Note: Descriptions are shown in the official language in which they were submitted.
-1
ANTIPARASITIC AGENTS
DESCRIPTION
This invention relates to antiparasitic agents and
in particular to compounds related to the avermectins and
milbemycins but having a novel substituent group at the 25-
position and to a process for their preparation.
The avermectins are a group of broad spectrum anti-
parasitic agents referred to previously as the C-076
compounds. They are produced by fermenting a strain of the
microorganism Streptomyces avermitilis ATCC 31267, 31271 or
31272 under aerobic conditions in an aqueous nutrient medium
containing inorganic salts and assimilable sources of carbon
and nitrogen. The morphological and cultural properties of
the strains ATCC 31267, 31271 and 31272 are described in
detail in British Patent Specification No. 1573955 of Merck,
published August 28, 1980, which also describes the isolation
and the chemical structure of the eight individual components
which make up the C-076 complex. The milbemycins are
structurally related macrolide antibiotics lacking the sugar
residues at the 13-position. They are produced by
fermentation, for example as described in British Patent
Specification No. 1390336 of Sankyo, published February 26,
1975 and European Patent Application Publication No. 0170006
of American Cyanamid, published June 27, 1979.
In our European Patent Application Publication No.
0214731 we disclose that by adding certain specified
carboxylic acids, or derivatives thereof, to the fermentation
of an avermectin producing organism it is possible to obtain
~.340~7~
-2-
novel compounds, related to the avermectins but having an
unnatural substituent group at the 25-position in place of the
isopropyl or sec-butyl group which is normally present.
The novel compounds produced are characterised in
that the substituent group at the 25-position is alpha-
branched i.e. the carbon atom attached to the C-25 ring
position is a secondary carbon atom linked to two further
carbon atoms.
In our European Patent Application A 0284176
published September 28, 1988 we describe and claim new mutant
strains of the microorganism Streptomyces avermitilis lacking
branched-chain 2-oxo acid dehydrogenase activity. Said
strains have been deposited on January 16, 1987 in the
American Type Culture Collection, Rockville, Maryland under
the designations Streptomyces avermitilis ATCC 53567 and ATCC
53568.
We have now discovered that, by using these new
mutant strains of Streptomyces avermitilis it is possible to
obtain a further range of novel avermectin derivatives, not
previously obtainable, wherein the C-25 substituent is linked
by an unbranched (primary) carbon atom. The novel compounds
are highly active antiparasitic agents having particular
utility as anthelmintics, ectoparasiticides, insecticides and
acaricides. The compounds can be subjected to conventional
chemical transformation reactions to obtain further novel
semi-synthetic derivatives. Thus, according to the present
invention there are provided compounds having the formula (I):
x
13~~1~7~
3
R~
R4 CH3 _ ~ ~,, C H3
~Ow CH 2 R2
CH3
O
OH~
O
O R3 CH3
wherein the broken line at the 22-23 position represents an
optional double bond and wherein either R1 is H or OH and the
double bond is absent, or, the double bend is present and Rl is
absent;
R2 is H, Cl-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, alkoxyalkyl or
alkylthioalkyl containing from 1 to 6 carbon atoms in each alkyl
or alkoxy group, wherein any of said alkyl, alkoxy alkenyl or
alkynyl groups may be substituted by one or more halo atoms; or a
C3-C8 cycloalkyl or C5-C8 cycloalkenyl group, either of which may
optionally be substituted by methylene or one or more Cl-C4 alkyl
groups or halo atoms; or a 3 to 6 membered oxygen or sulphur
containing heterocyclic ring which may be saturated, or fully or
partially unsaturated and which may optionally be substituted by
one or more C1-C4 alkyl groups or halo atoms; or a group of the
formula SR5 wherein RS is Cl-C8 alkyl, C2-C$ alkenyl,
C2-C8 alkynyl, C3-C8 cycloalkyl, C5-C8 cycloalkenyl, phenyl or
~.34~Q~~
4
substituted phenyl wherein the substituent is C1-C4 alkyl,
C1-C4 alkoxy or halo,
R3 is hydrogen or methyl;
and R4 is H or a 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyl-
oxy group of the formula:
H3C~ H3
CH30 CH
with the proviso that when R4 and R1 are both H and the double
bond is absent, R2 is not H or CH3.
