Note: Descriptions are shown in the official language in which they were submitted.
3143
1~37~i~i9
BRIEF SUMMARY OF THE INVENTION
The no~el antibiotic of the in~ention, U-44,590 is
obtained by culturing StreptomYces platensis ~ar. clarensis,
NRRL 8035, in an aqueous nutrient medium under aerobic
conditions. Various derivati~es of U-44,590 can be made -
as disclosed infra. U-44,590 and its derivati~es ha~e
the property of ad~ersely affecting the growth of Gram-
negative and Gram-positi~e bacteria, for example, _repto-
coccus hemolyticus, Klebsiella pneumonia, Salmonella Sp.,
Serratia marcescens, Pasteurell~ multocida, Hemophilus Sp..
Proteus morqani and Proteus rettqeri. Accordingly, U-44,590
and its deri~ati~es can be used alone or in combination
with other antibiotic agents to pre~ent the growth of or
reduce the number of bacteria, as disclosed abo~e, in
~arious en~ironments.
U-44,590 and its deri~ati~es are also acti~e against
DNA ~iruses, for example, the Herpes ~irus and, thus. can
be used to control such ~irus where its presence is not
desired.
DETAILED DESCRIPTION OF THE INVENTION
Chemical and Physical Properties of U-44,590
Elemental AnalYsis:
Calcd. for C~H15N305:
C, 44.o8; H, 6.17; N, 17.13.
Found:
C, 44.14; H, 6.o8; N, 17.36.
Mo!ecular Weiqht: 245 (Determined by mass spectrometry)
Meltinq Point Ranqe: 141 - 142 C.
SPecific Rotation: [a]D = -5 (c, 0.9030 in H20)
Solubilities: Highly soluble in water, and lower alcohols,
-2-
' '; """ '
:
: - - -: - , - ~ , ~,
--~ 3143
1 ~ 7 ~ ~ 9
for example, methanol and ethanol; relatively insoluble . . . .-
in Me2CO, EtOAc, hydrocarbons, CHzC12 and CHC13.
Infrared AbsorPtion SPectra: U-44,590 has a characteristic
infrared absorption spectrum when suspended in a mineral
oil mull. Peaks are observed at the following wave lengths : -
expressed in reciprocal centimeters:
Band Frequency
(Wa~e Numbers) IntensitY
3440 M -
3400 M .: .
334 M rO
3190 M
3080 M
3000 W .-~
2960 (N = Nujol) S
, . . . .
2930 (N) S ;. .
2860 (N) S - :
1695 sh. S
1683 S
1510 M
1503 M , : .-
1483 . M :
1463 ~N) S :` ;
1440 S
1421 I M -
1407 M
1396 M
1375 (N) W .:
1350 M ..
1315 W
3143
.,
1~;)37~9
Band Frequency
(Wave Numbers) Intensity
1300 . W~
1280 W
1276 W
1262 W
1243 S
1230 sh. M
1195 W
1165 W
1133 W
1093 M
1085 W
1060 S
1011 S
985 M
971 W -
943 M .
885 W
872 W
' 849 W -
826 W . .
792 M
755 M
735 I W
Note: sh means a shoulder band.
U-44,590 also has a characteristic infrared absorption ..
spectrum when pressed in a KBr disc. Peaks are obser~ed at ~:
the fol1Owing wa~e lengths expressed in reciprocal centi- :
meters: `
.. ~
: 4 ~ .
~-, .
- . - . . :, . . ~: . - . . .
, ~ ~ : . , , -. ..
- - . , . . : . : .. . ~ . .
., . ~ . ,
314:~
.
1~3786~9
Barld F r eq uenc y
(Wave Numbers) Intensity
3440 S
5200 M
3080 M
~000 W
2970 W
2960 W
2935 W
2920 W
2870 W
1697 sh. S
1685 S
1510 M
1482 M
1461 S
1437 M
1420 M
1406 M
1396 M
1349 W
1310 W
1298 W
1290 W
1275 sh. M
1263 M
1243 S
1195 W
1165 W
1133 W
3 1097 .
~ .
