Note: Descriptions are shown in the official language in which they were submitted.
;
162/VLR85
- 1 - 18307Y ,~
: : ~ TITLE ~F THE rNvENTIoN
NOVEL ANTHELMINTIC MILBEMYCIN ANALOGS OF ~OVEL ~ :
MICROORGANISMS
: 15 ~CROSS REFERENCE:TO RELATED APPLICATIONS
: This application is a eontinuation-in-part of ~ -~
;~ our copending application Serial Number 694,165,~f:iled
1 May 1991.
20 BACKGROUND:OF THE:INVENTION : ~ :
The instant~novel~compounds are related to~ .
tHe mi:lbemycin compounds:disclosed in U.S. ~atent
: 3,950,360.:~Howeve~r the~instant compounds
possess~significant~struc~tu:ral d:ifferences which ~`
~ ~ 25~ read}1y differen~iate:them from t~e prior art c~ompounds. ~`
; ~ : S ~ RY OF:T~E INV$NTION
This~invention:is concerned with novel
chemical compounds~. In particular, it is concerned
: 3~ :with novel macrocyclic ~lactoneB which are produced by
: the fermentati:on~of a nutrient medium with a ~train of
: the microor:ganism~Stre~tomvces hy~rQscopicus MA-6825,
: : MA-6864 or MA-6865.
: .
~: .,
~:
162/DLR85 - 2 - 18307IA
Thus, it is an object of thiæ invention to provide for
such novel compounds, and a method for preparing such
products microbiologically. It is a further object of
this invention to provide for the recovery and
purification of such compounds from the fermentation
broth. These substances have anti-
parasitic and insecticidal activity, in particu~ar
anthelmintic, acaracidal and nematocidal activity, and
it is, thus, an additional object of this invention to
provide novel antiparasitic and insecticidal
compositions containing the disclosed compounds.
Further objects of this invention will become apparent ~`
from the following description of this invention.
DESCRIPTION OF THE INVENTION
In accordance with this invention, novel
substances are described, which are prepared by growing
under controlled conditions, a previously undescribed
strain of microorganism, Streptomvces hy~roscopicus
MA-6825, MA-6864 or MA-6865. The compounds are
obtained by fermentation and recovered in ubstantially
pure form as described herein.
Based on taxonomic studies, the micro-
organism capable of producing these compoundæ iæ of a
new strain of the microorganiem St~eptomy~Q~
hv~ros~opicus. The cultures are designated MA-6825,
MA-6864 and MA-6865 in the culture collection of Merck
& Co., Inc., Rahway, N.J. A sample o~ these cultures,
capable of producing the herein described compounds,
have been deposlted on 28 January 1991 under the
Budapest Treaty on the International Recognition of the
Deposit of Microorganismæ for the Purpoæes of Patent
Procedure in the permanent culture collection of the
, .
162/DLR85 - 3 - 18307IA
American Type Culture Collection at 12301 Parklawn
Drive, Rockville, Md. 20852, and have been assigned the
accession numberæ ATCC 55144 for MA-6825, ATCC 55145
for MA-6864 and ATCC 55146 for MA--6865.
The morphological and cultural character-
istics of Strepto~vces hvgroscopicu~ MA-6825 are ~et
forth below:
We have compared culture MA-6825 with
lD Sankyo's milbemycin patent strain S~reptomvces
hygroscopicus subsp aureolacrimosis, MA-4830. The
following is a general description of these strains.
Observations of growth, general cultural
characteristics and carbon source utilization were made
in accordance with the methods of Shirling and Gottleib
~Internat. J. System. Bacteriol. 16: 313-340).
Chemical composition of the cells was determined using
the methods of Lechevalier and Lechevalier (in
Actinomycete Taxonomv, A. Dietz and D.W. Thayer, Ed.
Society for Industrial Microbiology, 1980). Coloration
of the culture was determined by comparison with color
standards contained in the Inte~r-Society Color
Council-National Bureau of Standards Centroid Color
Charts (U.S. Dept. of Commerce National Bureau of
Standards supplement to NBS Circular 553, 1985).
DNA-DNA homology of the strains was determined by the
method descri~ed by Kurtzman, et al. (Int. J. Syst.
Bacteriol. 30: 208-216).
162/DLR85 - 4 - 18307IA
Analysis of Cell Wall Composition - MA-4830
Peptidoglycan contains L-diaminopimelic ;
acid. Whole cell carbohydrate analysis reveals glucose ;;
and traces of xylose. MA-6825 - Peptidoglycan contains
L-diaminopimelic acid. Whole cell carbohydrate
analysis reveals glucose and traces of xylose.
:
General Growth Charac~eristics - MA-4830
Good growth on yeast extract-malt extract
(YME), glucose asparaginase (GAs), inorganic
salts-starch (ISS), oatmeal (Oat), sigma water (SW),
Czapek (CZ), Peptone Iron agars. Culture also grows
well in yeast extract dextrose (YED) broth. ~A-6825 -
.Good growth on YME, GAs, ISS, Oat, SW, CZ, Peptone Iron ~;~
agars. Culture also grows well in YE~ broth. Growth
occurs at 27 and 37C for both strains. `
. :.
Colonv morphology - MA-4830 - (On YME_21 d~
Substrate mycelium is medium olive brown (95
m. Ol Br) and leathery in texture. Colonies are
opaque, raised and erose. Aerial mycelium is initially
white, turning to light brown~gray (63 1 br Gy). Spore
mass i3 gray to black. MA-6825 - Substrate mycelium is
brown gray (64 br Gy) and leathery. Colonies are
25 opaque, raised and erose. Spore mass is gray to black. .
MicromorphQlogv - MA-4830
Aerial mycelium (0.76 ~m dia.) arises from a
substrate mycelium and i8 branched and flexous. In
mature cultures, the aerial mycelium commonly
terminates in tightly coiled spirals. Aerial growth
tends to coalesce into amorphous masses as the culture
... . . .. . .. .
