~
C U CL
~ o U ~:
c~ s ~ V
O ~ E 1~ c ~! ~ o Q~ c ~
_ ~ ._ ~ c ~ o CU ~) ~ _ ff~ C ~ O ~ - L > V~ V E
n) o v L
.a 4- ro v-- O n) ~ V ~1 11 11 ^^
~ C U)---- ~-- * ~) E
C~: ' "5 O ~
, , . . . .
..... .
_~
C ~_ >~
, a) u. v 3 >
q~ 3 -- ~n 3 a) ~ O
3 0 ~- O O ~ >-~ O ~ ~
~;n ~--r~ r~ ~ rn --~ 3 ~
3-- rv ~ 2 1:) r~-- ~ 3 o O
o a) ~ o l_ :~ o -- a~ ~ o .` * z 0--* ~ tv v~
Ir ~ C7) rv ~ ~ ~ C ~ ~ .~ r~ O O
Ir O ~ v~ ~ r~ ~n ro ru ~ ~ Ul ., O _ ~ ~ ~ _ ~ ~._ ~
O v v a~ ~ aJ QJ ~ rL~ r _ >~ ~ r -- -- ~n-- rD ~ ~ ~ ~ ~ ~
c r._ ~ ~ O O ~- O O a) -- Q~ O rD---~--Lt 3 3 J l:L rs ~ ~ ~ ~ S ~- ~ ~ ~ S ~L, J J
. ~ ~
1~
.. _ . '.
a ._ : - tU ~ r I 5.
-~ o I O tn F ~ E ~ n n r 7) ~ CJ~ E rn ~
3 _ ~ ~:;1 .rl N ~ CU ~ ~ ~ N (~1~ 0 ~ ~::~ N
I_ I O ~ D N NCO O Ot~D O tr~X) C~t~totx) t~ 5-l ~, Z ~ N N CU .1 ~1 .
11~ 0 o - "'.. "'-.~ '. '.' ~ . . -,.
~ . ~
O ' f~
. ~ . ~ ~ S
~ ~ --3 :; . a ~ ~ E 3 . . ., ~ ~ f~ .
V U7 fO ~ ' ' ' a~ . ~D OC tJ~ O
L cn ., a a~ v fV ~ `
' L d~ ~U ~ fV .~
E ~O -- ~ cr ~ ~ .- ~ ~ ~ .
_ f~ ~ J ~ tl~
~0 Z v tJJ _ v -- O - D
3 ~-- ~ ~ ~ O ~n >
~: ~
aE O fV a) ~J tV ~ ~J n) '`V fU-~ O a) ~, O V ~ .C ~ 1 U.0 > -,:
~ ~ ~ rJ~ ~ U) E -- ; . .
e ~ tO ~ O O tV~- to-- ta E~ . 3t0 ~ l J<~ p t~
' , ' ~ . ,:, : ~ .' I C
,_ .' ~V D ~ r~ rJ Cn C) 4 i ~ ~N,~ ,~ ,U
' ` ~
, _ U ~ ~1 ~1 ~t ,1 1~ tO r~ ~ ~ tV
t) tl~ D O ~ Il) 1- ~
t~ tD ~ ~ I ~I N N CU CU CU N
.
:.
~3~6~
C .~, .
~ ~C ~ "
C ~ C ~ L. C ~
. ~ i o_~ 3~ 3:
-- C ~ C ~ U~ r, ~ . .
_ 3 ~ ~ 3 ._ u~ ~ v o ~ o c 3~
~: 3 ~ ~ o 3 ~ 3 a~
E c~ o D ~ ~ c.a---O > O ,~ ~
10 __ (n o Q) a3 a) c o- (1~ 0 0-------- ~ V
z a ~ ~ > ~ cn 3_ >~ >-- ~ a) o Q~ o - --$ ~ 0 --lr o ~ o o o ~ ~ .o ~ cn o ~ ~ r v ~ V n~
Lr _ V~ V) ~ O 11 o .~ . ~ JJ ~ a) v v v JJ V ~ ~ ~ -- ~ C
~1 V ~ ,~, r r ~- ~ ~ S C r~ ,C 5~
~. . ~ -- o ~ o O t~ ~ ~
W ~ c:~ J J I O ~ I J ~ J ~ c E ~:
3 . ~ :
' ' L L~ o O
, . . _ ~ .
O E E E cJ~ F ~, E E ~:n E E E ~ n E
7 O ~ .J ~ ~ CU ~ ~ ~0 C~ Lr\ o ~ o ~ a~ o ~
Z ~) ~n F ~:
'~ . ' ' ' ~ ~ Z .' ~
.
. .
u~
~ O
LL t~
,, : ~ * , .~,V~.
r I ~ -~
V~ ~ ~ ~ ~
v~
._ t", L ~ Ul ~ a V
L E o ~ ~ u~ Ct g ~ ..
~Q n~ c > ~ ~
_ z -- c v ~ ~ 0 E Q 3 c O
~ u ~ c ~ c ~ ~ ~ ro - :
C~l C v 3 Q cn ~ o E ~ ~ O tn ~
a) ~ _ o ~ ~ o ~ : ~ :
-- :IE O O L V ~
.~3 ~ ~ al . Y ~cn ~ o >
--~ ~ E ~ ~ ~~ ~-- E ~ c . 1-- C ~ 3 O ~ v~
E a:~ v ~ c~ a J ~ o Q ~3 :
O L . O ~ E
O I LLI ~ J C
u~c~ L ~ L
~ ,. ._ ~ ~ O
" v ~, O c ~ ~ : -O,~ L V ~ . .. a 0-- V
~ o o ~ .S ~ U O ~ ~
I~ O O CJ~
,~-~ t ) ~ ~ * *
., ~22--~, ' ~' , , , ~ .
, . :
,, : ..
, , ~14~
,` '.
.
~ 3.
~ .~ ~ r ._ CL ~ 3 ~' V~ ~ Q) t - ~'J a) O ~J
. c ~ ~ c ~ --~--~ E ~--a) ~ , ~ u u~
~ ~ ~ ~ ?~ ~ 3 V~ ~ 3 J Q J o c ~ ~ h o v Ir ~ O ~ ~
, ~ ~Itr ~ QW S u~
. In . .~ v C
._ V~ ~ C~
. c ._ ~ Q 3 . ~ C: ~ ~` ~1) C :~ O ,~ C~ ni ~a) o~ ~ v I ~ C-- ~-- ~ ~ O
_. ~ Ln ~ I r~
:~ cr . ~ ~ J .
, ~ ~ u~
,, . ~lZ ~ Q ~ I ~ ~. CL I ~. Q.. ~
,., ' ,.,. . .
, ' ~ ,V~ ~ ~ O ~ ~ '"' -~' . , ~U 'c O_~C ~
~; - ~ ~!J ._ ~,J V ~ ~ Q ~ ~L O O
J ~ Vo ~ ~ V
._ C ~ C
~] ~ ~ , ~ U ~, 2 ~ ~2 : ~
~ ~ Q~ , .
,'~t V ~- '> IU C V .
Ul al ._ _ ~ ~ Q -:
.~ ~ U~ ~ N U~ C
.~ O ~ O -- c ~ cn o ~ q)~ o ~ cn Q~
:,1, V _ _ _ ~ C C ' E . ~ u ~ O ~
v t~ , z ~ n,, ~ , ~ 0 0 ~;, ,,. .1~,, ,:
. I cC~ ~ E c ~ ~7 ac a. o ~ c~: ~ o u~
~U a~ ' :1 .
O ~ 0 ¦~-- E 1~ .
~ . O ._ o E
-o ~ u E
., ~ Q~ Y
:E: IL ~ ~ In . .
-23~
, " , ,~, _,.. ... ,, .,~,._. --,, .,. " . , ~ ~ .. , ., . . , . , .. ,, .. . ,.. , .. , ,, . . . _ . _._ .. _ ._._ . . _._, _.. _ _._ , __._ _ .
.
,X
~ C ~ ~ C ~ ~
6r~ .~ ~ O
_ ~ L O ~ t~
a.~ c c Q) I U~ C
C ~O r~ ~ ~ ~ ~_ c~
t~_ L I IC-- 1-- C '~:J ' 5) 0 ~ C-~ ~ r CJ~
_ ~--D ~n rn ~ D i~ r N ~ U
. ~ o J ~ 1 0 C ~ O ~
,; tr ~ o ~ ~ a~ o ~ ~ o ~ a~ ~ V t~
enlz ~ ul Q o o l--~ CL ~ ~ X ~ ~ Q
u~ ~D V ~3 3 '~ O~ 'Q3 IL ~ ~ Q) O ~ C
~ O N ~ N ~ N
v ~ a~ c-~
3 ~ ~ C~ 3~
, _ _ ~ o O ~ D r~ U O
Q ~1 ~ ~ ~ ~ ~ ~ o o_ ~ c~ u~ ~ E~
_ ~ 1~ 0 1~ O t~ ~ ~ r3 ~ ~ ~ ~ v f "', -:
I~IZ r~ . X u~ 73 ~: ~ . :~ ~ ~' .:.
, ~
t~ v~
~ _ : . ~ 1~: ~ O N N -- ~ O ~ ~Y .
: . . ~J 0 ~D C~ ~ c r - i~ . ~ ~ . ~ .~
~ ~ t~ t~ ~ - 3 '3 ~ ~ ~ ~ CL 3 c Q
~ ~ --~ ~U o o v).~ ~ r n o : . ~ 11 J ~ w _ o ~
Q tn a: ~ O r 3_ ~ z~J ~3~ 3~ ~ x u Q . ~
_ . ~ ~ N
C 1~
~ ._ _ ~ ~ -:) Q
Oao ~1l ~n ~: U~S ~ V
V~ ~ QJ 1 ~, C -~ ~0 ~V ~ r ;~
C ~ ~ Q
~~ ~ ~ e ~ ~ 3 --~~' ~ r 3 V_ , w ~--D ~ 0 Q U _ e~liz ~ ~ ~ x ~ I
"
:' ' ' ~3 ': ' ~o ~ ~ O
VO V ~ o U) ~ CL e~ cL ~ ~ ~ ~ o ~n .
il~ ~
~J lg ..
9 0 .
e ~ 4 V C~ ~ ~ ~ :
~3~ ~ 5~ :
j ID ~ ~ 0 , C C C
S E
~ S ~-- w " ~ E S~
3J ~ D J ~ E :
1~ I i ,~
V C~J ' C L. ~~_ C
~ 0 ~ 1~
_ _ J ~ ~ ~ CL~:1 V
U~ Q tl~ C I C C I 0 ~5 -C ce: ~ ) v 0 . Z ~ ~ > ~
l J Q J CLJ t~
. ' ..
¦_ . . . ..U~ L~ ., r~ ' ' (~ ,. .
.) .. o r J- C
, CO .~ !~ .-- ~ V
E ~t~ ~- C C .-- c O > J 3 ~-- ~ 2 .--ul Q
r ._ ~ ~ 3 j~
U~ ~ V_ >,_ I ~ ._ ~ v) S~ Q O ~ ~J ~. . - .
1~ ITl C Q)>~
,, O ' q~ ~ v I ~ ~ ~ ~ I
E 1:: E C O E O E
î~ Q~ ~ ~D
_ ~ îT) D
lU I
I C
Q) ~ '-- 0 Cl E c~-~E
, ., ~ ~ -. '"
~ ~ o .~ . Ul ~_ ,_ U~
~I) v E u x ", c aJ Q~ O
c a. ~
C 9 ~ o, a~ ~ D aJ
~: ~ ~ S: ~
.. . .
D J
'1~5~
., ' ..
, V) >.
. ~ .
C ~ rD U~ ~ o .~ ~ ~ ~ U~ .. ._ v U~ ~ C~ a _ ~ ", , _ ~ . C
J ~ O C~ ~ _ t~ ~_ O ~D ~ V~ '' In , . ..
~ .
C~
>~
~ C~
~ , ~
* -- E : .
', Q~ .UI O ~ C~ C
C ~n- v C) I ~
~O ~ C L_ ro ~1 C\l ~ ~ ~ ~7 C
V =_ l C ._ .
J C~ Y O C) ~: ~
:~, c ~nlz ~ Q I n.~ ~
O D
Ul "~ . .
0 V\
, J ~ ~ Q) > J ~ U
x: Y ~ c ,, I . 1~ - c~
:'. l ~ ,~v ~ ~ E Q
I ~ V~ ~ ~ C, ;~
I ~ ~ C~ O ~ I'D Ln I _ C~ V ~
C ~ C) ~ C J.J I
~ u ~
_ r~---- >-- ~L~
,,:O ~1 o ~ o ~
5: ~O
~ a) c)~ o .-C:~ ~ E c:~- ~ ~ y ,~ :
~1_ .
,,: ~U~ ~
a~ u E ~ a,~
8 ~ ~ 1" ID v u~ . .
. O J~ rD ~ Ct t ~ , ,, ~ ~,. .
C~ ~ ~ ~ .. Z
~ C ~ ~ y _ ~, ~ ~ a) ~ c S~ Q 10 ,~ v~ 11 11 -- C .
ro Q Cl . t~J O .4.
~- ~s a o .
-~ 31 ~q~5~
C ~
V ~ _,_ ~ _.._~
O J F ~J) ~ E E E E E E
a~ ~ to u ~.) (l\ u tJ~¦Z N N 9 D N I N tU I ~N I ~ N I
V ~S ,: :
(~.VJ D
. ~n _~ ~ ~ ~` ~
~_ ~a~ cr~ O ~--~
' V~ ~V Lt'~
. ~ 11)~ ~ ~ 'J' ^-- ~ ~ --` cn ~_, ..
Q~ ~13_ J E~ E~ E~ E~ E~
~ .C~
r_ ~ C~
O. ~IZN ~1 I N -1 I N ~t I N ~1 I C\l ~1 . ~ _ ' : .
,u~ . E , . -v >~~n - . Q cc ~t O
, Q) O Q~ ~D
~, ~ Ev 1 CUv ~~11 N
~n ~_ J E ~:n E ~ E E E E E
~ c~: a~ u ~ a~ a) u ~ u a~ a - 1~ 0~lZ c~
1~ . .
~,~ . .u o ,_, ' ~ ~ ~ E cn ._ ~ ~ Q~ q ,, ~ u~ u) ~
al ._ ,_ ~) Il~ 1~ N
v c~ U~ O N ~ O O
Q~ <1~ 1~ .~ v ~ ' D '~a ~ :o _- v_~ ~ ~ ~"~
Q U J ~
n~ ~:n a~ n u O ~¦ > Z C~J N I tl~ "~
O ' . , .