,
- 3143
-
1(~378~39
Band Frequency
(Wa~e Numbers) Intensity
10'd5 M
1 o60 M
1010 M
985 W
971 W
942 M
883 W
870 W
847 W
827 W
791 M
754 W
733 W
15 Note: sh means a shoulder band.
Infrared band intensities, throughout this disclosure,
are indicated as "S", "M", and "W" respectively and are
approximated in terms of the backgrounds in the vicinity
of the bands. An "S" band is of the same order of inten-
sity as the strongest in the spectrum; "M" bands are be-
tween 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
transmission scal~.
The following is considered to be the structure of
U-44.590: -~-
', :
.. .
~ ;
.' ' : .
3143
1~378~9
~ .
Hl 3 sN-CH3
50 ~ N~
..
HOH2 ~ ~
~1 ~ ':, ' .
OH `
Thus, U-44,590 can be referred to by the tri~ial name
5,6-dihydro-5-azathymidine, or by its chemical name 1-
(2-deoxy-~-D-erythro-pentofuranosyl)-5,6-dihydro-5-methyl
s-triazine-2,4(lH,3H)-dione.
15Antibacteria ! Acti~itY of U-44.590 .
Orqanism No. of Inhibition (~q/ml)
- Strains
StaPhYlococcus aureus 1 ~1000 .
StrePtococcus hemolyticus 1 15.2
DiPlococcus Pneumoniae 1 500.
Klebsiella Pneumoniae 5 2.0 - >1000
Salmonella sp. 4 15.6 - >1000
Serratia marcescens 2 125
Pseudomonas aeruqinosa 5 >1000 ;`
Pasteurella multocPda 1 125
HemoPhilus sp. 5 31.2 - >1000
.,
Proteus ~ulaaris 3 >1000
Proteus mirabilis 3 ~1000 :
Proteus morqani 3 62.5 - 250
Proteus rettqeri 3 31.2 - >1000
- ; , . " :. -
,, - , . . ..
., . - . , ,.,: -' - . : ' ' '
314-S
~o~7~
The above antibacterial spectrum was obtained by a
standard agar dilution test with the following media and
conditions:
Difco Brain Heart Infusion Medium was used for all
5 test bacteria except P. multocida and HemoPhilus species
which were grown in Difco Blood Agar Base with 5g de-
fibrinated rabbit blood. All were grown aerobically at
37 C . ( except HemoPhilus species, grown anaerobically)
16-18 hours. Inocula were grown overnite (16-18 hours)
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 vivo testing of U-44,590 in mice infected with
selected microorganisms is as follows:
Activity (CD50 in mg/kg)
Mice, IP
Challenge Subcutaneous Oral
- Orqanism LD5~ Route Route --
Salmonella flexneri 40 38 (25-57) 62 .5
Escherichia coli 79 141 (116-172) 218 (154-307)
Proteus mirabilis1259 152 (96-240) lol (66-156) ~ -
Proteus ~ulqaris 79 lOo (66-152) <62.5 `
1 50 ~3
s hemo-
lvticus 100 - >160
ANTIVIRAL A~TIYITY O~ U-44,590
The following is an examp1e of the antiviral activity
of antibiotic U-44,590. The antibiotic is administered sub-
cutaneously to mice which are inoculated intravenously with
HerDes simDlex virus. Treatment is initiated two hours prior
to viral infection and is followed by treatment four times
-8-
'. ~
: . . - . - . - . . - - . ., -
. . .
- . - - . . . . . .
3143
1~378~39
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. Group 1 is treated with -
saline, Group 2 with 400 mg./kg./dose (mkd) U-44,590, ~ ~
Group ~ 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, 4 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 LD5bs, is inoculated ~
into the tail ~ein at 10 a.m. on day 0. Paralysis and --
death are recorded daily.
Hind leg paralysis usually preceded death by 1^2 ~-
days. All mice died that became paralyzed. Death pattern
of the 4 groups, as shown in the cur~es which follow,
illustrates the dose response obtained. Statistical
analysis of the results at day 11 indicates that all 3 ~
treated groups are significantly different from the con~
trol group (1).
:' ~'.' "
;~ ~'"'
,.,: . . :
---` 31 43
1~)378~9
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3143
~0378i~9
THE MlcRooRGANlsM
The microorganism used for the production of U-44,590 ~
is Stre~PtomYces platensis \~ar. clarensis, NRRL 8035. A ~ -
subculture of this microorganism can be obtained from the ~ -
5 permanent collection of the Northern Regional Research
Laboratory, U.S. Department of Agriculture, Peoria, Illinois,
U.S.A. -
The microorganism of this in~Jention was studied and
characterized by Alma Dietz of the Upjohn Research Labora-
tories.
