Derivatives of avermectin and milbemycin

Compounds of formula (I): ##STR1## wherein R.sup.1 is hydrogen or optionally protected hydroxy; R.sup.2 is alkoxy, optionally protected hydroxy, oxo or optionally O-substituted oximino; R.sup.3 is hydrogen, optionally protected hydroxy, or a group 4'-(.alpha.-L-oleandrosyl)-.alpha.-L-oleandrosyloxy or .alpha.-L-oleandrosyloxy wherein the terminal hydroxy group is optionally protected; one or R.sup.4 and R.sup.5 is hydrogen and the other is methyl; and one of R.sup.6 and R.sup.7 is hydrogen and the other is methyl; with the proviso that (a) when R.sup.1 is optionally protected hydroxy, R.sup.3 is hydrogen, and (b) when R.sup.2 is not methoxy or optionally protected hydroxy, R.sup.1 and R.sup.3 are both hydrogen, are new and useful as anthelmintic agents.

The following Examples illustrate the present invention. VM 44864 and VM 
44866 are the compounds of formula (IV) wherein R.sup.1 is hydrogen, 
R.sup.3 and R.sup.8 are both hydrogen, R.sup.9 is (Z) but-2-en-2-yl, and 
R.sup.2 is methoxy and hydroxy respectively. VS 47709 is the compound of 
formula (III) wherein R.sup.1 and R.sup.3 are both hydrogen and R.sup.2 is 
methoxy. 
Compound 6 of GB-A-2 170 499 is the compound of formula (A) wherein Ra is 
methoxy, R.sup.b is hydrogen, R.sup.c is 2,4-dimethylpentanoyloxy and 
R.sup.d is methyl. "X" represents the following structure: 
##STR15## 
EXAMPLE 1 
(a) 22-Keto VM 44864 
To a solution of oxalyl chloride (0.05 ml) in dry dichloromethane (1 ml) at 
-70.degree., under a nitrogen atmosphere, was added dropwise a solution of 
dimethylsulphoxide (0.05 ml) in dry dichloromethane (1 ml). To this 
solution was added dropwise a solution of VM 44864 (8 mg), prepared as 
described in Example 2 of EP-A-0 254 583 (U.S. Ser. No. 076,274), in dry 
dichloromethane (0.5 ml) and the resulting solution stirred at -70.degree. 
for 1.5 h. A solution of triethylamine (0.2 ml) in dry dichloromethane (1 
ml) was added, the mixture allowed to warm to room temperature whilst 
stirring for 1.25 h, and the mixture poured onto a 1:1 mixture of cold 
water and ether. The layers were separated, the aqueous layer extracted 
with ether and the combined ether extracts washed with water, brine, and 
dried (MgSO.sub.4). The ether was evaporated and the product purified by 
preparative thin layer chromatography (Analtech silica taper plate eluted 
with methanol/dichloromethane 2:98) to give the title compound (1.4 mg), 
max (CH.sub.3 OH) 244 nm, m/z (FAB Na.sup.+ /Noba) (relative intensity) 
619 [MNa].sup.+ (100%). 
(b) 22-oximino derivative of VM 44864 
To a solution of 22-keto VM 44864 (18 mg) in methanol (5 ml) was added 
dropwise a solution of hydroxylammonium chloride (24 mg) is water (1 ml). 
The resulting solution was stirred at room temperature for 15 minutes 
before evaporation at reduced pressure to remove the methanol. Water (15 
ml) and ether (15 ml) were added and the ether layer was evaporated, 
extracted with water (15 ml), dried (MgSO.sub.4) and evaporated. The crude 
product was purified by preparative thin layer chromatography (silica 
eluted with 595 MeOH/CH.sub.2 Cl.sub.2) to give the title compound (10 mg) 
m/.sub.z (FAB Na.sup.+ /Noba) (relative intensity)634 [MNa].sup.+ 100% 
.delta..sub.c (CDCl.sub.3), 173.8, 156.8, 142.4, 139.8, 137.3, 135.9, 
133.7, 124.1, 123.5, 120.5, 119.5, 118.5, 98.0, 81.9, 80.4, 77.6, 76.9, 
68.7, 68.4, 68.3, 57.8, 48.5, 45.7, 36.4, 35.9, 35.8, 34.5, 32.9, 27.1, 
22.4, 19.9, 17.7, 15.6, 13.2, 10.9 ppm. 