.: . :; . . . : ::
162/DLR85 - 5 - 18307IA
ages. MA~6825 - Aerial mycelium (0.76 ~m dia.) arises ~-
from a substrate mycelium and is branched and flexous.
In mature culturels, the aerial mycelium commonly
terminates in tightly coiled spirals. Aerial growth
tends to coalesce into amorphous ma6ses in older
cultures.
Miscellaneous phvsiolo~ical reactions - ~A-4830
No melanoid pigments produced in tryptone
yeast extract broth, H2S negative in peptone-iron
agar. Culture produces a bright yellow, diffusible,
non pH-dependent pigment on ~ME and ISS agar. Carbon
~-D-lactose, ~-D-lactose, D-maltose, D-mannitol,
D-mannose, D-raffinose, L-rhamnose, sucrose, D-xylose;
poor utilization of D-arabinoset L-arabinose; no
utilization of xylose. MA-6825 - No melanoid pigments
prodùced in tryptone yeast extract hroth, H2S negative
in peptone-iron a~ar. Starch hydrolyzed. Culture
produces a bright yellow, diffusible, non pE-dependent
pigment on YME and ISS agar. Carbon source utilization
pattern is as follows: good utilization of
L-arabinose, cellobiose, D-fructose, inositol,
a-D-lactose, ~-D-lactose, D-maltose, D-mannitol,
D-mannose, D-raffinose, L-rhamnose, sucrose, D-xylose;
poor utilization of D-arabinose; no utilization of
~xylose.
.,
DNA Homolo~y
These s~rains were found to exhibit 69~70%
homology at Tm-25.
162/DLR85 - 6 - 18307IA
Dia~nosis
The results of these studies show that strain
MA-6825 exhibits a similarity in morphological and
physiological characteristics to ~~reptomyces
hygroscopicus subsp. aureolacrimo~ (MA-4830~. There
are differences between these strains in the
carbohydrate utilization patterns and some
physiological differences were noted in the response of
MA-6825 to certain suhstrates. Most notably, this
culture was able to sporulate on D-fructose, D-maltose,
D-mannose, D-raffinose, and a-D-glucose (control plate)
whereas MA-4830 did not. DNA homology data indicates
that the strains are related at the species level, but,
distinct from one another. The relationship between
these strains and Streptomyces violaceusni~er ~Str.
hygroscopicus is a subjective synonym of Str ~ ~:
violaceusniger, ~ergey's Manual of Systematic
Bacteriology, Vol 4, 1989) remains to be established.
Both MA-6825 and MA-4830 e~hibit marked differences in
- 20 carbohydrate utilization patterns from most other
validly named æubspecies of Str. hygroscopicus.
(Bergeyis Manual of Determinative Bacteriology, 8th
Ed., 1974) Only Str. hygroscopicus subsp ossamyceticus
shown a high similarity to either MA-6825 or MA-4830
and can be deffere~tiated on the basis of the
production of melanoid pigments.
- . . .- ~ :.
162/DLR85 - 7 - 18307IA
Carbohydrate utilization pattern of :
MA-4830 and ~A-6825 at 21 days
,
;
5 Carbon source Utilization byUtilization by
MA-4830 MA-6825 .
_
D-arabinose 2 2
L-arabinose 2 3 :
10: cellobiose 3 3
D-fructose : 3 3
: inositol 3 3
a-D-lactoSe 3 3
~-D-lactose 3 3
: 15 D-maltose 3 3
: D-mannito~ 3 3
: D-mannose ~ 3 3
D-raffinose 3 3
: L-rhamnose 3 ; 3
20: sucrose ~ 3 : 3
D-xylose 3 3:
L-xylose~ 0 ~ 0
a-D-glucose (control): 3 : : 3 ~.
,
: 25 :. ::~
: 3 = good utilization, 2 = moderate utilization, 1 = : .
poor utilization,~:0 = no utilization.
: : ~ :
: ~ ~
:~ :Note - MA-6825 was:found to sporulate on D-fructose,
: 30 ~D-maltosej D-mannos:e, D-raffinose, and ~-D-glucose,
MA-4830 did not respond similarly.
~.
.,,
:,
E ~ o ~ ~ L
~n E ~ ~ O ~ ~ n r~
L ~ L r~ ~ r- . L
L ~r O r- C O
L E ~ E E ~ 3 E e~ o r~
~ L . L ~ " L ~ ~ ~ ~
O o o
r- ~~ o
~n v ~ ~J ~" '~ ~
~ ~ 3 L L ~ o e ~e
,~ r- ~ r ~, O e
~ ~ C C ~ ~
~ L: ~ O n~ e 7 ~ ~
_ : :
1 5 ~ '
~ L ~ ~ ~ L : ~~ o ~ n
e- ~v C ~0 'C ~ CL ~ ~ ~0
L LO LO
,~ L ~ ~ L V~ L
~ ~ ~ 2 0 L L o ~ _ O ._ O ._ O ~ L _ ~
r- ~ C ; ~ O o ~ ~L~
~ ~ ,0 o ,~, t : ~ ~ ~ ~ ~ E
~ ~ ~2 5 ~ ~ ~ L ~ 3 ~ c
~ ~f L ~. L U~ e cL e ~ ~ L~ ~ O
~ L ~ _ ~ L
~: : m a~ ~
3 0 L ~ O O o O L o ~
~0
ë ~ I
C
L llS ~ L
L ¢~ L10 : V
162/DLR85 - 9 - 18307IA
The morphological and cultural characteris-
tics of MA-6864 and MA-6865 are ~ubstantially the same
as those of MA-6825 and thus all the cultures have been
characterized as Streptomyces _~~,roscopicus. The only
difference are that culture MA-6865 has slightly less
vigorous growth in Czapek o~ significant cultures but
this difference is not sufficient to render MA-6865 a
separate species from MA-6825 and MA-6864. In
addition, somewhat better production of the compounds
of this invention has been achieved with the ~A-6865
culture then with the other cultures.