A new soil isolate with hygroscopic spore masses, but
with s~ooth, hat-shaped (crescent) or braz ll-nut-shaped
(elliptical) spores, has been found to differ in certain
characteristics from the type culture strePtomyces platensis. F
An outstanding difference of the new culture is the produc-
tion of antibiotic U-44,590. The new isolate can be recog-
nized as a ~ariant of StreptomYces platensis by its cultura1,
- . .
microscopic, and biochemical characteristics. Therefore, it
is proposed that this new isolate be designated StrePtomYces
Dlatensis ~ar. clarensis Dietz \~ar. no\~a. Rule 7 of the
: .
International Code of Nomenclature of Bacteria ~ Internation-
al Code of Nomenclature of Bacteria. 1966. Edited by the
Editorial Board of the Judicial Commission of the Interna- ~ -~
tional Committee on Nomenciature of Bacteria. Int. J.
Syst. Bacteriol. 1~: 459-490] was applied in designating
the ~ariety eptthet.
strePtomYces platensis ~ar. clarensis is compared with ~ -
the type species BtrePtomyces platensis Pittenger and Gott-
lieb [Shirling, E.B., and D. Gottlieb. 1968. Cooperati\~e
description of type cultures of Streptomyces III. Addi-
-11 - .
- . .
- . . , . - . , . .. ~
3143
1~378~9
tiona1 species descriptions from first and second studies.
Int. J. Syst. Bacteriol. 18:279 392] [Tresner, H.D., E.J.
Backus, and Jean A. Hayes. 1967. Morphological spore
types in the StreptomYces hyqroscop_cus-like complex.
Appl. Microbiol. 15:637-639] NRRL 2369, and two recently
characterized strains: BtrePtomyces platensis NRRL 3593
[E~ans, Ralph Henry Jr., and Samuel Owen Thomas. 1971.
Antibiotics AH272oe and AH272~2 and process for producing
same. U.S. Patent 3,592,925] and Streptomyces platensis
NRRL 3761 [Okuda, Tomohau, and Shigemi Awatagouchi. 1973.
Antibiotics YL 704 and preparation thereof. U.S. Patent
3,718,742]-
Color characteristics: Aerial growth white to yellow to
gray. Moist black hygroscopic patches on some media.
Melanin-negati~e. Appearance on Ektachrome [Dietz, A.
1954. Ektachrome transparencies as aids in actinomycete
classification. Ann. N.Y. Acad. Sci. 60:152-154] is
gi~en in Table 1. Reference color characteristics are
gi~en in Tables 2 and 3. The new culture may be placed
in the White (W), Yellow (Y), and Gray (GY) color series
of Tresner and Backus [Tresner, H.D., and E.J. Backus.
1963. System of color wheels for streptomycete taxonomy.
Appl. Microbiol. 11 :335-338].
Microscooic characteristics: Spore chains in tight spirals
uncoiling to long open spirals. Spore chains spiral (S) in
the sense of Pridham et al. [Pridham, T.G., C.W. Hesseltine,
and R.G. Benedict. 1958. A guide for the classification of
streptomycetes according to selected groups. Placement of
strains in morphological sections. Appl. Microbiol. 6:52-79].
Spore hat-shaped (crescent) or Brazil-nut (elliptical) shaped.
-12-
~ . : . :: , . . .
3143
~ ~ 7 8 ~ ~
Spores are the platensis-type of Tresner and Backus [Tresner, I
H.D., E.J. Backus, and Jean A. Hayes. 1967. Morphological
spore types in the Streptomyces hyqroscopicus-like complex.
Appl. Microbiol. 15 637-639]. Spore silhouette smooth by
direct obser~ation with the electron microscope. Spore
surface ridged with surface markings by the carbon replica-
tion technique of Dietz and Mathews [Dietz, A. and J. Mathews.
1962. Taxonomy by carbon replication. I. An examination
of StrePtomyces hyqroscopicus. Appl. Microbiol. 10:258-26~].
o CU!_ ural and biochemical characteristics: See Table 4, infra.