U Q~ O
Q ~ E ~ ~ ~ n ~ ~ tL L~ L ~ ~ Q ~
Yj Q~ . ' ', .' .
~ v ,,:
E u u .-._ v~ a~ v ~-~ ~ c ~ )~ X--~
Q~ ~ _ ~ Q~ O I aJ a~
:~: vv o u~ ~ c Q) a~ ~- o Q q) V~ v v o,, ~ cQ U V ~ v ': 0 C~ ~ ~
~ n~ v -'~ a~ v -17- :
:, . . . .... . . ... . . ..
,u~ .' ~ ~o~ ~~
c _l ~ ~J) E Cl) E
~ u .' ~ o~ -- u ~ a) ~ u a ~Iz ., *
~, oo V\ .~ -: o~ ._ _ , ,1 ~ t~
O L~ C
ILI ~ ~ ~
.~ _ E ~ E ~ 1~ E
~ ~ J ~ ~ ~ ~
u~ ~: a~ u u u ~ ~ c 0 ~ ~ . ~ ~ -CJ O) D ~ O . :~
, ~ ~nlz ~C~I I C`J CU I CU C~l I E
_ ~ ,_ ~a ~ ~ .~ O
,u~ r c ~ 0 0 c c . O a) ~D J U
a~ E ~ 1~\ lu .
V ~ (~ C~l ~~
11~ X . _~ . E V) E E ~ ~ E ' C o a:: ~ c In O ~nlz ,_ ._ _ U -~
_ E ~ :
& U7 ,v) ~0 ~ ~ i~ V~
~' ~ ~ ~ ~0 ,~
- O _ _ E cn E ~ ~c~ c I:L ~J J ~
.' tn Ix: ~:n u-- ~ u ~ u ~--~ . ~ ~ ~ ~`~ ~ .. L
r~1 a z ~ w ,' ,,~ ~ :
,,, o C o ~Q ~ E C ~ ~,0 :~
~::
., ~ -:
a l_ Q : .
O .~ ~ , , V-- c ~ O
S: 3 8 " ~ a~ .
w Q ~ V ~~ ._ c ~, -- V~ O C
C Q1 ~ I ~ 0 0 U~
~) ~ ~ D tO
Q~ ~ ~ ~ ~ v Q ~ to ~:L O V
v-- o ~ ~ ~ O
:.. ,".o -- v -18 ~
. . ; .' . ' ': .
~ ~:
~, ~ E
a ~D c~l .
0~ E E
~ ~ .~ ;J
_ _l E~ E ~ E E E ` E ~ E
a~ ~ ~1~ co cl) coE ~ E
~ ~ ~ ~ C~ar,o ~o : ~ , ~IZ C~ I N 00 1 C1~ C~ I ~ C~ I ~ ~ I .
. . ....... ...
V~ .,.
.' * ~ E E cn E
*~ a~ 1~
:~ Ll~v L~ O O O N
n~ ~\ . ~
c~_ ~ E ~E E E ~E cn E
~. Ct~ ~0C~ ~ 1~ 0t~O ~
U~
, _ ~ . ' ~.
._ u :~ E
~V ._ v cu E E a~o cn E tn ~ (~ N ~ ::r ~ O N u~
.. tD ~ _ ~It~ Ci~ ~ ` Ci~ O~
_ ~Cl _I " E ~ F E ~ E
zc~: ~ ~n E E E ~ ~ E ~ E E
Q~ ~ ~ ~ cr s ~ O O O
- .~nlz c~
,"~ . . ' .:
- v E
., . ., ,u) v) ,' . ~ L~ l:ln 1:~1 ~) ~ .. . .
. ~ Q~ ~ ~ ~ C~ C`l cn c~l , .
.1 ~ ~ ~ O ,I E C~ E cnc~
~. ~ ~ ~ oO ~ ~.~ o .; O ~ _ ~
~ V ~ ~Q ~J J N ~ O N ~ O o~
~-- :u~¦ ~ Z -i 00 ~ N ) ~ ~ a ) I ~ ~ a~ I C~ ~ ,, / .~
. , , O ~ E c ~
~ : . .
: ~: . ', ' .
~J
i o . I : , :
:~ w_ . ~,, ~, c S~ ~ v : :
0 ~ :~
v~ ~ v ~ . .
~ _ t~ X X--CU ~ E ~ y O
Q~ ~ ' . ~ v w Q
c n s~ V ~
n -~ ~ C. ~ u 11 n~ N U~ J E---I~ 'Clt a~
~' .
-19 ~
; .
/
~ ' , ' , ,; , , , ' ' :
., ~ , , 31 1~;~
:
v) .-c ~l ~ ~ ~ o _ ~ ` E ` EE E '1:1 ~ E ~ E ~7~ 1: C
~ O cr. ~ O C'J ~
nlz aoa~
~, ~ .
,u~ O
: * C I~ C
If~ ~ lS~ E E --~u ~ ~ cr~
U~ _ J ~` E E E E ` C
u~ E 1~7 ~) CJ)~Jl ~n q3 ~ ~: ~r, o ~ o c~
' ,~ tl~ ~ ' 3 . ,,_ .
~ ~ L. O
~ ~ 1~
.~ ~ U ~ ~ , ~0 ~ :
E ~ u~
. . a~ .. _ ~ ~ ~ cn ~o cn E Q~
C~.l Ln ~ ~ J E
1~ _ ~ t~
_ ~ ~I _l ~ ` E ~` ~ E v~ `
:, CL a~ t:~ E E E c~i E E ~n ~D.--Z . ~ . ~ O Crl,~D ~ 0 u~ ~12 O
O v E
~~ QJ .U~
~:: ~ L~ cn Q~ ~1 ~ N ~
v ~ O ~nCU ~ E ~D 1S~
11~ t~ 00 L~ ~ Ir~ a`~ cu u~ .
O _ _ c~
. ~ : Q ~ J q E ~ E E E .~
u~ ot: E ~I E cn ~)~ ~, V ~ ~J CU O t--O ~D O
._ V~ Z aD ~1 l CO oo ~ (u C5~ ~ ` ~ :
V) ._ . g a~ :
Q~ C -~ E ~ ~, I O C
r~o ~ E C J~ . ~ , ~ .
t:~ O
a ~3 0 U~
C U CL
~ o U ~:
c~ s ~ V
O ~ E 1~ c ~! ~ o Q~ c ~
_ ~ ._ ~ c ~ o CU ~) ~ _ ff~ C ~ O ~ - L > V~ V E
n) o v L
.a 4- ro v-- O n) ~ V ~1 11 11 ^^
~ C U)---- ~-- * ~) E
C~: ' "5 O ~
, , . . . .
..... .
_~
C ~_ >~
, a) u. v 3 >
q~ 3 -- ~n 3 a) ~ O
3 0 ~- O O ~ >-~ O ~ ~
~;n ~--r~ r~ ~ rn --~ 3 ~
3-- rv ~ 2 1:) r~-- ~ 3 o O
o a) ~ o l_ :~ o -- a~ ~ o .` * z 0--* ~ tv v~
Ir ~ C7) rv ~ ~ ~ C ~ ~ .~ r~ O O
Ir O ~ v~ ~ r~ ~n ro ru ~ ~ Ul ., O _ ~ ~ ~ _ ~ ~._ ~
O v v a~ ~ aJ QJ ~ rL~ r _ >~ ~ r -- -- ~n-- rD ~ ~ ~ ~ ~ ~
c r._ ~ ~ O O ~- O O a) -- Q~ O rD---~--Lt 3 3 J l:L rs ~ ~ ~ ~ S ~- ~ ~ ~ S ~L, J J
. ~ ~
1~
.. _ . '.
a ._ : - tU ~ r I 5.
-~ o I O tn F ~ E ~ n n r 7) ~ CJ~ E rn ~
3 _ ~ ~:;1 .rl N ~ CU ~ ~ ~ N (~1~ 0 ~ ~::~ N
I_ I O ~ D N NCO O Ot~D O tr~X) C~t~totx) t~ 5-l ~, Z ~ N N CU .1 ~1 .
11~ 0 o - "'.. "'-.~ '. '.' ~ . . -,.
~ . ~
O ' f~
. ~ . ~ ~ S
~ ~ --3 :; . a ~ ~ E 3 . . ., ~ ~ f~ .
V U7 fO ~ ' ' ' a~ . ~D OC tJ~ O
L cn ., a a~ v fV ~ `
' L d~ ~U ~ fV .~
E ~O -- ~ cr ~ ~ .- ~ ~ ~ .
_ f~ ~ J ~ tl~
~0 Z v tJJ _ v -- O - D
3 ~-- ~ ~ ~ O ~n >
~: ~
aE O fV a) ~J tV ~ ~J n) '`V fU-~ O a) ~, O V ~ .C ~ 1 U.0 > -,:
~ ~ ~ rJ~ ~ U) E -- ; . .
e ~ tO ~ O O tV~- to-- ta E~ . 3t0 ~ l J<~ p t~
' , ' ~ . ,:, : ~ .' I C
,_ .' ~V D ~ r~ rJ Cn C) 4 i ~ ~N,~ ,~ ,U
' ` ~
, _ U ~ ~1 ~1 ~t ,1 1~ tO r~ ~ ~ tV
t) tl~ D O ~ Il) 1- ~
t~ tD ~ ~ I ~I N N CU CU CU N
.
:.
~3~6~
C .~, .
~ ~C ~ "
C ~ C ~ L. C ~
. ~ i o_~ 3~ 3:
-- C ~ C ~ U~ r, ~ . .
_ 3 ~ ~ 3 ._ u~ ~ v o ~ o c 3~
~: 3 ~ ~ o 3 ~ 3 a~
E c~ o D ~ ~ c.a---O > O ,~ ~
10 __ (n o Q) a3 a) c o- (1~ 0 0-------- ~ V
z a ~ ~ > ~ cn 3_ >~ >-- ~ a) o Q~ o - --$ ~ 0 --lr o ~ o o o ~ ~ .o ~ cn o ~ ~ r v ~ V n~
Lr _ V~ V) ~ O 11 o .~ . ~ JJ ~ a) v v v JJ V ~ ~ ~ -- ~ C
~1 V ~ ,~, r r ~- ~ ~ S C r~ ,C 5~
~. . ~ -- o ~ o O t~ ~ ~
W ~ c:~ J J I O ~ I J ~ J ~ c E ~:
3 . ~ :
' ' L L~ o O
, . . _ ~ .
O E E E cJ~ F ~, E E ~:n E E E ~ n E
7 O ~ .J ~ ~ CU ~ ~ ~0 C~ Lr\ o ~ o ~ a~ o ~
Z ~) ~n F ~:
'~ . ' ' ' ~ ~ Z .' ~
.
. .
u~
~ O
LL t~
,, : ~ * , .~,V~.
r I ~ -~
V~ ~ ~ ~ ~
v~
._ t", L ~ Ul ~ a V
L E o ~ ~ u~ Ct g ~ ..
~Q n~ c > ~ ~
_ z -- c v ~ ~ 0 E Q 3 c O
~ u ~ c ~ c ~ ~ ~ ro - :
C~l C v 3 Q cn ~ o E ~ ~ O tn ~
a) ~ _ o ~ ~ o ~ : ~ :
-- :IE O O L V ~
.~3 ~ ~ al . Y ~cn ~ o >
--~ ~ E ~ ~ ~~ ~-- E ~ c . 1-- C ~ 3 O ~ v~
E a:~ v ~ c~ a J ~ o Q ~3 :
O L . O ~ E
O I LLI ~ J C
u~c~ L ~ L
~ ,. ._ ~ ~ O
" v ~, O c ~ ~ : -O,~ L V ~ . .. a 0-- V
~ o o ~ .S ~ U O ~ ~
I~ O O CJ~
,~-~ t ) ~ ~ * *
., ~22--~, ' ~' , , , ~ .
, . :
,, : ..
, , ~14~
,` '.
.
~ 3.
~ .~ ~ r ._ CL ~ 3 ~' V~ ~ Q) t - ~'J a) O ~J
. c ~ ~ c ~ --~--~ E ~--a) ~ , ~ u u~
~ ~ ~ ~ ?~ ~ 3 V~ ~ 3 J Q J o c ~ ~ h o v Ir ~ O ~ ~
, ~ ~Itr ~ QW S u~
. In . .~ v C
._ V~ ~ C~
. c ._ ~ Q 3 . ~ C: ~ ~` ~1) C :~ O ,~ C~ ni ~a) o~ ~ v I ~ C-- ~-- ~ ~ O
_. ~ Ln ~ I r~
:~ cr . ~ ~ J .
, ~ ~ u~
,, . ~lZ ~ Q ~ I ~ ~. CL I ~. Q.. ~
,., ' ,.,. . .
, ' ~ ,V~ ~ ~ O ~ ~ '"' -~' . , ~U 'c O_~C ~
~; - ~ ~!J ._ ~,J V ~ ~ Q ~ ~L O O
J ~ Vo ~ ~ V
._ C ~ C
~] ~ ~ , ~ U ~, 2 ~ ~2 : ~
~ ~ Q~ , .
,'~t V ~- '> IU C V .
Ul al ._ _ ~ ~ Q -:
.~ ~ U~ ~ N U~ C
.~ O ~ O -- c ~ cn o ~ q)~ o ~ cn Q~
:,1, V _ _ _ ~ C C ' E . ~ u ~ O ~
v t~ , z ~ n,, ~ , ~ 0 0 ~;, ,,. .1~,, ,:
. I cC~ ~ E c ~ ~7 ac a. o ~ c~: ~ o u~
~U a~ ' :1 .
O ~ 0 ¦~-- E 1~ .
~ . O ._ o E
-o ~ u E
., ~ Q~ Y
:E: IL ~ ~ In . .
-23~
, " , ,~, _,.. ... ,, .,~,._. --,, .,. " . , ~ ~ .. , ., . . , . , .. ,, .. . ,.. , .. , ,, . . . _ . _._ .. _ ._._ . . _._, _.. _ _._ , __._ _ .
.