In the above definition, alkyl groups containing 3
or more carbon atoms may be straight or branched chain. Halo
means fluoro, chloro, bromo or iodo.
The C-076 complex comprises eight distinct but
closely related compounds described as C-076 Ala, Alb, A2a,
A2b, Bla, Blb, B2a and B2b. The "a" series of compounds
refers to the natural avermectin wherein the 25-substituent is
(S)-sec-butyl and the "b" series to those wherein the 25-
substituent is isopropyl. The
~.34~6'~8
- 5 -
designations "A" and "B" refer to avermectins wherein the 5-
substituent is methoxy or hydroxy, respectively, and the numeral
"1" refers to avermectins wherein a double bond is present at the
22-23 position, and numeral "2" to avermectins having a hydrogen
at the 22-position and hydroxy at the 23 position.
In this application, the "a" and "b" identifiers have
been dropped. Identifiers A1, A2, B1 and B2 have been retained to
refer to non-natural avermectins having the structural features
corresponding to those of the natural avermectins as noted above.
Preferred compounds of the formula (I) are those wherein
R4 is 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy.
Also preferred are compounds of the formula (I) wherein
R2 is SR5 and R5 is methyl or ethyl.
In another group of preferred compounds R2 is methyl,
isopropyl or sec-butyl.
In a further group of preferred compounds R2 is branched
C3-C8 alkyl group substituted by one or more halo atoms, parti-
cularly 1-(trifluoromethyl)ethyl.
In accordance with the invention the compounds of
formula (I) wherein R1 is OH and the double bond is absent or
wherein the double bond is present and R1 is absent and R4 is
4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy are prepared by
fermenting a Streptomyces atrerrtiitilis mutant organism ATCC 53567
or 53568, as described in European Patent Application A 0284176,
in the presence of the appropriate carboxylic acid of the formula
R2CH2C02H, wherein R2 is as previously defined, or a salt, ester,
134078
6
or amide thereof or oxidative precursor therefor. The acid is
added to the fermentation either at the time of inoculation or at
intervals during the fermentation. Production of the compounds of
formula (I) may be monitored by removing samples from the
fermentation, extracting with an organic solvent and following the
appearance of the compound of formula (I) by chromatography, for
example using high pressure liquid chromatography. Incubation is
continued until the yield of the compound of formula (I) has been
maximised, generally for a period of from 12 to 16 days.
A preferred level of each addition of the carboxylic acid or
derivative thereof is between 0.05 and 4.0 grams per litre. The
best yields of the compounds of formula (I) are obtained by
gradually add~.ng the acid to the fermentation, for example by
daily additions of the acid or derivative thereof over a period of
several days. The acid may be added as a salt, such as the sodium
or ammonium salt, or as an ester, such as the methyl or ethyl
ester or as an amide, but is preferably added as the free acid.
Alternative substrates which may be used iH the fermentation are
derivatives which are oxidative precursors for the carboxylic
acids; thus, for example suitable substrates would be alcohols of
the formula R2(CH2)nOH or amine derivatives of the formula
R2(CH2)nNH2, wherein n is 2, 4 or 6, substituted lower alkanoic
acids of the formula 'R2(CH2)nC02H wherein n is 3 or 5 or
aldehydes of the formula R2(CH2)nCHO wherein n is 1, 3 or 5 and R2
is as previously defined. The media used for the fermentation may
be a conventional complex media containing assimilable sources of
carbon, nitrogen and other trace elements.
~..
~~~oo~s
After fermentation for a period of several days at a
temperature preferably in the range of from 24 to 33°C, the
fermentation broth is centrifuged or filtered and the mycelial
cake is extracted with acetone or methanol. The solvent extract
is concentrated and the desired product is then extracted into a
water-immiscible organic solvent, such as methylene chloride,
ethyl acetate, chloroform, butanol or methyl isobutyl ketone. The
solvent extract is concentrated and the crude product containing
the compounds of formula (I) is further purified as necessary by
chromatography, for example using preparative reversz phase, high
pressure liquid chromatography.