Carbon utilization: Growth on carbon compounds was deter-
mined in the synthetic medium of Pridham and Gottlieb [Prid-
ham, T.G., and D. Gottlieb. 1948. The utilization of car-
bon compounds by some Actinomycetales as an aid for species
determination. J. Bacteriol. 56 107-114], Table 5 and in
the synthetic medium of Shirling and Gottlieb [Shirling, E.B.
and D. Gottlieb. 1966. Methods for characterization of ~-
StrePtomYces species. Int. J. Syst. Bacteriol. 16:31~ 340],
Table 6.
TemPerature: The cultures grew well at 18 - 37 c. on Ben-
nett's, Czapek's sucrose, maltose-tryptone, and Hickey-Tres-
ner agars. Optimum growth was at 24 - 37 c . The new culture
and the type culture did not grow at 45 - 55 c. The cultures
designated NRRL 35CJ3 and NRRL 3761 grew at 45 c. but not at
55 C.
Antlblotlc-Producinq properties: See Table 7, infra.
Source: Soil
TYPe culture: StrePtomYces platensis Pittenger and Gottlieb
NRRL 2364.
TYpe ~ariety: StrePtomYces platensis ~ar. platensis NRRL 2364.
- .. . . .. . . .. . .
,. - - - . ................ ., . ~..... . . . . ... .
. - . .... . . . . . . . .
~1 4~
1()37~89
VarletY: StrePtomYces P!atensis \~ar. cl~rensis Dietz
- \~ar. no\~a.
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.. ... . . . . ... .
The new compound of the in~ention is produced when j :
the elaborating organism is grown in an aqueous nutrient
medium under submerged aerobic conditions. It is to be
understood, also, that for the preparation of limited
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 include glucose, brown sugar, sucrose, glycerol, ~ -
starch, cornstarch, lactose, dextrin, molasses, and the
like. Preferred nitroger sources include cornsteep liquor,
yeast, autolyzed brewer's yeast with milk solids, soybean -
meal, cottonseed meal, cornmeal, milk solids, pancreatic
digest of casein, fish meal, distillers' solids, animal i~
peptone liquors, meat and bone scraps, and the like. Com- ~ .
binations of these carbon and nitrogen sources can be used -
ad~antageously. Trace metals, for example, zinc, magnesium,
manganese, cobalt, iron, and the like, need not be added
to the fermentation media since tap water and unpurified
ingredients are used as components of the medium prior to
sterilization of the medium.
Production of the compound of the in~ention can be
effected at any temperature conduci~e to satisfactory
growth of the microorganism, for example, between about
o o o o
18 and 40 C., and preferably between about 20 and ~2 C.
Ordinarily, optimum production of the compound is obtained
in about 5 to 15 days. The medium normally remains neutral
during the fermentation. The final pH is dependent, in
part, on the buffers present, if any and in part on the
:. -
-34-
~ .
.
- ., - - - . - . -
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 of 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 ~egetati~e
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 variety 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-
tion 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 recovered from the culture medium by
separation of the ~ycelia and undissol~ed solids by con-
~entional means, such as by filtration or centrifugatton.
lhe antibiotic is reco~ered from the filtered or centri-
fuged broth by adsorption on acti~ated carbon. The acti-
~ated carbon is then washed with water to remo~e some
impurities. This is followed by-elutions with acetone:
~';. ~ . '
-35~
., .
-.
3143
~378~9
water solutions which remo~e the antibiotic from the
acti~ated carbon. The acetone in the eluates i5 remo~ed,
ad~antageously by e~aporation, and the remalnlng aqu~ou~
residue is lyophil ized to afford a crude preparation of - - ;-
antibiotic U-44,590.
A preferred purification procedure is to subject a -~ -
crude preparation of U-44,59P, as described abo\~e, to
chromatography on sil ica gel from which U-44,590 is
eluted. Fractions which show acti~Jity
against the bacterium Klebsiella Pneumoniae on a standard
agar plate test, are pooled and taken to dryness to yield
a relati~ely pure preparation of U-44,590. Further purifi
catton is achie~ed by acetylation to a crystalline diacetate -
deri~ative of U-44,590. Zemplen [G. Zemplen and E. Pacsu, o ~ -
Ber., 62, 161~ (1929~] de-esterification (trans-esterifi-
cation) with sodium methoxide in methanol, and neutraliza-
tion of the catalytic amount of base with carbon dioxide t' ,~, ;,
gi~es the free antibiotic U-44,590 which crystallizes ~ -
readily from methanol-ethyl acetate to gi~e a pure prepara~
tion of U-44,590.