,X
~ C ~ ~ C ~ ~
6r~ .~ ~ O
_ ~ L O ~ t~
a.~ c c Q) I U~ C
C ~O r~ ~ ~ ~ ~_ c~
t~_ L I IC-- 1-- C '~:J ' 5) 0 ~ C-~ ~ r CJ~
_ ~--D ~n rn ~ D i~ r N ~ U
. ~ o J ~ 1 0 C ~ O ~
,; tr ~ o ~ ~ a~ o ~ ~ o ~ a~ ~ V t~
enlz ~ ul Q o o l--~ CL ~ ~ X ~ ~ Q
u~ ~D V ~3 3 '~ O~ 'Q3 IL ~ ~ Q) O ~ C
~ O N ~ N ~ N
v ~ a~ c-~
3 ~ ~ C~ 3~
, _ _ ~ o O ~ D r~ U O
Q ~1 ~ ~ ~ ~ ~ ~ o o_ ~ c~ u~ ~ E~
_ ~ 1~ 0 1~ O t~ ~ ~ r3 ~ ~ ~ ~ v f "', -:
I~IZ r~ . X u~ 73 ~: ~ . :~ ~ ~' .:.
, ~
t~ v~
~ _ : . ~ 1~: ~ O N N -- ~ O ~ ~Y .
: . . ~J 0 ~D C~ ~ c r - i~ . ~ ~ . ~ .~
~ ~ t~ t~ ~ - 3 '3 ~ ~ ~ ~ CL 3 c Q
~ ~ --~ ~U o o v).~ ~ r n o : . ~ 11 J ~ w _ o ~
Q tn a: ~ O r 3_ ~ z~J ~3~ 3~ ~ x u Q . ~
_ . ~ ~ N
C 1~
~ ._ _ ~ ~ -:) Q
Oao ~1l ~n ~: U~S ~ V
V~ ~ QJ 1 ~, C -~ ~0 ~V ~ r ;~
C ~ ~ Q
~~ ~ ~ e ~ ~ 3 --~~' ~ r 3 V_ , w ~--D ~ 0 Q U _ e~liz ~ ~ ~ x ~ I
"
:' ' ' ~3 ': ' ~o ~ ~ O
VO V ~ o U) ~ CL e~ cL ~ ~ ~ ~ o ~n .
il~ ~
~J lg ..
9 0 .
e ~ 4 V C~ ~ ~ ~ :
~3~ ~ 5~ :
j ID ~ ~ 0 , C C C
S E
~ S ~-- w " ~ E S~
3J ~ D J ~ E :
4'~ ~ K 2 ~_ :
,~.: :
, : . . . .
,r~ t~
E
V~ g ~ .
._ :~ a In ~ ~ a~ ~
C ~ V >
~ ~O ~ C
V C-- ~7 a~ 1~ a~ 3 0 v _ O ~ ~ E
J ~ w ~ ~ ~ IV
_ QJ L
~31Z ~ ~ U
~ .
~n ~n '.:
3 ~
g 1~ o ~ ,>~ ~ s~) ..
~ a~ o~ ~
1~ Q) ~ O O 1- --:~ 1~ ~ 3 3 3 0 ~9 _ ~ v v ~ o ~ a . J o ~
~ ~ _ ~
~n t~l~2 C- ~-Q _~J 3 ~ :~-~0 ~ : ~
O . ' , ' ' , ' .
. . . .
,_ a~
Ul ~ ~ ~: 0 _ ~ 3 ., o~ U~ V) U `~--~ C -- O . .:
O C :~
-- v C n~
. . ~ . v ~ C ~ C
E 1~ CJ ~o ~ ~ r ~ ~ ,o O ~J
.: ~1 ~ oc U ~ E
. ;. ,~ j ., ., ,., . ~ Q~ ~ ~ v cs: ~ ~ ~ ~ ~ ~ C ~ c ~ ~ -- a) c--0 ~ Q)~ 3 a. o ~
Z R~ Q ~ ~
. '` ` ~3 . `:
.
. . ~So .
' . 'O . . ...
113~ y ' 7 1~ c u E ~ t~
O ~ ~ W
0 a~ 3~ 3 3 6~' ' ~ 1 ~ ~ J . ~ >
-- >
o CS: ~ O n~ 3 ~~ 19 0 L
J~ Z ~ ~_ ~ ~ n ~ ~ (:1. J ~ O
1~
~; . .-.
` ' ~ 0~
, . ~ . ; .
~:
V
0 ~
, ~ . L .U ~ o U~ ~ -"~ 113 Il~ ~ v ~ ~ :
~ E~ ~T
~ ~ a ~ w v c ~
V~ ~ r O ~
~8~ ~ ID ~0 ~) C 1~ ~ " ~
~ a ~ a~
9~V :E ala ~ ~ æ : ~;
.~ . .
~. . .
~25~ :-'':.:~
- ~ .
' ' 311~
E
n~ >
U QJ ~ ~ O
", ~ V V ~
u~ -- v ~ C ~D ~
._ ~ c1~ ~~ 3 u In Q~ V ~ O 1 ~: ,~ > C E~ C
G) ~D
u ~ C
0 1~ 3 ~ 0 3 -- u)` ~
_ ~ C C ` >
C~ J u ~ iD OQ~ ~o o~ V
~: to ~ _ _ V .--~ ~~ ~ ~ 0 Q~ O ~ O ~
U~l~ ~ Qt~S CL Q ~ ~ ~ Z 3 O ~ ~ >
V ".~ ~ ~s ~--In U~ ~ c ~ a) ~ C V
u . 3 ~ a~n 3 ~ 3 v~ I > ~ v Q~ I a) a.l o ~ >. ~C >.
~- ~ a~ ~ 0-- I u ~D
` O 0 3 1~ ~ 3--- Cl) O~-- -- aJ
~ _ V ~ Q) o ~ C ` ~ ~ ~
I:L J Q ~ U-- ~ a~ a~ ~ '-- ~ V
u a~ Q~ _ _ v, --._ ~y ~1~---- ~ ~ ~ n~ r~ a) o ~ o ~: ~
~lz ~ ~J~_~SC~D,s ~ ~z3 a~ a~a) ., > v~ >
~ ~ -- ~ c -- a) o ~ .
_. ~ J ,~ ._ v c~ r c v) ~ Q ~ ~ v v ~ ~
._ 2 co~ I cr~ ~ 3 o > 3 u~ u~ C a~ O a~E O~0 Q) ~ ~ v C ;~ r~ C _C n~ C V~ ~ ~ V) I_ ~ . JJ 1~ 3 J'-- o ~--- u~ c ~ v ~ C.
E ~ N o>~--- 3 o ~ V ~ -~ __ ~ ~ c c c > a~ >
~ a ~ v ~ > Q) a) ~ o - v--o -~ ~ ~---- _ V ~
~ ~ ~ a) o ~ ~ o o o ' U~l Z C~ ~ O ~ I ~ S ~ Q Q~ S ~J V ,~--Z t~ V
O
V ~ V >
O ~ -- V -- t,)-_ . ~ ~ ~ V
_ D ~D ~s) tl~
-- U ~ 1 C ~ t~ r ~c_C
O O ~ v .--._ v CC) 3 ~ ~n 3 - 3 a) O - 3 ~
~ _ . a) ~1 ~ c ~ c . aJ ~ ~ ~ V ~ -v~:L o l ~v > ~_ ~ ~ U Q~ U U 3 ~3 ~: ~ V _ _ -- -- ~ ~ O '~
::. L ~1~ ~ ~0 I C~ ~ Qa-~ 3 ~1 ., .
C . .
O
v O ~ E c ~ ~ o tn ~ o u~ t~ o ~o E
,_ :
_ ~ :': : -' ' ,,. _ : ~ ,,:
I ~,~ V C ~
~ O U~ C
U t~ Q~ ~ . .
I ~_ ~ -. ..
nj ~ ~
, 1 ~o ` ~ ~ ~ ~ .`. .
E '5 C ~ W
C._ V _ ~ O ~ ~ ~3 0 ~
E
9 ~ rL Z , .
.:
, ~26 .
.
' 314~
. ~ '.
o ~ a~
.Ul .~
~n ~ v Q) ~ LJ a~ ~3 C G) O
v t-- ~ ~ ~ ~ a> -~ ~ cr c ~ v r v v _ a~ v ~
Q J --o 3 c 1 2 C~ ~ O ~ ~ O ~ ~' ~ O' ' ~-- O ~_ -- O : .
~lZ ~ z~_ ~z v ~ z J
CI a~
oV
3 ~ EV E
-- ~ ~_1 o ~- o JJ
V) ~ JO C V o ~
c ~ --~ v v ~ v : :, Q~ C~ ~) O OO O :, :
v 15~ ~ ~ v D OJ ~ O
r v r ~J ~ IJ '' _ O --O V ~ ~ ~ ~ ~
Cl J U-- ~ 3 --~- U 3 1 ~ `
v l t~ ~al o V ~ ~ O v ~ v ~ O' -- ~
2 1_ z~ Z~ Z `
Q11 Q~
, ,~
. L. ~U ~ L- ~ ~
U'~ ~ :J-- W ~ .LI V
._ o o ul ~ v -- o c 3 ~---n C -- -- E O C ~ --V o o C
~ C ~t ~U--- 0 1~ C L C
~ a ~0 o ~ ul_ v o ~ ~ ~ o :~ v ~ ~ V ~ ~ 3 a~
F 1~1 C~J Q O c v u ~ E "
~5 ~ Q) a~ 1- D v rD o O ~ U O
C J t) ~1 C) a) 3 a~ V~ ~
n: ~ ~ -j v ~ c~ v U o v v u~ ~J71Z I ~ 7z~ 1- u~ O 2 G~ a U Q,)-_ v~l ~ ~: v ~ ._ _ ~ ~S V -- a~ c 3 ~ ~
~- U U Ir~ ~ ~ O ~ ~ V O O C
C C ~ ~ ~ Lr C L e ~n ~ a~ o _ o O ~ ~ ~
v ~ m ~ .~ ~ v3 a)~ a~ J
~ 1~ ~ ,~ v ~ 3 _ _ . a~
J~ CL IJ v c ~ 3 L. a~ V ~ ~ v ~ ~
~ ~ o ~ o v U~ ~ O ~ J~ V
~ ._ ~ ~ ~O -- ~
z ~ 2 ~~--u l.~ l~ Z~
I~
~C
e o .
m ~ -D v ~ c JJ ~ O
._ Q~ .
r ~ . :: .
_u . ~ .:
. ~ . ~ v ~ .~
v : :,. -~" . r ` - O ~: ~: O ~
r , C ..
.. _ ~ ~J ~ ~ ~J ', ' ~' ::
~ 11J ~ C O ~ ~ .-L~
_ a~ r .. ...
~_ L.
.. 5:1-- . 'Cl ~ C V
~ aE Z tf~ Z : ., ,, ..
. .
' .
. .
314~
.
':
a~.- ' ~ o In _ v ._ V~ ~ ~
C ~ Q QI
v ~D N--~ ~ ~ ~ C
_ ~-- O
~ ~_ V---C~ ` ~ V
t~7 aL N
O--. ,- C
U V
a) ._ -- cL e--~n Q~ a~
C ~ QQI
Lt~ CL
U rD C
_ .. ~ ~ v.
u~
,_ ~ ~ ~ ~
c ~nlz ~) t~ N
Q~
a IU _ C .V~ U , ...
u~ ul a~
O C ~ CL~
I ~ ~O Cl~
C
C N ~, cr~
~: ::1 0 I
V ~Iz U) Z Q
~ _ ._ I
S) 0~ C 0 , U) 'Ul ~ --U ~
U ~--_ 3 CL s~O
--U~ V~ Il~ Q~
V C C i~ CL Q) Q O t-- . :
U~ O ~ O ' U
._ V . ~ IU -- T
~ (U n~ ~ ~ C CL
Q~ _ _ ~ o Q U ~ ~ ~ ~ ~ O
Z ~ J 0- N
' ' C'~ ~ C U) ' . '"'~':
I O 1.) ' '.. :
u-- ._ ~o w E /~ ~ O ~
E ._ -0 , .::
_.c o , ,,:
.~ ~ O
.
~ . . ': - :- .
v : ~ `~
c ~J r J, 0 ~ . ~ C~
U ~ r ~: E
V
. . ~ E O v~ ~ . c:
,~ a: E ~:
. ::
._ .:-: .
': '~'. '' . ~
o28- ' ~' : . .' :' :' . ., ' ',' '.
c: ~l ~
o~ ~ . ~ I I + ~ ~ + ~ f ~ 1 + ~ ~ ~ .
t--, . ," ,11) t<~
Q~ ' ' a J
:~
o~
VIIZ
iO
:1 ~
~n a~ ._ ,..... 1~ ~
V ~ O~ . ~ ~ ~ ~ ~ ~ + ~ ~ ~ +
~ ~ ~J L~
_ O
,~ ' . W _ _l ~!
i ~ z , .
E~
~' ' .' ' , ~ . , .
` ' ' U~ V CL Ul 3n c ~ ,~
0 4- a~ ~ + + ~ ~ + ~ + ~ + ~ ~ t ~ ~ ~ ~ , :~ O " 1~ .
.19 ~ 1~ ~J
. ' .: ~ ~ 3 a .
.~ lz , ~ ~ , , j ~
- ~ v ~ .~ ~::
~ ul ~n U~
"~ ~ C
+ ~ ~ ~ + ~ +
r lo `
~,~ ~ J . - `
- ' . . ~ i:l: . ' . , ::
~ . , . ~ .
~ 1:~ `
- N . 59~ - ;-~
' ' 9,9 a 0 5~ ~ ~ O ~ ~ Ul ~9 ~
u~ o o ~ a~ ~ ~ ~ Q~ . :
~3 F o~a O ~ f~ ~ C ~
- _ Ç3 ~ C ~--~ C O O O O ~ n ~ ~ ~ 3~ ~ x ~
~ . - . - ~ ~ ~ ~ ~ a~ ~: o ~ ~:3lJI~ 01~1~1~
~ O ~ Q O ~
~, ' ' ' `
'' ' ', .. '' ' ~' :' ' , ,, .. , . . , , . ' ',' ,;: ", ' .~.' ' ' ~ . , ~ . . :
" . . ' : , : . ', . '. ' ~', . , . ' . . , . ' ' , ~-. ~ O
Q) ~.--~ Ull ~ V
S Cl~
~D ~ ~ ^ ~ C
UV ~ + I + + + I I I I 1, 1, + + ~._ ._~ r~ ~ ~ E
V _ 0~ 0 '-J ~ v, a c~ a~
c ~l U~ V~ U~
.D ~ Q~ -U~l .~
V ._ V ~ , . .
cc ~ ~ E v~
._~ ~ ~ ' O O
~ ~ ~ + + + - - - - I I + + ~ ~ ~
u~~ ~ ~ O ~J
Q)~ J C:l t- , ~ Q~ O
! ~ ~ --~1 u~lz c ~
, ~ V
~n O ~
._ V~ ~ C ~ ,.
