Macrolide compounds

Compounds of formula (1) ##STR1## wherein R represents a sugar residue or an acylated derivative thereof; PA1 R.sup.1 represents a methyl, ethyl or isopropyl group; PA1 Y.sup.1 is --CH.sub.2, Y.sup.2 is --CH-- and X represents ##STR2## [wherein R.sup.2 represents a hydrogen atom or a group OR.sup.6 (where OR.sup.6 is a hydroxyl group or a substituted hydroxyl group having up to 25 carbon atoms) and R.sup.3 represents a hydrogen atom, or R.sup.2 and R.sup.3 together with the carbon atom to which they are attached represent >C.dbd.0, >C.dbd.CH.sub.2 or >C.dbd.NOR.sup.7 (where R.sup.7 represents a hydrogen atom, a C.sub.1-8 alkyl group or a C.sub.3-8 alkenyl group) and the group >C.dbd.NOR.sup.7 is in the E configuration] or --Y.sup.1 --X--Y.sup.2 -- represents --CH.dbd.CH--CH-- or --CH.sub.2 --CH.dbd.C--; and PA1 R.sup.4 represents a group OR.sup.6 as defined above and R.sup.5 represents a hydrogen atom, or R.sup.4 and R.sup.5 together with the carbon atom to which the are attached represent >C.dbd.0 or >C.dbd.NOR.sup.8 (where R.sup.8 is as defined above for R.sup.7), and salts thereof. The compounds may be used to control nematode, acarine, insect or other pests.

This invention relates to novel macrolide compounds having antibiotic 
activity, to processes for their preparation and to compositions 
containing them. 
Thus, according to one aspect of the present invention we provide the 
compounds of formula (1) 
##STR3## 
wherein R represents a sugar residue or an acylated derivative thereof; 
R.sup.1 represents a methyl, ethyl or isopropyl group; 
Y' is --CH.sub.2 --, Y.sup.2 is --CH-- and X represents 
##STR4## 
[where R.sup.2 represents a hydrogen atom or a group OR.sup.6 (where 
OR.sup.6 is a hydroxyl group or a substituted hydroxyl group having up to 
25 carbon atoms) and R.sup.3 represents a hydrogen atom, or R.sup.2 and 
R.sup.3 together with the carbon atom to which they are attached represent 
&gt;C.dbd.O, &gt;C.dbd.CH.sub.2 or &gt;C.dbd.NOR.sup.7 (where R.sup.7 represents a 
hydrogen atom, a C.sub.1-8 alkyl group or a C.sub.3-8 alkenyl group) and 
the group &gt;C.dbd.NOR.sup.7 is in the E configuration] or --Y.sup.1 
--X--Y.sup.2 -- represents --CH.dbd.CH--CH-- or --CH.sub.2 --CH.dbd.C--; 
and 
R.sup.4 represents a group OR.sup.6 as defined above and R.sup.5 represents 
a hydrogen atom, or R.sup.4 and R.sup.5 together with the carbon atom to 
which the are attached represent &gt;C.dbd.O or &gt;C.dbd.NOR.sup.8 (where 
R.sup.8 is as defined above for R.sup.7), and salts thereof. 
The group R.sup.6 when present in compounds of formula (I) may represent an 
acyl group e.g. a group of the formula R.sup.9 CO-- or R.sup.9 OCO-- or 
R.sup.9 OCS-- (where R.sup.9 is an aliphatic, araliphatic or aromatic 
group, for example an alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl or aryl 
group), a formyl group, a group R.sup.10 which is as defined above for 
R.sup.9, a group R.sup.11 SO.sub.2 -- (where R.sup.11 is a C.sub.1-4 alkyl 
or C.sub.6-10 aryl group), a silyl group, a cyclic or acyclic acetal 
group, a group --CO(CH.sub.2).sub.n CO.sub.2 R.sup.12 (where R.sup.12 is a 
hydrogen atom or a group as defined above for R.sup.9 and n represents 
zero, 1 or 2) or a group R.sup.13 R.sup.14 NCO-- (where R.sup.13 and 
R.sup.14 may each independently represent a hydrogen atom or a C.sub.1-4 
alkyl group). 
Where R.sup.9 or R.sup.10 are alkyl groups, they may be for example 
C.sub.1-8 alkyl groups, e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, 
i-butyl, t-butyl or n-heptyl which alkyl groups may also be substituted. 
Where R.sup.9 is a substituted alkyl group it may be substituted by, for 
example, one or more, e.g. two or three, halogen atoms (e.g. chlorine or 
bromine atoms), or a carboxy, C.sub.1-4 alkoxy (e.g. methoxy, ethoxy), 
phenoxy or silyloxy group. Where R.sup.10 is a substituted alkyl group it 
may be substituted by a cycloalkyl e.g. cyclopropyl group. 
Where R.sup.9 and R.sup.10 are alkenyl or alkynyl groups, they preferably 
have 2-8 carbon atoms and where R.sup.9 and R.sup.10 are cycloalkyl 
groups, they may be for example C.sub.3-12 cycloalkyl, such as C.sub.3-7 
cycloalkyl, e.g. cyclopentyl groups. 
Where R.sup.9 and R.sup.10 are aralkyl groups, they preferably have 1-6 
carbon atoms in the alkyl moiety, and the aryl group(s) may be carbocyclic 
or heterocyclic and preferably contain 4-15 carbon atoms e.g. phenyl. 
Examples of such groups include phen C.sub.1-6 alkyl e.g. benzyl groups. 
Where R.sup.9 and R.sup.10 are aryl groups, they may be carbocyclic or 
heterocyclic and preferably have 4-15 carbon atoms e.g. phenyl. 
When R.sup.6 is a group R.sup.11 SO.sub.2 --, it may be for example a 
methylsulphonyl or p-toluenesulphonyl group. 
Where R.sup.6 represents a cyclic acetal group, it may for example have 5-7 
ring members as in the tetrahydropyranyl group. 
When R.sup.6 represents a silyl group or R.sup.9 contains a silyloxy 
substituent, the silyl group may carry three groups which may be the same 
or different, selected from alkyl, alkenyl, alkoxy, cycloalkyl, aralkyl, 
aryl and aryloxy groups. Such groups may be as defined above and 
particularly include methyl, t-butyl and phenyl groups. Particular 
examples of such silyl groups are trimethylsilyl and t-butyldimethylsilyl. 
When R.sup.6 represents a group --CO(CH.sub.2).sub.n CO.sub.2 R.sup.12, it 
may for example be a group --COCO.sub.2 R.sup.12 or --COCH.sub.2 CH.sub.2 
CO.sub.2 R.sup.12 where R.sup.12 represents a hydrogen atom or a C.sub.1-4 
alkyl group (e.g. methyl or ethyl). 
When R.sup.6 represents a group R.sup.13 R.sup.14 NCO--, R.sup.13 and 
R.sup.14 for example may each independently be a hydrogen atom or a methyl 
or ethyl group. 
When R.sup.7 or R.sup.8 represents a C.sub.1-8 alkyl group it may be for 
example a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl 
group, and is preferably a methyl group. 
When R.sup.7 or R.sup.8 represents a C.sub.3-8 alkenyl group it may be for 
example an allyl group. 
When R is a sugar residue it may be, for example, a mono- or disaccharide. 
Examples of monosaccharides include pyranose and furanose sugars e.g. 
glucose, mannose, fructose, galactose, allose, gulose, talose, xylose, 
threose, lyxose, erythrose, altrose, ribose, arabinose, idose, 
2-deoxyglucose, glucosamine, galactosamine, desosamine, mycaminose, 
angolosamine, forosamine, megosamine, chalcose, aldgarose, mycinose, 
mycosamine, mycarose, cladinose, oleandrose and 3-demethyloleandrose. 
Examples of disaccharides include 
.alpha.-L-oleandrosyl-.alpha.-L-oleandrose and 
.alpha.-3-demethyloleandrosyl-.alpha.-3'-demethyloleandrose. 
Particular examples of the radical R include 
.alpha.-L-oleandrosyl-.alpha.-L-oleandrose, L-oleandrose, D-desosamine, 
D-mycaminose, D-angolosamine, D-forosamine, L-megosamine, D-chalcose, 
D-aldgarose, D-mycinose, D-mycosamine, L-mycarose and L-cladinose. 
When R is an acylated sugar residue the sugar may be as described above and 
the acyl group may be as defined for the group R.sup.6 above. 
Compounds of formula (1) containing an acidic group may form salts with 
bases. Examples of such salts include alkali metal salts such as sodium 
and potassium salts. 
Compounds of formula (1) in which R represents an .alpha.-L-oleandrose or 
.alpha.-L-oleandrosyl-.alpha.-L-oleandrose group are preferred. 
In the compounds of formula (1) R.sup.1 preferably represents an isopropyl 
group. 