Antibiotic U-44,590 is acti~e against strePtococcus
hemolyticus and, thus, can be used to dlsinfect inStruments~ -~
utenslls or surfaces when contaminated with this microorgan- `~t
ism, where the inacti~ation of this microorganism is desir-
able. Also, U-44,990 is acti~e against Escherichia coli
~nd can be used to reduee, orrest, and/or cradlcate ~llme
production in papermill systems because of its antibacterial
ctlon against this bacterium. Antibiotlc U-4~,590 can also
be used to prolong the llfe of cultures of `rrlchomonas
foetus, Trichomonas hominis, and Trichomonas ~aainalis
-36- . ,: ' ' ,.
-~
~ 3
.
~o37~89
by freeing them of Escherichia coli contamination. Furt~ r.
U-44J590 can be used to inhibit the growth of E. coli in
hospital flower ~ases where it has been reported to exist
and present a hazard to hospital patients. See Clinical
Medicine, February, 1974, Page 9.
No~el acyl deri~atives of U-44,590, as disclosed
herein, can be used for the same antibiotic purposes as
U-44,590 in en~ironments possessing means to deacylate
the compound to U-44,590. Thus, the acyl deri~ati~es of
U-44,590 can be used to treat laboratory mice infected
with a Gram-negati~e bacteria, for example E. coli, as
disclosed herein. Further, acyl derivati~es of U-44,590
can be used, ad~antageously, to upgrade U-44,590. This
- is 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 proces~
and products of the present in~ention but are not to be
construed as l;miting. All percentages are by weight and ~-
sol~ent mixture proportions are by ~olume unless other-
wise noted.
Exam~le 1
Part A. Fermentation <
A soil stock df StrePtomYces platensis ~ar. clarensis.
NRRL ôO35 is used to inoculate a series of 500-ml. Erlen- ~ ~ -
meyer flasks, each containing 100 m!. of sterile seed
medium consisting of the following ingredients~
Glucose monohydrate ~ 10 Gm/l ~
Bacto Peptone (Dif 10 Gm/l ;
-37~ ;
. .
Al. ~
314
1()378~9
- Bacto Y~ast Extract (Difco) 2.5 ~im/l
Deionized water Balance --
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 inoculate
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 10~ ml. -
of fermentation medium. The fermentation medium consists ~ -
of the following ingredients:
- Brer Rabbit ~ olasses
(RJR Foods Inc., N.Y.J N.Y. 10017) 20 ml ~ -
Yeast Extract (Difc~ Detroit,
Michigan 1 Gm/l ~--
Glucose monohydrate 10 Gm/l
Dextrin (Corn Products Co. Inter- -
national Inc., International Plaza -~
Englewood Cliffs, New Jersey 07632jlO 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
28 C. on a Gump rotary shaker operating at 250 r.p.m. ;
w;th a 2 1/2 inch stroke. Ucon antifoam (a synthetic
- defoamer supplied by Union C;arbide, N.Y., N.Y.) jS used
if needed. Har~est is usually after 5 to 12 days of
fermentation.
~he antibiotic titer of the fermentation beer can
be monitored by an agar plate disc assay using the bacter-
ium Klebsiella pneumoniae. This bacterium is inoculated
into the assay agar (Streptomycin Assay Agar, BBL, Cockeys-
~ille, Maryland, 21030) of the following composition: -
- ~'
A~ ~
..... , ,. ,,, , , , ~ "
~14~
,
~ 0 37 ~ ~ 9
Beef Extract 1.5 G~/~
Yeast Extract 3.0 Gm/l
Gelysate Peptone, supplied by
Baltimore Biological 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~
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
-15 po~ency of the antibiotic sample. Thus, a 20 mm. zoneof inhibition using a 12.7 mm. paper disc to which o.o8 ml.
of antibiotic sample has been applied is expressed as one
- bio unit per ml. (1 BU/ml.).
Part B. Reco~_rY ;
, . . " .