C ._ ~ O
a v, ._ "~ . ~ ..
c ~
~~ `D ----~ E ~--v _V ~ + +~t + ~ + + + ~ ~ ~ ~
aro N
U)CL ~ ._._ ~)C~
.. u~lz n. ~ E--~ ; :
... , ~ . . , .
. . ~ '.
W.U! ~ -,.:~
u~ ~n ~ ..
C C r~ ., ~ a~ o + I + + + ~ i + + +
_ _ O
C U J v i Z O O N .
~* ~--O
1~ 8 Il~ N N V v O ~
V
O V ~ ~ ~ ~, ._ .
~ V
: ~ O O ~ ~ ~ ~V ~ C o ~
~ O ~ ~ O O O O ' ' r ~ ~~- O c E~ v ~ z - ~ O ~: ~-- v c ~ ~ ~
~O~ ~--c~--~OOOX~0~~~ '"''''' _._ ~ ~ u ~ o u~ ~-- c ul ~ O ~ ~7 ~ ~ ~ .
~ ~C ~ 7 0 ~
- N '-- ~ llDI--~ ~ ~ 2: Z Z 2 z z z ~
~--- ~ . ' .. -.
_ _ t~
t~ ~ ~ ~ID 1~00 t:~O .I N ~ L~ O . . -.
. . , :
., -3a- . :'-~.',.: .
:'. '', .
.. ,' .. .
. , -, ,. . - - , ~ . :
~1 43 35~ dP
~::
._ 3 c V) O V
C ~i ~ 3 ~ ~ cn O ~1 + + + +
n~ ~ cn + +~1~ + ++1+
U~ _ ~_ Q J .~J) o -- ,.
* r . .
V~ ~ ~ ~
._ o._ 3 Ul _U) O J
C __ 1~ 3 ~.:
V ~-V In ~ + + + ~ + +
~D O~ ~ ~ ~ + +~1+ + +~1+ , '.
Q ~o CL J .~n o .:
U~ C ~ IY -- O
~ (~ ~ lZ 0 ~ , ~ C~ . '~
.~ ~ .C . ,.
a) ,_ _~. v ':
~n~ ~ :
~11 1_ v ~ ~ + + ~ ,~ + +
~ n ~ C~ ~ ~ + ~ + + + + +I +
Q J ~O ~ :
-- O :' olz -- O
O ~ ' . ~.
. - ' ' ' .
os --1-1 v ~ U~v ~. O ~_ + + + ~
(~n~ 1~ w VCJI ' + ~ + + + ++I+
~ _ ~ -C -a ~ > ~: _ . ,~ ~.
._ .-,,: .
N :
' _ . : ' ._ `~ 3 V'1 _ _ ', `: : ~
Q~
~: . ' l O o o o ~ a) Q~ F a~ E IJ ~ C O--V O (L~ u) -~ ~ ~ ~ ::1 ~ -- ~ Vl ~?---~ U~ O
'i: , O ,_ .. ,~ Z .U Ul O ~ t: V O C
a:: .Id ~ V -O ~ n~ o~ c ~ ' ' ' ---- ~ ~ ~ ~ ~ E
z l~n E ul E C~ C! cr O X O ~ L~ IU
o Q~ O a ~ c ~
I_~ ~ L~ J~ Qlo ~ l .
,. . ~
3~3~ ~
~o o , ~:~ ~
.,, ,a ~ o ~ ,, ~
. .
. ~ a~
g,, U~ Z
U~
~ , .~
a~ o -:~ o :.
.,.
.,, .,, U~
N
,i , .
::J rd ~ h , ~ U
:I K . ,~
C~ Z .~ .
U~ . ,~
~1 U
~ . O ;,~.,;"""
~1 U~
P m .
~ . I ,C ~
Q~ ~ J~ tr7 ~, E3 ~ ~ _1 .
~3 ~
~ ~; U~
~ cn æ ,~
U~ ..
~, ~o~
~1 tn u~
O ~ . ,....... ~,.
~a ~ ~ ~ cO ~
O
$ rl IJ N ~
I;~rl O
N ~
~ O
~ O ~ ' .~ 3 H
8 ~ ~ - ~.~ N N t~ ~
O
N-,l ~1 ~\ O ~ ~h O - : .-1 ~1 ~ ~
g p ~: lC .~:, ~.. .....
.: .
: ' -- , .
.:' _ 3~
~, .
~ . ~
` ' 314~
~ ~ ' ~1 + ~`
J .
~ :
~nlz U~
a~
V .U~ .'~
~ Ul : :
C~ C ~ ~ ~
' ' Q) O~ . .
U~ ~ Il~ . . .
._ t~ ~ ~ + ' :
a~ ~:1 J ' "
I~ ~
~ . ' .' .
U~
a) ~,3 .U~
.
. ... :
+
-o ~ --~ ~:
. . ...
. .Q , . , .
; ~o ~7 u~ u7 c~ r ~ Q~ O
- Q ~ 0~:) '` '. O ~ . ~ .
~ 2 ; . ~. .
C . ~. .
`~¢ . '' '~
. . : '',.-. .
. .
ql ....
. , ~ ,.1~ U
'~ : - : ~ ~ ~ . . .
~ .0 ~
~ a) ~I N
X X ~ J
` ~tc ~ O ~: . ec ~
.
.', --3~- ~
~. .
-, ... ~ - , - ,. :
' ' 3143 ,~ 3Z~~
The new compound of the in~ention ;s produced when the elab~rating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of 7imited amounts surface cultures and bottles can be employed.
The organism is grown in a nutrient medium containing a carbon source, for example~ an assimilable carbohydrate, and a nitrogen source~ for example, an assimilable nitro~
gen compound or proteinaceous material. Preferred carbon : `
sources inclùde glucose, brown sugar~ sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the ~;
like. Preferred nitrogen sources include cornsteep liquvr, yeast, autolyzed brewerls yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solidsJ pancreatic digest of casein, fish meal, dist:illersl solids, animal peptone liquors) meat and bone sc:raps, and the like. Com-binations of these carbon and nit:rogen sources can be used ad~antageously. Trace metals, for example, zino, magnesium, manganese~ cobalt, iron~ and the like, need not be added to the fermentation media since tap water and unpurified `~ ingreJients are used as components of the medium prior to sterilization of the medium.
Production of the compound o~ the in~ention can be effected at any temperature conduci~e to satisfactory --growth of the microorganism, for example, be~ween about 18 and 40 C~, and preferably between about 20 and ~2 C.
Ordinarily, optimum production of the compound is obtained In about S to 15 days. The medium nonmally remains neutral during the fermentation. The final pH is dependentJ in 30 part~ on the buf~ers present, if any and in part on the ,~
-~ 3143 initial pH of the culture medium.
When growth is carried out in large ~essels and tanks, it is preferable to use the ~egetati~e form, rather than the spore Form, of the microorganism for inoculation to a~oid a pronounced lag in the production of the new com-pound and the attendant inefficient utilization ~f the equipment. Accordingly, it is desirable to produce a ~ege-tati~e inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N2 agar plug, or a slant culture. When a young, acti~e ~egetative inoculum has thus been secured, it is transferred asepti-cally to large ~essels or tanks. The medium in which the ~- -~egetati~e 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 ~ariety of procedures can be employed in the isola~-tion and purification oF the compound of the subject in~en-tion, for example, sol~ent extraction~ partition chromato~
graphy, silica gel chromatography, liquid-liquid distribu-t;on in a Craig apparatus, absorption on resins, and crystallization from sol~ents.
In a preferred reco~ery process the compound of the subject in~ention is reco~ered from the culture medium by separation of the mycelia and undissol~ed solids by con-~entional means, such as by filtration or zentrifugatTon.
The antibiotic is reco~ered from the filtered or centri-~uged broth by adsorption on acti~a~ed carbon. The acti ~ted carbon is then washed with water to remo~e some ~0 impurities. This is followed by elu~ions with ace~one:
-35~
: :' water solutions which remo~e the antibiotic from the 3ct~ated carbon. The acetone in the eluates is remo~ed, ad~antageously by evaporatiOn, and the remalnlng aqu~ou~
residue is lyophilized to afford a crud~ preparation of antibiotic U-44,590.
A preferred pur i f i cation procedure is to subject a crude preparation of U 44,~90, as described abo~e, to chromatography on siliea gel from which U 44,590 i5 eluted. Fract;ons which show acti~ity against the bacterium Klebsiella Pneumoniae on a standard agar plate test, are pooled and taken to dryness to yield a relatî~ely pure preparation of U-44,590. Further purifi- :
eatlon is achie~ed by acetylation to a crystalline diacetate ,. . derl~ative of U-44,590. Zemplen EG. Zemplen and E. Pacsu, BerO~ 62 1613 (1929)] de-esterification (trans-esterifi~
. cation) with sodium methoxide in methanol~ and n~utraliza- ` `
- . tlon of the catalytic amount o~ base with carbon dioxide - ``
: gt~es the free antibiotic U-44,590 which crystallizes . - r@ad~ly fram methano1-ethy~ acetate to give ~ pure prep~ra-; . 20 ~lon of U-~4,590 , AntlbTotk U~44,590 ls acti~e against ~e~Q~ ~
and, thus, can be used to dTsinfeet instrumentS~
utensils or surfaces when contaminated with ehis microorgan- . ; -llsm9 where the înacti~ation of this microorganism is desir . . 25~ abte~ Also~ u~44.sgo is àcti~e against Escherichia coli ~fld e~n be used to r~duee~ ar~5t, ~nd~oi er~dlc~te ~1 Ime ~- . product;oR in p~permill systems because ~sf its antibacterial ~et~orl agatnst th~s bacter;um. Antlbiotic ll~44v590 can also .
~ use~ ~o prolong ehc llfe of culture~ ~f Trlchomon~s 3a ~, ~ hominis. a~d ~ 5~
' ~ . -, ' .
::, :
.. . .: . .. :
:~3~
by freeing them vf Esch~-ri~hia coli contaminaLion. Further.
U-44l590 can be used to inhibit the growth of E. coli in hospital flower ~ases where it has been reported to exis-t and present a hazard to hospital patients. See Clinical Medicine, February, 1974, Page 9.
No~el acyl deri~ati~es of U-44,590, as disclosed herein, can be used for the same antibiotic purposes as u_l~4,5go in en~ironments possessing means to deacyla~e the compound to U-44~590. Thus, the acyl deri~ati~es of U-44~590 can be used to treat laboratory mice in~ected with a Gram-negati~e bacteria, for example E. coli, as disclosed herein. Further, acyl deri~ati~es of U-44~590 can be used, ad~antageously, to upgrade U-44,590. This ` i5 accomplished by acylating U-44,590, reco~ering the acylated compound relati~ely free of impurities then de-acylating the acylated U-44,590 to gi~e U-44,590 in a more purified form.
The following examples are illustrati~e of the proc~sS
and products of the present in~ention but are not to be construed as limiting. All percentages are by weight and solYent mixture proportions are by ~olume unless other-wise no~ed.
Exarnple 1 Part A. Fermentation A soil stock of StrePt~myces platensis ~ar. clarens~s.
NRRL 8035 is used to inoculate a series of 500-ml. Erlen-meyer flasks, each containing 100 ml. of sterile seed medium consisting of the following ingredients:
Glucose monohydrate~ 10 Gm/l Bacto Peptone ~Difco) 10 Gm/l , ~.
~37~
. `' `.` ' :, ., . . , .. ~,, .. , ~: ,. .
.~:, . . , , ., . - . .. -.:
B~cto Ye3st Extract (DiFc~ 2.5 rim/l Deionized water ~aiance The flasks are grown for 2 days at 28 C. on a Gump rotary shaker operating at 250 r.p.m.
Seed inoculum, described abo~e, is used to inocula~e a series of 500 ml. Erlenmeyer flasks each con-taining 100 ml. of sterile fermentation medium. The inoculation rate is 5 ml. of seed inoculum per 100 ml.
of fermentation medium. The fermentation medium consistS - -1~ of the follo~ing ingredients: ~ -Brer Rabbit ~ olasses (RJR Foods Inc., N.Y., N.Y. 10017) 20 ml Yeast Extract (Difc~ Detroit, Michigan 1 Gm/l Gluco~e monohydrate 10 Gm/l 15 Dextrin (Corn Products Co. Inter-national Inc., International Plaza, Englewood Cli~Fs, New Jersey 07632)10 Gm/l Proteose Peptone #3 (Difco) 10 Gm/l Tap water q.s. Balance The presterilization pH is 7Ø The inoculated fermentation flasks are incubated at a temperature of o 28 C. on a Gump rotary shaker operating at 250 r.p.m.
~with a 2 1/2 inch stroke. Ucon antifoam (a synthetic de~oamer supplied by Union Carbide, N.Y., N.Y.) is used if needed.- Har~est 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 bacter-ium Klebsiella pneum~niae. This bacterium is inoculated -in~o the assay agar (Streptomycin Assay Agar, BBL, Cockeys- -~ , Maryland, 21030) of the following composition: ~ ~
. ` ' ~.-38- ;
Beef ~xtract 1.5 Gm/l Yeast Extract 3.0 G,m/l Gelysat~ Peptone, supplied by Baltimore 8iological Laboratories 6.o Gm/l Agar 15.0 Gm/l Deionized water Balance adjust pH to 7 9 Sterilize at 121 C. (15# steam pressure) for 15 minutes.
Phosphate buffer (O.lN pH 6.o) is used as the diluent. The agar plates are incubated at 37 C. for 16-18 hours~ Presence of antibiotic U-44,590 is e~i-denced 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 antibiotic sample. Thus, a 20 mm. zone of inhibition using a 12.7 mm. paper disc to which 0.08 ml.
o~ antibiotic sample has been applied is expressed as one bio unit per ml. (1 BU/ml.).
Part B. ~eco~ery Whole fermentation beer (ca 1600 ml. assaying 5 BU/ml. against K. pneumoniae), obtained as described abo~e, is filtered using diatomaceous earth as a filter ;~
aid. The filter cake is washed with water. The clear beer and wash (1800 ml.) is then passed through an acti- `
~ated carbon column. The column measures 2.8 x 44 cm.
and contains 126 grams of acti~ated carbon. The carbon column is washed with 1750 ml. water and the wash is dls-carded. The column is then washed with 1 liter each of a 1%~ 2~ and 5% acetone:water concentrationO These eluates are al~o discarded. The column is then eluted , -39~
. . ~, .. ~ .. . . .