The product is generally obtained as a mixture of the
compounds of formula (I) wherein R4 is 4'-(alpha-L-oleandrosyl)-
alpha-L-oleandrosyloxy, R1 is OH and the double bond absent or Rl
is absent and the double bond is present and wherein R3 is H or
CH3; however the proportions can vary depending on the particular
carboxylic acid employed and the conditions used in the
fermentation.
We have found that a range of carboxylic acids as defined by
R2CH2C02H may be added to the fermentation to yield avermectins
having a novel substituent group at the 25-position. Examples of
particular acids which may be employed include the following:
methylthioacetic acid
ethylthioacetic acid
3-methylbutyric acid
3-trifluoromethyl butyric acid
3-methylpentanoic acid
n-butyric acid
8 1340fi78
cyclopentane acetic acid
thiophene-3-acetic acid
and propionic acid.
In one particular and preferred aspect of the invention, the
fermentation is performed in the presence of methylthioacetic acid
to yield predominantly the compound of formula (I) wherein RI is
OH, the double bond is absent, R2 is SCH3, R3 is CH3 and R4 is
4'-(alpha-L-oleandrosyl)-alpha-4-oleandrosyloxy, referred to
herein as 25-methylthiomethyl avermectin A2.
In another preferred aspect of the invention, the
fermentation is performed in the presence of propionic acid to
yield predominantly the compound of formula (I) wherein RI is OH,
the double bond is absent, R2 is CH3, R3 is CH3 and R4 is
4'(-(alpha-L-oleandrosyl)-alpha-4-oleandrosyloxy, referred to
herein as 25-ethyl avermectin A2.
In a further preferred aspect of the invention the
fermentation is performed in the presence of 3-methylbutyric acid
to yield predominantly the compound of forcqula (I) wherein R1 is
absent, the double bond is present, R2 is isopropyl, R3 is H and
R4 is 4'-(alpha-L-oleandrosyl)-alpha-4-oleandrosyloxy, referred to
herein as 25-isobutyl avermectin B1.
In a further preferred aspect of the invention, the
fermentation is performed in the presence of 3-trifluoromethyl
butyric acid to yield predominantly the compounds of formula (I)
wherein RI is OH, the double bond is absent, R2 is I-(trifluoro-
methyl)ethyl, R4 is 4'-(alpha-L-oleandrosyl)alpha-4-oleandrosyloxy
and R3 is CH3 or H, referred to herein as 25-(2-trifluoromethyl-
propyl)avermectin A2 and B2 respectively.
13~06~~
9
Compounds of the formula (I) wherein the double bond is
present and Rl is absent may alternatively be prepared from the
corresponding compound of formula (I) wherein R1 is OH and the
double bond is absent by a dehydration reaction. The reaction is
performed by first selectively protecting the hydroxyl groups at
the 5 and 4" positions, e.g. as the t-butyldimethylsilyloxy acetyl
derivative, then reacting with a substituted thiocarbonyl halide,
such as (4-methylphenoxy)thiocarbonyl chloride, followed by
heating in a high boiling point solvent, e.g. trichlorobenzene, to
effect the dehydration. The product is finally deprptected to
give the unsaturated compound. These steps together with
appropriate reagents and reaction conditions are described in
United States patent 4328335.
The compounds of formula I wherein R3 is H may also be
prepared from the corresponding compounds wherein R3 is CH3 by
demethylation. This reaction is achieved by treating the
5-methoxy compound, or a suitably protected derivative thereof,
with mercuric acetate and hydrolysing the resulting 3-acetoxy enol
ether with dilute acid to give the 5-keto compound. This is then
reduced using, for example, sodium borohydride to yield the
S-hydroxy derivative. Appropriate reagents and reaction
conditions for these steps are described in United States patent
4423209. '
The compounds of formula I wherein R1 is H and the double
bond is absent can be prepared from the corresponding compound
wherein the double bond is present and Rl is absent, by selective
catalytic hydrogenation using an appropriate catalyst. For
13~' ~~7~
example the reduction may be achieved using tris(triphenyl-
phosphine)rhodium (I) chloride as described in European Patent
Application Publication No. 0001689.