(c) Lactone VS 47709 
To a solution of pyridine (1 ml) in dichloromethane (20 ml) at -30.degree. 
was added triflic anhydride (0.2 ml) followed by the 22-oximino derivative 
of 22-keto VM 44864 (37 mg). After stirring at -30.degree. for 2 hours, 
the solution was allowed to warm to 5.degree. and poured onto 2M 
hydrochloric acid. After shaking with the acid, the dichloromethane layer 
was separated, dried (MgSO.sub.4), and evaporated. The crude product was 
purified by preparative thin layer chromatography (silica eluted with 
ether) to give the title lactone (5mg). The 13.sub.C n.m.r. (CDCl.sub.3) 
spectrum showed the following signals, 172.9, 168.4, 142.6, 139.9, 139.1, 
136.4, 123.6, 119.5, 118.8, 118.0, 80.6, 77.8, 76.7, 76.6, 68.1, 66.1, 
57.8, 48.4, 45.6, 36.8, 35.9, 34.6, 34.1, 22.3, 19.9, and 15.7. 
EXAMPLE 2 
(a) 5-TBDMS-protected VM 44866 
A mixture of VM 44866 (10.9 mg), prepared as described in Example 3 of 
EP-A-0 254 583 (U.S. Ser. No. 076,274), imidazole (20 mg), 
t-butyldimethylsilylchloride (30 mg) and anhydrous dimethylformamide (5 
ml) was stirred at 20.degree. C. for 16 h in an atmosphere of nitrogen. To 
the mixture was added t-butyldimethylsilylchloride (30 mg), imidazole (20 
mg) and stirring continued at 21.degree. C. for 5 h. The mixture was 
poured onto water (10 ml) and extracted with ether (2.times.5 ml). The 
combined ether extracts were washed with water (10 ml), dried (MgSO.sub.4) 
and evaporated to give an oil. This residue was purified by preparative 
thin layer chromatography (silica; ether/hexane 30/70) to give two 
products. 
Product.1. 5-t-butyldimethylsilyl VM44866 (6.0 mg); t.l.c. R.sub.f =0.33 
ether/hexane 1:1 SiO.sub.2 ; M/.sub.z (FAB Na.sup.+ /Noba) (relative 
intensity) 721 (100%) [MNa].sup.+ 
Product.2. 5,22-di-t-butyldimethylsilyl VM44866 (1.0 mg); t.l.c. R.sub.f= 
0.66 ether/hexane 1:1 SiO.sub.2 ; M/.sub.z (FAB Na.sup.+ /Noba) (relative 
intensity) 835 (100%) [MNa].sup.+ 
5-TBDMS-protected 22-keto VM 44866 
A solution of oxalyl chloride (0.12 ml) in dry dichloromethane (2 ml) at 
-70.degree. C. under nitrogen was treated dropwise with a solution of 
dimethylsulphoxide (0.1 ml) in dry dichloromethane (2 ml) and then 
dropwise with a solution of 5-t-butyldimethylsilyl VM 44866 (46 mg) in dry 
dichloromethane (1.0 ml). The resulting solution was stirred at 
-70.degree. for 1.5 h before being treated dropwise with a solution of 
triethylamine (0.4 ml) in dry dichloromethane (1 ml). The reaction mixture 
was stirred for 1 h without cooling and poured onto a mixture of cold 
water and ether (1:1). The aqueous layer was extracted with ether. The 
combined organic layers were washed with water, dried (MgSO.sub.4) and 
evaporated. The residue was chromatographed over silica using 
dichloromethane increasing to dichloromethane/methanol 98:2 to give the 
title compound (16 mg) which was deprotected as described below. 