An important group of compounds of formula (1) is that in which Y.sup.1 is 
--CH.sub.2 --, Y.sup.2 is --CH-- and X represents 
##STR5## 
Particularly important compounds of this type are those in which R.sup.2 
is a hydrogen atom or a hydroxy, ethoxy or acetyloxy group and R.sup.3 is 
a hydrogen atom or R.sup.2 and R.sup.3 together with the carbon atom to 
which they are attached represent &gt;C.dbd.O, &gt;C.dbd.CH.sub.2 or 
&gt;C.dbd.NOCH.sub.3. 
A further important group of compounds of formula (1) is that in which 
R.sup.4 is a hydroxy, methoxy or acyloxy (e.g. acetyloxy) group or R.sup.4 
and R.sup.5 together with the carbon atom to which they are attached 
represent &gt;C.dbd.NOCH.sub.3. R.sup.4 preferably represents a hydroxyl 
group. 
Important active compounds according to the invention are those of formula 
(1) in which: 
R represents a .alpha.-L-oleandrosyl-.alpha.-L-oleandrose group, R.sup.1 is 
an isopropyl group, Y.sup.1 is --CH.sub.2 --, Y.sup.2 is --CH--, X 
represents &gt;C.dbd.NOCH.sub.3, R.sup.4 is a hydroxy group and R.sup.5 is a 
hydrogen atom; and 
R represents a .alpha.-L-oleandrosyl-.alpha.-L-oleandrose group, R.sup.1 is 
an isopropyl group, Y.sup.1 is --CH.sub.2 --, Y.sup.2 is --CH--, X 
represents --CH.sub.2 --, R.sup.4 is a hydroxy group and R.sup.5 is a 
hydrogen atom. 
As indicated previously, compounds of the invention have antibiotic 
activity e.g. antihelminthic activity, for example against nematodes, and 
in particular, anti-endoparasitic and anti-ectoparasitic activity. 
Compounds of the invention may also be of use as intermediates in the 
preparation of other active compounds. 
The antibiotic activity of the compounds of formula (I) may, for example, 
be demonstrated by their activity in vitro against free living nematodes 
e.g. Caenorhabditis elegans. 
Ectoparasites and endoparasites infect humans and a variety of animals and 
are particularly prevalent in farm animals such as pigs, sheep, cattle, 
goats and poultry (e.g. chickens and turkeys), horses, rabbits, 
game-birds, caged birds, and domestic animals such as dogs, cats, guinea 
pigs, gerbils and hamsters. Parasitic infection of livestock, leading to 
anaemia, malnutrition and weight loss is a major cause of economic loss 
throughout the world. 
Examples of genera of endoparasites infecting such animals and/or humans 
are Ancylostoma, Ascaridia, Ascaris, Aspicularis, Brugia, Bunostomum, 
Capillaria, Chabertia, Cooperia, Dictyocaulus, Dirofilaria, Dracunculus, 
Enterobius, Haemonchus, Heterakis, Loa, Necator, Nematodirus, 
Nematospiroides (Heligomoroides), Nippostrongylus, Oesophagostomum, 
Onchocerca, Ostertagia, Oxyuris, Parascaris, Strongylus, Strongyloides, 
Syphacia, Toxascaris, Toxocara, Trichonema, Trichostrongylus, Trichinella, 
Trichuris, Triodontophorus, Uncinaria and Wuchereria. 
Examples of ectoparasites infecting animals and/or humans are arthropod 
ectoparasites such as biting insects, blowfly, fleas, lice, mites, sucking 
insects, ticks and other dipterous pests. 
Examples of genera of such ectoparasites infecting animals and/or humans 
are Ambylomma, Boophilus, Chorioptes, Culliphore, Demodex, Damalinia, 
Dermatobia, Gastrophilus, Haematobia, Haematopinus, Haemophysalis, 
Hyaloma, Hypoderma, Ixodes, Linognathus, Lucilia, Melophagus, Oestrus, 
Otobius, Otodectes, Psorergates, Psoroptes, Rhipicephalus, Sarcoptes, 
Stomoxys and Tabanus. 
Furthermore, the compounds of formula (I) are also of use in combating 
insect, acarine and nematode pests in agriculture, horticulture, forestry, 
public health and stored products. Pests of soil and plant crops, 
including cereals (e.g. wheat, barley, maize and rice) vegetables (e.g. 
soya), fruit (e.g. apples, vines and citrus) as well as root crops (e.g. 
sugarbeet, potates) may usefully be treated. Particular examples of such 
pests are fruit mites and aphids such as Aphis fabae, Aulacorthum 
circumflexum, Myzus persicae, Nephotettix cincticeps, Nilparvata lugens, 
Panonychus ulmi, Phorodon humuli, Phyllocoptruta oleivora, Tetranychus 
urticae and members of the genera Trialeuroides; nematodes such as members 
of the genera Aphelencoides, Globodera, Heterodera, Meloidogyne and 
Panagrellus; lepidoptera such as Heliothis, Plutella and Spodoptera; grain 
weevils such as Anthonomus grandis and Sitophilus granarius; flour beetles 
such as Tribolium castaneum; flies such as Musca domestica; fire ants; 
leaf miners; Pear psylla; Thrips tabaci; cockroaches such as Blatella 
germanica and Periplaneta americana and mosquitoes such as Aedes aegypti. 
According to the invention we therefore provide the compounds of formula 
(I) as defined above, which may be used as antibiotics. In particular, 
they may be used in the treatment of animals and humans with 
endoparasitic, ectoparasitic and/or fungal infections and in agriculture, 
horticulture, or forestry as pesticides to combat insect, acarine and 
nematode pests. They may also be used generally as pesticides to combat or 
control pests in other circumstances, e.g. in stores, buildings or other 
public places or location of the pests. In general the compounds may be 
applied either to the host (animal or human or plants or other vegetation) 
or to the pests themselves or a locus thereof. 
The compounds of the invention may be formulated for administration in any 
convenient way for use in veterinary or human medicine and the invention 
therefore includes within its scope pharmaceutical compositions comprising 
a compound in accordance with the invention adapted for use in veterinary 
or human medicine. Such compositions may be presented for use in 
conventional manner with the aid of one or more suitable carriers or 
excipients. The compositions of the invention include those in a form 
especially formulated for parenteral (including intramammary 
administration), oral, rectal, topical, implant, ophthalmic, nasal or 
genito-urinary use. 
The compounds of formula (I) may be formulated for use in veterinary or 
human medicine according to the general methods described in UK Patent 
Specification 2166436. 
The total daily dosages of the compounds of the invention employed in both 
veterinary and human medicine will suitably be in the range 1-2000 
.mu.g/kg bodyweight, preferably from 50-1000 .mu.g/kg and these may be 
given in divided doses, e.g. 1-4 times per day. 
The compounds according to the invention may be formulated in any 
convenient way for horticultural or agricultural use and the invention 
therefore includes within its scope compositions comprising a compound 
according to the invention adapted for horticultural or agricultural use. 
Such formulations include dry or liquid types, for example dusts, 
including dust bases or concentrates, powders, including soluble or 
wettable powders, granulates, including microgranules and dispersible 
granules, pellets, flowables, emulsions such as dilute emulsions or 
emulsifiable concentrates, dips such as root dips and seed dips, seed 
dressings, seed pellets, oil concentrates, oil solutions, injections e.g. 
stem injections, sprays, smokes and mists. 
Generally such formulation will include the compound in association with a 
suitable carrier or diluent. Such carriers and diluents are as described 
in UK Patent Specification 2166436. 
In the formulations, the concentration of active material is generally from 
0.01 to 99% and more preferably between 0.01% and 40% by weight. 
Commercial products are generally provided as concentrated compositions to 
be diluted to an appropriate concentration, for example from 0.001 to 
0.0001% by weight, for use. 
The rate at which a compound is applied depends upon a number of factors 
including the type of pest involved and the degree of infestation. 
However, in general, an application rate of 10 g/ha to 10 kg/ha will be 
suitable; preferably from 10 g/ha to 1 kg/ha for control of mites and 
insects and form 50 g/ha to 10 kg/ha for control of nematodes. 
For use in veterinary medicine or for horticultural and agricultural use it 
may be desirable to use whole fermentation broth, as a source of the 
active compound. It may also be suitable to use dried broth (containing 
mycelia) or to use mycelia separated from the broth and pasteurised or 
more preferably, dried e.g. by spray-, freeze-, or roller drying. If 
desired the broth or mycelia may be formulated into compositions including 
conventional inert carriers, excipients or diluents as described above. 
The antibiotic compounds of the invention may be administered or used in 
combination with other active ingredients. 
In particular, the antibiotic compound of the invention may be used 
together with other antibiotic compounds. This may occur, for example, 
where whole fermentation broth is used without prior separation of 
compounds of the invention or where crude fermentation products are 
reacted according to the fermentation process of the invention without 
prior or subsequent separation; this may be preferable for example in 
agricultural use of a compound, where it is important to maintain low 
production costs. 