The compounds of formula (L) wherein R4 is H are prepared
from the corresponding compounds wherein R4 is 4'-(alpha-L-
oleandrosyl)-alpha-L-oleandrosyloxy by removing the 4'-(alpha-L-
oleandrosyl)-alpha-L-oleandrose group by mild hydrolysis with an
acid in an aqueous organic solvent to yield the aglycone having a
hydroxy group at the 13-position; this is then halogenated, for
10 example by reaction with a benzene sulphonyl halide, to yield the
13-deoxy-13-halo derivative which is finally selectively reduced,
for example using tributyltin hydride. In order to avoid
unwanted side reactions it is desirable to protect any other
hydroxy groups which may be present, for example using a tert-
butyldimethylsilyl group. This is then readily removed after
the halogenation or reduction step by treatment with methanol
containing a trace of acid. All these steps together with
appropriate reagents and reaction conditions for their performance
are described in European Patent Application Publication No.
0002615 of Merck, published June 27, 1979.
The compounds of the invention are highly active anti-
parasitic agents having particular utility as anthelmintics,
ectoparasiticides, insecticides and acaricides.
Thus the compounds are effective in treating a variety of
conditions caused by endoparasites including, in particular,
helminthiasis which is most frequently caused by a group of
parasitic worms described as nematodes and which can cause severe
economic losses in swine, sheep, horses and cattle as well as
,,:~.'~ i
1.34007
11
affecting domestic animals and poultry. The compounds are also
effective against other nematodes which affect various species of
animals including, for example, Dirofilaria in dogs and various
parasites which can infect humans including gastro-intestinal
parasites such as Ancylostoma, Necator, Ascaris, Strongyloides,
Trichinella, Capillaria, Trichuris, Enterobius and parasites which
are found in the blood or other tissues and organs such as '
filiarial worms and the extra intestinal stages of Strongyloides
and Trichinella.
The compounds are also of value in treating ect~oparasite
infections including in particular arthropod ectoparasites of
animals and birds such as ticks, mites, lice, fleas, blowfly,
biting insects and migrating dipterous larvae which can affect
cattle and horses.
The compounds are also insecticides active against household
pests such as the cockroach, clothes moth, carpet beetle and the
housefly as well as being useful against insect pests of stored
grain and of agricultural plants such as spider mites, aphids,
caterpillars and against migratory orthopterans such as locusts.
The compounds of formula (I) are administered as a
formulation appropriate to the specific use envisaged and to the
particular species of host animal being treated and the parasite
or insect involved. For use as an anthelmintic the compounds may
be administered orally in the form of a capsule, bolus, tablet or
preferably a liquid drench, or alternatively, they may be
administered by injection or as an implant. Such formulations are
prepared in a conventional manner in accordance with standard
veterinary practice. Thus capsules, boluses or tablets may be
~~40~7~
12
prepared by mixing the active ingredient with a suitable finely
divided diluent or carrier, additionally containing a
disintegrating agent and/or binder such as starch, lactose, talc,
magnesium stearate etc. A drench formulation may be prepared by
dispersing the active ingredient in an aqueous solution together
with dispersing or wetting agents etc. and injectable formulations
mad be prepared in the form of a sterile solution which may
contain other substances, for example, enough salts or glucose to
make the solution isotonic with blood. These formulations will
vary with regard to the weight of active compound depending on the
species of host animal to be treated, the severity and type of
infection and the body weight of the host. Generally for oral
administration a dose of from about 0.001 to 10 mg per Kg of
animal body weight given as a single dose or in divided doses for
a period of from 1 to 5 days will be satisfactory but of course
there can be instances where higher or lower dosage ranges are
indicated and such are within the scope of this invention.
As an alternative the compounds may be administered with the
animal feedstuff and for this purpose a concentrated feed additive
or premix may be prepared for mixing with the normal animal feed.
For use as an insecticide and for treating agricultural pests
the compounds are applied as sprays, dusts, emulsions and the like
in accordance with standard agricultural practice.
For human use the compounds are administered as a
pharmaceutically acceptable formulation in accordance with normal
medical practice.
134~J~'~8
13
The invention is illustrated by the following Examples in
which Examples 1 to 8 are Examples of the preparation of compounds
of the formula (I), Example 9 is an example of a drench
formulation and Examples 1~ and lI illustrate the antiparasitic
and insecticidal activity of the compounds.