(c) 22-Keto VM 44866 
A solution of 5-t-butyldimethylsilyl-22-keto VM 44866 (16 mg) in methanol 
(5 ml) was treated with p-toluenesulphonic acid (10 mg) and the mixture 
stirred for 1.5 h at 21.degree. C. The mixture was then evaporated to 
dryness before addition of water (10 ml) and ether (10 ml). After 
separation, the ether layer was washed with water (10 ml), dried 
(MgSO.sub.4) and evaporated to give an oil. This residue was purified by 
preparative thin layer chromatography (silica: dichloromethane/methanol 
98:2) to give the title compound (10 mg) M/.sub.z (FAB Na.sup.+ /Noba) 
(relative intensity) 605 (100%) [MNa].sup.+ .delta..sub.c (CDCl.sub.3), 
202.5, 173.7, 142.7, 139.7, 137.9, 137.7, 133.2, 124.9, 123.5, 120.3, 
120.2, 118.0, 99.2, 81.8, 80.2, 79.2, 69.1, 68.5, 68.4, 67.8, 48.5, 45.7, 
43.3, 37.1, 36.3, 36.0, 34.6, 33.9, 22.4, 19.9, 18.2, 15.6, 13.3, 10.9, 
ppm. 
(d) 5-TBDMS-22-hydroxyimino VM 44866 
The title compound was prepared from 5-TBDMS-22-keto VM 44866 in a similar 
procedure to that described in Example 1(b). 
(e) 5-TBDMS-de-O-methyl VS 47709 and 5-de-O-methyl VS 47709 
To a stirred solution of 5-TBDMS-22-hydroxyimino VM 44866 (1.5 g) and 
triethylamine (1.0 g) in dichloromethane (10 ml) at 0.degree. C. was added 
portionwise 4-nitrobenzenesulphonyl chloride (0.94 g). The mixture was 
allowed to warm to room temperature before the addition of water (10 ml). 
The dichloromethane layer was separated, washed with water (10 ml), dried 
(MgSO.sub.4) and evaporated. To a solution of the residue, in 
tetrahydrofuran (50 ml), was added 4-toluenesulphonic acid (1.0 g) and the 
mixture was stirred at room temperature for 3 h. The mixture was then 
evaporated to dryness before addition of dichloromethane (25 ml) and 
aqueous sodium bicarbonate solution (25 ml). After separation, the 
dichloromethane layer was washed with water (20 ml), dried (MgSO.sub.4) 
and evaporated to give an oil. This residue was purified by column 
chromatography (silica eluted with hexane/ether) to give the title 
products. 
5-TBDMS-de-O-methyl VS 47709 (200 mg), m/z (FAB Na.sup.+ /Noba) (relative 
intensity) 581 [MNa].sup.+ (100%) and 
5-de-O-methyl VS 47709 (450 mg), m/z (FAB Na.sup.+ /Noba) (relative 
intensity) 467 [MNa].sup.+ (100%), .delta..sub.C (CDCl.sub.3) 172.3, 
168.6, 142.6, 139.4, 138.9, 137.7, 123.3, 120.0, 118.7, 117.6, 80.2, 79.4, 
76.5, 68.0, 67.4, 65.9, 48.1, 45.4, 36.7, 35 7, 34.3, 33.8, 22.1, 19.7, 
15.5 ppm. 
EXAMPLE 3 
Oxidation of compound 6 of GB-A-2 170 499 (10mg) by the procedure described 
in Example 1 yielded the 22-keto derivative (5 mg) which was purified by 
preparative tlc (Silica eluted with ether/hexane, 60:40) .sup.m /z (FAB, 
Na.sup.+ /Noba) 707 [MNa].sup.+. HPLC Retention time 15 min (25 
cm.times.4.6 mm ultrasphere ODS, Methanol/water (90:10), 1 ml/min.). The 
22-keto derivative (5 mg) was dissolved in methanol (5 ml) and an excess 
of an aqueous solution of hydroxylamine hydrochloride added. The solution 
was stored at 5.degree. for 2 days and hplc, using the above conditions, 
indicated the presence of two new products, the `syn` and `anti` isomers 
of the title compound with retention times of 8.7 and 9.1 mins. The 
mixture was poured into water, extracted with dichloromethane, and the 
extracts dried (MgSO.sub.4) and evaporated. The crude product was used 
directly in the following reaction. 