Whole fermentation beer (ca 1600 ml. assaying 5
- BU/ml. against K. ~neumoniae), obtained as described
abo~e, Ts filtered using diatomaceous earth as a filter ; ~ -
aid. The filter cake is washed with water. The clear ~ ;
beer and wash (1800lml.) 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 dis-
carded. The column is then washed with 1 liter each of
a 1%, 2% and 5% acetone:water concentration. These
eluates are also dTscarded. The column is then eluted
"..~ ,
~c, ' ' ' '
3143
1~37E~9
with 1 liter each of a 10~, 25,~ and 50~6 acetone:water
concentration. These eluates, which contain antibiotic
U-44~590~ are pooled and the acetone is removed on a
rotatory e~aporator at :~i0 C~/15 mm. Hg. The resulting
acetone-free preparation is shell-frozen to an aqueous
residue and then lyophilized, yield~ 3~55 grams assaying
2 BU/mg. of U-44~590 against K. PneUmOniae. This prep-
aration, labeled for con~enience as Solid 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~ ). The column measures 3.8 x 88 mm.
Solid Q, obtained as described abo~e, is added on the top
of the column and the column is then eluted with methanol:
chloroform (1:1 v/v) . Acti~/e fractions, as determined
by the above-described K. pneumoniae assay, are pooled r`
and the solvent is remo~ed from said pooled fractions
by use of a rotatory e~/aporator at ~O C~/15 mm. Hg.;
yield, 8~o mg. assaying 7~5 BU/mg. of antibiotic U-44,590.
Part C. Purification No. 1
A preparation of antibiotic U-44,590, obtained as
described abo~e in Part B., is subjected to chromatography
on silica gel using the solvent system ethyl acetate:
methanol (6:1 v/~) to gi~e a purer preparation containing
U-44J590~ The procedure for this purification step is as
follows:
A column of silica gel (Merck-Darmstadt, 115 grams/
gram of the U-44,590 preparation being chromatographed)
in ethyl acetate: methanol (6:1 ~/~) is prepared by pour-
~0 ing a slurry of silica in the sol~ent into the column to
~40~
- . . ~ . ~ . ......................... ...
.
--~ 3143
~37~9
gi~e a height-diameter ratio of 10:1 after being packed.
The U-44,590 preparation, obtained as described above in
Part B, is dissol~ed in methanol~ silica is added (three
times the weight of the U-44~590 preparation used), and
5 this is then taken down to a dry powder on a rotatory
e~aporator at 40 /15 mm. Hg. The resulting dry solid is
added to the top of the silica column through a small head
of the sol~ent ethyl acetate:methanol (6:1 ~/~). After a
forerun of 4 liters~ 50 ml. fractions are collected and
assayed for acti~ity against K. pneumoniae. Active 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. pneumoniae (K.p.) acti~ity and
15 solids from a usual run are as follows:
Fraction Zone of Wt. of 501 id
Number Inhibition in Fraction : -
(using 12.7 (mqm)
mm. discs)
110 16 34.5
li5 30
120 33 30.9
125 ~5
1~0 37 40.8
135 37
140 36 35-7 ~ -
145 35
150 35 27.0
155 34
160 ~ 21.7
165 ~2
170 131 21.8
175 30
lôO 29 21.8
185 28
190 28 17.9
195 28
200 27 17.5
205 27
210 26 14.4
215 26
220 26 12.1
225 26
' .
-41 -
-, : . .. ,. : .
'. ' ' ; ~ ' ' -
., ,. ~ . ; ..
, ` . ; ,~
3143
~378i~
Fract;on Zone of Wt. of solid
Number Inhibition in Fraction
(using 12.7 (mqm)
mm. discs~
230 26 10.0
235 25
240 25 11.5
245 24
250 24 11.7
255 23
260 23 12.6
265 23
270 23 15.4
Fractions 12O-18OJ incl. are pooled and taken to dryness
on a rotatory e~aporator at 40 /7 mm. Hg. to gi~e a syrup,
wt. 2.669, assaying 54 K.p. BU/mg (Fractions 181-240,
incl. gi~e a syrup, 830 mg. ~2 BU/mg.,and fractions
241-300, incl. gi~e a syrup, wt. 710 mg., assaying 11 .
BU/mg.). The standard assayed 4 BU/mg. against the usual
assay for this standard of 6 BU/mg.