~~~oo7g
14
EXAMPLE 1
25-Ethyl avermectin A2
A frozen inoculum (2 ml) of a culture of Streptomyces
avermi~ilis mutant organism ATCC 53568 was inoculated into 50 mls
of a medium containing starch (1 g), Pharmamedia (Trademark)
(0.75 g), ardamine pH (0.25 g), and calcium carbonate (0.1 g) in a
300 ml flask and incubated at 28°C for 2 days. This inoculum (50
ml) was transferred to a second inoculum flask (1 litre)
containing starch (20 g), Pharmamedia (15 g), ardamine pH (5 g)
and calcium carbonate (2 g) and incubated at 28°C fox a further 2
Gays. This inoculum was used to inoculate 60 litres of a medium
containing starch (6 kg), magnesium sulphate (60 g), Pharmamedia
(300 g), dipotassium hydrogen phosphate (60 g), ferrous sulphate
(0.6 g), calcium carbonate (420 g), glutamic acid (36 g), zinc
sulphate (0.06 g) and manganous sulphate (0.06 g) contained in a
60 litre fermenter. The fermentation was incubated at 29°C, with
agitation at 350 r.p.m. and aeration at 60 litres per minute.
Sodium propionate (140 g) was added after 9,6 hours and again after
192 hours (54 g). After 288 hours the mycelium was removed by
filtration and extracted with acetone (2 x 50 litres) followed by
ethyl acetate (50 litres). The acetone extract was concentrated
to approximately 10 litres and extracted with the above ethyl
acetate extract in three portions. The resulting ethyl acetate
layers were combined and evaporated to give a brown oil (112 g).
13~0~7~
is
The above oil was dissolved in 160 ml of a mixture of
methanol and water (95:5) and extracted with n-hexane (2 x
300 ml), the hexane extracts were discarded and the methanol layer
was evaporated to give a brown oil (87 g). The latter was
dissolved in methyleae chloride (250 ml) and stirred with silica
gel (80 g) and charcoal (30 g) for 1 hour. The silica and
charcoal were removed by filtration through Arbacel and the
filtrate was evaporated to give a yellow oil (53 g). The latter
was dissolved is methylene chloride (2.2 litres) and stirred with
1 0 alumina (I90 g) for two hours. The alumina was removed by
filtration and the filtrate stirred with more alumina (64 g) for a
further hour. The alumina was removed by filtration and the
combined filter cakes from both filtrations were stirred with
chloroform (1.3 litres) for 45 minutes and then the alumina was
removed by filtration. The filtrate was evaporated to give a pale
yellow oil (12.5 g) which was dissolved in diethyl ether and added
to a column of silica gel (400 g). The column was eluted with
diethyl. ether and 100 ml fractions were co~,lected. Fractions
21-28 were combined and the solvent evaporated to yield partially
20 purified material (150 mg). The product was dissolved in methanol
(0.5 ml) and chromatographed on a C18 Zorbax ODS (Trademark,
Dupont) column (21 mm x 25 cm) eluting with a mixture of methanol
and water (77:23) at 'a flowrate of 9 mls. per minute. The
relevant fractions were combined and the solvent evaporated to
yield the compound of formula (I) wherein R1 is OH, the double
bond is absent, R2 and R3 are both CH3 and R4 is 4'-(alpha-L-
Trade-mark
1~~(~~7~
16 .
oleandrosyl)-alpha-L-oleandrosyloxy as a white powder, m.p
146-153°C. The structure of the product was confirmed by fast
atom bombardment mass spectrometry performed on a VG Model
7070E mass spectrometer using a sample matrix of triethylene
glycol with solid sodium chloride, (M + Na)+ observed at m/e 899
(theoretical 899).
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 570, 295, 277, 275, 183, 165, 145, 127, 1I3, 95
and 87. .