The crude oxime from above was dissolved in dichloromethane (5 ml) and 
triethylamine (1 ml). 4-Nitrobenzenesulphonyl chloride (30 mg) was added 
in portions until hplc indicated that all the oxime had reacted The 
solution was evaporated, the residue dissolved in methanol and 
4-toluenesulphonic acid (30 mg) added. The reaction mixture was stored at 
5.degree. for 15 h, evaporated and the residue dissolved in 
dichloromethane. The solution was washed with aqueous sodium bicarbonate 
solution, dried and evaporated. Preparative tlc (silica eluted with 80:20 
ether/petrol) gave a pure sample of VS 47709 (1 mg), .sup.m /z (FAB 
Na.sup.+ /Noba) 481 [MNa].sup.+. 
EXAMPLE 4 
(24R,25R)-22,23-Didehydro-24,25,dimethyl-X 
To a solution of (3R*,4R*)-3-methyl-4-(tetrahydropyran)-2-yloxy)-1-pentyne 
(110 mg, 0.6 mmole) in THF (5 ml) at -78.degree. C. under nitrogen was 
added butyllithium (1.6M in hexane; 0.32 ml, 0.5 mmole) and the mixture 
was stirred at -78.degree. C. for 11/2 h. A solution of VS 47709 (90 mg, 
0.2 mmole) in THF (1 ml) was added to the mixture which was then stirred 
at -78.degree. C. for 3 h. The reaction was quenched with dilute aqueous 
ammonium chloride (5 ml) and the mixture was then allowed to warm to 
0.degree. C. The aqueous phase was extracted with diethyl ether 
(2.times.20 ml) and the combined organic extracts were washed with water 
(10 ml), dried (MgSO.sub.4) and evaporated to give a crude product (150 
mg). 
A solution of this crude product (150 mg) in ethyl acetate (20 ml) was 
hydrogenated over Lindlar catalyst (90 mg) for 16 h. The mixture was 
filtered through celite and evaporated to an oil. 
To the unpurified product was added methanol (10 ml) and dilute 
hydrochloric acid (1M, 1 ml). The mixture was stirred for 1 h and 
evaporated to dryness to give the crude product. The residue was purified 
by preparative t.l.c. (silica plates eluted with ethyl acetate:hexane 
30:70) to give the title compound (10 mg); m/z (FAB Na.sup.+ /NOBA) 
(relative intensity) 563 [M+Na].sup.+ (100%); .delta..sub.C (CDCl.sub.3) 
174.0, 142.5, 139.7, 137.0, 136.0, 135.5, 127.5, 123.5, 120.8, 119.4, 
118.3, 96.2, 80.3, 77.4, 77.0, 68.6, 68.3, 68.2, 65.6, 57.8, 48.5, 45.6, 
40.4, 36.3, 35.9, 34.9, 32.9, 22.3, 19.9, 17.9, 15.5, 12.1 ppm. 
EXAMPLE 5 
(24S,25R)-22,23-Didehydro-24,25-dimethyl-X and 
(24R,25S)-22,23-didehydro-24,25-dimethyl-X 
In a similar manner to Example 4, 
(3R*,4S*)-3-methyl-4-(tetrahydropyran-2-yloxy)-1-pentyne (152 mg. 0.84 
mmole) and VS 47709 (100 mg, 0.21 mmole) gave two products (24S,25R) and 
(24R,25S)-22,23-didehydro-24,25-dimethyl-X as a mixture. Purification by 
preparative HPLC gave (24R,25S)-22,23-didehydro-24,25-dimethyl-X (3.9 mg); 
m/z (FAB Na.sup.+ /Noba) (relative intensity) 563 [MNa.sup.+ ] (100%); and 
(24S,25R)-22,23-didehydro-24,25-dimethyl-X (22.0 mg); m/z (FAB Na.sup.+ 
/Noba) (relative intensity) 563 [MNa.sup.+ ] (100%); .delta..sub.C 
(CDCl.sub.3) 173.8, 142.6, 142.4, 139.7, 137.1, 135.9, 135.4, 127.9, 
123.5, 120.7, 119.4, 118.3, 95.7, 80.3, 77.4, 76.9, 69.7, 68.4, 68.3, 
68.2, 57.8, 53.4, 48.5, 45.6, 40.3, 36.3, 36.1, 35.9, 34.8, 22.3, 19.9, 
19.0, 16 6, 15.5 ppm. 