, with 1 liter each of a 10%, 25~ and 5~p acetone:water concentration. ~hese eluates, which contain antibiotic U-44,590, are pooled and the acetone is remo~ed on a rotatory e~aporator at ~0 C./15 mm. Hg. The resulting
,~.: :
, : . . . .
,r~ t~
E
V~ g ~ .
._ :~ a In ~ ~ a~ ~
C ~ V >
~ ~O ~ C
V C-- ~7 a~ 1~ a~ 3 0 v _ O ~ ~ E
J ~ w ~ ~ ~ IV
_ QJ L
~31Z ~ ~ U
~ .
~n ~n '.:
3 ~
g 1~ o ~ ,>~ ~ s~) ..
~ a~ o~ ~
1~ Q) ~ O O 1- --:~ 1~ ~ 3 3 3 0 ~9 _ ~ v v ~ o ~ a . J o ~
~ ~ _ ~
~n t~l~2 C- ~-Q _~J 3 ~ :~-~0 ~ : ~
O . ' , ' ' , ' .
. . . .
,_ a~
Ul ~ ~ ~: 0 _ ~ 3 ., o~ U~ V) U `~--~ C -- O . .:
O C :~
-- v C n~
. . ~ . v ~ C ~ C
E 1~ CJ ~o ~ ~ r ~ ~ ,o O ~J
.: ~1 ~ oc U ~ E
. ;. ,~ j ., ., ,., . ~ Q~ ~ ~ v cs: ~ ~ ~ ~ ~ ~ C ~ c ~ ~ -- a) c--0 ~ Q)~ 3 a. o ~
Z R~ Q ~ ~
. '` ` ~3 . `:
.
. . ~So .
' . 'O . . ...
113~ y ' 7 1~ c u E ~ t~
O ~ ~ W
0 a~ 3~ 3 3 6~' ' ~ 1 ~ ~ J . ~ >
-- >
o CS: ~ O n~ 3 ~~ 19 0 L
J~ Z ~ ~_ ~ ~ n ~ ~ (:1. J ~ O
1~
~; . .-.
` ' ~ 0~
, . ~ . ; .
~:
V
0 ~
, ~ . L .U ~ o U~ ~ -"~ 113 Il~ ~ v ~ ~ :
~ E~ ~T
~ ~ a ~ w v c ~
V~ ~ r O ~
~8~ ~ ID ~0 ~) C 1~ ~ " ~
~ a ~ a~
9~V :E ala ~ ~ æ : ~;
.~ . .
~. . .
~25~ :-'':.:~
- ~ .
' ' 311~
E
n~ >
U QJ ~ ~ O
", ~ V V ~
u~ -- v ~ C ~D ~
._ ~ c1~ ~~ 3 u In Q~ V ~ O 1 ~: ,~ > C E~ C
G) ~D
u ~ C
0 1~ 3 ~ 0 3 -- u)` ~
_ ~ C C ` >
C~ J u ~ iD OQ~ ~o o~ V
~: to ~ _ _ V .--~ ~~ ~ ~ 0 Q~ O ~ O ~
U~l~ ~ Qt~S CL Q ~ ~ ~ Z 3 O ~ ~ >
V ".~ ~ ~s ~--In U~ ~ c ~ a) ~ C V
u . 3 ~ a~n 3 ~ 3 v~ I > ~ v Q~ I a) a.l o ~ >. ~C >.
~- ~ a~ ~ 0-- I u ~D
` O 0 3 1~ ~ 3--- Cl) O~-- -- aJ
~ _ V ~ Q) o ~ C ` ~ ~ ~
I:L J Q ~ U-- ~ a~ a~ ~ '-- ~ V
u a~ Q~ _ _ v, --._ ~y ~1~---- ~ ~ ~ n~ r~ a) o ~ o ~: ~
~lz ~ ~J~_~SC~D,s ~ ~z3 a~ a~a) ., > v~ >
~ ~ -- ~ c -- a) o ~ .
_. ~ J ,~ ._ v c~ r c v) ~ Q ~ ~ v v ~ ~
._ 2 co~ I cr~ ~ 3 o > 3 u~ u~ C a~ O a~E O~0 Q) ~ ~ v C ;~ r~ C _C n~ C V~ ~ ~ V) I_ ~ . JJ 1~ 3 J'-- o ~--- u~ c ~ v ~ C.
E ~ N o>~--- 3 o ~ V ~ -~ __ ~ ~ c c c > a~ >
~ a ~ v ~ > Q) a) ~ o - v--o -~ ~ ~---- _ V ~
~ ~ ~ a) o ~ ~ o o o ' U~l Z C~ ~ O ~ I ~ S ~ Q Q~ S ~J V ,~--Z t~ V
O
V ~ V >
O ~ -- V -- t,)-_ . ~ ~ ~ V
_ D ~D ~s) tl~
-- U ~ 1 C ~ t~ r ~c_C
O O ~ v .--._ v CC) 3 ~ ~n 3 - 3 a) O - 3 ~
~ _ . a) ~1 ~ c ~ c . aJ ~ ~ ~ V ~ -v~:L o l ~v > ~_ ~ ~ U Q~ U U 3 ~3 ~: ~ V _ _ -- -- ~ ~ O '~
::. L ~1~ ~ ~0 I C~ ~ Qa-~ 3 ~1 ., .
C . .
O
v O ~ E c ~ ~ o tn ~ o u~ t~ o ~o E
,_ :
_ ~ :': : -' ' ,,. _ : ~ ,,:
I ~,~ V C ~
~ O U~ C
U t~ Q~ ~ . .
I ~_ ~ -. ..
nj ~ ~
, 1 ~o ` ~ ~ ~ ~ .`. .
E '5 C ~ W
C._ V _ ~ O ~ ~ ~3 0 ~
E
9 ~ rL Z , .
.:
, ~26 .
.
' 314~
. ~ '.
o ~ a~
.Ul .~
~n ~ v Q) ~ LJ a~ ~3 C G) O
v t-- ~ ~ ~ ~ a> -~ ~ cr c ~ v r v v _ a~ v ~
Q J --o 3 c 1 2 C~ ~ O ~ ~ O ~ ~' ~ O' ' ~-- O ~_ -- O : .
~lZ ~ z~_ ~z v ~ z J
CI a~
oV
3 ~ EV E
-- ~ ~_1 o ~- o JJ
V) ~ JO C V o ~
c ~ --~ v v ~ v : :, Q~ C~ ~) O OO O :, :
v 15~ ~ ~ v D OJ ~ O
r v r ~J ~ IJ '' _ O --O V ~ ~ ~ ~ ~
Cl J U-- ~ 3 --~- U 3 1 ~ `
v l t~ ~al o V ~ ~ O v ~ v ~ O' -- ~
2 1_ z~ Z~ Z `
Q11 Q~
, ,~
. L. ~U ~ L- ~ ~
U'~ ~ :J-- W ~ .LI V
._ o o ul ~ v -- o c 3 ~---n C -- -- E O C ~ --V o o C
~ C ~t ~U--- 0 1~ C L C
~ a ~0 o ~ ul_ v o ~ ~ ~ o :~ v ~ ~ V ~ ~ 3 a~
F 1~1 C~J Q O c v u ~ E "
~5 ~ Q) a~ 1- D v rD o O ~ U O
C J t) ~1 C) a) 3 a~ V~ ~
n: ~ ~ -j v ~ c~ v U o v v u~ ~J71Z I ~ 7z~ 1- u~ O 2 G~ a U Q,)-_ v~l ~ ~: v ~ ._ _ ~ ~S V -- a~ c 3 ~ ~
~- U U Ir~ ~ ~ O ~ ~ V O O C
C C ~ ~ ~ Lr C L e ~n ~ a~ o _ o O ~ ~ ~
v ~ m ~ .~ ~ v3 a)~ a~ J
~ 1~ ~ ,~ v ~ 3 _ _ . a~
J~ CL IJ v c ~ 3 L. a~ V ~ ~ v ~ ~
~ ~ o ~ o v U~ ~ O ~ J~ V
~ ._ ~ ~ ~O -- ~
z ~ 2 ~~--u l.~ l~ Z~
I~
~C
e o .
m ~ -D v ~ c JJ ~ O
._ Q~ .
r ~ . :: .
_u . ~ .:
. ~ . ~ v ~ .~
v : :,. -~" . r ` - O ~: ~: O ~
r , C ..
.. _ ~ ~J ~ ~ ~J ', ' ~' ::
~ 11J ~ C O ~ ~ .-L~
_ a~ r .. ...
~_ L.
.. 5:1-- . 'Cl ~ C V
~ aE Z tf~ Z : ., ,, ..
. .
' .
. .
314~
.
':
a~.- ' ~ o In _ v ._ V~ ~ ~
C ~ Q QI
v ~D N--~ ~ ~ ~ C
_ ~-- O
~ ~_ V---C~ ` ~ V
t~7 aL N
O--. ,- C
U V
a) ._ -- cL e--~n Q~ a~
C ~ QQI
Lt~ CL
U rD C
_ .. ~ ~ v.
u~
,_ ~ ~ ~ ~
c ~nlz ~) t~ N
Q~
a IU _ C .V~ U , ...
u~ ul a~
O C ~ CL~
I ~ ~O Cl~
C
C N ~, cr~
~: ::1 0 I
V ~Iz U) Z Q
~ _ ._ I
S) 0~ C 0 , U) 'Ul ~ --U ~
U ~--_ 3 CL s~O
--U~ V~ Il~ Q~
V C C i~ CL Q) Q O t-- . :
U~ O ~ O ' U
._ V . ~ IU -- T
~ (U n~ ~ ~ C CL
Q~ _ _ ~ o Q U ~ ~ ~ ~ ~ O
Z ~ J 0- N
' ' C'~ ~ C U) ' . '"'~':
I O 1.) ' '.. :
u-- ._ ~o w E /~ ~ O ~
E ._ -0 , .::
_.c o , ,,:
.~ ~ O
.
~ . . ': - :- .
v : ~ `~
c ~J r J, 0 ~ . ~ C~
U ~ r ~: E
V
. . ~ E O v~ ~ . c:
,~ a: E ~:
. ::
._ .:-: .
': '~'. '' . ~
o28- ' ~' : . .' :' :' . ., ' ',' '.
c: ~l ~
o~ ~ . ~ I I + ~ ~ + ~ f ~ 1 + ~ ~ ~ .
t--, . ," ,11) t<~
Q~ ' ' a J
:~
o~
VIIZ
iO
:1 ~
~n a~ ._ ,..... 1~ ~
V ~ O~ . ~ ~ ~ ~ ~ ~ + ~ ~ ~ +
~ ~ ~J L~
_ O
,~ ' . W _ _l ~!
i ~ z , .
E~
~' ' .' ' , ~ . , .
` ' ' U~ V CL Ul 3n c ~ ,~
0 4- a~ ~ + + ~ ~ + ~ + ~ + ~ ~ t ~ ~ ~ ~ , :~ O " 1~ .
.19 ~ 1~ ~J
. ' .: ~ ~ 3 a .
.~ lz , ~ ~ , , j ~
- ~ v ~ .~ ~::
~ ul ~n U~
"~ ~ C
+ ~ ~ ~ + ~ +
r lo `
~,~ ~ J . - `
- ' . . ~ i:l: . ' . , ::
~ . , . ~ .
~ 1:~ `
- N . 59~ - ;-~
' ' 9,9 a 0 5~ ~ ~ O ~ ~ Ul ~9 ~
u~ o o ~ a~ ~ ~ ~ Q~ . :
~3 F o~a O ~ f~ ~ C ~
- _ Ç3 ~ C ~--~ C O O O O ~ n ~ ~ ~ 3~ ~ x ~
~ . - . - ~ ~ ~ ~ ~ a~ ~: o ~ ~:3lJI~ 01~1~1~
~ O ~ Q O ~
~, ' ' ' `
'' ' ', .. '' ' ~' :' ' , ,, .. , . . , , . ' ',' ,;: ", ' .~.' ' ' ~ . , ~ . . :
" . . ' : , : . ', . '. ' ~', . , . ' . . , . ' ' , ~-. ~ O
Q) ~.--~ Ull ~ V
S Cl~
~D ~ ~ ^ ~ C
UV ~ + I + + + I I I I 1, 1, + + ~._ ._~ r~ ~ ~ E
V _ 0~ 0 '-J ~ v, a c~ a~
c ~l U~ V~ U~
.D ~ Q~ -U~l .~
V ._ V ~ , . .
cc ~ ~ E v~
._~ ~ ~ ' O O
~ ~ ~ + + + - - - - I I + + ~ ~ ~
u~~ ~ ~ O ~J
Q)~ J C:l t- , ~ Q~ O
! ~ ~ --~1 u~lz c ~
, ~ V
~n O ~
._ V~ ~ C ~ ,.
C ._ ~ O
a v, ._ "~ . ~ ..
c ~
~~ `D ----~ E ~--v _V ~ + +~t + ~ + + + ~ ~ ~ ~
aro N
U)CL ~ ._._ ~)C~
.. u~lz n. ~ E--~ ; :
... , ~ . . , .
. . ~ '.
W.U! ~ -,.:~
u~ ~n ~ ..
C C r~ ., ~ a~ o + I + + + ~ i + + +
_ _ O
C U J v i Z O O N .
~* ~--O
1~ 8 Il~ N N V v O ~
V
O V ~ ~ ~ ~, ._ .
~ V
: ~ O O ~ ~ ~ ~V ~ C o ~
~ O ~ ~ O O O O ' ' r ~ ~~- O c E~ v ~ z - ~ O ~: ~-- v c ~ ~ ~
~O~ ~--c~--~OOOX~0~~~ '"''''' _._ ~ ~ u ~ o u~ ~-- c ul ~ O ~ ~7 ~ ~ ~ .
~ ~C ~ 7 0 ~
- N '-- ~ llDI--~ ~ ~ 2: Z Z 2 z z z ~
~--- ~ . ' .. -.
_ _ t~
t~ ~ ~ ~ID 1~00 t:~O .I N ~ L~ O . . -.
. . , :
., -3a- . :'-~.',.: .
:'. '', .
.. ,' .. .
. , -, ,. . - - , ~ . :
~1 43 35~ dP
~::
._ 3 c V) O V
C ~i ~ 3 ~ ~ cn O ~1 + + + +
n~ ~ cn + +~1~ + ++1+
U~ _ ~_ Q J .~J) o -- ,.
* r . .