The above description is illustrative of
strains of Streptomvces hygroscopicus MA-6825, MA-6864
and MA-6865 which can be employed in the production of
the instant compounds. However, the present invention
also embraces mutants of the above described
microorganisms. For example, those mutants which are
obtained by natural selection or those produced by
mutating agents including ionizing radiation such as
- 20 ultraviolet irradiation, or chemical mutagens such as
nitrosoguanidine or the like treatments are also
included within the ambit of this~invention.
The instant compounds are produced during the
aerobic fermentation of suitable aqueous nutrient media
2~ under conditions described hereinafter, with a
producing strain of Streptomyces hvgroscopicus MA~6825, -~
MA-6864 and MA-6865. Aqueous media such as those usied
for the production of many antibiotic substances are
suitable for use in this process for the production of
this macrocyclic compound. Such nutrient media contain
sources of carbon and~nitrogen assimilable by the
microorgani~m and generally low lev~ls of inorganic
salts. In addition, the fermentation media may contai~
162/DLR85 - 10 - 18307IA
small amounts of inorganic salts and traces of metals
necessary for the growth of the microorganisms, and
production o~ the desired compound. These are usually
present in sufficient concentrations in the complex
S sources of carbon and nitrogen, which may be used as
nutrient sources, but can, of course, be added
separately to the medium if desired.
In general, carbohydrates such as sugars, for
example dextrose, sucrose, maltose, lactose, dextran,
cerelose, corn meal, oat flour, and the like, and
starches are suitable sources of assimi~lable carbon in
the nutrient media. The exact quantity of the carbon
source whieh is utilized in the medium will depend, in
part, upon the other ingredients in the medium, but it
is usually found that an amount of carbohydrate between
0.5 and 5% by weight of the medium is satisfactory.
These carbon sources can be used individually or
several such carbon sources may be combined in the same
medium.
Various nitrogen sources such as yeast
hydrolysates~ yeast autolysates, yeast cells, tomato
paste, corn meal, oat flour, soybean meal, casein
hydrolysates, yeast extracts, corn steep liquors,
distillers solubles, cottonseed meal, meat extract and
2S the like, are readily assimilable by Streptomvces
hygroscopi~us MA-6825, MA-6864 and MA-6865 in the
production of the instant compounds. The various
sources of nitrogen can be used alone or in combination
in amounts ranging from 0.2 to 6% by weight of the
medium.
. . . .
162/DLR85 ~ 18307IA
Among the nutrient inorganic salts, which can
be incorporated in the culture media are the customary
salts capable of yielding sodium, potassium, magnesium,
ammonium, calcium, phosphate, sulfate, chloride,
carbonate, and like ions. Also included are trace
metals such as cobalt, manganese, and the like.
It should be noted that the media described
hereinbelow and in the Examples are merely illustra-
tive of the wide variety of media, which may be
lo employed, and are not intended to be limitative.
The following are Examples of media suitable - .
for growing strains of Streptomvces hy~roscopicus :
MA-6825, MA-6864 and MA-6865.
Medium 1
Dextrose 1.0 g '
Dextrin (Fisher) 10.0 g
Beef Extract (Difco 3.0 g
Yeast Autolysate (Ardamine pH, 5.0 g .
Yeast Prod.)
NZ ~mine Type E (Sheffield) 5.0 g -
MgS04 7~20
: Phosphate Buffer 2 ml
: ~ CaC03 ; 0.5 g
: 25 dE2 1000 ml
pH 7.0-7.2
Phosphate Bu~fer: KH2P04 91.0 g
Na2HP04 95.0 g
dH20 1000 ml
p~ 7.0
'~:
.. ,: ,. . ~ .:: , ,
. .
162/DLR85 - 12 - 18307IA
Medium 2
Yeast E~tract (Difco) 4.0 g
Malt Egtract (Difco) 10.0 g
Dextrose 4.0 g
dH20 1000 ml
Agar 20 g
pH 7.2
Medium 3
lo Basal
Sucrose 103 g
K2SO4 0.25 g
Glucose 10 ~ ::
L-Asparagine 1.8 g
Casamino Acids (Difco) 0.1 g
MgC12 6H2 10.12 g
Trace Element Mix A 2 ml
dH20 to 700 ml
Agar : 22.0 g -~
Post-sterilization additions, per 700 ml Basal:
;
: l~OO:ml of CaC12 solution (29.5 g/1000 ml dH2O)
: 100 ml of KH2P04 solution (0.5 g/1000 ml dH2O~ .
100 ml:of Tes solution (0.3 g Tris HCl + 0.1 g
EDTA + :
: ~ 0.14 g NaCl in 1000 ml d~20, adjust to pH 8.03
:
':
, . . . . . . . . .
162/DLR85 - 13 - 18307IA
_rac.e Element Mix A Composition:
Fe(S04)3 7H20 250 mg
MnCl2.4H20 500 mg
CuC12.2H2O 25 mg
CaCl2.2E2O 1~00 mg
H3BO3 50 mg :
(N~4)6M7024 4~20 20 mg
ZnSO4.7H2O. 100 mg
Co(N03)2 6H20 20 mg .
0.lN HCl 1000 ml
,~ , .
Medium 4
Dextrin (Fisher) 40 g
Distillers Solubles (Grain Processing 7 g
Corp.)
Yeast Extract (Oxoid) 5 g
CoCl2.6H2O : 50 mg
dH2 1000 ml
pH 7.3
~0 ~:
Medium 5
Dex~rose : 45 g
~Peptonized Milk (Sheffield); ~4 g
~ Ardamine pH (Yeast Products, Inc.) 2.5 g :
Polyglycol 2000 (Dow) 2.5 ml
~2 : 1000 ml
pH 7.0
~: 30
:,-
,
.