The compounds according to the invention may be prepared by a number of 
processes as described in the following where R.sup.1, R.sup.4, R.sup.5, 
X, Y.sup.1 and Y.sup.2 are as defined for general formula (1) unless 
specified otherwise. In some of these processes it may be necessary to 
protect a hydroxyl group at the 5-, 13- and/or 23-position in the starting 
material prior to effecting the reaction described. In such cases it may 
then be necessary to deprotect the same hydroxyl group once the reaction 
has occurred to obtain the desired compound of the invention. Conventional 
methods of protection and deprotection may be used, for example, as 
described in `Protective Groups in Organic Synthesis` by Theodora W. 
Greene (Wiley-Interscience, New York 1981) and `Protective Groups in 
Organic Chemistry` by J. F. W. McOmie (Plenum Press, London 1973). Thus, 
for example, an acyl group such as an acetyl group may be removed by basic 
hydrolysis e.g. using sodium hydroxide or potassium hydroxide or ammonia 
in an aqueous alcohol such as methanol. 
Thus, according to another aspect of the invention, we provide a process 
for preparing a compound of formula (1) which comprises incubating a 
compound of formula (2) 
##STR6## 
in a suitable medium in the presence of a microorganism or an enzyme 
derived therefrom or a preparation derived from a microorganism containing 
the enzyme of interest capable of effecting the conversion. 
Suitable microorganisms and extracts thereof for use in the process 
according the invention may be identified by preliminary small scale tests 
designed to demonstrate ability of a microorganism or an extract thereof 
to convert compounds of formula (2) to compounds of formula (1). The 
formation of the compounds of formula (1) may be confirmed by suitable 
chromatographic analysis (e.g. high performance liquid chromatography) of 
the reaction mixture. 
We have found microorganisms of the genus Streptomyces and extracts thereof 
to be particularly suitable for use in the process according to the 
present invention. 
Particular Streptomyces microorganisms for use in the process according to 
the invention include strains of Streptomyces avermitilis, Streptomyces 
venezuelae, Streptomyces violaceoniger, Streptomyces erythaeus, 
Streptomyces spinichromogenes var. kujimyceticus, Streptomyces 
narbonensis, Streptomyces antibioticus, Streptomyces felleus, Streptomyces 
chryseus, Streptomyces flocculus, Streptomyces griseoflavus, Streptomyces 
lavendulae, Streptomyces eurythermus, Streptomyces hygroscopicus, 
Streptomyces halstedii, Streptomyces albogriseolus, Streptomyces cirratus, 
Streptomyces deltae, Streptomyces platensis, Streptomyces fungicidicus 
var. espinomyceticus, Streptomyces mycarofaciens, Streptomyces rimosus, 
Streptomyces djakartensis, Streptomyces platensis subsp malvinus, 
Streptomyces ambofaciens and Streptomyces fradiae and mutants of these 
strains. 
Particularly suitable Streptomyces microorganisms for use in the process 
according to the invention include strains of Streptomyces avermitilis 
e.g. Streptomyces avermitilis ATCC 31272 and Streptomyces avermitilis ATCC 
31780 and mutants thereof. 
Mutants of the above strains may arise spontaneously or may be produced by 
a variety of methods including those described in UK Patent Specification 
2166436. 
Other microorganisms which may be used in the process according to the 
invention include fungi and plant cell preparations. 
Examples of particular fungi for use in the process according to the 
invention include Roccellaria mollis, Roccellaria galapagoensis, 
Schismatomma accedens, Ascochyta pisi, Cladosporium herbarum, Septoria 
nodorum and Stemphylium botryosum. 
Examples of plant cell preparations for use in the process according to the 
invention include Phaseolus aureus, Petroselinum hortense, Glycine max, 
Phaseolus vulgaris, Nicotiana tabacum, Dioscorea deltoidea, Datura 
innoxia, Digitalis purpurea and Digitalis lanata. 
The bioconversion may also be effected using an organism containing the 
genetic material of one of the aforementioned microorganisms that 
participates in the synthesis of the compound of formula (1). Such 
organisms may be obtained using genetic engineering techniques including 
those outlined by D. A. Hopwood in `Cloning genes for Antibiotic 
Biosynthesis in Streptomyces Spp.: Production of a hybrid antibiotic` 
p409-413 in Microbiology 1985, Ed. L. Lieve, American Society of 
Microbiology, Washington D.C. 1985. Such techniques may be used in a 
similar manner to that described previously for cloning antibiotic 
biosynthetic genes, including the biosynthetic genes for actinorhodin 
(Malpartida, F. and Hopwood, D. A. 1984, Nature 309, p462-464), 
erythromycin (Stanzak, R. et al, 1986, Biotechnology, 4, p229-232) and an 
important enzyme involved in penicillin and cephalosporin production in 
Acremonium chrysogenum (Sansom, S. M. et al, 1985, Nature, 318, p191-194). 
Suitable enzymes for use in the process according to the present invention 
may be derived from an extremely wide range of sources. The aforementioned 
Streptomyces microorganisms, however, represent a particularly suitable 
source of enzymes capable of converting compounds of formula (2) into 
compounds of formula (1). 
In one embodiment of the process according to the invention, the conversion 
of a compound of formula (2) into a compound of formula (1) may be 
effected by feeding the compound of formula (2) e.g. in a suitable solvent 
into a fermentation medium comprising the aforementioned microorganism in 
the presence of assimilable sources of carbon, nitrogen and mineral salts. 
Assimilable sources of carbon, nitrogen and minerals may be provided by 
either simple or complex nutrients. Sources of carbon will generally 
include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, 
sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, 
alkanes and vegetable oils. Sources of carbon will generally comprise from 
0.5 to 10% by weight of the fermentation medium. 
Sources of nitrogen will generally include soya bean meal, corn steep 
liquors, distillers solubles, yeast extracts, cottonseed meal, peptones, 
ground nut meal, malt extract, molasses, casein, amino acid mixtures, 
ammonia (gas or solution), ammonium salts or nitrates. Urea and other 
amides may also be used. Sources of nitrogen will generally comprise from 
0.1 to 10% by weight of the fermentation medium. 
Nutrient mineral salts which may be incorporated into the culture medium 
include the generally used salts capable of yielding sodium, potassium, 
ammonium, iron, magnesium, zinc, nickel, cobalt manganese, vanadium, 
chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, 
chloride and carbonate ions. 
An antifoam may be present to control excessive foaming and added at 
intervals as required. 
The compound of formula (2) in a solvent such as a water miscible organic 
solvent (e.g. an alcohol such as methanol or propan-2-ol, a diol such as 
propan-1,2-ol or butan-1,3-ol, a ketone such as acetone, a nitrile such as 
acetonitrile, an ether such as tetrahydrofuran or dioxan, a substituted 
amide such as dimethylformamide or a dialkylsulphoxide such as 
dimethylsulphoxide) may be added at the beginning of the cultivation, or 
more usually, when the growth of the microorganism is under way, e.g. 2 to 
4 days after the start of the cultivation. 
Cultivation of the organism will generally be effected at a temperature of 
from 20.degree. to 50.degree. C., preferably from 25.degree. to 40.degree. 
C., and will desirably take place with aeration and agitation e.g. by 
shaking or stirring. The medium may initially be inoculated with a small 
quantity of a suspension of the sporulated microorganism but in order to 
avoid a growth lag a vegetative inoculum of the organism may be prepared 
by inoculating a small quantity of the culture medium with the spore form 
of the organism, and the vegetative inoculum obtained may be transferred 
to the fermentation medium, or, more preferably to one or more seed stages 
where further growth takes place before transfer to the principal 
fermentation medium. The fermentation will generally be carried out in the 
pH range 4.0 to 9.5, preferably 5.5 to 8.5 when a Streptomyces organism is 
present and preferably 4.0 to 8.5 when a fungus is present. 
Once the compound of formula (2) has been added to the culture, usually 
with gentle mixing, the cultivation is continued such that the desired 
product is accumulated. The presence of the product in the fermentation 
broth may be determined by monitoring extracts of the broth by high 
performance liquid chromatography, and uv spectroscopy at 238 nm. 
The product(s) may be isolated from the whole fermentation broth by 
conventional isolation and separation techniques as described in UK Patent 
Specifications 2166436 and 2176182. 
When plant cells are used as part of the fermentation process it is 
preferable for the cultivation to be carried out using a plant medium 
containing a plant cell growth regulator such as indole acetic acid, 
naphthalene acetic acid, indole butyric acid, 2,4-dichlorophenoxyacetic 
acid, kinetin or benzylamino purine at a temperature of from 15.degree. to 
35.degree. C. with the pH maintained within the range 5.0 to 7.5. Ammonium 
salts and nitrates also constitute the preferred sources of nitrogen 
present in the fermentation medium. Sucrose, fructose and glucose also 
constitute the preferred sources of carbon present in the fermentation 
medium. 