V~ ~ ~ ~
._ o._ 3 Ul _U) O J
C __ 1~ 3 ~.:
V ~-V In ~ + + + ~ + +
~D O~ ~ ~ ~ + +~1+ + +~1+ , '.
Q ~o CL J .~n o .:
U~ C ~ IY -- O
~ (~ ~ lZ 0 ~ , ~ C~ . '~
.~ ~ .C . ,.
a) ,_ _~. v ':
~n~ ~ :
~11 1_ v ~ ~ + + ~ ,~ + +
~ n ~ C~ ~ ~ + ~ + + + + +I +
Q J ~O ~ :
-- O :' olz -- O
O ~ ' . ~.
. - ' ' ' .
os --1-1 v ~ U~v ~. O ~_ + + + ~
(~n~ 1~ w VCJI ' + ~ + + + ++I+
~ _ ~ -C -a ~ > ~: _ . ,~ ~.
._ .-,,: .
N :
' _ . : ' ._ `~ 3 V'1 _ _ ', `: : ~
Q~
~: . ' l O o o o ~ a) Q~ F a~ E IJ ~ C O--V O (L~ u) -~ ~ ~ ~ ::1 ~ -- ~ Vl ~?---~ U~ O
'i: , O ,_ .. ,~ Z .U Ul O ~ t: V O C
a:: .Id ~ V -O ~ n~ o~ c ~ ' ' ' ---- ~ ~ ~ ~ ~ E
z l~n E ul E C~ C! cr O X O ~ L~ IU
o Q~ O a ~ c ~
I_~ ~ L~ J~ Qlo ~ l .
,. . ~
3~3~ ~
~o o , ~:~ ~
.,, ,a ~ o ~ ,, ~
. .
. ~ a~
g,, U~ Z
U~
~ , .~
a~ o -:~ o :.
.,.
.,, .,, U~
N
,i , .
::J rd ~ h , ~ U
:I K . ,~
C~ Z .~ .
U~ . ,~
~1 U
~ . O ;,~.,;"""
~1 U~
P m .
~ . I ,C ~
Q~ ~ J~ tr7 ~, E3 ~ ~ _1 .
~3 ~
~ ~; U~
~ cn æ ,~
U~ ..
~, ~o~
~1 tn u~
O ~ . ,....... ~,.
~a ~ ~ ~ cO ~
O
$ rl IJ N ~
I;~rl O
N ~
~ O
~ O ~ ' .~ 3 H
8 ~ ~ - ~.~ N N t~ ~
O
N-,l ~1 ~\ O ~ ~h O - : .-1 ~1 ~ ~
g p ~: lC .~:, ~.. .....
.: .
: ' -- , .
.:' _ 3~
~, .
~ . ~
` ' 314~
~ ~ ' ~1 + ~`
J .
~ :
~nlz U~
a~
V .U~ .'~
~ Ul : :
C~ C ~ ~ ~
' ' Q) O~ . .
U~ ~ Il~ . . .
._ t~ ~ ~ + ' :
a~ ~:1 J ' "
I~ ~
~ . ' .' .
U~
a) ~,3 .U~
.
. ... :
+
-o ~ --~ ~:
. . ...
. .Q , . , .
; ~o ~7 u~ u7 c~ r ~ Q~ O
- Q ~ 0~:) '` '. O ~ . ~ .
~ 2 ; . ~. .
C . ~. .
`~¢ . '' '~
. . : '',.-. .
. .
ql ....
. , ~ ,.1~ U
'~ : - : ~ ~ ~ . . .
~ .0 ~
~ a) ~I N
X X ~ J
` ~tc ~ O ~: . ec ~
.
.', --3~- ~
~. .
-, ... ~ - , - ,. :
' ' 3143 ,~ 3Z~~
The new compound of the in~ention ;s produced when the elab~rating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of 7imited amounts surface cultures and bottles can be employed.
The organism is grown in a nutrient medium containing a carbon source, for example~ an assimilable carbohydrate, and a nitrogen source~ for example, an assimilable nitro~
gen compound or proteinaceous material. Preferred carbon : `
sources inclùde glucose, brown sugar~ sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the ~;
like. Preferred nitrogen sources include cornsteep liquvr, yeast, autolyzed brewerls yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solidsJ pancreatic digest of casein, fish meal, dist:illersl solids, animal peptone liquors) meat and bone sc:raps, and the like. Com-binations of these carbon and nit:rogen sources can be used ad~antageously. Trace metals, for example, zino, magnesium, manganese~ cobalt, iron~ and the like, need not be added to the fermentation media since tap water and unpurified `~ ingreJients are used as components of the medium prior to sterilization of the medium.
Production of the compound o~ the in~ention can be effected at any temperature conduci~e to satisfactory --growth of the microorganism, for example, be~ween about 18 and 40 C~, and preferably between about 20 and ~2 C.
Ordinarily, optimum production of the compound is obtained In about S to 15 days. The medium nonmally remains neutral during the fermentation. The final pH is dependentJ in 30 part~ on the buf~ers present, if any and in part on the ,~
-~ 3143 initial pH of the culture medium.
When growth is carried out in large ~essels and tanks, it is preferable to use the ~egetati~e form, rather than the spore Form, of the microorganism for inoculation to a~oid a pronounced lag in the production of the new com-pound and the attendant inefficient utilization ~f the equipment. Accordingly, it is desirable to produce a ~ege-tati~e inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N2 agar plug, or a slant culture. When a young, acti~e ~egetative inoculum has thus been secured, it is transferred asepti-cally to large ~essels or tanks. The medium in which the ~- -~egetati~e 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 ~ariety of procedures can be employed in the isola~-tion and purification oF the compound of the subject in~en-tion, for example, sol~ent extraction~ partition chromato~
graphy, silica gel chromatography, liquid-liquid distribu-t;on in a Craig apparatus, absorption on resins, and crystallization from sol~ents.
In a preferred reco~ery process the compound of the subject in~ention is reco~ered from the culture medium by separation of the mycelia and undissol~ed solids by con-~entional means, such as by filtration or zentrifugatTon.
The antibiotic is reco~ered from the filtered or centri-~uged broth by adsorption on acti~a~ed carbon. The acti ~ted carbon is then washed with water to remo~e some ~0 impurities. This is followed by elu~ions with ace~one:
-35~
: :' water solutions which remo~e the antibiotic from the 3ct~ated carbon. The acetone in the eluates is remo~ed, ad~antageously by evaporatiOn, and the remalnlng aqu~ou~
residue is lyophilized to afford a crud~ preparation of antibiotic U-44,590.
A preferred pur i f i cation procedure is to subject a crude preparation of U 44,~90, as described abo~e, to chromatography on siliea gel from which U 44,590 i5 eluted. Fract;ons which show acti~ity against the bacterium Klebsiella Pneumoniae on a standard agar plate test, are pooled and taken to dryness to yield a relatî~ely pure preparation of U-44,590. Further purifi- :
eatlon is achie~ed by acetylation to a crystalline diacetate ,. . derl~ative of U-44,590. Zemplen EG. Zemplen and E. Pacsu, BerO~ 62 1613 (1929)] de-esterification (trans-esterifi~
. cation) with sodium methoxide in methanol~ and n~utraliza- ` `
- . tlon of the catalytic amount o~ base with carbon dioxide - ``
: gt~es the free antibiotic U-44,590 which crystallizes . - r@ad~ly fram methano1-ethy~ acetate to give ~ pure prep~ra-; . 20 ~lon of U-~4,590 , AntlbTotk U~44,590 ls acti~e against ~e~Q~ ~
and, thus, can be used to dTsinfeet instrumentS~
utensils or surfaces when contaminated with ehis microorgan- . ; -llsm9 where the înacti~ation of this microorganism is desir . . 25~ abte~ Also~ u~44.sgo is àcti~e against Escherichia coli ~fld e~n be used to r~duee~ ar~5t, ~nd~oi er~dlc~te ~1 Ime ~- . product;oR in p~permill systems because ~sf its antibacterial ~et~orl agatnst th~s bacter;um. Antlbiotic ll~44v590 can also .
~ use~ ~o prolong ehc llfe of culture~ ~f Trlchomon~s 3a ~, ~ hominis. a~d ~ 5~
' ~ . -, ' .
::, :
.. . .: . .. :
:~3~
by freeing them vf Esch~-ri~hia coli contaminaLion. Further.
U-44l590 can be used to inhibit the growth of E. coli in hospital flower ~ases where it has been reported to exis-t and present a hazard to hospital patients. See Clinical Medicine, February, 1974, Page 9.
No~el acyl deri~ati~es of U-44,590, as disclosed herein, can be used for the same antibiotic purposes as u_l~4,5go in en~ironments possessing means to deacyla~e the compound to U-44~590. Thus, the acyl deri~ati~es of U-44~590 can be used to treat laboratory mice in~ected with a Gram-negati~e bacteria, for example E. coli, as disclosed herein. Further, acyl deri~ati~es of U-44~590 can be used, ad~antageously, to upgrade U-44,590. This ` i5 accomplished by acylating U-44,590, reco~ering the acylated compound relati~ely free of impurities then de-acylating the acylated U-44,590 to gi~e U-44,590 in a more purified form.
The following examples are illustrati~e of the proc~sS
and products of the present in~ention but are not to be construed as limiting. All percentages are by weight and solYent mixture proportions are by ~olume unless other-wise no~ed.
Exarnple 1 Part A. Fermentation A soil stock of StrePt~myces platensis ~ar. clarens~s.
NRRL 8035 is used to inoculate a series of 500-ml. Erlen-meyer flasks, each containing 100 ml. of sterile seed medium consisting of the following ingredients:
Glucose monohydrate~ 10 Gm/l Bacto Peptone ~Difco) 10 Gm/l , ~.
~37~
. `' `.` ' :, ., . . , .. ~,, .. , ~: ,. .
.~:, . . , , ., . - . .. -.:
B~cto Ye3st Extract (DiFc~ 2.5 rim/l Deionized water ~aiance The flasks are grown for 2 days at 28 C. on a Gump rotary shaker operating at 250 r.p.m.
Seed inoculum, described abo~e, is used to inocula~e a series of 500 ml. Erlenmeyer flasks each con-taining 100 ml. of sterile fermentation medium. The inoculation rate is 5 ml. of seed inoculum per 100 ml.
of fermentation medium. The fermentation medium consistS - -1~ of the follo~ing ingredients: ~ -Brer Rabbit ~ olasses (RJR Foods Inc., N.Y., N.Y. 10017) 20 ml Yeast Extract (Difc~ Detroit, Michigan 1 Gm/l Gluco~e monohydrate 10 Gm/l 15 Dextrin (Corn Products Co. Inter-national Inc., International Plaza, Englewood Cli~Fs, New Jersey 07632)10 Gm/l Proteose Peptone #3 (Difco) 10 Gm/l Tap water q.s. Balance The presterilization pH is 7Ø The inoculated fermentation flasks are incubated at a temperature of o 28 C. on a Gump rotary shaker operating at 250 r.p.m.
~with a 2 1/2 inch stroke. Ucon antifoam (a synthetic de~oamer supplied by Union Carbide, N.Y., N.Y.) is used if needed.- Har~est 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 bacter-ium Klebsiella pneum~niae. This bacterium is inoculated -in~o the assay agar (Streptomycin Assay Agar, BBL, Cockeys- -~ , Maryland, 21030) of the following composition: ~ ~
. ` ' ~.-38- ;
Beef ~xtract 1.5 Gm/l Yeast Extract 3.0 G,m/l Gelysat~ Peptone, supplied by Baltimore 8iological Laboratories 6.o Gm/l Agar 15.0 Gm/l Deionized water Balance adjust pH to 7 9 Sterilize at 121 C. (15# steam pressure) for 15 minutes.
Phosphate buffer (O.lN pH 6.o) is used as the diluent. The agar plates are incubated at 37 C. for 16-18 hours~ Presence of antibiotic U-44,590 is e~i-denced 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 antibiotic sample. Thus, a 20 mm. zone of inhibition using a 12.7 mm. paper disc to which 0.08 ml.
o~ antibiotic sample has been applied is expressed as one bio unit per ml. (1 BU/ml.).
Part B. ~eco~ery Whole fermentation beer (ca 1600 ml. assaying 5 BU/ml. against K. pneumoniae), obtained as described abo~e, is filtered using diatomaceous earth as a filter ;~
aid. The filter cake is washed with water. The clear beer and wash (1800 ml.) is then passed through an acti- `
~ated carbon column. The column measures 2.8 x 44 cm.
and contains 126 grams of acti~ated carbon. The carbon column is washed with 1750 ml. water and the wash is dls-carded. The column is then washed with 1 liter each of a 1%~ 2~ and 5% acetone:water concentrationO These eluates are al~o discarded. The column is then eluted , -39~
. . ~, .. ~ .. . . .
, with 1 liter each of a 10%, 25~ and 5~p acetone:water concentration. ~hese eluates, which contain antibiotic U-44,590, are pooled and the acetone is remo~ed on a rotatory e~aporator at ~0 C./15 mm. Hg. The resulting
5 acetone-free preparation is shel l-frozen to an aqueous residue and then Iyophilized~ yield, 3.55 grams assaying 2 BU/mg. of U-44,590 against K. pneumoniae. Th is prep- -aration, labeled for con~enience as Soiid A, is then subjected to further reco~ery 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). The column measures 3.8 x 88 mm.
SQI id A, obtained as described abo~e, is added ~n the top of the column and the coiumn is then eluted with methanol:
chloroform (1:1 v/v). Acti~e fractions, as determined by the abo~e-described K. pneumoniae assay, are pooled and the solvent is remo~ed from said pooled fractions by use of a rotatory e~aporator at ~0 C./15 mm. Hg.;
yi~ld, 8~o mg. assaying 7.5 BU/mg. of antibiotic U-44,590.
~ Part C. Pu _fication No. 1 A preparation of anti~iotic U-~4,590J obtained as described abo~e in Part B., is subjected to chromatography on s;lica sel using the s~l~ent system ethyl acetate:
methanol (6~ ) to gi~e a purer preparation containing -;~
U-44,590. The procedure for this purification step is as ' ~t~l l ows: " ,: . -.
a ~olumn of silica gel (Merek-Darmstadt, 115 grams/
gram o~ ~he U-44,590 prepara~ion being chr~natographed~
tn ethyl acetate:- methanol (6~ ) is prepared by pour-~0 ing a slurry of silica in ~he sol~ent into the column to : ' ~
- ~40~
. ' ~ .
gi~e a height-diam~ter ratio of 10:1 after being packc~.