162/DLR~5 - 14 - 18307IA
Medium 6
Dextrose 2.0~/~
Yeas~ Extract (Difco) 2.0
Casamino Acids (Difco) 2 0
KN03 0.2
MgS04~7H20 0.05
NaCl 0.05
FeS04~7~20. 0.0025
CaC12o7H20 0.002
ZnSO407H20 0 . 001
MnS04~H20
d~2o 1000 ml
pH 7.0 with NaOH
Medium 7
Dextrose 0.1% :
Soluble Starch (Fisher) 1.0 :~
Beef Extract (Difco) 0.3
Yeast Autolysate (Ardamine pH : :-
Yeast Products) 0.5
NZ Amine Type E (Sheffield) 0.5
MgS04-7H~0 0.005
K~2P04 O.Olg2
Na2-~IPo4 0 . 0190 ,~,
CaC03* 0.05
dH20 1000 ml ~:
p~ 7.0-7.2 with NaOH
* Added after p~ adjustment :
162/DLR85 - 15 - 18307IA
The fermentations employing Streptomyces
hygrosco~icus MA-6825, MA-6864 and MA-6865 can be
conducted at temperatures ranging from about 20C to
about 40C. For optimum results t it is most convenient -~
to conduct these fermentations at a temperature in the
range of from about 24C to about 30C. Temperatures
of about 27-2BC are most preferred. The p~ of the ~:
nutrient medium suitable for producing the instant
compounds can vary from about 5.0 to 8.5 with a
preferred range of from about 6.0 to 7.5.
Small scale fermentations are conveniently
carried out by placing suitable quantities of nutrient
medium in a flask employing known sterile techniques,
inoculating the flask with either spores or veg~tative
cellular growth of Streptomvces hv~roscopicus MA-6825,
MA-6864 or MA-6865, loosely stoppering the flask with
cotton and permitting the fermentation to proceed in a
constant room temperature of a~out 28~C on a rotary
shaker at from 95 to 300 rpm for about 2 to 10 days.
For larger scale work, it is preferable to conduct the
fermentation in suitable tanks provided with an
agitator and a means of aerating the fermentation
medium. The nutrient medium is made up in the tank and
after sterilization is inoculated with a source of ~ :~
25 vegetative cellular growth of Streptomy~es :
hy~roscopicus MA-6825, MA-6864 or MA-6865. The
fermentation is allowed to continue for from 1 to 8 :
days while agitating and/or aerating the nutrient
medium at a temperature in the range of from about 24O
to 37C. The degree of aeration is dependent upon
162/DLR85 - 16 - 18307IA
several factors such as the size of the fermentor,
agitation speed, and the like. Generally the larger
scale fermentations are agitated at about 95 to 500 RPM
and about 50 to 500 liters per minute of air.
The novel compounds of this invention are
found primarily in the mycelium on termination of the
Streptomvces hy~roscopicu~ MA-6825, ~A-6864 and MA-686
fermentations and may be removed and separated
therefrom as described below.
The separation of the novel compounds from
the whole fermentation broth and the recovery of said
compounds is carried out by solvent extraction and
application of chromatographic fractionations with
various chromatographic techniques and solvent systems.
}5 The instant compounds have slight solubility
in water, but are soluble in organic solvents. This
property may be conveniently employed to recover the
compound from the fermentation broth. Thus, in one
recovery method, the whole fermentation broth is
20 combined with approximately an equal volume of an `
organic solvent. While any organic solvent may be
employed, it is preferable to use a water immiscible
solvent such as ethyl acetate, methylene chloride,
chloroform, methyl ethyl ketone and~the like.
Generally several extractions are desirable to achieve
maximum recovery. The solvent removes the instant
compounds as well as other substances lacking the
antiparasitic activity of the instant compounds. If
the solvent is~a water immiscible one, the layers are
separated and the organic solvent is evaporated under
reduced pressure. Ir the solvent is water miscible, it
: ;~
162/DLR85 - 17 - 18301IA
can be extracted with a water immiscible solvent to
separate the entrained water. This solvent can then be
concentrated under reduced pressure. The residue is
placed onto a chromatography column containing
s preferably, silica gel. The column retains the desired
products and some impurities, but lets many oP the
impurities, particularly the nonpolar impurities, pass
through. The column is washed with a moderately polar
organic solvent such as methylene chloride, chloroform
o or hexane to further remove impurities, and is then
washed with a mixture of methylene chloride, chloroform
or hexane and an organic solvent of which acetone,
ethyl acetate, methanol, and ethanol and the like are -
preferred. The solvent is evaporated and the residue
further chromatographed using column chromatography,
thin layer chromatography, preparative layer
chromatography, high pressure liquid chromatography and
the like, with silica gelj aluminum oxide, dextran gels
and the like, as the chromatographic medium, with
20 variouF solvents and combinations of~solvents as the ,~
eluent. Thin layer, high pressure, liquid and
preparative layer chromatography may be employed to
detect the presence of, and to isolate the instant
compounds. The use ofi the~foregoing techniques as well
as others known to those skilled in the art, will
afford purified compositions containing the instant
compounds. The presence of the desired compounds is
determined by analyzing the various chromato~r~phic
fractions for biological activity against selected ~'
parasites, or physicochemical characteristics. The
```\
162/DLR85 - 18 - 18307IA
structures of the instant compounds have been
determined by detailed analysis of the various spectral
characteristics of the compounds, in particular their
nuclear magnetic reso~ance, mass, ultraviolet a~d
infrared spectra.
Based on these experimental data, the instant
compounds are believed to have the following structural
formulae:
'
~ 0~,0 ,,,
¦l OH-.
lS I
OH ~,~
OM~
,
3C NMR Data in CDC13 Solution: Chemical shifts for
13C spectra recorded in CDC13 solution are given in ppm
reIative to tetramethylsilane (TMS) at zero ppm using
the solvent peak at 77.00 ppm as an i~ternal standard:
11.35, 17.50, 17.87, 18.98, I9.01, 19.29, 19.34, 27.67,
34.36 x 2~ 35.59, 36.48j 36.77, 37.25, 40.08, 40.96,
48.96, 56.75, 57.86, 67.02, 68.50, 71.4~, 75.72~. 76.80,
: 82.78, 97.54, 118.30, 125.44, 126.07, 129.30, 135.05,
30 138.56 5 139.04, 141.01, 173.43, 176.14.
lH NMR data-See Figure 1
162/DLR85 - 19 - 18307IA
MS: This compound has the molecular formula C36E5409 :
(calc 630.3768; found 630.3782) and forms a di-TMS
derivative. Characteristic fragment ions in the EI
spectra are observed at m/z 153/181 (defining C17-C25),
488 ([M-142], defining Cl-C5 with a C5-0-methyl, 542
([M-88] for loss of a C4 acid moiety) 265 (C16H2503,
calc. 265.1804, ~ound 265.1797; defining C13-C25
bearing oxygen at C13). The lack of an ion at m/z 151
coupled with the TMS count to indicate a ~-type
milbemycin.