EXAMPLE 2
25-Methylthiomethyl avermectin A2
A frozen inoculum (2 ml) of a culture of Streptomyces
' avermitilis mutant organism ATCC 53568 was inoculated into 50 mls
of a medium containing starch (1 g), Pharmamedia (Trademark)
(0.75 g), ardamine pH (0.25 g) and calcium carbonate (0.1 g) in a
300 ml flask and incubated for 2 days at 2$°C on a reciprocal
shaker operating at 180 r.p.m. An inoculum from this flask
(25 ml) was transferred to a 3 litre flask containing 600 mls
1~40~78
17
of the above medium (all ingredients fro rata) and was incubated
for two days at 28°C with agitation on a reciprocal shaker
operating at 180 r.p.m. The product from this flask (40 ml) was
used to inoculate a 3 litre fermenter containing 2.5 litres of a
medium consisting of starch (250 g), magnesium sulphate (2.5 g).
Pharmamedia (12.5 g), dipotassium hydrogen phosphate (2.5 g),
ferrous sulphate (0.025 g), calcium carbonate (1.75 g), glutamic
acid (1.5 g), zinc sulphate (0.0025 g), and manganous sulphate
(1.5 g). This fermentation was incubated at 28°C with agitation
at 1000 r.p.m. Methylthioacetic acid (1 g) was added at 96 hours
and the fermentation continued for a further 11 days. Then the
mycelium was removed by filtration and extracted with acetone (2 x
2 litres) followed by ethyl acetate (2 litres). The acetone
extract was concentrated to approximately 400 mls. and extracted
with the ethyl acetate extract in three portions. The resulting
ethyl acetate layers were combined and evaporated to give a brown
oil (4 g) which was dissolved in diethyl ether and applied to a
column of silica gel (100 g). The column was eluted with diethyl
ether and 50 ml fractions were collected. Fractions 11-18 were
combined and evaporated to yield partially purified material which
was further purified by chromatography on a C18 Zorbax ODS
(Trademark, Dupont) column (21 mm x 25 cm) eluting with a mixture
of methanol and water (77:23) at a flowrate of 9 mls. per minute.
The relevant fractions were combined and evaporated to yield the
compound of formula (I) wherein Rl is OH, the double bond is
absent, R2 is SCH3, R3 is CH3
13~4~78
18
and R4 is 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy as a
white powder m.p. 105-112°C. The structure of the product was
confirmed by fast atom bombardment mass spectrometry was performed
on a VG Model 7070E mass spectrometer using a sample matrix of
triethylene glycol with solid sodium chloride. (M + Na)+ observed
at m/e 931 (theoretical 931).
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 327, 309, 243, 225, 215, 145, I27, 113, 95 and 87.
EXAMPLE 3
25-(2-Trifluoromethyl)propyl avermectins A2 and B2
The procedure of Example 1 was followed but using
3-trifluoromethyl butyric acid as substrate instead of sodium
propionate. The relevant combined fractions from silica gel
chromatography containing the crude A2 derivative were
chromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21
mm x 25 cm) eluting with a mixture of methanol and water (75:25)
at a flowrate of 9 mls/min. Fractions 167-179 were combined and
evaporated to yield the compound of formula (I) wherein Rl is OH,
the double bond is absent, R2 is 1-(trifluoromethyl)ethyl, R3 is
CH and R4 is 4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a white
3
powder, m.p. 130-136°C. The structure of the product was
confirmed by fast atom bombardment mass spectrometry, performed on
a VG Model 7070E mass spectrometer using a sample matrix of
triethylene glycol with solid sodium chloride. (M + Na)+ observed
at m/e 981 (theoretical 981).
1340~7g
19
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 652, 377, 359, 293, 275, 265, 257, 247, 223,
179, 145, 127, 113, 111 and 87.
The relevant fractions from silica gel chromatography
containing the crude B2 derivative were combined and
chromatographed on a C-18 Dynamax (Trademark Rainin) column (41.4
mm x 25 cm) eluting with a mixture of methanol and water (73:27)
at a flowrate of 60 mls/min. Relevant fractions were combined to
yield the compound of formula (I), wherein R1 is OH,.the double
bond is absent, R2 is 1-(trifluoromethyl)ethyl, R3 is H and R4 is
4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a white powder, m.p.
158-160°C. The structure of the product was confirmed by fast
atom bombardment mass spectrometry performed on a VG Model 7070E
mass spectrometer using a sample matrix of triethylene glycol with
solid sodium chloride. (M + Na)+ observed at m/e 967 (theoretical
967).