The U-44,590 preparation, obtained as described abo~e in Part B, is dissol~ed 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 e~ap~rator at 40 /15 mm. Hg. The resulting dry solid is added to the top o~ the silica column through a small head of the sol~ent ethyl acetate:methanol (6:1 ~/~). After a forerun of 4 litersJ 50 ml. fractions are collected and assayed for acti~ity against K. pneumoniae. Acti~e frac-tions are also tested for solids content. Fractions great-er than 50 BU/mg are pooled and then taken to dryness in a rotatory e~aporator at 40 C./7 mm. Hg. to yield a syrup.
Fractions and their K. ~neumoniae (K.p.) acti~ity and ;
solids from a usual run are as follows: - -Fraction Zone of Wt. of solid Number Inhibition in Fraction ~ (using 12.7 (mqm) mm. discs) 115 ~0 1~0 33 3-9 130 37 40.8 :~
36 ~5-7 150 - ~5 27.0 155 ~4 -160 ~ 21.7 165 ~2 170 31 21.8 175 3~
1~ 29~ 21.8 185 2~
190 28 17.9 195 ~8 205 ~7 14.4 215 ~6 220 26 1201 :
.
~ `' '.
. ~ ' .
.. ....
. .
~ .
, 3143 5J$i~ d Fract;or- Zone of Wt. of solid Nulnb~r Inhibition in Fraction (using 12.~ (mgm) mm. discs) 230 26 1 o.o 2~5 25 240 25 11.5 250 24 11.7 260 23 12.6 270 25 15.4 Fractions 120-180, incl. are pooled and taken to dryness on a rotatory e~/aporator at 40 /7 mm. Hg. to gi~e a syrup, wt. 2.66y, assaying 54 K.p. BU/mg (Fractions 181-240, incl. gi\re 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 8U/mg. against the usual assay for this standard of 6 BU/mg.
Part D. Purification No. 2 The prepa rations of U-44,590 obtained as described ~ -in Part_C., can be further purifïed to a preparation of essentially pure U-44,590 by passage o\~er another silica gel column using this time the sol\~ent system meth~nol: -methylene chloride (1:8 \~ ). The procedure i~ as follows:
A U-44,590 preparation, as obtained in Part C~, (2.28 grams) is dissol~led in methanol and 7 grams of silica gel~
as described in Part C.. is added. The sol~lent from this mixture is remo\~ed on a rotary e\~aporator at 40 C. /7 mm.
` Hg The resulting solid is added to a column of silica gel ~750 9., 4.8 x 96 cm, hold-up \lolume 1500 ml., made up ;n MeOH-GH2Cl2 (1:8 ~ ]. A forerun (1100 ml.) is collect-ed, followed by 50 ml. fractions. Fractions 141-2009 in-;~iO clusi\~e, weigh 390 mg. when taken to dryness in the form of ~ : ' , .
...
il~ 5~
a syrup. Thi~i material is sh~wn co be almost pure U-L~4,5~0 by thin layer chromatography (tlc).
I'he tlc is conducted on silica gel plates using the sol~ent system MeOH-CH2Cl2 (1:9 v/~). Zones of the anti-biotic are detected by spraying the plates with 104/MnO4spray, and with 50% aq. H2S04 followed by heating at 110 C. for ca 10 min. The Rf of the acti~e material in this sol~ent system is 0.11.
Part_E. Purification No; ~
The preparation of antibiotic U-44,590 obtained in ~' Part D can be further purified by acetylation of the prep- -aration 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 dissol~ed in pyridine (300 ml). and to this solution.stirred magnetic-ally.is added acetic anhydride (150 ml) o~er the course of 45 min. After st~nding o~ernight at room temperature, ~olatile materials are remo~ed as completely as possible -on a rotatory e~aporator at 40 /15 mm. Hg., and finally under high ~acuum, to gi~e a tan syrup.
This syrup is stirred with CH2Cl2 (200 ml), and a colorless, flocculent precip;tate is remo~ed by filtration and washed with CH2Cl2 until the washings are colorless. ~ ' 1'he precipî~ate îs discarded. The comb;ned fîltrate and washings are'washed with aqueous HCl (N/20, 100 ml) twice, 'the aqueous layer beîng acidic a~ter the second wash. The '~
aquenus layers are d'iscarded. The organic phase is then washed with water (100 ml), saturated aqueous NaHC0~ (100 ml), ~0 again with water (100 ml)/ and dried (Na~S0~). The aqueous . ~: ~
` :~ ~43~ ~:
~' ~
layers are discarded.
Removal of sol~ent on a rotatory e~/aporator at 40 and 15 mm Hg. gi~es a dark syrup (21.10 g), which is dissol\~ed in EtOAc (50 ml) by warming on a steam-bath. On cooling.
5 crystallization occurs; the solid is remo\~ed by filtration, washed with EtûAc, and dried at 60 /15 mm. Hg., to gi\~e essentially pure ~',5'-di-0-acetylated U-44,590 (12.01 9., o m.p. 12 3-124.5 C.). Recrystal1ization from the same sol~ent gi~es 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_44J590 by the Zemplen procedure which is as follows.
The crystalline diacetate U-44,474 (24.90 9) is stirred magnetically in methanol (400 ml)J and methanoiic sodium 15 methoxide (Stauffer Chem. Co. 25~, 5 drops) is added. Stir-ring is continued till the solid has dissol\~ed (Drierite tube), and the solution allowed to stand at room temperature for about 2 hours. Solid carbon dioxide, in small pieces, - ;s then added cautiously, with stirring, to neutralize the 20 methoxide, and the sol\~en~ is remo~ed on a rotatory e~apor-ator at 40 and 15 mm. Hg., giving a colorless oil.
The residue is dissol~ed in methanol (50 ml) by warm-ing on a steambath and diluted with ethyl acetate (50 ml).
Crystallization occurs on cooling. The solid (12.39 9) is 25 collected on a sintered filter at the pump. washed ~ h methanol, ~nd dried in a vacuum o~/en at 60 /15 mm. Hg.
Antibiotic U-44..590 crystallizes in colorless prismatic needles, m.p. 141-142 . Remc)~al of sol~ent fran the fi1-trate plus washings on the e~/aporator and crystallization 30 From methanol-ethyl acetate gi~es additional material , -1~4-~ .
. , 3~
,~ r;~
r~
(1.91 g, m.p. 140.5-141.5 j.
Example 2 The acylating procedure described in Example 1, Part E can be substituted by acylating U-44,590 with any readily-a~ailable acylating agent to qi~e aeylated U-44,590. This acylated U-44,590 product can then be deacylated by methods well known in the art to yield a purified preparation of U-44,590. Readily-a~ailable acylating agents which can be ùaed to acylate U-44,590, and which are within the scope of this in~ention. are as disclosed in U.S. Patent 3~426,012. Columns 5 and 6.
Example ~
As disclosed in Example 2, \~arious acylates of U-44,590 can be made, and these acylates are useful to upgrade U-44,590. By following the procedure of Example 1, Part E, the 3',5l-di-esters of U -44 7 590 ~re formed.
The 5'-mono-esters can be formed by standard procedure~
~sing a minimum amount of acylating agent.
The 3'-mono-esters and ~o~phate ~n be formed by tritylating U-44,590 to gi~e the 5'-tr;tyl dori~ati~e, acylating this compound with the desired acylating agent, selected -from those disclosed abo~e, to gi~e the ~I-mono-ester 5~-trityl deri~ati~e, which ~hen can be converted to the 31-mono-ester by ren~o~al of the trityl group. The tritylat;on proc~dure disclosed in U.S. Patent ~i~426,012. Columna 1~ and 5~ or other standard tritylation procedures can be employed.
rhe trityl group can be remo~ed by using the procedure .
diaclo~ed in IJ~S~ Patent ~426~012, Column 6.
.. . .
.
~Q
. ~ '' . ' ':
~ -.' ' , 31~
~ S~
Example 4 Th~ 5'-phosphate of U-44,590 can be prepared by procedures as disclosed in th~ work of D. Mitsunobu~
K. Kato, and J. Kimura [J. Amer. Chem. Soc.~ ~, 6510 (1969)]. This compound can be used for the same pur-poses as U-4~,590.
The compounds, described abo~e, being the deri~a-ti~es or U-44,590 which are within the scope of the ~ub- '~
ject in~ention,can be shown by the following structural formula O
H- ~ N-CH3 ~l J
\ / , . .. ~ ..
, 5 , , `' R'OHz ~ \
~' \L / .:
,~R ~
wherein R and R' are selected frorn the group consisting of a carboxylic acid acyl radical of frorn 2 to 18 carbon atoms,.
', inclus;~e; or a halo-~ nitro-,. hydroxy-,, amino-, cyano-J
c ' thiocyano-, and 1 ower a 1 kox y -s ubstituted hydrocarbon carbox-i yl ic acid acyl radical of from 2 to 18 carbon atorns. inclu-si~; R is hydrogen and R' is ac defined abo~e or phosphatei ' ~ or Rl is hydrogen and R is a carboxylic acid acyl rad;cal of `` fr~m 2 ~o 18 carbon atoms,l inclusi~e; or a halo-. nitro-~ .
` ~ .
hydroxy-, amino-~. cyano-J th iocyano-~ and lower al koxy-substituted hydrocarbon carboxylic acid acyl radical of from -~
2 to 18 carbon atoms~ inclusi~e,,. or phosphate. `~ , ~ ~': , , `',' , -46- ;~
.
.
-- .
Additional charactcrization data for U-44.47'"
prepared as disclosed in Example 1~ Part E, is as follow~:
Elemental Analysis:
Calcd. for C1 3Hl.~ N30 Found:
-CJ47~41; H, 5.82; N, 12.76; 0, 34.01 Molecular Weight: 329 (Determined by mass spectrometry) Infrared Absorption Spectra: U 44,474 has a characteristic infrared absorption spectrum when suspended in a rnineral oil mull. Peaks are obser~ed at the following wa~e lengths expressed in reciprocal centimeters:
Band Frequency (Wa~e Numbers~ 5 ~080 M
~960 (~il) S
2930 (oil) S
2B60 (oil) S
1750 S ~:
1~02 5 1468 (oil) . S ` -~
1379 (~il) S ~ :
1327 W . .~
1318 W .~:
1 ~1 0 W -12.98 W . ~ ..
-47~ ~
~' ~
.~
:~`i3.~
Band ~ requenc (Wa~,~ Nurn~ Intens itV
12~;0 S
111~ W
1097 S .
1057 . M
10~0 S
957 . M
9~6 W
: 775 M
755 M . ~ :
721 (~il) W
A silica gel (Merck-Darmstadt Cat 7734) column is prepared from 420 grams of silica gel packed in methanol:
chloroform (1:1 ~/V). The column measures 3.8 x 88 mm.
SQI id A, obtained as described abo~e, is added ~n the top of the column and the coiumn is then eluted with methanol:
chloroform (1:1 v/v). Acti~e fractions, as determined by the abo~e-described K. pneumoniae assay, are pooled and the solvent is remo~ed from said pooled fractions by use of a rotatory e~aporator at ~0 C./15 mm. Hg.;
yi~ld, 8~o mg. assaying 7.5 BU/mg. of antibiotic U-44,590.
~ Part C. Pu _fication No. 1 A preparation of anti~iotic U-~4,590J obtained as described abo~e in Part B., is subjected to chromatography on s;lica sel using the s~l~ent system ethyl acetate:
methanol (6~ ) to gi~e a purer preparation containing -;~
U-44,590. The procedure for this purification step is as ' ~t~l l ows: " ,: . -.
a ~olumn of silica gel (Merek-Darmstadt, 115 grams/
gram o~ ~he U-44,590 prepara~ion being chr~natographed~
tn ethyl acetate:- methanol (6~ ) is prepared by pour-~0 ing a slurry of silica in ~he sol~ent into the column to : ' ~
- ~40~
. ' ~ .
gi~e a height-diam~ter ratio of 10:1 after being packc~.
The U-44,590 preparation, obtained as described abo~e in Part B, is dissol~ed 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 e~ap~rator at 40 /15 mm. Hg. The resulting dry solid is added to the top o~ the silica column through a small head of the sol~ent ethyl acetate:methanol (6:1 ~/~). After a forerun of 4 litersJ 50 ml. fractions are collected and assayed for acti~ity against K. pneumoniae. Acti~e frac-tions are also tested for solids content. Fractions great-er than 50 BU/mg are pooled and then taken to dryness in a rotatory e~aporator at 40 C./7 mm. Hg. to yield a syrup.
Fractions and their K. ~neumoniae (K.p.) acti~ity and ;
solids from a usual run are as follows: - -Fraction Zone of Wt. of solid Number Inhibition in Fraction ~ (using 12.7 (mqm) mm. discs) 115 ~0 1~0 33 3-9 130 37 40.8 :~
36 ~5-7 150 - ~5 27.0 155 ~4 -160 ~ 21.7 165 ~2 170 31 21.8 175 3~
1~ 29~ 21.8 185 2~
190 28 17.9 195 ~8 205 ~7 14.4 215 ~6 220 26 1201 :
.
~ `' '.
. ~ ' .
.. ....
. .
~ .
, 3143 5J$i~ d Fract;or- Zone of Wt. of solid Nulnb~r Inhibition in Fraction (using 12.~ (mgm) mm. discs) 230 26 1 o.o 2~5 25 240 25 11.5 250 24 11.7 260 23 12.6 270 25 15.4 Fractions 120-180, incl. are pooled and taken to dryness on a rotatory e~/aporator at 40 /7 mm. Hg. to gi~e a syrup, wt. 2.66y, assaying 54 K.p. BU/mg (Fractions 181-240, incl. gi\re 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 8U/mg. against the usual assay for this standard of 6 BU/mg.
Part D. Purification No. 2 The prepa rations of U-44,590 obtained as described ~ -in Part_C., can be further purifïed to a preparation of essentially pure U-44,590 by passage o\~er another silica gel column using this time the sol\~ent system meth~nol: -methylene chloride (1:8 \~ ). The procedure i~ as follows:
A U-44,590 preparation, as obtained in Part C~, (2.28 grams) is dissol~led in methanol and 7 grams of silica gel~
as described in Part C.. is added. The sol~lent from this mixture is remo\~ed on a rotary e\~aporator at 40 C. /7 mm.
` Hg The resulting solid is added to a column of silica gel ~750 9., 4.8 x 96 cm, hold-up \lolume 1500 ml., made up ;n MeOH-GH2Cl2 (1:8 ~ ]. A forerun (1100 ml.) is collect-ed, followed by 50 ml. fractions. Fractions 141-2009 in-;~iO clusi\~e, weigh 390 mg. when taken to dryness in the form of ~ : ' , .