Part D. Purification No. 2
The preparations of U-44,590 obtained as described -
.
in Part C., can be further purified to a preparation of
essentially pure U-44,590 by passage o~er another silica `-
gel column using this time the sol~ent system methanol:
methylene chloride (1:8 ~/~). The procedure is as follows:
A U-44,590 preparation, as obtained in Part C~, (2.28
grams) is dissol~ed in methanol and 7 grams of silica gely
as described in Part C., is added. The sol~ent from this
mixture is remo~ed lon 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 ~olume 1500 ml., made up
in MeOH-CH2CI2 (1:8 ~/~)]. A forerun (1100 ml.) is collect- -
ed, followed by 50 ml. fractions. Fractions 141-200, in- ~:
clusi~e, weigh 390 mg. when taken to dryness in the form of
.''~' .
-42-
. : -- ,
31 4
~C~37t~9
a syrup. This material is shown to be almost pure U-44,5~30
by thin layer chromatography (tlc).
The tlc is conducted on silica gel plates using the
sol~ent system MeOH-CH2Cl2 (1:9 ~/~). Zones of the anti-
biotic are detected by spraying the plates with l04/MnO4spray, and with 50% aq. H2S04 fo1lowed by heating at
110 C. for ca 10 min. The Rf of the active material
in this sol~ent system is 0.11.
Part E. Purification No. 3
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 9.) of U-44,590 preparation, prepared
as described in Part D and assaying 160 BU/mg,is dîssolved
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 standing o~ernight at room temperature, ~ -
~olatile materials are remo~ed as completely as poss;ble ~
o
20on a rotatory e~aporator at 40 /15 mm. Hg., and finally ~ -
under high ~acuum, to gi~e a tan syrup.
This syrup ;s stirred with CH2Cl2 (200 ml), and a
colorless, flocculent precipitate is remo~ed by filtration ;
and washed with CH2Cl~ until the washings are colorless
25Ihe precipîtate is Idiscarded. The comb;ned filtrate and
washings are washed with aqueous HCl (N/20, 100 ml) twice,
the aqueous layer being ac;dic after the second wash. The
aqueous layers are discarded. The organic phase is then
washed with water (100 ml), saturated aqueous NaHC03 (100 ml),
30again with water (100 ml), and dried (Na2S0~). The aqueous
~43-
.-; .~ . ..
'`. : ' ' , . :'' .
3143
~0;~7~89
layers are discarded.
Remo~al of sol~ent on a rotatory e~aporator at 40 and
15 mm. Hg. gi~es a dark syrup (21.10 9), which is dissolved
in EtOAc (50 ml) by warming on a steam-bath. On cooling,
crystallization occurs; the solid is remo~ed by filtration, -`-
washed with EtOAc, and dried at 60 /15 mm. Hg., to gi~e
essentially pure 3',5'-di-0-acetylated U-44,590 (12.01 9.,
m.p. 123-124.5 C.). Recrystallization from the same
sol~ent gi~es U-44,590 diacetate, ha~ing a melting point
o
124-125 C. This compound is then labeled U-44,474.
U-44,474 is deacetylated to afford essentially pure
U-44,590 by the Zemplen procedure which is as follows.
The crystalline diacetate U-44,474 (24.90 9) is stirred
magnetically in methanol (400 ml), and methanolic sodium ~-
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~
is then added cautiously, with stirring, to neutralize the
methoxide, and the sol~ent is remo~ed on a rotatory e~apor- '~
ator at 40 and 15 mm. Hg., gi~ing 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
collected on a sintered filter at the pump, washed with ;-
methanol, and dried in a ~acuum o~en at 60 /15 mm. Hg.
Antibiotic U-44,590 crystallizes in colorless prismatic
needles, m.p. 141-142 . Remo~al of sol~ent from the fil-
trate plus washings on the e~aporator and crystallization ;
from methanol-ethyl acetate gi~es additional material
-44- `
... .
. 3î 43
~()37t~Y~9
(1.91 g, m.p. 140.5-141.5 )~
Example 2
The acylating procedure described in Example 1,
Part E can be substituted by acylating U-44,590 with any ~ ~
readily-a~ailable acylat;ng agent to gi~e acylated ~ -
U-44,590. This acylated U-44,590 product can then be
deacylated by methods well known in the art to yield
a purified preparation of U-44,590. Readily-a~ailable ~ '
acylating agents which can be used 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'g5'-di-esters of U-449590 are formed.
.:.
The 5'-mono-esters can be formed by standard procedures
using a minimum amount of acylating agent.
The 3'-mono-esters andphosphate can be formed by tritylating
U-44,590 to gi~e the 5'-trityl deri~ative, acylating this
compound with the desired acylating agent, selected from
those disclosed abo~e, to gi~e the 3'-mono-ester 5l-trityl
der;~ati~e, which then can be con~erted to the ~'-mono-ester
by remo~al of the trityl group. The tritylation procedure
disclosed in U.S. ~atent ~,426,012. Columns 4 and 5. or
other standard tritylation procedures can be employed.
rhe trityl group can be remo~ed by using the procedure
disclosed in U.S. Patent ~426,012, Column 6.
' .
.
,..,, ' .:.-, . .- . . ...
-45-
.
.
314
lU37~
ExamPle 4
The 5'-phosphate of U-44,590 can be prepared by
procedures as disclosed in the work of D. Mitsunobu,
K. Kato, and J. Kimura [J. Amer. Chem. Soc., ~, 6510
5 (1969)]. This compound can be used for the same pur-
poses as U-44,590.
The compounds, described abo\~e, being the derh/a-
ti\/es of U-44,590 which are within the scope of the sub-
ject in~ention, can be shown by the following structural
formula 0
~\ '
H-l~ N-CH3
o~\ N J . . ..
~/ \ . .
~ l
R ~ ; I
20 wherein R and Rl are selected frorn the group consisting of
a carboxylic acid acyl rad;cal of frorn 2 to 18 carbon atoms.
inclusi~e; or a halo-, nitro-, hydroxy-, amino-, cyano-,
thiocyano-, and lower alkoxy-substituted hydrocarbon carbox- .
ylic acid acyl radical of from 2 to 18 carbon atoms, inclu-
25 si~/e; R i5 hydrogenl and R' is as defined abo\~e or phc,sphate;
or R' ;s 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 al koxy-
substituted hydrocarbon carboxylic acid acyl radical of from
30 2 to 18 carbon atoms, inclusi\~e, or phosphate.
-46-
- , , . ; : . , - ~ : .
,. -
. ' . . - ., ~:~
::
:
_~ 3143
1 ~ 7 ~ ~ g
Additional characterïzation data for U-44,474,
prepared as disclosed in Example 1, Part E, is as follows:
Elemental Analysis: ~ .
Calcd. for C13H~N307 :.
Found: : -
C,47.41; H, 5.82; N, 12.76; 0, 34.01.
Molecular Weiqht: 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 Frequenc
(Wa~e Numbers) Intensity
3210 M .
3080 M
2960 (~il) S .
2930 (o;l) S
2860 (oil) S : .
1750 S ~:
1732 S
1702 S
1520 S ~. .
1468 (oil) S :'
1411 M
1379 (oil) I S
1358 W `.` :
1327 W .
1318 W .
1310 W
1298 W
:~,
-47~
. ~ . . . . . . . . . .
1~37~)~9
Band Frequency
(Wave N_mbers) IntensitY
1280 M
1250 S
1227 S
1188 M
1148 W
1113 W
1097 S
lOy M
1030 S
1011 M :
1000 M ~
987 M . -
964 M ~ ~ -
957 M
950 M
936 W
892 M
863 M
828 M
791 M
775 M
755 M
740 I W `
~5 721 (oil) W .
675 W :.
668 W ,~
U-44,474 also has a characteristic infrared absorption
spectrum when pressed in a KBr disc. Peaks are obser~ed at
the following wa~e lengths expressed in reciprocal centi-
-48-
.
- ,
31L~
~ 7~;59 ~ -
meters:
Band Frequency
!wa~e Numbers) Intensity ~ -
3420 (water) W
3210 M
3080 M
2970 W
2960 W
2930 W
2880 W
2830 W
1750 S
1732 S
1703 S
1517 M
- 1468 S
1410 M
1382 M
1370 M
1248 S
1227 S
1186 M
1145 W
1095 M
1055 I M
1070 M
1011 M
999 M
986 M
3 963 M
~.,~ ,....
-49- :
. ~ .
-
3143
10;~7~9
Band Frequenc
(Wa\~e Numbe rs ~ I ntens i ty
949 M
9~2 W
890 M
863 M
825 M
79 0 M
773 M
754 M ~ r
7~9 W
668 W
-5o-
- . . . .
- .
: . .