Electron impact mass spectrometry was,performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 638, 377, 359, 293, 275, 265, 261, 257, 247, 223,
145, 127, 113, 111, 95 and 87.
EXAMPLE 4
25-Ethylthiomethyl avermectin A2
The procedure of Example 1 was followed but using
ethylthioacetic acid as substrate instead of sodium propionate.
After chromatography on a Zorbax ODS (Trademark, Dupont) column
fractions 24-72 were combined to yield the compound of formula (I)
1344~7~
wherein R1 is OH, the double bond is absent, R2 is ethylthio, R3
is CH3 and R4 is 4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a
white powder, m.p. 265-270°C (dec). The structure of the product
was confirmed by fast atom bombardment mass spectrometry performed
on a VG Model 7070E mass spectrometer using a sample matrix of
triethylene glycol with solid, sodium chloride. (M + Na)+ observed
at,m/e 945 (theoretical 945).
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 616, 473, 341, 323, 257, 239, 229, zll, I87, 179,
145, 113 and 87.
EXAMPLE 5
25-Isobutvl avermectin B1
The procedure of Example 1 was followed but using
3-methylbutyric acid as substrate instead of sodium propionate.
The relevant fractions from silica gel chromatography were
combined and chromatographed on a C18 Zorbax ODS (Trademark,
Dupont) column (21 mm x 25 cm) eluting with a mixture of methanol
and water (81:19) at a flowrate of 9 mls/min. Fractions 93-98
were combined and evaporated to yield the compound of formula (I)
wherein R1 is absent, the double bond is present, R' is isopropyl,
R3 is H and R4 is 4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy, as a
white powder, m.p. 120-123. The structure of the product was
confirmed by fast atom bombardment mass spectrometry performed on
a VG Model 7070E mass spectrometer using a sample matrix of
1~4~6~~
21
triethylene glycol with solid sodium chloride. (M + Na)+ observed
at m/e 895 (theoretical 895).
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 565, 319, 305, 221, 193, 169, 145, 127, 113 and
87.
EXAMPLE 6
25-(2-Methylbutyl) avermectins A2 and B1
The procedure of Example I was followed but using
3-methylpentanoic acid as substrate instead of sodium propionate.
The relevant fractions from silica gel chromatography containing
the crude A2 derivative were combined and chromatographed on a
C-18 Zorbax ODS (Trademark Dupont) column (21 mm x 25 cm) eluting
with a mixture of methanol and water (80:20) at a flowrate of 9
mls/min. Relevant fractions were combined to yield the compound
of formula (I), wherein Rl is OH, the double bond is absent, R2 is
sec-butyl, R3 is methyl and R4 is 4'-(alpha-L-oleandrosyl)-L-
oleandrosyloxy as a white powder, m.p. 120-I25°C. The structure
of the product was confirmed by fast atom bombardment mass
spectrometry performed on a VG Model 7070E mass spectrometer using
a sample matrix of triethylene glycol with solid sodium chloride.
(M + Na)+ observed at m/e 941 (theoretical 941).
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 337, 319, 253, 235, 225, 207, 179, 145) 113 and
87.
1340~7~
22
The relevant fractions from silica gel chromatography
containing the crude B1 derivative were combined and
chromatographed on a C-18 Ultrasphere (Trademark Beckman) column
(10 mm x 25 cm) eluting with a mixture of methanol and water
(85:15) at a flowrate of 4 mls/ min. Relevant fractions were
combined to yield the compound of formula (I), wherein Rl is H,
tha double bond is present, R2 is sec-butyl, R3 is H and R4 is
4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a white powder, m.p.
158-164°C. The structure of the product was confirmed by fast
atom bombardment mass spectrometry performed on a VG. Model 7070E
~hass spectrometer using a sample matrix of triethylene glycol with
solid sodium chloride. (M + Na)+ observed at m/e 909 (theoretical
909).
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 319, 235, 207, 183, 145, 113, 95 and 87.