: '
~ o~,o
¦ I OH -
II ~
0~ ~'; -: '
H O
13C NMR Data in CDC13 Solution: Chemical shifts ~or
: 13C spectra recorded in CDC13 solution are given in ppm
relative to tet:ramethylsilane (TMS) at zero ppm using
the s:olvent peak at 77.00 ppm as an internal standard:
~: 10.91, 17.87, 18.60, 18.97, 19.00, 19.34, 19.95, 27.67,
34.33 34.50, 35.59, 36.47, 36.73, 40,07, 41.04, 45.63,
67.00, 67.67, 68.49 x 2, 71.43, 79.23, 80.24, ~.3.26,
30 97.55, 118.11j 120.02, 124.55, 126.02, 135.49, 137.90, :-
140.96, 173.58, 176.~5.
1~ NMR ~ata-See Figure 2
, ., ~ . ........... . . .. .... . ...... .
.- , , :, , . , , ,. . .: . . : :-
162/DLR85 - 20 - 18307IA
MS: This compound has the molecular formula C35~5009
(calc 614.3455; found 614.3460) and forms a di-TMS
derivative. Characteristic fragment ions in the EI
spectra are observed at m/z 153/181 (defining C17-C25),
486 (~M-128] defining Cl-C5 with a C5-hydroxyl), 525
([M-88] for loss of a C4 acid moiety), 265 (defining
C13-C25 bearing oxygen at C13), and 151 (defining
C6-C12 and an a-type milbemycin).
~ 0~,0
il OH . .~
III
O ~~
OH
`:~
, ~ . " . . .. ..
162/DLR85 - 21 - 18307IA
3C NMR Data in CDC13 Solution: Chemical shifts for
3C spectra ~ecorded in CDC13 solution are given in ppm
relative to tetramethylsilane (TMS) at zero ppm using
the solvent peak at 77.00 ppm as an internal standard.
Carbons in the vicinity of the 2-methyl position of the
ester side chain exhibit distinct chemical shifts for
the two diastereomers whic integrate for approximately ~:
half the intensity of carbon resonances belonging to
o carbons which axe remote from the ester side-chain and
are enclosed in parenthesis and denoted as (x .5):
(10.93 x 5)1 (ll.oo x .5), 17.87, (18.58 x .5), (18.62
x .5), 19.33, 19.95, 22.37, (25.83 x .5), (26.84 x .5), -
27.67, 34.48, 35.59, 36.47, 36.73, (39.93 ~ .5), 39.98,
~1.02, (41.38 x .5), 43.79, 45.63, 66.99, 67.67, 6~.48
x 2, 71.42, 79.23, 80.24 (83.19~x .5), (83.29 x .5),
97.55, 118.11~ 120.02, (124.54 x .5), (126.06 x .5),
(126.15 x .5), (135.46 x .5), (135.51 x .5), (I37.78 x , .:
~: .5),~ (137.86 x .5), 137.9, 140.95, 173.6 x 2.
0
lH NMR data-See Figure 3
MS: ~This compound has the molecular formula C36H5209
: (calc 628.3611;::found 628.3645) and forms a di-TMS;
25 derivative. Characteri~stic fragment ions in the EI '~
spectra observed~at m/z 153/181 (defining C17-C25), 500
: ([M-128] defining Cl-C5 with a C5-hydroxyl), 526
([M-102] for loss of a C5 acid moiety)~ 265 (defining
C13-C25 bearing oxygen at C13), and 151 (defining
C6-C12 and an a-type milbemycin).
,
. ~ . ~ . . . . .~, . . .. .. . ..
.. .. . .
162/DLR85 - 22 - 18307IA
oJ~
CH3 ~~ ~CH3
,~ 'O CH3 :~:
CH3 11
~ 0~,0
~3
: 13C NMR Data in CDC13 Solution: Chemical shifts for
13C spectra recorded in CDC13 solution are given in ppm
relative to tetramethylsilane (TMS) at zero ppm using
15~ ~the solvent peak at 77.00 ppm as an internal standard:
13.05,~15.56, 17.70, 18.84, 22.26, 22~4~, 22.57, 25.~6, .
34.54, 35.97, 36;.13, 36.16, 37.98, 41.309 42.24, 42.92, :~
45.53, 48.51, 64.65, 64.72, 6:8.29~68.51,~68.53,:69.38, ~ -
75.42, 75.48, 79.01, 80.34, 99.~99~, 120.3~ 0.55, ~: :
121.88, 123.36, 127:.11, 128.61 x 2, 129.29 x 2, I34.00 :
:~ ~ (weak),:136.36, 137.4 (weak), 139.18, 143.11, 171.30, : ~:
:~ ~ 172.:98, 177.91. ~ : ~
:,
: ~ lH NMR~data-See Flgure 4
25~
MS:: This compound has the molecular weight 806 by
FAB-MS. EI exhibits a pseudo-molecular ion at m/z 670
which~:~corresponds to [M-136] wherein the 136 m~iety is
:
~ 30
:
,~ -
:: ~ : ,
:~ ~ : : :
.,
162/DLR85 - 23 - 18307IA
phenylacetic acid as indicated by fragment ions at m/z
136 and 91 (as in compound V). HR-~I-MS affords
C38E54010 (calc 670.372; found 670.382) which plus
phenylacetiC acid (CgHgO2~ yields C46H6212 for t e 1'
5 molecular formula. Fragment ions indicate a milbemycin :
a5 analog wherein a phenyl acetic acid moiety is
present at the C4 methyl posi~ion.