...
il~ 5~
a syrup. Thi~i material is sh~wn co be almost pure U-L~4,5~0 by thin layer chromatography (tlc).
I'he tlc is conducted on silica gel plates using the sol~ent system MeOH-CH2Cl2 (1:9 v/~). Zones of the anti-biotic are detected by spraying the plates with 104/MnO4spray, and with 50% aq. H2S04 followed by heating at 110 C. for ca 10 min. The Rf of the acti~e material in this sol~ent system is 0.11.
Part_E. Purification No; ~
The preparation of antibiotic U-44,590 obtained in ~' Part D can be further purified by acetylation of the prep- -aration 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 dissol~ed in pyridine (300 ml). and to this solution.stirred magnetic-ally.is added acetic anhydride (150 ml) o~er the course of 45 min. After st~nding o~ernight at room temperature, ~olatile materials are remo~ed as completely as possible -on a rotatory e~aporator at 40 /15 mm. Hg., and finally under high ~acuum, to gi~e a tan syrup.
This syrup is stirred with CH2Cl2 (200 ml), and a colorless, flocculent precip;tate is remo~ed by filtration and washed with CH2Cl2 until the washings are colorless. ~ ' 1'he precipî~ate îs discarded. The comb;ned fîltrate and washings are'washed with aqueous HCl (N/20, 100 ml) twice, 'the aqueous layer beîng acidic a~ter the second wash. The '~
aquenus layers are d'iscarded. The organic phase is then washed with water (100 ml), saturated aqueous NaHC0~ (100 ml), ~0 again with water (100 ml)/ and dried (Na~S0~). The aqueous . ~: ~
` :~ ~43~ ~:
~' ~
layers are discarded.
Removal of sol~ent on a rotatory e~/aporator at 40 and 15 mm Hg. gi~es a dark syrup (21.10 g), which is dissol\~ed in EtOAc (50 ml) by warming on a steam-bath. On cooling.
5 crystallization occurs; the solid is remo\~ed by filtration, washed with EtûAc, and dried at 60 /15 mm. Hg., to gi\~e essentially pure ~',5'-di-0-acetylated U-44,590 (12.01 9., o m.p. 12 3-124.5 C.). Recrystal1ization from the same sol~ent gi~es 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_44J590 by the Zemplen procedure which is as follows.
The crystalline diacetate U-44,474 (24.90 9) is stirred magnetically in methanol (400 ml)J and methanoiic sodium 15 methoxide (Stauffer Chem. Co. 25~, 5 drops) is added. Stir-ring is continued till the solid has dissol\~ed (Drierite tube), and the solution allowed to stand at room temperature for about 2 hours. Solid carbon dioxide, in small pieces, - ;s then added cautiously, with stirring, to neutralize the 20 methoxide, and the sol\~en~ is remo~ed on a rotatory e~apor-ator at 40 and 15 mm. Hg., giving a colorless oil.
The residue is dissol~ed in methanol (50 ml) by warm-ing on a steambath and diluted with ethyl acetate (50 ml).
Crystallization occurs on cooling. The solid (12.39 9) is 25 collected on a sintered filter at the pump. washed ~ h methanol, ~nd dried in a vacuum o~/en at 60 /15 mm. Hg.
Antibiotic U-44..590 crystallizes in colorless prismatic needles, m.p. 141-142 . Remc)~al of sol~ent fran the fi1-trate plus washings on the e~/aporator and crystallization 30 From methanol-ethyl acetate gi~es additional material , -1~4-~ .
. , 3~
,~ r;~
r~
(1.91 g, m.p. 140.5-141.5 j.
Example 2 The acylating procedure described in Example 1, Part E can be substituted by acylating U-44,590 with any readily-a~ailable acylating agent to qi~e aeylated U-44,590. This acylated U-44,590 product can then be deacylated by methods well known in the art to yield a purified preparation of U-44,590. Readily-a~ailable acylating agents which can be ùaed to acylate U-44,590, and which are within the scope of this in~ention. are as disclosed in U.S. Patent 3~426,012. Columns 5 and 6.
Example ~
As disclosed in Example 2, \~arious acylates of U-44,590 can be made, and these acylates are useful to upgrade U-44,590. By following the procedure of Example 1, Part E, the 3',5l-di-esters of U -44 7 590 ~re formed.
The 5'-mono-esters can be formed by standard procedure~
~sing a minimum amount of acylating agent.
The 3'-mono-esters and ~o~phate ~n be formed by tritylating U-44,590 to gi~e the 5'-tr;tyl dori~ati~e, acylating this compound with the desired acylating agent, selected -from those disclosed abo~e, to gi~e the ~I-mono-ester 5~-trityl deri~ati~e, which ~hen can be converted to the 31-mono-ester by ren~o~al of the trityl group. The tritylat;on proc~dure disclosed in U.S. Patent ~i~426,012. Columna 1~ and 5~ or other standard tritylation procedures can be employed.
rhe trityl group can be remo~ed by using the procedure .
diaclo~ed in IJ~S~ Patent ~426~012, Column 6.
.. . .
.
~Q
. ~ '' . ' ':
~ -.' ' , 31~
~ S~
Example 4 Th~ 5'-phosphate of U-44,590 can be prepared by procedures as disclosed in th~ work of D. Mitsunobu~
K. Kato, and J. Kimura [J. Amer. Chem. Soc.~ ~, 6510 (1969)]. This compound can be used for the same pur-poses as U-4~,590.
The compounds, described abo~e, being the deri~a-ti~es or U-44,590 which are within the scope of the ~ub- '~
ject in~ention,can be shown by the following structural formula O
H- ~ N-CH3 ~l J
\ / , . .. ~ ..
, 5 , , `' R'OHz ~ \
~' \L / .:
,~R ~
wherein R and R' are selected frorn the group consisting of a carboxylic acid acyl radical of frorn 2 to 18 carbon atoms,.
', inclus;~e; or a halo-~ nitro-,. hydroxy-,, amino-, cyano-J
c ' thiocyano-, and 1 ower a 1 kox y -s ubstituted hydrocarbon carbox-i yl ic acid acyl radical of from 2 to 18 carbon atorns. inclu-si~; R is hydrogen and R' is ac defined abo~e or phosphatei ' ~ or Rl is hydrogen and R is a carboxylic acid acyl rad;cal of `` fr~m 2 ~o 18 carbon atoms,l inclusi~e; or a halo-. nitro-~ .
` ~ .
hydroxy-, amino-~. cyano-J th iocyano-~ and lower al koxy-substituted hydrocarbon carboxylic acid acyl radical of from -~
2 to 18 carbon atoms~ inclusi~e,,. or phosphate. `~ , ~ ~': , , `',' , -46- ;~
.
.
-- .
Additional charactcrization data for U-44.47'"
prepared as disclosed in Example 1~ Part E, is as follow~:
Elemental Analysis:
Calcd. for C1 3Hl.~ N30 Found:
-CJ47~41; H, 5.82; N, 12.76; 0, 34.01 Molecular Weight: 329 (Determined by mass spectrometry) Infrared Absorption Spectra: U 44,474 has a characteristic infrared absorption spectrum when suspended in a rnineral oil mull. Peaks are obser~ed at the following wa~e lengths expressed in reciprocal centimeters:
Band Frequency (Wa~e Numbers~ 5 ~080 M
~960 (~il) S
2930 (oil) S
2B60 (oil) S
1750 S ~:
1~02 5 1468 (oil) . S ` -~
1379 (~il) S ~ :
1327 W . .~
1318 W .~:
1 ~1 0 W -12.98 W . ~ ..
-47~ ~
~' ~
.~
:~`i3.~
Band ~ requenc (Wa~,~ Nurn~ Intens itV
12~;0 S
111~ W
1097 S .
1057 . M
10~0 S
957 . M
9~6 W
: 775 M
755 M . ~ :
721 (~il) W
6~8 W
U-44,474 also has a characteristic infrared absorption :~
spectrum when pressed in a KBr disc. Peaks are obser~ed at ~0 the following wa~e lengths expressed in reciprocal centi- .
' -4~ -,,, , : . :
me Ler s:
Band Frequenc Wa ~c Numbc r s~ I n ten s i t y 3420 (wa ter ) W
29:~0 W
102880 . W
1468 . S
l ;~i7 0 M
201248 ~
118~
W
~51055 ` M
1()11 M
. .. .
986 M : ~:
.: . ' , .
-49~
.''.' '"
Band Fre~u~ncy ,(Wa~ Numbe rs :
9~ W
751~ M
66~ W
.
This application is a division of copending Canadian appl.ication Serial No. 225,567, filed April 24, 1975. . .
, . ':
' ` ':
. -~50~ . :
:~ .
, :. :
U-44,474 also has a characteristic infrared absorption :~
spectrum when pressed in a KBr disc. Peaks are obser~ed at ~0 the following wa~e lengths expressed in reciprocal centi- .
' -4~ -,,, , : . :
me Ler s:
Band Frequenc Wa ~c Numbc r s~ I n ten s i t y 3420 (wa ter ) W
29:~0 W
102880 . W
1468 . S
l ;~i7 0 M
201248 ~
118~
W
~51055 ` M
1()11 M
. .. .
986 M : ~:
.: . ' , .
-49~
.''.' '"
Band Fre~u~ncy ,(Wa~ Numbe rs :
9~ W
751~ M
66~ W
.
This application is a division of copending Canadian appl.ication Serial No. 225,567, filed April 24, 1975. . .
, . ':
' ` ':
. -~50~ . :
:~ .
, :. :
Claims (4)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing a compound of the formula:
wherein R and R1 are selected from the group consisting of a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy--, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive;
R is hydrogen and R1 is as defined above; or R1 is hydrogen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; which comprises acylating a compound of the formula:
with an acylating agent selected from the group consisting of lower-alkoxy carbonyl halides and the acid halides and acid anhydrides of hydrocarbon carboxylic acids and hydro-carbon carboxylic acids substituted with halo-, nitro-, hydroxy-, amino-, cyano-, and thiocyano- groups.
wherein R and R1 are selected from the group consisting of a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy--, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive;
R is hydrogen and R1 is as defined above; or R1 is hydrogen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; which comprises acylating a compound of the formula:
with an acylating agent selected from the group consisting of lower-alkoxy carbonyl halides and the acid halides and acid anhydrides of hydrocarbon carboxylic acids and hydro-carbon carboxylic acids substituted with halo-, nitro-, hydroxy-, amino-, cyano-, and thiocyano- groups.
2. A process, according to claim 1, wherein a compound of the formula I
is acetylated to give a diacetyl compound of the formula II
II
is acetylated to give a diacetyl compound of the formula II
II
3. Compounds of the formula wherein R and R1 are selected from the group consisting of a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; R is hydrogen and R1 is as defined above;
or R1 is hydrogen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive, whenever prepared or produced by the process defined in claim 1 or by the obvious chemical equivalent.
or R1 is hydrogen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy-substituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive, whenever prepared or produced by the process defined in claim 1 or by the obvious chemical equivalent.
4. A compound, U-44,474, characterizable by the formula II in claim 2, whenever prepared or produced by the process defined in claim 2 or by the obvious chemical equivalent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA303,945A CA1056822A (en) | 1974-05-20 | 1978-05-24 | Acyl and phosphate derivatives of 5,6-dihydro-5-azathymidine |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US471322A US3907779A (en) | 1974-05-20 | 1974-05-20 | 5,6 Dihydro-5-azathymidine and derivatives |
| CA225,567A CA1037889A (en) | 1974-05-20 | 1975-04-24 | Antibiotic 5,6-dihydro-5-azathymidine from streptomyces platensis |
| CA303,945A CA1056822A (en) | 1974-05-20 | 1978-05-24 | Acyl and phosphate derivatives of 5,6-dihydro-5-azathymidine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1056822A true CA1056822A (en) | 1979-06-19 |
Family
ID=27163924
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA303,945A Expired CA1056822A (en) | 1974-05-20 | 1978-05-24 | Acyl and phosphate derivatives of 5,6-dihydro-5-azathymidine |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1056822A (en) |
-
1978
- 1978-05-24 CA CA303,945A patent/CA1056822A/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5278052A (en) | Process for the simultaneous production of benanomicins A and B | |
| CA1037889A (en) | Antibiotic 5,6-dihydro-5-azathymidine from streptomyces platensis | |
| AU602327B2 (en) | Novel glycopeptide antibiotics | |
| GB1589536A (en) | Antitumor anthracycline antibiotics | |
| EP0185456B1 (en) | Cl-1577d and cl-1577e antibiotic/antitumor compounds, their production and use | |
| EP0300273B1 (en) | AB-006 antibiotics and process for their production | |
| Isono et al. | Studies on homomycin. II | |
| US4536398A (en) | Antiviral agents | |
| US4138481A (en) | Antibiotic BL580Δ and method of use | |
| GB1563432A (en) | Antitumor anthracycline glycosides | |
| US3592925A (en) | Antibiotics ah272alpha2 and ah272beta2 and process for producing same | |
| CA1056822A (en) | Acyl and phosphate derivatives of 5,6-dihydro-5-azathymidine | |
| EP0110155B1 (en) | Novel anthracycline derivatives, a process for preparing the same by a microorganism strain, the novel strain streptomyces cyaneus, and use of the anthracycline derivatives as medicaments | |
| EP0048660B1 (en) | New antibiotic substance, its process of production and pharmaceutical composition containing the same | |
| EP0171677B1 (en) | Novel anthracycline antibiotics | |
| US3118812A (en) | Antibiotics lankavamycin and lankavacidin and process of production | |
| US5109122A (en) | Antibiotics, dexylosylbenanomicin B | |
| US4189537A (en) | Process of producing antibiotic BL580Δ with Streptomyces hygroscopicus | |
| CA1236785A (en) | ANTIBIOTIC LL-D42067.beta. | |
| US4202886A (en) | Antibiotic SF-1942 substance and process for production of the same | |
| EP0236110A2 (en) | Animal growth promoting agents | |
| CA1134299A (en) | Ravidomycin and process of preparation | |
| US3692777A (en) | 5h-pyrrolo {8 2,1-c{9 {8 1,4{9 {0 benzodiazepin-5-ones | |
| CA1046965A (en) | Naphthyridinomycin antibiotics from streptomyces | |
| EP0055299A1 (en) | Antibiotic y-16482 alpha and/or antibiotic y16482 beta, process for their preparation, and medicinal composition containing the same |