EYAMPLE 7 ,
25-n-Propyl avermectin A2
The procedure of Example 1 was followed but using n-butyric
acid as substrate instead of sodium propionate. The relevant
fractions from silica gel chromatography were combined and
chromatographed on a C-18 Dynamax (Trademark Rainin) column (41.4
mm x c25 cm) eluting with a gradient of methanol and water from
(75:25) to (100:0) over 170 minutes at a flowrate of 40 mls/min.
One minute fractions were collected and fractions 36 and 37 were
combined to yield the compound of formula (I), wherein Rl is OH,
the double bond is absent, R2 is ethyl, R3 is methyl and R4 is
~~~oo~ts
23
4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a white powder, m.p.
150-155°C. The structure of the product was confirmed by fast
atom bombardment mass spectrometry performed on a VG Model 7070E
mass spectrometer using a sample matrix of triethylene glycol with
solid sodium chloride. (M+Na)+ observed at m/e 913 (theoretical
913).
Electron impact mass spectrometry was performed using a VG
Model 7070 F mass spectrometer. The m/e values for the principal
fragments were: 584, 309, 291, 225, 207, 197, 179, 145, 113 and
87.
EXAMPLE 8
25-Cyclonentylmethyl avermectins B1 and B2
The procedure of Example 1 was followed but using
cyclopentane acetic acid instead of sodium propionate. The
relevant fractions from silica gel chromatography containing the
crude B1 derivative were combined and chromatographed on a C-18
Dynamax (Trademark Rainin) column (41.4 mm,x 25 cm) eluting with a
mixture of methanol and water (84:16) at a flowrate of 60 mls/min.
Relevant fractions were combined to yield a compound of formula
(I), wherein R1 is absent, the double bond is present, R2 is
cyclopentyl, R3 is H and R4 is 4'-(alpha-L-oleandrosyl)-L-
oleandrosyloxy, as a 'white powder, m.p. 140-146°C. The structure
of the product was confirmed by fast atom bombardment mass
spectrometry performed on a VG Model 7070E mass spectrometer using
a sample matrix of triethylene glycol with solid sodium chloride.
(M + Na) observed at m/e 921 (theoretical 921).
..-
24
Electron impact mass spectrometry was performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 592, 331, 295, 257, 247, 218, 195, 145, 127, 113,
111, 95 and 87.
The relevant fractions from silica gel chromatogrpahy
containing the crude B2 derivative were combined and
chromatographed on a C-18 Ultrasphere (Trademark Beckman) column
(10 mm x 25 cm) eluting with a mixture of methanol and water
(80:20) at a flowrate of 4 mls/min. Relevant fractions were
combined to yield the compound of formula (I), wherein R1 is OH,
the double bond is absent, R2 is cyclopentyl, R3 is H and R4 is
4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a white powder, m.p.
155-165°C. The structure of the product was confirmed by fast
atom bombardment mass spectrometry performed on a VG Model 7070E
mass spectrometer using a sample matrix of triethylene glycol with
solid sodium chloride. (M + Na)+ observed at m/e 939 (theoretical
939).
Electron impact mass spectrometry was,performed using a VG
Model 7070F mass spectrometer. The m/e values for the principal
fragments were: 349, 335, 331, 289, 265, 261, 257, 247, 237, 219,
195, 179, 145, 127, 113, 111, 95 and 87.
EXAMPLE 9
Drench Formulation
The product of any one of the preceding Examples was
dissolved in polyethylene glycol (average molecular weight 300) to
give a solution containing 400 micrograms/ml for use as a drench
formulation.
25
Anthelmintic Activity
Anthelmintic activity was evaluated against Caenorhabditis
elegans using the in vitro Screening test described by K. G.
Simpkin and G. L. Coles in Parasitology, 1979, 79, 19. The
products of Examples 1 to 8 all killed 100% of the worms at a well
concentration of 0.1 micrograms per ml.
EXAMPLE 11
Insecticidal Activity ,
' Activity against the larval stage of the blowfly Lucilia
cuprina (Q strain) is demonstrated using a standard procedure in
which first instar larvae are kept in contact with filter paper
treated with test compound. The test compound is first applied to
the paper as an acetone solution. The treated filter papers are
then placed into tubes containing I ml of newborn calf serum and
the first instars are added. The products of Examples 1 to 8
killed 100% of the larvae when applied to the filter paper at a
level of 1 milligram per square metre.