~3
CH3 ll
~ 0~,0 ;':'
V ~
<O ~ ~ :
.
'~ .
`
,,
~ 25 ~ :
,-
:'
,~ ; .. .. ~,:, : , :
162/DLR85 - 24 - 18307IA
3C NMR Data in CDC13 Solution: Chemical shifts for
13C spectra ~ecorded in CDC13 solution are given in ppm
relative to tetramethylsilane (TMS) at zero ppm using
the solvent peak at 77.00 ppm as an internal standard:
15.49, 17.87, 1~.37, 22.Z8, 27.71, 34.68, 35.66, 35.97,
36.52, 36.61, 41.13, 41.18, 45.70, 48.53, 63.25, ~7.48,
68.15, 68.60, 68.90, 71.34, 77.95, 80.72, 97.55,
120.~5, 120.95, 122.78, 123.40, 127.23, 128.64 x 2,
129.29 ~ 2, 133.77, 136.78, 136.96, 138.98, 143.01,
171.30, 173.10.
lH NMR data-See Figure 5
MS. This compound has the molecular weig~t 662 by FAB `
and EI-MS. HR-EI-MS yielded the molecular formula
C39H5009 (found 662.3466, calculated 662.3455). The -~
pattern of characteristic fragment ions in the EI
spectrum indicated an al type Milbemycin with a 135
amu ester moiety at the C4 C~3 group. This moiety was .
assigned by MMR and MS as phenylacetic acid~
characteri~tic~phenylacetyl fragments at m/z 136,
C8H802 (Calc 136.0524; found 136.05Z2), and m/z 91
(C7H7, calc for 91.0548; found 91.0548). : :
The inætant compounds are:potent endo-and :
25 ecto-anti~arasitic~agents against parasites :
particularly helminths, ectoparasites, insects, and
acarides, infecting ~man, animals and plants, thus
having utility~ in human and animal health, a~riculture
and pest control in household and commercial areas.
: 30 The disease or group of diseases described
generally as helminthiasis is due to infection of an
animal host with parasitic worms known as helminths.
Helminthiasis i8 a prevalent and serious economic
problem in domesticated animals such as swine, sheep,
,.
'', ' : '
,: '.` ~,
162/DLR85 - 25 - 18307IA
horses, cattle, goats, dogs, cats, fish, buffalo,
camels, llamas, reindeer, laboratory animals, fur-
bearing animals, zoo animals and exotic species and
poultry. Among the helminths, the group of worms
described as nematodes causes widespread and often
times serious infection in various species of animals.
The most co~mon genera of nematodes infecting the
animals referred to above are Haemonchus,
Trichostrongvlus, Ostertagia, Nematodirus, Cooperia,
Ascaris, Bunostomum, Oesophagostomum, Chabertia,
Trichuris, Stron~vlus, Trichonema, Dictvocaulus,
Capillaria, Eabronema, Druschia, ~eterakis, T~xocara,
Ascaridia, Oxyuris, Ancvlostoma, Un~inaria, Toxascaris
and Parascaris. Certain of these, such as Nematodirus,
Cooperia, and Oesophagostomum attack primarily the
intestinal tract while others, such as Haemonchus and
Ostertagia, are more prevalent in the stomach while
still others such as Dictyocaulus are found in the
lungs. Still other parasites may be located in other
20 tissues and organs of the body such as the heart and .
blood vessels, subcutaneous a~d lymphatic tissue and
the like. The parasitic infections ~nown as
helminthiases lead to anemia, malnutrition, weakness,
weight loss, severe damage to the walls of the
intestinal tract and other tissues and organs and, if
left untreated, may result in death of the infected
host. The compounds o~ this invention have
unexpectedly high activity against these parasites, and
in addition are also active against Dirofilaria in dogs
and cats, Nematospiroides, Syphacia, Aspiculuris in
rodents, arthropod ectoparasites of animals and birds
such as ticks,~mltes, lice, fleas, blowflies, in sheep
162/DLR85 - 26 - 18307IA
Lucilia sp., biting insects and such migrating diperous
larvae as Hypoderma sp. cattle, ~astrophilus in horses,
and Cuter~bra sp. in rodents and nuisance flies
including blood feeding flies and filth flies.
The instant compounds are also useful against
parasites which infect humans. The moæt common genera
of parasites of the gastro-intestinal tract of man are
Ancylostoma, Necator, Ascaris, StrQngyloides,
Trichinella, Capillaria, Trichuris, and ~nterobius.
Other medically important genera of parasites which are
found in the blood or other tissues and organs outside
the gastrointestinal tract are the filiarial worms such
as Wuchereria, Brugia, Onchocerca and Loa, Dracunuculus
and extra intestinal stages of the intestinal worms
Strongvloides and Trichinella. The compounds are also
of value against arthropods parasitizing man, biting
insects and other dipterous pests causing annoyance to
man.
The compounds are also active against
household pests such as the cockroach, Bla~ella sp.,
clothes moth, Tineola sp., carpet beetle, At~genus -
p., the housefly Musca domestica as well as fleas,
house dust mites, termltes and ants. ~;~
~ The compounds are also useful against insect
pests of stored grains such as Tribolium sp., Tenebrio
sp. and of agricultural plants such as aphids,
(Acyrthiosiphon sp.); against migratory orthopterans ~
such as locusts and immature stages of insects living :
on plant tissue. The compounds are useful as a
nematocide for the control of soil nematodes and plant
parasites such as Meloidogyne sp. which may be of
162/DLR85 - 27 - 18307IA
',
importance in agriculture. The compounds are also
highly usefui in tseating acerage infested with fire
ant nests. The compounds are scattered above the
infested area in low levels in bait formulations whic~
are broght back to the nest. In addition to a
direct-but-slow onset toxic effect on the fire ants,
the compound has a long-term effect on the nest by
sterilizing the queen which effectively destroys the
nest.