In a further embodiment of the process according to the invention, the 
conversion of a compound of formula (2) into a compound of formula (1) may 
be effected by combining and incubating a compound of formula (2) e.g. in 
a suitable solvent (e.g. a water miscible organic solvent as previously 
defined) with a preparation of the enzyme of the invention and an 
appropriate sugar, desirably in a buffer solution, at, for example, 
0.degree. to 60.degree., preferably 20.degree. to 40.degree. e.g. about 
28.degree. C. The reaction will generally be carried out in the pH range 
3.5 to 8.5 e.g. 5.5 to 7.5. When the reaction is complete, i.e. when the 
compound of formula (2) is no longer converted to the compound of the 
invention (as determined by monitoring extracts of the reaction mixture by 
high performance liquid chromatography and uv spectroscopy at 238 nm) the 
product is recovered by conventional isolation and separation techniques 
as described in UK Patent Specifications 2166436 and 2176182. 
The enzyme for use in the process of the present invention may be prepared, 
for example, by culture of a microorganism which produces the enzyme in a 
nutrient medium. Suitable nutrient media and fermentation conditions for 
the preparation of the enzyme include those previously described for the 
preparation of a compound of formula (1) from a compound of formula (2) in 
the presence of a microorganism. The time at which the required enzymic 
activity reaches a maximum will, of course, vary according to the 
microorganism used and, hence, the optimum cultivation time is desirably 
determined independently for each strain employed. 
For microorganisms where the enzyme is extracellular, the liquid culture 
medium or the filtrate after removal of whole cells may be used as a 
source of enzyme. Where the enzyme is cell-bound it may be released for 
use by conventional methods such as sonication, grinding with glass beads, 
homogenisation, treatment with lytic enzymes or with detergents, after 
suspension of the cells in a suitable buffer. 
The resulting preparation, either with or without removal of cell debris, 
may be used as a source of enzyme. It is preferred, however, to purify the 
enzyme further by conventional means. Batch or column chromatography with 
ion-exchange celluloses or affinity adsorbents or other adsorbents e.g. 
hydroxylapatite may conveniently be employed. In addition, the enzyme may 
be concentrated or further purified by molecular sieve techniques e.g. 
ultrafiltration or salting out. In general, during purification 
procedures, it is desirable to maintain the pH within the range 3 to 11. 
The enzyme may be employed in an immobilized form, e.g. by insolubilisation 
or entrappment thereof on or in a suitable matrix. Thus an extract of the 
enzyme may be bound or linked to an otherwise inert inorganic or organic 
polymer, entrapped on or in a fibre, or on or in a membrane or polymer 
such as polyacrylamide gel, adsorbed on an ion-exchange resin, crosslinked 
with a reagent such as glutaraldehyde, or occluded in an envelope such as 
a bead. Immobilized enzymes may advantageously be employed both in batch 
processes, after which the enzyme may be reused, and continuous flow 
processes wherein substrates pass through a column containing the 
immobilized enzyme. 
In a particular embodiment of the fermentation process compounds of formula 
(1) in which R.sup.4 is a hydroxy group are conveniently prepared from 
corresponding compounds of formula (2) in a suitable medium in the 
presence of a strain of Streptomyces avermitilis capable of effecting the 
conversion. 
The fermentation process described above will generally produce the 
compounds of formula (1) in which R is a sugar residue. Acylated 
derivatives thereof may be prepared from the products of the fermentation 
process using standard N- and/or O-acylating conditions. 
Intermediate compounds of formula (2) may be prepared by reducing a 
compound of formula (3) 
##STR7## 
followed, if necessary, by removal of any protecting groups present. 
The reduction may be effected for example using a reducing agent such as a 
borohydride, for example an alkali metal borohydride such as sodium 
borohydride or a lithium alkoxyaluminium hydride such as lithium 
tributoxyaluminium hydride. 
The reaction involving a borohydride reducing agent takes place in the 
presence of a solvent such as an alkanol e.g. isopropyl alcohol or 
isobutyl alcohol conveniently at a temperature in the range of -30.degree. 
to +80.degree. C. e.g. at 0.degree. C. The reaction involving a lithium 
alkoxyaluminium hydride takes place in the presence of a solvent such as 
an ether e.g. tetrahydrofuran or dioxan conveniently at a temperature in 
the range of -78.degree. to 0.degree. C. e.g. at -78.degree. C. 
Intermediate compounds of formula (3) may be prepared by oxidising a 
compound of formula (4) 
##STR8## 
[wherein R.sup.4 is as defined in formula (1) (except that it cannot 
represent a hydroxyl group)] followed, if necessary, by removal of any 
protecting groups present. 
Suitable oxidising agents for the conversion include dialkylsulphoxides 
e.g. dimethylsulphoxide, in the presence of an activating agent such as 
N,N'-dicyclohexylcarbodiimide or an acyl halide, e.g. oxalyl chloride. The 
reaction may conveniently be effected in a suitable solvent such as a 
halogenated hydrocarbon e.g. methylene chloride at a temperature in the 
range of -80.degree. to +50.degree. C. 
Intermediate compounds of formula (4) may be prepared by oxidising a 
compound of formula (5) 
##STR9## 
The oxidation may be effected for example with an oxidising agent such as 
selenium dioxide, preferably in the presence of an activator such as a 
peroxide, e.g. tert-butyl hydroperoxide. The reaction may conveniently be 
effected in an inert solvent such as a halogenated hydrocarbon e.g. 
dichloromethane, an ester, e.g. ethyl acetate or an ether, e.g. 
tetrahydrofuran, at a temperature in the range of 0.degree. to 50.degree. 
C., preferably at room temperature. 
Alternatively, a compound of formula (5) may be treated with an oxidising 
agent described above in formic acid at a temperature of from 20.degree. 
to 100.degree. C. to provide a compound of formula (6) 
##STR10## 
which, upon acid hydrolysis, e.g. using hydrochloric acid provides a 
compound of formula (4). 
Intermediate compounds of formula (6) in which Y.sup.1 is --CH.sub.2 --, 
Y.sup.2 is --CH-- and --X-- represents &gt;C.dbd.NOR.sup.7 (wherein R.sup.7 
is as previously defined) may, if desired, be prepared from a 
corresponding compound of formula (6) in which Y.sup.1 is --CH.sub.2 --, 
Y.sup.2 is --CH-- and --X-- represents &gt;C.dbd.O by reaction with a reagent 
H.sub.2 NOR.sup.7. 
The oximation reaction may conveniently be effected at a temperature in the 
range -20.degree. to +100.degree. C., e.g. -10.degree. to +50.degree. C. 
It is convenient to use the reagent H.sub.2 NOR.sup.7 in the form of a 
salt, for example an acid addition salt such as the hydrochloride. When 
such a salt is employed the reaction may be carried out in the presence of 
an acid binding agent. 
Solvents which may be employed include alcohols (e.g. methanol or ethanol), 
amides (e.g. N,N-dimethylformamide, N,N-dimethylacetamide or 
hexamethylphosphoramide), ethers (e.g. cyclic cyclic ethers such as 
tetrahydrofuran or dioxan, and acylic ethers such as dimethoxyethane or 
diethylether), nitriles (e.g. acetonitrile), sulphones (e.g. sulpholane) 
and hydrocarbons such as halogenated hydrocarbons (e.g. methylene 
chloride), as well as mixtures of two or more such solvents. Water may 
also be employed as a cosolvent. 
When aqueous conditions are employed the reaction may conveniently be 
buffered with an appropriate acid, base or buffer. 
Suitable acids include mineral acids, such as hydrochloric or sulphuric 
acid, and carboxylic acid such as acetic acid. Suitable bases include 
alkali metal carbonates and bicarbonates such as sodium bicarbonate, 
hydroxides such as sodium hydroxide, and alkali metal carboxylates such as 
sodium acetate. A suitable buffer is sodium acetate/acetic acid. 
Intermediate compounds of formula (5) in which Y.sup.1 is --CH.sub.2 --, 
Y.sup.2 is --CH--, and X represents 
##STR11## 
(where R.sup.2 represents a hydrogen atom or a group OR.sup.6 and R.sup.3 
represents a hydrogen atom or R.sup.2 and R.sup.3 together with the carbon 
atom to which they are attached represent &gt;C.dbd.O), R.sup.4 is a group 
OR.sup.6 and R.sup.5 is a hydrogen atom are known compounds described in 
UK Patent Specifications 2166436 and 2176182. 
Intermediate compounds of formula (5) in which --Y.sup.1 --X--Y.sup.2 -- 
represents --CH.dbd.CH--CH-- or CH.sub.2 --CH.dbd.C--, R.sup.4 is a group 
OR.sup.6 and R.sup.5 is a hydrogen atom are known compounds described in 
European Patent Specification 215654. 
Intermediate compounds of formula (5) in which Y.sup.1 is --CH.sub.2 --, 
Y.sup.2 is --CH-- and X represents &gt;C.dbd.CH.sub.2 may be prepared by 
reaction of a corresponding compound of formula (5) in which X is &gt;C.dbd.O 
with an appropriate Wittig reagent e.g. a phosphorane of formula 
(R.sup.13).sub.3 P.dbd.CH.sub.2 (where R.sup.13 is C.sub.1-6 alkyl or 
aryl, e.g. monocyclic aryl such as phenyl). Suitable reaction solvents 
include ethers such as tetrahydrofuran or diethyl ether or a dipolar 
aprotic solvent such as dimethylsulphoxide. The reaction may be carried 
out at any suitable temperature e.g. at 0.degree. C. 