PREATION 1 
(3R*,4R*)-3-Methyl-1-pentyn-4-ol 
Prepared as described in the literature from trans-2,3-epoxybutane and 
lithium acetylide, ethylenediamine complex in dimethylsulphoxide (J Am 
Chem Soc (1970) 101, 5367). 
PREATION 2 
(3R*,4R*)-3-Methyl-4-(tetrahydropyran-2-yloxy)-1-pentyne 
To dihydropyran (1.3 g) cooled in an ice bath was added 
(3R*,4R*)-3-methyl-1-pentyn-4-ol (1.3 g) followed by concentrated 
hydrochloric acid (1 drop) and the mixture was stirred for 16 h at room 
temperature. Potassium carbonate (100 mg) was added and the mixture 
stirred for 15 minutes. Diethyl ether (10 ml) was added and the mixture 
was filtered before evaporation to an oil. The residue was distilled to 
give the title compound (1.2 g) (b.p. 130.degree.-5.degree. C. at 10 mm 
Hg). 
PREATION 3 
(3R*,4S*)-3-Methyl-1-pentyn-4-ol 
To a suspension of lithium acetylide ethylenediamine complex (4 g, 44 
mmole) in dimethyl sulphoxide (10 ml) was added cis-2,3-epoxybutane (2.9 
g, 40 mmole) and the mixture was stirred under nitrogen for 10 days at 
room temperature. The reaction was quenched with water (25 ml) and 
extracted with diethyl ether (3.times.50 ml). The combined ethereal 
extracts were dried (MgSO.sub.4) and evaporated to give an oil. The 
residue was distilled to give the title compound as a colourless oil (1.2 
g) (b.p. 35.degree.-45.degree. C. at 30 mm Hg). 
PREATION 4 
(3R*,4S*)-3-Methyl-4-(tetrahydropyran-2-yloxy)-1-pentyne 
In a similar manner to Preparation 2 (3R*,4S*)-3-methyl-1-pentyn-4-ol gave 
the title compound as a colourless oil (1.2 g) (b.p. 
115.degree.-20.degree. C. at 40 mm Hg). 
REFERENCE EXAMPLE 1 
Culture 
Streptomyces sp. NCClB 12509 as described in EP-A-0 254 583 (U.S. Ser. No. 
076,274) was maintained as vegetative mycelium stored in liquid nitrogen. 
1st Stage Seed 
100 mls Medium Y was sterilised in 500ml Erlenmeyer flasks closed with 
cotton gauze caps. 
______________________________________ 
Medium Y contained:- 
*Special Peptone 0.25% 
*Lab Lemco 0.25% 
*Tryptone 0.25% 
*Neutralised Soya Peptone 
0.25% 
*Malt Extract 0.25% 
Soluble Starch 0.25% 
Glucose monohydrate 0.25% 
Glycerol 0.25% 
.0.Trace elements solution 
10 ml/liter 
pH was unadjusted 
.0.The trace elements solution contained:- 
CaCl.sub.2.2H.sub.2 O 1.0% 
MgCl.sub.2.6H.sub.2 O 1.0% 
NaCl 1.0% 
FeCl.sub.3 0.3% 
ZnCl.sub.2 0.05% 
CuCl.sub.2.2H.sub.2 O 0.05% 
MnSO.sub.4.4H.sub.2 O 0.05% 
CoCl.sub.2.6H.sub.2 O 0.05% 
______________________________________ 
(*These products were supplied by Oxoid Ltd., Basingstoke, Hants. UK) 
One flask of Medium Y was inoculated with the contents (1.5 ml) of one 
ampoule of preserved culture. The flask was incubated at 25.degree. C. on 
a gyratory shaker at 240 rpm for 48 hours. 
2nd Stage Seed 
Seed Medium C was sterilised in 100 ml amounts in 500 ml Erlenmeyer flasks 
closed with cotton gauze caps. 
______________________________________ 
Medium C contained: 
Arkasoy 50 1.0% 
Glucose monohydrate 2.0% 
Spray dried Corn Steep Liquor 
0.5% 
NaCl 0.3% 
pH adjusted to 6.8. 
______________________________________ 
(Arkasoy 50 was supplied by British Arkady Co. Manchester, UK). 
(Corn Steep Liquor was supplied by Roquette UK Ltd., Tunbridge Wells, 
Kent, UK). 
Flasks of medium C were inoculated with 4% of the 1st stage seed inoculum. 
These were incubated at 26.degree. C. on a gyratory shaker at 240rpm for 
72 hours. 
3rd Stage Seed 
15 liters of Medium C together with Polypropylene glycol (P2000) 0.1% 
.sup.v /v were sterilised in a 20 liter Biolafitte fermenter (Biolafitte, 
Poissey, France) The fermenter was fully baffled and fitted with three 
vaned-disc impellers. Sterile air was supplied at 1 v.v.m and agitation 
was at 200 rpm. 
The contents of two second-stage seed flasks were used to inoculate one 20 
liter fermenter and incubation, at 26.degree. C., was for 48 hours. 
4th Stage Seed 
100 liters Medium C was sterilised at 121.degree. C. in a 150 liter Braun 
fermenter (B. Braun, Melsungen, W.Germany). 0.1% Antifoaming agent was 
included with the medium and consisted of 10% pluronic L81 in soybean oil. 
(Pluronic L81 was supplied by Blagden-Campbell Chemical Co., Croydon, UK). 
The fermenter was fully baffled and fitted with three vaned-disc impellers. 
Agitation was at 120 rpm and sterile air supplied at 1 v.v.m. The 
fermenter was inoculated with 4 liters of the 3rd stage seed and incubated 
at 28.degree. C. for 48 hours. 
Fermentation Stage 
3000 liters of Medium F1 was sterilised in a 4500 liter Bioengineering 
fermenter (Bioengineering-Wald, Switzerland). 
______________________________________ 
Medium F1 contained:- 
Arkasoy 50 1.0% 
Glucose monohydrate 2.0% 
Dextrin 07005 2.0% 
Casein 0.2% 
MgSO.sub.4 0.1% 
CaCO.sub.3 0.5% 
10% Pluronic L81 in soybean 
0.1% 
oil 
______________________________________ 
(Dextrin 07005 was supplied by Corn Products Ltd., Manchester, UK) 
(Casein was supplied by Oxoid Ltd, Hants., UK). 
The fermenter was fully baffled and fitted with three, vaned-disc 
impellers. Sterile air was supplied at 0.5 v.v.m. 
The fermenter was inoculated with 100 liters of fourth stage seed inoculum 
and incubated at 26.degree. C. The agitation rate was maintained at the 
following rates: 
______________________________________ 
0-2 days 50 rpm 
2-3 days 75 rpm 
3 days-harvest 100 rpm 
______________________________________ 
The fermentation ran for 17 days and extra shots of antifoaming agent were 
added on demand. 
Isolation Procedure 
At harvest whole broth was transferred to a separate stirred vessel and 10% 
v/v butan-1-ol was added. The mixture was stirred for 16 hours at 
7.degree. C. Thereafter the whole broth (2339 liters) was fed at 5 
liters/minute, together with butan-1-ol at 1.7 liters/minute through an 
in-line static mixer to a Westfalia SA7-03-076 liquid/liquid/solid 
centrifugal separator (Westfalia Separator Ltd., Oelde, W. Germany). The 
accumulated solids were discharged intermittently as required. 
The raffinate and mycelial solids were combined and submitted to a second, 
similar extraction with butan-1-ol. Combined butanol extracts were 
concentrated in vacuo to 20 liters. 40 liters petroleum spirit 
(60.degree.-80.degree.) was added to preciptitate pigmented impurities 
which were removed by centrifugation. The supernatant was concentrated in 
vacuo to 15 liters. 
Chromatographic Purification 
The concentrate was chromatographed on a column (600 mm diameter.times.200 
mm) of silica gel (Riedel de Haen, Seelze, W. Germany, 32-63 .mu.m), 
eluting with a step gradient of ethyl acetate in petroleum spirit 
(60.degree.-80.degree.). Fractions obtained using &gt;8% ethyl acetate, 
containing VM 47704 and other active material were set aside. All of the 
remaining milbemycin--containing fractions were rechromatographed on a 
similar column, resulting in the separation of a further quantity of VM 
47704 and other active material which was combined with that already 
obtained and evaporated to an oily concentrate (208 g). 
This was again chromatographed on silica gel (150 mm.times.180 mm column), 
eluting with a step gradient of ethyl acetate in petroleum spirit 
(60.degree.-80.degree.), using 3.3 liters pure petroleum spirit, followed 
by 6.6 liters each of 15%, 25%, 30%, 35% and 40% v/v ethyl acetate in 
petroleum spirit. The first 25 liters of eluate was discarded, thereafter 
fractions of 2 liters were collected. Fractions 3-8 were combined and 
concentrated to an oil. 
This material was chromatographed on a column (75 mm diameter.times.500 mm) 
of Sephadex.RTM. LH 20 (Pharmacia, Milton Keynes, UK) using methanol as 
eluant. All fractions containing milbemycin components were combined. 
Further purification was achieved using chromatography on reverse-phase 
silica. Matrex.RTM. C.sub.18 silica, 20-45 .mu.m, 60A pore size (Amicon, 
Stonehouse, UK) was used for all subsequent columns. 
Product obtained from the Sephadex.RTM. LH 20 column was chromatographed on 
a 100 mm diameter.times.180 mm column of C.sub.18 silica, eluting with 85% 
methanol. The first 1 liter of eluant was discarded, thereafter 
20.times.100 ml fractions, all containing milbemycin components were 
collected and combined. 
The solution so obtained was chromatographed on a similar column, eluting 
firstly with 85% methanol, then with 87.5% methanol. After discarding the 
first 1 liter of eluate, 36 fractions each of 85 ml, containing active 
material other than VM 47704 were collected. Thereafter elution was with 
87.5% methanol with eluted VM 47704 admixed with other active material. 
The latter fractions were chromatographed on a 100 mm diameter.times.260 mm 
column (designated 22I), eluting with 85% methanol. 100 ml fractions were 
collected. Enrichment of VM 47704 was observed in fractions 42-55. 
Fractions 35-41 contained additional product. 
Fractions 42-55 from column 22I were chromatographed on a similar column 
(designated 22J), eluting with 82 5% methanol. The first 7.2 liters was 
discarded then 100 ml fractions were collected. The majority of the VM 
47704 was found in fractions 66-85 and further, less pure, product was 
present in fractions 57-65. 
The less pure fractions from columns 22I and 22J were combined and 
chromatographed on a column (80 mm diameter .times.600 mm) using 81% 
methanol as eluant. The first 9.3 liters were discarded then 100 ml 
fractions were collected. Fractions 76-86 containing predominantly VM 
47704 were combined with fractions 66-85 from column 22J and 
chromatographed on the same column, using 79% methanol as eluant. The 
first 20 liters were discarded then 100 ml fractions were collected. 
Fractions 34-55 were evaporated to dryness to give 209 mg VM 47704. 
Characterisation Data for VM 47704 
Mass (Electron Impact Mass Spectroscopy) [M]+=684; .delta.13.sub.C 
(CDCl.sub.3) 173.0, 172.9, 142.9, 139.1, 137.1, 136.4, 134.0, 123.6, 
123.3, 121.7, 120.54, 120.47, 98.8, 81.9, 80.3, 79.0, 71.5, 68.8.,68.4, 
67.9, 64.6, 64.1, 48.4, 45.5, 43.2, 36.8, 36.4, 36.3, 35.9, 34.6, 32.0, 
25.6, 22.4, 22.2, 17.4, 15.5, 13.1, 10.9. 
REFERENCE EXAMPLES 2 AND 3 
The seed stages and fermentation stages were carried out as in Reference 
Example 1, except that in the fermentation stage the agitation was 
maintained at the following rates: 
______________________________________ 
0-3 days 50 rpm 
3 days-harvest 100 rpm 
______________________________________ 
The fermentation broth was harvested after 404 hours. Whole broth was 
discharged from the fermenter and saturated with butanol. Product was 
extracted with 1/3 volume butanol using an in-line static mixer followed 
by the Westfalia liquid/liquid/solid separator. The broth was processed at 
5 l /min resulting in ca 80% yield. A second, similar, extraction was 
performed to give essentially quantitative recovery. 
Combined butanol extracts were combined, concentrated to low volume (19 1) 
and 2 volumes of petroleum spirit (60.degree.-80.degree.) were added to 
precipitate pigmented impurities. Reconcentration resulted in 10.8 1 brown 
oil which was chromatographed on 28 kg silica gel packed in a 600 mm 
diameter column. Elution was carried out using increasing concentrations 
of ethyl acetate (up to 50%) in petrol. 
Fractions containing VM 48130 and VM 48633 were identified and subjected to 
further chromatography. 
VM 48130 (Reference Example 2) and VM 48633 (Reference Example 3) 
A 1.8g fraction containing VM 48130 and VM 48633 was subjected to 
preparative reverse phase HPLC using a Dynamax-60A C-18 column 
(500.times.21.4 mm, Rainin Instrument Company, USA), eluted with a 
methanol:water gradient at 10 ml/min (82:18 methanol:water rising to 100% 
methanol over 140 min), monitored by UV spectroscopy at 244 nm. VM 48130 
and VM 48633 were detected in the same fractions which were pooled and the 
solvent evaporated to yield 97.4 mg of material. This was further purified 
by preparative silica HPLC using a Dynamax-60A Si column (250.times.21.4 
mm, Rainin Instrument Company, USA), eluted with a hexane:acetone gradient 
(87:13 to 82:18 over 120 min at 10 ml/min). Fractions were collected and 
identified by TLC (silica gel plates run with 60:40 hexane:acetone). 
This produced substantially pure VM 48633 (4.9 mg; .lambda..sub.max 
(CH.sub.3 OH) 244 nm, mass (FAB Na.sup.+ /NOBA) [MNa].sup.+ =705, 
.delta.13.sub.c (CDCl.sub.3) 173.2, 166.4, 157.8, 142.9, 139.2, 137.2, 
136.8, 134.1, 123.7, 123.4, 121.4, 120.6, 120.5, 115.6, 98.9, 81.9, 80.3, 
79.1, 71.6, 68.8, 68.5, 68.0, 64.7, 63.5, 48.5, 45.6, 36.9, 36.45, 36.37, 
36.0, 34.6, 32.1, 27.5, 22.3, 20.3, 17.5, 15.5, 13.1, 10.9 ppm. 
Fractions containing VM 48130 (10.6 mg) required final purification by 
semipreparative HPLC (Hypersil 5 .mu.m ODS 250.times.10 mm column, HPLC 
Technology Ltd), eluted with a methanol:water gradient (77:23 
methanol:water, rising to 85.15 over 60 min, flow 3 ml/min), monitored by 
UV spectroscopy at 244 nm. This yielded 4.1 mg of substantially pure VM 
48130; .lambda..sub.max (CH.sub.3 OH) 244 nm, mass (FAB, Na.sup.+ /NOBA) 
[MNa].sup.+ =693; .delta.13.sub.c (CDCl.sub.3) 177.8, 173.7, 142.8, 139.5, 
137.9, 137.4, 133.1, 124.9. 123.4, 120.4, 120.3, 118.1, 100.2, 80.2, 79.2, 
76.0, 75.4, 68.5, 68.1, 67.7, 48.5, 45.7, 37.7, 36.21, 36.14, 36.0, 34.6, 
34.3, 22.3, 19.9, 19.2, 19.0, 15.6, 13.2, 13.0, 10.8 ppm. 
They have retention times of 12.3 min (VM 48130) and 12.9 (VM 48633) when 
subjected to HPLC under the following conditions: Ultrasphere ODS 5 .mu.m 
column 250.times.4.6 mm (Altex) eluted with methanol:water (85.15) at a 
flow rate of 1 ml/min and monitored by UV spectroscopy at 244 nm. 
Temperatures given in the Examples are in Celsius unless otherwise 
indicated.