The compounds of this invention may be
administered in formulations wherein the active
compound is intimately admixed with one or more inert
ingredients and optionally indlucing one or more
additional active ingredients. The compounds may be
used in any composition known to those skilled in the
art for administration to humans and animals, for ~;
application to plants and for premise and area .
application to control household pests in either a
residential or commercial setting. For application to
humans and animals to control internal and e~ternal
parasites, oral formulations,~in solid or liquid or
parenteral liquid, implant or depot injection forms may
be used. For topical application dip, spray, powder, -
dust, pour-on, spot-on, jetting fluid, shampoos,
collar, tag or harness, may be used.
For agricultural premise or area applications, liquid :
spray, powders, dust, or bait forms may be used. In
~addition "feed-through" forms may be used to control
nuisance flies that feed or breed in animal waste. The
compounds are formulated, such as by encapsula-
tion, to lease a residue of acti~e agent in the animal
waæte which controls filth flies or other arthropod
pests.
"
162/DLR85 - 28 - 18307IA
These compounds may be administered orally in
a unit dosage form such as a capsule, bolus or tablet,
or as a liquid drench where used as an anthelmintic in
mammals. The drench is normally a solution, suspension
5 or dispersion of the active ingredient usually in water
together with a suspending agent such as bentonite and
a wetting agent or like excipient. Generally, the
drenches also contain an antifoaming agent. Drench
formulations generally contain from about 0.001 to 0.5%
l~ by weight of the active compound. Preferred drench
formulations may contain from 0.01 to 0.1% by weight.
The capsules and boluses comprise the active ingredient
admi~ed with a carrier vehicle such as starch, talc,
magnesium stearate, or di-calcium phosphate.
Where it is desired to administer the instant
compounds in a dry, solid unit dosage form, capsules,
boluses or tablets containing the desired amount of ~ -
active compound usually are employed. These dosage
forms are prepared by intimately and uniformly mixing
the active ingredient with suitable ~inely divided
diluen~s, fillers, disintegrating agents, and/or
binders such as starch, lactose, talc, magnesium
stearate, vegetable gums and the like. Such unit
dosage formulations may be varied widely with respect
to their total weight and content of the antiparasitic
agent depending upon factors such as the type of host
animal to be treatedi the severity and type of
infection and the weight of the host.
When the active compound is to be adminis-
tered via an animal feedstuff, it is intimately
dispersed in the feed or used as a top dressin~ or in
the form of pellets or liquid which may then be added
- , .
162/DLR85 - ~9 - 18307IA
to the finished feed or optionally fed separately.
Alternatively, feed based individual dosage forms may
be used such as a chewable treat. Alternatively, the
antiparasitic compounds of this invention may be
administered to animals parenterally, for example, by
intraruminal, intramuscular, intravascular, intratra-
cheal, or subcutaneous injection in which the active
ingredient is dissolved or dispersed in a liquid `:
carrier vehicle. For parenteral administration, the
active material is suitably admixed with an acceptable
vehicle, preferably of the vegetable oil variety such
as peanut oil, cotton seed oil and the like. Other
parenteral vehicles such as organic preparation using
solketal, glycerol formal, propylene glycol, and
15 aqueous parenteral formulations are also used. The -
active compound or compounds are dissolved or suspended
in the parenteral formulation for adminis-
tration; such formulations generally contain from
0.0005 to 5% by wei~ht of the active compound.
Although the antiparasitic agents of this
invention find their primary use in the treatment
and/or prevention of helminthiasis, they are also
useful in the prevention and treatment of diseases
caused by~other parasites, for example, arthropod ;~
parasites such as ticks, lice, fleas, mites and other
biting arthropods in domesticated animals and poultry.
They are also effective in treatment of parasitic
diseases that occur in other animals includin~ humans.
The optimum amount to be employed for best results
will, of course, depend upon the particular compound
employed, the species of animal to be treated and the
type and severity of parasitic infection or
.
, . ,
162/DLR85 - 30 - 18307IA
infestation. Generally good results are obtained with
our novel compounds by the oral adminis-
tration of from about 0.001 to 10 mg per kg of animal
body weight, such total dose being given at one time or
in divided doses over a relatively short period of time
such as 1-5 days. With the preferred compounds of the
invention, excellent control of such parasites is
obtained in animals by administering from about 0.025
to 0.5 mg per kg of body weight in a single dose.
Repeat treatments are given as required to combat
re-infections and are dependent upon the species of
parasite and the husbandry techniques being employed.
The techniques for administering these materials to
animals are known to those skilled in the veterinary
field.
When the compounds described herein are
administered as a component of the feed of the animals,
or dissolved or suspended in the drinking water,
compositions are provided in which the active compound
or compounds are intimately dispersed in an inert
carrier or diluent. By inert carrier is meant one that
will not react with the antiparasitic agent and one
that may be administered safely to animals.
Preferably, a carrier for feed administration is one .
that is, or may be~ an in~redient of the animal ration.
Suitable compositions include feed premixes
or supplements in which the active ingredient is
present in relatively large amounts and which are
suitable for direct feeding to the animal or for
addition to the feed either directly or after an
intermediate d:ilution or blending step. Typical
carriers or diluents suitable for such compositions
include, for e~ample, distillers~ dried grains, corn
... . . , " ~ , . . .. . . .
16~/DLR85 - 31 - 18307IA
meal, citrus meal, fermentation residues, ground oyster
shells, wheat shorts, molasses solubles, corn cob meal,
edible bean mill feed, soya grits, crushed limestone
- and the like. The active compounds are intimately
dispersed throughout the carrier by methods such as
grinding, stirring, milling or tumbling. Compositions ;~
containing from about 0.005 to 2.0% weight of the
active compound are particularly suitable as feed
premixes. Feed supplements, which are fed directly to
the animal, contain from about 0.0002 to 0.3% by weight
of the active compounds.
Such supplements are added to the animal feed
in an amount to give the finished feed the con-
centration of active compound desired for the treatment
and control of parasitic diseases. Although the
desired concentration of active compound will vary
depending upon the factors pre~iously mentioned as well
as UpOII the particular compound~employed, the compounds
of this invention are usually fed at concentrations of
between 0.00001 to 0.002% in the feed in order to
achieve the desired anti- parasitic result.