Intermediate compounds of formula (5) in which Y.sup.1 is --CH.sub.2 --, 
Y.sup.2 is --CH--, X represents &gt;C.dbd.NOR.sup.7 (where R.sup.7 is as 
previously defined), R.sup.4 is a group OR.sup.6 and R.sup.5 is a hydrogen 
atom or R.sup.4 and R.sup.5 together with the carbon atom to which they 
are attached represent &gt;C.dbd.O, or intermediates in which X represents a 
group 
##STR12## 
(where R.sup.2 is a hydrogen atom or a group OR.sup.6 and R.sup.3 is a 
hydrogen atom) or X represents &gt;C.dbd.NOR.sup.7 or --Y.sup.1 --X--Y.sup.2 
-- represents --CH.dbd.CH--CH-- or --CH.sub.2 --CH.dbd.C-- and R.sup.4 and 
R.sup.5 together with the carbon atom to which they are attached represent 
&gt;C.dbd.NOR.sup.8 may be prepared from the corresponding 5 and/or 23 keto 
compounds of formula (1) by reaction with a reagent H.sub.2 NOR.sup.7 
using the oximation reaction conditions previously described. It will be 
appreciated that in the preparation of a 5,23-bisoxime of formula (5) from 
a corresponding 5,23-diketone the groups &gt;C.dbd.NOR.sup.7 and 
&gt;C.dbd.NOR.sup.8 will be equivalent. 
Intermediates of formula (5) in which R.sup.4 and R.sup.5 together with the 
carbon atom to which they are attached represent &gt;C.dbd.O may be prepared 
by oxidation of the corresponding 5-hydroxy compounds in which R.sup.4 is 
a hydroxy group. 
The reaction may be effected with an oxidising agent serving to convert an 
allylic secondary hydroxyl group to an oxo group, whereby a compound of 
formula (5) is produced. 
Suitable oxidising agents include, for example, transition metal oxides, 
such as manganese dioxide, and atmospheric oxygen in the presence of a 
suitable catalyst such as a finely divided metal e.g. platinum. 
The oxidising agent will generally be used in excess over the 
stoichiometric quantity. 
The reaction may conveniently be effected in a suitable solvent which may 
be selected from a ketone, e.g. acetone; an ether, e.g. diethyl ether, 
dioxan or tetrahydrofuran; a hydrocarbon, e.g. hexane; a halogenated 
hydrocarbon e.g. chloroform or methylene chloride; or an ester, e.g. ethyl 
acetate. Combinations of such solvents either alone or with water may also 
be used. 
The reaction may be carried out at a temperature of from -50.degree. C. to 
+50.degree. C., preferably from 0.degree. to 30.degree. C. 
In a further process, the compounds of (1) in which OR.sup.6 is a hydroxyl 
group may be prepared from a corresponding compound of formula (1) in 
which OR.sup.6 is a substituted hydroxyl group. The conversion will 
usually be carried out in the context of removing a protecting group such 
as referred to above. 
In a yet further process, the compounds of formula (1) in which OR.sup.6 is 
a substituted hydroxyl group may generally be prepared by reacting the 
corresponding 5- and/or 23-hydroxy compound with reagent serving to form a 
substituted hydroxyl group. 
The reaction will in general be an acylation, sulphonylation, 
etherification, silylation or acetalation, and the reaction may be carried 
out according to the general methods described in UK Patent specification 
2176182. 
Salts of acids of formula (1) may be prepared by conventional means, for 
example by treating the acid with a base or converting one salt into 
another by exchange of ion. 
The invention is further illustrated by the following Preparations and 
Examples wherein the compound of formula (5) above in which R.sup.1 is 
isopropyl, Y.sup.1 is --CH.sub.2 --, Y.sup.2 is --CH--, X represents 
##STR13## 
(where R.sup.2 is a hydroxyl group and R.sup.3 is a hydrogen atom), 
R.sup.4 is a hydroxy group and R.sup.5 is a hydrogen atom is referred to 
as `Factor A`. Compounds according to the invention are named with respect 
to Factor A. All temperatures are in .degree. C. 
INTERMEDIATE 1 
(13R)-Hydroxy-23-desoxy Factor A, 5-acetate 
23-Desoxy Factor A, 5-acetate (4.79 g, Example 112 in UK Patent 
Specification 2176182) was added to a stirred mixture of selenium dioxide 
(416 mg) and t-butyl hydroperoxide (3M in dichloromethane; 5 ml) in 
dichloromethane (30 ml). After stirring at room temperature for 30 h the 
reaction mixture was diluted with ethyl acetate (200 ml), washed with 
water and brine, and dried (Na.sub.2 SO.sub.4). The solvent was evaporated 
and the residue purified by chromatography (250 g silica gel, Merck 9385). 
Elution with ethyl acetate: light petroleum (1:4.fwdarw.1:2) afforded the 
title compound (560 mg) as a pale yellow foam. .nu..sub.max (CHBr.sub.3) 
3600, 3460 (OH), 1732 (OAc), 1712 (CO.sub.2 R), 993 cm.sup.-1 (C-O); 
.delta. (CDCl.sub.3) values include 0.69 (3H, t, J 5 Hz), 2.15 (3H,s), 
3.32 (1H,m), 3.72 (1H, d, J 10 Hz), 4.05 (1H, d, J 5 Hz), 5.52 (2H,m). 
INTERMEDIATE 2 
13-Keto-23-desoxy Factor A, 5-acetate 
A solution of dimethyl sulphoxide (92 .mu.l) in dichloromethane (1 ml) was 
added dropwise over 2 min to a solution of oxalyl chloride (57 .mu.l) in 
dichloromethane (2 ml) at -50.degree., under an atmosphere of nitrogen. 
After 5 min a solution of the compound of Intermediate 1 (213 mg) in 
dichloromethane (3 ml) was added dropwise over 2 min at -50.degree.. After 
30 min at -50.degree. to -45.degree., triethylamine (453 .mu.l) was added. 
After 5 min the cooling bath was removed and the reaction mixture was 
allowed to warm to room temperature during 30 min. The reaction mixture 
was partitioned between dichloromethane (50 ml) and water (50 ml). The 
organic phase was separated and the aqueous phase extracted with 
dichloromethane (25 ml). The combined organic extracts were washed with 2M 
hydrochloric acid (75 ml), saturated sodium bicarbonate solution (75 ml) 
and brine (75 ml), and dried (Na.sub.2 SO.sub.4). The solvent was 
evaporated and the residue purified by flash chromatography (35 g silica 
gel, Merck 9385). Elution with ethyl acetate: light petroleum (1:2) 
afforded the title compound (132 mg) as a white foam. 
[.alpha.].sub.D.sup.22 +256.degree. (c0.6, CHCl.sub.3); .delta. 
(CDCl.sub.3) values include 1.76 (3H,s), 1.82 (3H,s), 2.16 (3H,s), 3.40 
(2H,m), 5.09 (1H, d, J 9 Hz), 5.52 (2H,m), 6.26 (1H, t, J 8 Hz). 
INTERMEDIATE 3 
(13S)-Hydroxy-23-desoxy Factor A, 5-acetate 
A solution of sodium borohydride (0.2M in ethanol; 3.63 ml) was added 
dropwise to a solution of Intermediate 2 (431 mg) in ethanol (15 ml) at 
0.degree.. After 30 min at 0.degree., the reaction mixture was diluted 
with ethyl acetate, washed with 2M hydrochloric acid, saturated sodium 
bicarbonate solution and brine, and dried (Na.sub.2 SO.sub.4). The solvent 
was evaporated and the residue purified by flash chromatography (40 g 
silica gel, Merck 9385). Elution with ethyl acetate:light petroleum (1:3) 
afforded the title compound as a white foam (368 mg); .delta. (CDCl.sub.3) 
values include 0.69 (3H,d,J5 Hz), 0.93 (3H,d,J6 Hz), 1.04 (3H,d,J6 Hz), 
1.17 (3H,d,J6 Hz), 3.31 (1H,m), 4.00 (1H,s), 4.04 (1H,d,5 Hz), 5.52 
(2H,m). 
INTERMEDIATE 4 
(13S)-Hydroxy-23-desoxy Factor A 
Aqueous sodium hydroxide (1M; 87 .mu.l) was added to a stirred solution of 
Intermediate 3 (38 mg) in methanol (1 ml) at 0.degree.. After 1.5 h at 
0.degree., the reaction mixture was diluted with ethyl acetate (25 ml), 
washed with water and brine, and dried (Na.sub.2 SO.sub.4). The solvent 
was evaporated and the residue purified by flash chromatography (10 g 
silica gel, Merck 9385). Elution with ethyl acetate:light petroleum (1:2) 
gave the title compound as a white foam (31 mg); .nu.max (CHBr.sub.3) 
3540, 3460 (OH), 1704 cm.sup.-1 (CO.sub.2 R); .delta. (CDCl.sub.3) values 
include 0.69 (3H,d,J5 Hz), 0.95 (3H,d,J6 Hz), 1.06 (3H,d,J6 Hz), 1.18 
(3H,d,J6 Hz), 3.26 (1H,m), 3.96 (1H,d,J5 Hz), 4.01 (2H,s), 4.29 (1H,t,J5 
Hz). 
INTERMEDIATE 5 
(13R)-Formyloxy-23-keto Factor A,5-acetate 
To a slurry of selenium dioxide (120 mg) in formic acid (1 ml) stirring at 
60.degree. was added a solution of 23-keto Factor A, 5-acetate (420 mg, 
Example 18 in UK Patent Specification 2176182) in formic acid (3 ml). The 
reaction mixture was stirred at 60.degree. for 6 mins, then poured into 
water (150 ml) and extracted with diethylether (4.times.50 ml). The 
organic phase was dried (MgSO.sub.4) and solvent removed to give a brown 
solid which was purified by medium pressure column chromatography on 
silica (100 g Merck kieselgel 60; 230-400 mesh). Elution with 
dichloromethane:ethyl acetate (16:1) gave the title compound as a cream 
foam (103 mg); .nu.max (CHBr.sub.3) 3480 (OH) and 1714 cm.sup.-1 (ester 
and ketone); .delta. (CDCl.sub.3) includes 0.86 (d,6 Hz,3H), 0.97 (d,6 
Hz,3H), 1.02 (d,6 Hz,3 H), 1.07 (d,6 Hz,3H), 1.76 (s,3H), 3.32 (m, 1H), 
2.16 (s,3H), 4.06 (d,6 Hz,1H), 5.02 (d,10 Hz,1H), 5.53 (m,2H), 8.08 
(s,1H). 
INTERMEDIATE 6 
(13R)-Formyloxy-23(E)-methoxyimino Factor A,5-acetate 
To a solution of Intermediate 5 (80 mg) in methanol (8 ml) was added a 
solution of methoxyamine hydrochloride (29 mg) and sodium acetate (33 mg) 
in water (0.7 ml). The reaction mixture was stirred at room temperature 
for 3 h, then poured into ether (40 ml) and washed with water. The organic 
phase was dried (MgSO.sub.4) and solvent removed to give the title 
compound as a cream foam (79 mg); .delta. (CDCl.sub.3) includes 0.91 (d,6 
Hz,3H), 0.97 (d,6 Hz,3H), 1.02 (d,6 Hz,3H), 1.07 (d,6 Hz,3H), 1.76 (s,3H), 
2.16 (s,3H)m, 3.28 (d,15 Hz,1H), 1.91 (d,15 Hz,1H), 3.32 (m,1H), 3.83 
(s,3H), 4.06 (d,6 Hz,1H), 5.04 (d,10 Hz,1H), 5.54 (m,2H), 8.09 (s,1H). 
INTERMEDIATE 7 
(13R)-Hydroxy-23(E)-methoxyimino Factor A,5-acetate 
To a solution of Intermediate 6 (65 mg) in methanol (5 ml) was added 2N 
hydrochloric acid (0.1 ml). The reaction mixture was stirred at room 
temperature for 4 h, then poured into dichloromethane (60 ml) and washed 
with saturated sodium bicarbonate solution and water. The organic phase 
was dried (MgSO.sub.4) and solvent removed to give a foam which was 
purified by medium pressure column chromatography on silica (30 g, Merck 
Kieselgel 60, 230-400 mesh). Elution with dichloromethane:ethyl acetate 
(4:1) gave the title compound as a white foam (39 mg); 
[.alpha.].sub.D.sup.21 +126.degree. (C.dbd.0.22, CH.sub.2 Cl.sub.2). 
.delta. (CDCl.sub.3) includes 0.92 (d,6 Hz,3H), 0.96 (d,6 Hz,3H), 1.05 
(d,6 Hz,3H), 1.12 (d,6 Hz,3H), 1.77 (s,3H), 2.17 (s,3H), 3.29 (d,15 
Hz,1H), 1.91 (d,15 Hz,1H), 3.32 (m,1H), 3.70 (dd10,2 Hz,1H), 3.83 (s,3H), 
4.04 (d,6 Hz,1H), 5.54 (m,2H). 
INTERMEDIATE 8 
13-Keto-23(E)-methoxyimino Factor A,5-acetate 
To a solution of oxalyl chloride (0.24 ml) in freshly distilled 
dichloromethane (3.6 ml) stirring at -60.degree. under nitrogen was added 
a solution of dimethyl sulphoxide (0.4 ml) in freshly distilled 
dichloromethane (3.6 ml). The solution was cooled to -65.degree. and after 
5 mins and a solution of Intermediate 7 (770 mg) in dichloromethane (6 ml) 
was added. The cooling bath was allowed to warm to -60.degree. then the 
reaction mixture left a further 30 mins stirring at -60.degree. to 
-50.degree. C. 
Triethylamine (1.5 ml) was added and the reaction mixture allowed to warm 
to room temperature. The reaction mixture was then poured into 
dichloromethane (100 ml) and the solvent removed under vacuum. Diethyl 
ether (60 ml) was added and the triethylamine salt filtered off. The ether 
was removed under vacuum to give a foam which was purified by medium 
pressure column chromatography on silica (180 g, Merck Kieselgel 60, 
230-400 mesh). Elution with dichloromethane:ethyl acetate (14:1) gave the 
title compound as a beige foam (450 mg); .delta. (CDCl.sub.3) includes 
0.92 (d,6 Hz,3H), 0.96 (d,6 Hz,3H), 1.01 (d,6 Hz,3H), 1.18 (d,6 Hz,3H), 
1.76 (s,3H), 1.80 (s,3H), 2.16 (s,3H), 3.31 (d,15 Hz,1H), 1.93 (d,15 
Hz,1H), 3.39 (m,1H), 3.84 (s,3H), 4.08 (d,6 Hz,1H), 5.54 (m,2H), 6.22 (t,9 
Hz,1H). 
INTERMEDIATE 9 
(13S)-Hydroxy-23(E)-methoxyimino Factor A,5-acetate 
To a solution of Intermediate 8 (620 mg) in ethanol (25 ml) stirring at 
0.degree. was added a solution of sodium borohydride (4.9 ml of a 0.2M 
solution in ethanol). The reaction mixture was stirred 0.degree. for 30 
mins, then poured into ethyl acetate (400 ml) and washed with 2N 
hydrochloric acid, saturated sodium bicarbonate solution, water and brine. 
The organic phase was dried (MgSO.sub.4) and solvent removed to give a 
beige foam which was purified by medium pressure column chromatography on 
silica (180 g, Merck Kieselgel 60, 230-400 mesh). Elution with 
dichloromethane:ethyl acetate (10:1) gave the title compound as a white 
foam (502 mg); .delta. (CDCl.sub.3) includes 0.92 (d,6 Hz,3H), 0.97 (d,6 
Hz,3H), 1.05 (d,6 Hz,3H), 1.16 (d,6 Hz,3H), 1.76 (s,3H), 2.16 (s,3H), 3.29 
(d,15 Hz, 1H), 1.91 (d,15 Hz,1H), 3.32 (m,1H), 3.84 (s,3H), 4.00 (broad 
s,1H), 4.06 (d,6 Hz,1H), 5.53 (m,2H). 
INTERMEDIATE 10 
(13S)-Hydroxy-23(E)-methoxyimino Factor A 
To a solution of Intermediate 9 (291 mg) in methanol (7 ml) stirring at 
0.degree. was added dropwise a solution of sodium hydroxide (17 mg) in 
water (0.6 ml). The reaction mixture was stirred at 0.degree. for 2 h, 
then was poured into dichloromethane (75 ml) and washed with 2N 
hydrochloric acid (2.times.50 ml), water and brine. The organic phase was 
dried (MgSO.sub.4) and solvent removed to give a beige foam which was 
purified by medium pressure column chromatography on silica (80 g, Merck 
Kieselgel 60, 230-400 mesh). Elution with dichloromethane:ethyl acetate 
(4:1) gave the title compound as a white foam (228 mg). .delta. 
(CDCl.sub.3) includes 0.91 (d,6 Hz,3H), 0.96 (d,6 Hz,3H), 1.05 (d,6 
Hz,3H), 1.17 (d,6 Hz,3H), 1.88 (s,3H), 3.29 (d,15 Hz,1H), 1.92 (d,15 
Hz,1H), 3.27 (m,1H), 3.83 (s,3H), 3.96 (d,6 Hz,1H), 4.00 (broad s,1H), 
4.28 (t,6 Hz,1H).

EXAMPLE 1 
A slope of Streptomyces avermitilis ATCC 31272 was used to inoculate a 250 
ml shake flask containing the medium A (25 ml): 
______________________________________ 
gL.sup.-1 
______________________________________ 
D-Glucose 2.5 
Malt Dextrose MD30E 25.0 
Arkasoy 50 12.5 
Molasses 1.5 
KH.sub.2 PO.sub.4 0.125 
Calcium carbonate 1.25 
[3-(N-Morpholino)propanesulphonic acid] 
21.0 
Distilled water as required 
pH adjusted to 6.5 with H.sub.2 SO.sub.4 before autoclaving. 
______________________________________ 
The flask was incubated at 28.degree. for 2 days on a rotary shaker (250 
rpm) and a portion (5 ml) of this 2 day old culture was placed in a 50 ml 
shake flask and 50 .mu.l of a 20 mg/ml solution of 
(13S)-hydroxy-23(E)-methoxyimino Factor A in methanol added. The flask was 
incubated at 28.degree. for 5 days on a rotary shaker (250 rpm) after 
which time an equal volume of methanol was added. The shake flask and 
contents were shaken for 1 h, centrifuged to remove the cells and the 
supernatant evaporated in vacuo to 1 ml. 
180 .mu.l portions of the sample were fractioned on a column of Spherisorb 
S5 ODS-2 (100 mm.times.4.6 mm) with detection at 238 nm. A gradient 
solvent system was used at a constant flow of 3 ml/min between Solvent A 
(acetonitrile/water 1:1) and Solvent B (acetonitrile/water 65:35). The 
initial eluant contained 80% Solvent A and 20% Solvent B and after 10 min. 
100% Solvent B. From each injection, uv absorbing peaks with retention 
ratios relative to unchanged substrate of 2.13 and 3.45 were collected and 
those peaks with identical retention ratios were combined and evaporated 
to yield (a) a compound of formula (1) in which R is 
.alpha.-L-oleandrosyl-.alpha.-L-oleandrosyl, R.sup.1 is isopropyl, Y.sup.1 
is --CH.sub.2 --, Y.sup.2 is --CH--, X is &gt;C.dbd.NOCH.sub.3, R.sup.4 is a 
hydroxyl group and R.sup.5 is a hydrogen atom (retention ratio 2.13); Mass 
spectra (Electron impact) m/z include 907, 795, 764, 620, 618, 566, 476, 
408, 376 and 145 (negative CI, NH.sub.3) m/z 943 (M.sup.-), 925, 907 
(positive CI, NH.sub.3) m/z 961 (M.sup.+ NH.sub.4).sup.+, 944 (MH).sup.+ 
and (b) a compound of formula (1) in which R is 
.alpha.-L-oleandrosyl-.alpha.-L-oleandrosyl, R.sup.1 is isopropyl, Y.sup.1 
is --CH.sub.2 --, Y.sup.2 is --CH--, X is &gt;C.dbd.NOCH.sub.3, R.sup.4 is a 
methoxy group and R.sup.5 is a hydrogen atom (retention ratio 3.45); Mass 
spectra (Electron impact) m/z include 907, 827, 814, 795, 764, 670, 651, 
376, 275, 264, 263, 257, 145, 127, 113, 95 and 87 (negative CI, NH.sub.3) 
m/z 957 (M.sup.-), 907 (M--H.sub.2 O--CH.sub.3 OH)-- (positive CI, 
NH.sub.3) m/z 975 (M.sup.+ NH.sub.4).sup.+ 958 (MH).sup.+. 
EXAMPLE 2 
2.times.25 ml volumes of medium A in 250 ml shake flasks were inoculated 
directly from a slope of Streptomyces avermitilis ATCC 31780 and cultured 
at 28.degree. as a rotary shaker at a speed of 250 rpm, 2" throw for 2 
days. 
The contents of the flasks were used to inoculate 2.5 L of medium A in a 
3.5 L fermenter containing polypropylene glycol 2000 (1.25 ml). The 
culture was maintained at 28.degree. and agitated at 250 rpm and aerated 
at 1.25 L min.sup.-1. After 2 days, 13(S)-hydroxy-23-desoxy Factor A (234 
mg) in methanol (25 ml) was added followed by sinefungin (30 mg) in water 
(25 ml). At this stage the agitation and aeration rates were increased to 
500 rpm and 2.5 L min.sup.-1 respectively. 
After 4 days the fermentation liquor was centrifuged, the supernatant 
decanted and the cells were washed with water (0.5 L) and centrifuged 
again. The water wash was added to the supernatant and this was extracted 
with ethyl acetate (4.times.0.25 L). The combined ethyl acetate extracts 
were evaporated in vacuo to give an oil. The cells were extracted with 
methanol and the combined methanol extracts were washed with hexane 
(4.times.0.1 L with 0.1 L water added at each stage) and the aqueous layer 
was then extracted with methylene chloride (3.times.0.2 L) and the 
combined methylene chloride layers were evaporated in vacuo to an oil. The 
oil from extraction of supernatant was extracted with acetonitrile (25 ml) 
and this solution was added to the oil derived from the cells and 
filtered. The filtrate was applied to a column (26.times.2 cm) of Sephadex 
LH20 in acetonitrile and fractions (10 ml) were collected after a foreun 
of (50 ml). Fractions 2-18 were combined, evaporated in vacuo to an oil 
which was dissolved in acetonitrile (15 ml) and water (1 ml) and submitted 
to preparative chromatography on Spherisorb S50DS-2 (25.times.2 cm) in a 
solvent of acetonitrile/water (7:3) at a flow rate of 25 ml/min and 
detention at .lambda.238 nm. Fractions eluting between 43 to 46 min. from 
consecutive (1 ml) injections were combined, diluted with water (1:1) and 
pumped back onto the column and then eluted with acetonitrile. Evaporation 
of the acetonitrile in vacuo yielded an oil which was dissolved in 
methanol (2 ml) and passed down a column (100 ml) of Sephadex LH20 in 
methanol. Combination of fractions with .lambda..sub.max 244 nm and 
evaporation in vacuo again yielded an oil. This was once more dissolved in 
acetonitrile (4 ml) and fractionated (15 ml) on a column (25.times.2 cm) 
of Sephadex LH20 in acetonitrile. Combination of fractions 5-10, 
evaporation of these in vacuo, followed by lyophilisation of the residue 
from cyclohexane/acetone gave a compound of formula (1) in which R is 
.alpha.-L-oleandrosyl-.alpha.-L-oleandrosyl, R.sup.1 is isopropyl, Y.sup.1 
is --CH.sub.2 --, Y.sup.2 is --CH--, X is &gt;CH.sub.2, R.sup.4 is a hydroxy 
group and R.sup.5 is a hydrogen atom (19.3 mg) as a colourless solid, 
.lambda.(MeOH) 231.2 inf. (E.sub.1.sup.1 202), 240 inf. (E.sub.1.sup.1 
256), 244.6 (E.sub.1.sup.1 278), 250 nm inf. (E.sub.1.sup.1 206), 
.nu.CHBr.sub.3 3570, 3550, 1705, 1040, 980 cm.sup.-1. 
A 125 MHz .sup.13 C nmr spectrum in CDCl.sub.3 gave signals at about 
.delta.173.8, 139.5, 137.9, 137.8, 136.4, 134.6, 131.5, 124.5, 120.3, 
118.4, 117.9, 98.4, 97.5, 94.5, 82.4, 81.5, 80.4, 80.2, 79.2, 79.0, 78.1, 
76.0, 68.5, 68.3, 68.0, 67.6, 67.1, 56.3, 56.2, 45.6, 40.8, 39.6, 36.8, 
35.5, 34.4, 34.1, 31.5, 27.5, 26.5, 22.8, 22.6, 20.1, 19.8, 18.2, 17.6, 
17.5, 15.0 and 10.9. 
500 MHz .sup.1 H N.m.r. spectrum gave signals at about .delta.3.78 (1H, dq, 
10, 6), 3.82 (1H, dq, 10, 6), 3.18 (1H, t, 9), 3.22 (1H, t, 9) and 3.42 
(6H, s). 
MS (E.I.) 900 (M.sup.+) 882, 756, 738, 612, 594, 576, 484, 333, 315, 261, 
249, 221, 179, 145. 
EXAMPLE 3 
Streptomyces avermitilis ATCC 31272 was incubated with 
13(S)-hydroxy-23-desoxy Factor A according to the method of Example 1. 
180 .mu.l portions of the sample obtained were fractionated on a column of 
Spherisorb S5 ODS-2 (100 mm.times.4.6 mm) with detection at 238 nm using 
acetonitrile/water (65:35) adjusted to pH 4.2 with acetic acid as eluant. 
The uv absorbing peak with a retention ratio relative to unchanged 
substrate of 1.87 was collected from each injection and those peaks with 
identical retention ratios were combined and evaporated in vacuo to yield 
a compound of formula (1) in which R is .alpha.-L-oleandrosyl, R.sup.1 is 
isopropyl, Y.sup.1 is --CH.sub.2 --, Y.sup.2 is --CH--, X is &gt;CH.sub.2, 
R.sup.4 is a hydroxy group and R.sup.5 is a hydrogen atom (retention ratio 
1.87); Mass spectra (Electron impact) m/z include 756 (M.sup.+), 738, 720, 
594, 576, 333, 315, 261, 249, 221, 179, 151 and 145 (negative CI, 
NH.sub.3) m/z 756 (M.sup.-). 
The following are examples of formulations according to the invention. The 
term `Active Ingredient` as used hereinafter means a compound of the 
invention. 
MULTIDOSE ENTERAL INJECTION 
EXAMPLE 1 
______________________________________ 
% w/v Range 
______________________________________ 
Active ingredient 
2.0 0.1-6.0% w/v 
Benzyl alcohol 1.0 
Polysorbate 80 10.0 
Glycerol formal 50.0 
Water for Injections 
to 100.0 
______________________________________ 
Dissolve the active ingredient in the polysorbate 80 and glycerol formal. 
Add the benzyl alcohol and make up to volume with Water for Injections. 
Sterilize the product by conventional methods, for example sterile 
filtration or by heating in an autoclave and package aseptically. 
EXAMPLE 2 
______________________________________ 
% w/v Range 
______________________________________ 
Active ingredient 
4.0 0.1-7.5% w/v 
Benzyl alcohol 2.0 
Glyceryl triacetate 
30.0 
Propylene glycol 
to 100.0 
______________________________________ 
Dissolve the active ingredient in the benzyl alcohol and glyceryl 
triacetate. Add the propylene glycol and make up to volume. Sterilize the 
product by conventional pharmaceutical methods, for example sterile 
filtration, and package aseptically. 
EXAMPLE 3 
______________________________________ 
% Range 
______________________________________ 
Active ingredient 
2.0 w/v 0.1-7.5% w/v 
Ethanol 36.0 v/v 
Non-ionic surfactant 
10.0 w/v 
(e.g. Synperonic PE L44*) 
Propylene glycol 
to 100.0 
______________________________________ 
*Trademark of ICI 
Dissolve the active ingredient in the ethanol and surfactant and make up to 
volume. Sterilize the product by conventional pharmaceutical methods, for 
example sterile filtration, and package aseptically. 
EXAMPLE 4 
______________________________________ 
% Range 
______________________________________ 
Active ingredient 
2.0 w/v 0.1-3.0% w/v 
Non-ionic surfactant 
2.0 w/v 
(e.g. Synperonic PE F68*) 
Benzyl alcohol 1.0 w/v 
Miglyol 840** 16.0 v/v 
Water for Injections 
to 100.0 
______________________________________ 
*Trademark of ICI 
**Trademark of Dynamit Nobel 
Dissolve the active ingredient in the Miglyol 840. Dissolve the non-ionic 
surfactant and benzyl alcohol in most of the water. Prepare the emulsion 
by adding the oily solution to the aqueous solution while homogenising 
using conventional means. Make up to volume. Aseptically prepare and 
package aseptically. 
______________________________________ 
Aerosol spray 
% w/w Range 
______________________________________ 
Active Ingredient 0.1 0.01-2.0% w/w 
Trichloroethane 29.9 
Trichlorofluoromethane 
35.0 
Dichlorodifluoromethane 
35.0 
______________________________________ 
Mix the Active Ingredient with trichloroethane and fill into the aerosol 
container. Purge the headspace with the gaseous propellant and crimp the 
valve into position. Fill the required weight of liquid propellant under 
pressure through the valve. Fit with actuators and dust-caps. 
TABLET 
Method of Manufacture--Wet Granulation 
______________________________________ 
mg 
______________________________________ 
Active Ingredient 250.0 
Magnesium stearate 4.5 
Maize starch 22.5 
Sodium starch glycolate 
9.0 
Sodium lauryl sulphate 
4.5 
______________________________________ 
Microcrystalline cellulose to tablet core weight of 450 mg Add sufficient 
quantity of a 10% starch paste to the active ingredient to produce a 
suitable wet mass for granulation. Prepare the granules and dry using a 
tray or fluid-bed drier. Sift through a sieve, add the remaining 
ingredients and compress into tablets. 
If required, film coat the tablet cores using hydroxypropylmethyl cellulose 
or other similar film-forming material using either an aqueous or 
non-aqueous solvent system. A plasticizer and suitable colour may be 
included in the film-coating solution. 
VETERINARY TABLET FOR SMALL/DOMESTIC ANIMAL USE 
Method of Manufacture--Dry Granulation 
______________________________________ 
mg 
______________________________________ 
Active Ingredient 50.0 
Magnesium stearate 7.5 
Microcrystalline cellulose to tablet 
75.0 
core weight of 
______________________________________ 
Blend the active ingredient with the magnesium stearate and 
microcrystallise cellulose. Compact the blend into slugs. Break down the 
slugs by passing through a rotary granulator to produce free-flowing 
granules. Compress into tablets. 
The tablet cores can then be film-coated, if desired, as described above. 
______________________________________ 
Veterinary intrammary injection 
mg/dose Range 
______________________________________ 
Active Ingredient 150 mg 0.05-1.0 g 
Polysorbate 60 
3.0% w/w 
White Beeswax 
6.0% w/w to 3 g to 3 or 15 g 
Arachis oil 
91.0% w/w 
______________________________________ 
Heat the arachis oil, white beeswax and polysorbate 60.degree. to 
160.degree. C. with stirring. Maintain at 160.degree. C. for two hours and 
then cool to room temperature with stirring. Aseptically add the active 
ingredient to the vehicle and disperse using a high speed mixer. Refine by 
passing through a colloid mill. Aseptically fill the product into sterile 
plastic syringes. 
______________________________________ 
Veterinary slow-release bolus 
% w/v Range 
______________________________________ 
Active Ingredient 0.25-2 g 
Colloidal silicon 
dioxide 2.0 to required 
Microcrystalline to 100 fill weight 
cellulose 
______________________________________ 
Blend the active ingredient with the colloidal silicon dioxide and 
microcrystalline cellulose by using a suitable aliquot blending technique 
to achieve a satisfactory distribution of active ingredient throughout the 
carrier. Incorporate into the slow release device and give (1) a constant 
release of active ingredient or (2) a pulsed release of active ingredient. 
______________________________________ 
Veterinary oral drench 
% w/v Range 
______________________________________ 
Active Ingredient 
0.35 0.01-2% w/v 
Polysorbate 85 5.0 
Benzyl alcohol 3.0 
Propylene glycol 
30.0 
Phosphate buffer 
as pH 6.0-6.5 
Water to 100.0 
______________________________________ 
Dissolve the active ingredient in the Polysorbate 85, benzyl alcohol and 
the propylene glycol. Add a proportion of the water and adjust the pH to 
6.0-6.5 with phosphate buffer, if necessary. Make up to final volume with 
the water. Fill the product into the drench container. 
______________________________________ 
Veterinary oral paste 
% w/w Range 
______________________________________ 
Active Ingredient 
4.0 1-20% w/w 
Saccharin sodium 2.5 
Polysorbate 85 3.0 
Aluminium distearate 
5.0 
Fractionated coconut oil 
to 100.0 
______________________________________ 
Disperse the aluminum distearate in the fractionated coconut oil and 
polysorbate 85 by heating. Cool to room temperature and disperse the 
saccharin sodium in the oily vehicle. Disperse the active ingredient in 
the base. Fill into plastic syringes. 
______________________________________ 
Granules for veterinary in-feed administration 
% w/w Range 
______________________________________ 
Active Ingredient 2.5 0.05-5% w/w 
Calcium sulphate, hemi-hydrate 
to 100.0 
______________________________________ 
Blend the Active Ingredient with the calcium sulphate. Prepare the granules 
using a wet granulation process. Dry using a tray or fluid-bed drier. Fill 
into the appropriate container. 
______________________________________ 
Veterinary Pour-on 
% w/v Range 
______________________________________ 
Active Ingredient 2.0 0.1 to 30% 
Dimethyl sulphoxide 
10.0 
Methyl Isobutyl ketone 
30.0 
Propylene glycol (and pigment) 
to 100.0 
______________________________________ 
Dissolve the active ingredient in the dimethyl sulphoxide and the methyl 
isobutyl ketone. Add the pigment and make up to volume with the propylene 
glycol. Fill into the pour-on container. 
______________________________________ 
Emulsifiable Concentrate 
______________________________________ 
Active ingredient 50 g 
Anionic emulsifier 40 g 
(e.g. Phenyl sulphonate CALX) 
Non-ionic emulsifier 60 g 
(e.g. Synperonic NP13)* 
Aromatic solvent (e.g. Solvesso 100) to 1 liter. 
______________________________________ 
*Trademark of ICI 
Mix all ingredients, stir until dissolved. 
______________________________________ 
Granules 
______________________________________ 
(a) Active ingredient 50 g 
Wood resin 40 g 
Gypsum granules (20-60 mesh) 
to 1 kg 
(e.g. Agsorb 100A) 
(b) Active ingredient 50 g 
Synperonic NP13* 40 g 
Gypsum granules (20-60 mesh) 
to 1 kg. 
______________________________________ 
*Trademark of ICI 
Dissolve all ingredients in a volatile solvent e.g. methylene chloride, add 
to granules tumbling in mixer. Dry to remove solvent.