In using the compounds of this invention, the
individual compounds may be prepared and used in that
form. Alternatively, mixtures of the individual A~-
compounds may be used, or other active compounds not
related to the compounds of this invention.
The compounds of this invention are also
useful in combatting agricultural pests that inflict
damage upon crops while they are growing or while in
storage. The compounds are applied using known
techniques as sprays, dusts, emulsions and the like, to
: ' : :, , . : ' , ':
162/DLR85 - 32 - 18307IA
the growing or stored crops to effect protection from
such agricultural pests.
The following examples are provided in order
that this invention might be more fully understood;
they are not to be construed as limitative of the
invention.
EXAMPLE 1
A frozen vial of spores of the cuIture M~
6865 (ATCC 551~6) was inoculated into 50 ml of medium 1
(seed medium) in a 250 ml baf~led erlenmeyer flask and
incubated for three days at 28C with agitation on a
rotary shaker at 220 rpm with a 5 cm throw.
Two ml of this seed was inoculated into 40 ml
of medium 8 (growth medium) in each of 100, 250 ml
ba~fled erlenmeyer fl~asks with agitation on a rotary
shaker at 220 rpm with a 5 cm throw. Incubation was ~.
continued ~or 10 days.
Medium 8
Figs 30.0 g
Dextrin 15.0 g
25 Primary yeast 10.0 g
CoC12-6H2O 10.0 mg
Beta~cyclodextrin 10.0 g
Distilled H2O 1000 ml
.: ,
p~ 7.4 (adjust with NaOH)
162/DLR85 - 33 - 18307IA
ISOLATION
EXAMPLE 2
:
Whole broth (3.8 liters) was filtered and the
filtrate discarded. The cell cake was extracted with
two liters of acetone stirring ~or two hours, then
filtered. The filtrate plus wash ~olume was 2.4
liters. The fi~tered acetone extract was concentrated
to 450 ml. The concentrate was e~tracted with 2 X 450
ml of methy.lene chloride. A~ EPLC analysis of the
extracts showed incomplete extraction and the aqueous
layer was extractsd with 600 ml of methyl ethyl
ketone. HPLC analysis of the aqueous layer showed
complete extraction. The methylene chloride and methyl
ethyl ketone e~tracts were combined and concentrated to
an oily residue.
, :
EXAMPLE 3
The oily residue from Example 2 was taken up
in 10 ml o~ 3:2 methylene chloride:methanol, to a total
volume of 16 ml. ~The so~lution was chromato- graphed
on 1.6 liters of LH-20 using methanol at a flow rate of
20 ml/min. collecting~ 20 ml fractions. Fractions 40 ;;
th~ough 55 were combined on the basis of HPLC analysis
and~the combined fractionæ were concentrated to
dryness. Reæidue 2.1 grams.
: : : :
EXAMPLE 4
A one liter column o~ E. Merck silica-gel 60,
230-400 mesh, was prepared ln 5:1 hexane:acetone.
.. . .. . . .
~ ~ \
162/DLR85 - 34 - 18307IA
The 2.1 gram residue from Example 3 was taken up in 9:1
hexane:acetone, to a volume of 20 ml . The solution was
chromatographed using 5:1 hexane:acetone at a flow rate
of 20 ml/min. collecting 2 X 400 ml forecuts followed
by one hundred and eight 20 ml fractions. The
chromatography was continued based upon ~PLC using 3:1
hexane:acetone at the same flowrate collecting fifty 20
ml fractions were combined as follows:
.
Fractions k~bels
5 thru 11 A
12 thru 15 B
16 thru 30 C
31 thru 49 D
50 thru 59 E ~. .
60 thru 75 F
76 thru 103 G
114 thru 129 H
130 thru 139 J
,
EXAMPLE 5
Two hundred ml of Fraction D ~rom Example 4 ~ :
was concentrated to dryness. The residue was taken up
in 250 mcl of methanol and chromatographed on a DuPont
Zorbaæ ODS 2.21 X 25 cm column a 60C using a solvent
system of 87/13 methanol/water at a flow rate of 10
ml/min. The effluent strea~ was monitored at 243 nm
using a Gilson model 116 U.V. detector, equipped with
0.05 mm path length cell, and a setting of 20 AUFS.
The fraction with an Rt at i8.8 min corresponding to
the uv absorbance at 243 um was concentrated to dryness
yielding 7 mg. of compound I.
162/DLR85 - 35 - 18307IA
EXAMPLE 5
Two hundred and fifty ml of fraction ~ from .
Example 4 was concentrated to dryness. The residue was
taken up 250 mcl of methanol and chromatographed on a
DuPont Zorbax ODS 2.21 X 25 cm column at 60C using a
solvent system of 87/13 methanol/water at a flow rate
of 10 ml/min. The effluent stream was monitored at 243
nm using a Gilson model 116 U.V. dector equipped with a
0.05 mm path length cell and a setting of 4 AUFS. The
~raction with an Rt of 18.4 min corresponding to a uv
absorbance at 245 nm was concentrated to dryness
yielding g 2.2 mg. of compound II. The fraction with
an Rt at 23.7 min., also with the same uv absorbance
was concentrated to dryness yielding 1.6 mg of compound
III.
EXAMPLE 7
Three hundred ml of Fraction ~ from Example 4
was concentrated to dryneiss. The residue was taken up
in 250 mcl of methanol and chromatographed on a DuPont
Zorbax ODS 2.21 X 25 cm column at 600C using a solvent
system of 87/13 methanol/water at a flow rate of 10
ml/min. The effluent stream was monitored at 243 nm
using a Gilson model 116 U.V. detector equipped with a
0.05 mm path length cell and a setting of 2 AUFS. The
fraction with an Rt at 28.6 min. correspondin~ to a uv
absorbance at 243 nm was concentrated to dryness
yielding 1.8 mg. of compound IV. The fraction with a
Rt at 38.2 min, also with the same uv absorbance was
concentrated to dryness yielding 2.2 mg of compound V.
-:
