PH26844A - Antibiotic compounds - Google Patents

Description

’ This invention relates to new antibiotic compounds and to processes for their preparation. More particularly it relates to antibiotic compounds which may be obtained by fermentation of . Streptomyces organisms.

In one aspect this invention provides a novel class of substances, which we have designated Antibiotics 5541, and which may be prepared by growing under controlled conditions, a previously undescribed strain of microorganism. Antibiotics $541 have antibiotic and, in particular, anti-endoparasitic, anti-ectoparasitic, anti-fungal, insecticidal, nematicidal and acaricidal activity and are of special interest for use in agriculture, horticulture, animal and human health. The compounds may also be of use as intermediates in the preparation of further active compounds. The compounds may be \ obtained by fermentation and recovered in substantially pure form as + described herein.

Antibiotics 5541 are a group of related compounds hav ing the partial formula (I)

mp SE er Bh

OH i ’ CHy 1 TL Ws : ¥ = 0 iL kf} i 1X76 a 25 2. . i 15 17 IN| z § 5 12 R % “TN 18579 0 H — i . 7 RE

Lo (Hyg » 0 “y (D % \ q 1 Jig: i | of 2 k 8 = H 5: 7 3 3 0— : i . i CHy i § 0—S #3 f : 5 more particularly, the partial formula (11) E: ¢ v

Ok ; Cha 2 ot 3 = 0 eis x So ~ > CHy a i 10 0g = 2b 5 3 RAN H b> { CH / u 6 7 3 H “0s § FE i 0 0 H i : ~~ §

OH | _H (11) i g 0 1 % - CHj gir . } H g! 0—3 EF

F

. Six compounds having the partial formula (11) are more b- particularly described hereinafter. A : . : The present invention extends to the compounds, having the above p : partial formula, both individually and in combination. For certain E i ) i - ! ! WL | Fe

EEE FE

Say hin 37 1

Ce ——— ———— ee __ oo

TE

: uses, for example in agriculture or horticulture, or in veterinary hi ht ) medicine, it may be more suitable to use Antibiotics 5541 without he ; separation into individual components, put for other uses, for example Fi "in human medicine, it may be preferable to use individual compounds - on i The invention thus includes 3 compound of the invention when in i : admixture with at least one other compound of the invention, and also ii ¢ the individual compounds for example "in substantially pure form or oR substantially in the absence of other macrolide compounds. Ee i Antibiotics S541 as initially isolated can readily be separated i

Lo by chromatography on silica as hereinafter described into two iE ed”

Components having antibiotic €.G- ant i-helminthic activity and which ia : quench u.v. fluorescence at 254nm. gomponent I is characterised by & an Rf value in the range 0.70 to 0.75 and Component II by an Rf value : : in the range 0.39 to 0.46, the Rf values being determined by thin = layer chromatography of Merck 5735 silica 60 plates eluting with t chloroformzethyl acetate (3:1). components 1 and 11 (in which R? is ) & 1 -CH; and -H respectively) of Antibiotics g541 form 2a further feature i 3 : of this invention. RB

Br

Components { and II can themselves pe further purified and have ji yielded six compounds of partial formula (1) possessing antibiotic & e.g. ant i-helminthic activity. Thus, in a further aspect of the v invention we. provide compounds of general formula (rin) A! : 4 : b ! k

Cn ol i

: » . oy . - 4 -

A

. } ¥

OH i

Z CH fo } CHj H ot J Rt = 0 ok

EN : Lo chs 1 0 3 % ol i

WN Hi i CH i | ‘t, Rl a

I 0 0 H + : | OH 4 : = i! § : 0 | (111) iy ; z H i . H ths A 2 it

Re y . Bs . ’ 1% ~ le

S Re - Br, 4 = fo. © 5 in which R! is a methyl, ethyl or isopropyl group and R2 is a hydrogen 3 : atom or a methyl group. : al . We have designated the six compounds of formula (II1) as Factor A “He ; (Rlzisopropyl, RZ2z=hydrogen), Factor 8 (R!=methyl, R Z=methyl), Factor C 3 : (Rl=methyl, R2=hydrogen), Factor D (Rlzethvl, RZ%Zhydrogen), Factor E 3 , 10 (Rlzethyl, R2=methyl) and Factor F (Rlzisopropyl, RZ%=methyl). Factors x } A and C are particularly preferred. -

Factors B, E and F are obtained from Component I, while factors he . he fp

A, C and D are obtained from Component II. bs :

The compounds of this invention have antibiotic activity e.g. ) Ne 2 15 antihelminthic activity, for example against nematodes, and in R- i 3 kB - particular, anti-endoparasitic and anti-ectoparasitic activity. In Re: ky i general, the compounds are useful in combating parasites such as k i fi : . ¥ ee ' + i FO gh a Lens 3 or abe . aoa

Swi Ade - ee ——————— A ————————————————— ee

A Poe fy nik Risa ay $i hind Rh J ® » i Lad we rach ERR he ia siid ~ ; —e i i . _ 5 _ ; if y L

Ey “ gt : ectoparasites and endoparasites- fctgparasites and endoparasites VER i infect humans and 2 variety of animals and are particularly prevalent in fagm animals such as pigs, sheep, cattle, goats and poultry: horses el

AE \

Ho |! and domestic animals such 2S dogs and cats. parasitic infection of HEE : Nex ‘ ; t , Ls : : . al

Livestock, leading tO anaemia, malnutrition and weight loss is 2 major ol 3 p31 51 : cause of economic loss throughout the world. wl hy ky : id . . . . FIRE gxamples of genera of endoparasites infecting such animals ie 3 ! Brio

REV and/or humans are Ancylostoma, pscaridia, Ascaris, Aspicularis, LE : y Sl { gunostomum, Capillaris, Chabertia, Cooperia, Dictyocaulus, bh pirofilaria, Enterobius, Haemonchus, Heterakis, Necatol Nematodirus, Fog

Sh A :

Nematospiroides, Nippostronaylus: pesophagastomul, Ostertagia, val a

Oxyuris, parascaris, gtrongy lus, strong loides, gyphaciad, Toxascariss Ei

SxyuE22y =" strongylus, SESE Syphass2 ===" p73

Ri. 2g” By

Toxocara, Ir ichonema, Trichostrof lus, Trichinella, Trichuris, and ke { Toxocard, ————— Trichostrondy== RL —_— gray

Uncinaria. i et . LIAL, 1 15 Examples of ectoparasites infecting animals and/or humans are a arthropod ectoparasites such 8s biting insects, blowfly, fleas, lice, Wo . = ti mites, sucking insects, ticks and other dipterous pests. as

Examples of genera of such ectoparasites infecting animals her, . } Fei !

By and/or humans are Ambylomma, Boophilus, Coroptes, Culliphore, DamadeXx, i . . : . . 4

Damolinia, Gastrophilus, Haematobld, Haematopinus, Haemophysalis, ; Hyalomma, Linognathus, Lucilia, Me lophyaus. Qestr us, psorergaktes; i t . pe { - Pe: ! pooroptes, Rhipicephalvs: garcoptes and StomoxysS- i g The ‘compounds according to the invention nave been found tO be : i, j feat { effective both in vitro and in vivo against 3 range of endoparasites jr } —_— — i! 1 ie i 25 and ectoparasites. in particular, we have found that compounds of the is invention are active against parasitic nematodes such as Haemonchus Us ? i ; contortus, gstertagia circumcincta., [richostronay lus colubiformis, 7, 3 t i - pi

AR

- A . ty joe - 6 - 250

Py . : ads 4 § Dictyocaulus viviparis, Cooperia oncophera, OUstertagia ostertagi and B re:

EE — fg

A. . LL. . je pit]

Nippostrongylus braziliensis, and parasitic mites such as Sarcoptes Jia

NippostIORAY === === —_— Len sp. and Psoroptes sp. $e . dps

The compounds of the invention are therefore of use in treat ing AE : ERE animals and humans with endoparasitic and/or ectoparasitic | SRR infections. te

EX

Era:

The species of the parasite will vary according to the host and pied 500 N the predominant site of the infection. Thus, for example Haemonchus a

A 44 contortus, Ostertagia circumcincta and Trichostrongylus colubiformis {k : FEV generally infect sheep and are predominantly located in the stomach frie

Rd 3 and small intestine, whereas Dictyocaulus viviparus, Cooperia Ti mee i ; oncophora and Ostertagia ostertagi generally infect cattle and are par 4% v 3 {Re . i 1 Yel, predominantly located in the lung, intestine or stomach respectively. EEL 5% ; RAAT } Furthermore, compounds of the invention have been found to ih : Eo ! i possess ant j-fungal activity, for example, against strains of Candida a

Ee tt —— cx 8 i sp. such as Candida albicans and Candida glabrata and against yeast iN ! Rly, ; such as Saccharomyces carlsbergensis. 5 a ¢e AM

EY

} The compounds of the invention have also been found to be active IA ) 4 against the free living nematode Caenorhabditis elegans. ft : ee — — BE! t0 The compounds of the invention have also been found to be id . TY effective in combating insect, acarine and nematode pests in 8 gh pa agriculture, horticulture, forestry, public health and stored Wi di: products. Pests of soil and plant crops, including cereals (e.g. : Ls:

CBE wheat, barley, maize and rice) vegetables (e.g. saya), fruit (e.q. pid apples, vines and citrus) as well as root crops (e.g. sugarbeet, f 37: " Me : jv 3. ; potatoes) may usefully be treated. 7 ; ct i 0 : REAL : I 4 A ! 2dr’ i Pig 1% } fi! 3 [ALL i : Br 4 1a i

Sy

Lf

Fr Tracie % bi ty ee

So —— . aa a \ ” iN : In particular, we have found that the compounds of the invention are active against for example fruit .mites and aphids such as Aphis i fabae, Aulacorthum circumflexum, Myzus persicae, Nephotettix : cincticeps, Nilparvata lugens, Panonychus ulmi, Phorodon humuli, : j

Phyllocoptrut3 oleivora, Tetranychus urticae and members of the genera is { Trialeuroides; nematodes such 3s members of the genera Aphelencoides, ;

Globodera, Heterodera, Meloidagyne and Panagrellus; lepidoptera such as Heliothis, plutella and Spodoptera; grain weevils such as A

Anthonomyus grandis and Sitophilus granarius; flour beetles such as h

Tribolium castaneum; flies such as Musca domestica; fire ants; leaf E miners; Pear psylla; Thrips tabaci; cockroaches such as glatella 3 i germanica and periplaneta americana and mosquitoes such as Aedes 4 aegypti. 9

According to the invention we therefore provide compounds having 3 the partial formula (I) as defined above, which may be used as b antibiotics. In particular, they can be used in the treatment of i animals and humans with endoparasitic, ectoparasitic and/or fungal 3 infections and in agriculture, horticulture, of forestry 8S pesticides Pp to combat insect, acarine and nematode pests. They may also be used generally as pesticides to combat or control pests in other 4 3 i. circumstances, €.0- in stores, buildings or other public places or y location of the pests. In general the compounds may be applied either 3 to the host (animal or smn or plants or other vegetation) or to the J pests themselves OT a locus thereof. particularly preferred are h

Factors A,8,C,D,E and f as defined above. 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 3

Broa o k 5 SE x dia ki ila shal : a aha PET RR A Gi alii aa Te Chania ey id ii i di fats. : . = 8 = 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 1 administration), oral, rectal, topical or implant use. When 1 ~ formulated in a composition that is required to be sterile, for 1 example injections (including intramammary preparations), eye drops, k ointments and implants, the active ingredient itself may have been E manufactured aseptically or sterilised after manufacture by methods - such as gamma-irradiation or exposure to ethylene oxide. ’ :

The compounds according to the invention may be formulated for 3 use in veterinary or human medicine by injection and may be presented . i in unit dose form, in ampoules, or other unit-dose containers, or in 3 multi-dose containers, if necessary with an added preservative. The b compositions for injection may be in the form of suspensions, K, } solutions, or emulsions in oily or aqueous vehicles, and may contain i formulatory agents such as suspending, stabilising, solubilising j and/or dispersing agents. Alternatively the active ingredient may be j in sterile powder form for reconstitution with a suitable vehicle, i ! e.g. sterile, pyrogen-free water, before use. 0ily vehicles include polyhydric alcohols and their esters such as glycerol esters, fatty ‘ acids, vegetable oils such as arachis oil or cottonseed oil, mineral 3 oils such as liquid paraffin, and ethyl oleate and other similar 3 compounds. Other vehicles such as propylene glycol may also be used. f a ———————————————————————————_————— TT dm aii Skits fia 2 copaald Sabb Lak cb rir iis i 2 ee Zork Tf ARICA ak iad 2 Siskiab added ie aia & Ae Sane nl , 3 : - 9 —- : Compositions for veterinary medicine may also be formulated as intramammary preparations in either long acting or quick-release bases and may be sterile solutions or suspensions in aqueous or oily ; vehicles. The oily vehicles may for example by those described above is and may also contain a thickening or suspending agent such as soft or’ hard paraffins, beeswax, 12-hydroxy stearin, hydrogenated castar oil, 1 aluminium stearates, Or glyceryl monostearate. Conventional EE non-ionic, cationic or anionic surface active agents may be used alone Re or in combination in the composition. i

The compounds of the invention may also be presented for i veterinary or human use in a form suitable for oral administration, H : rt for example in the form of solutions, Syrups or suspensions, or 2 dry k/ powder for constitution with water or other suitable vehicle before 4 : use, optionally with flavouring and colouring agents. Solid i i: 13 compositions such as tablets, capsules, lozenges, pills, boluses, i powder, pastes or granules may also be used. ) E fi t Solid and liquid compositions for oral use may be prepared according . to methods well known in the art. Such compositions may also contain b : one or more pharmaceutically acceptable carriers and excipients which E - 20 may be in solid or liquid form. Examples of suitable pharmaceutically fr acceptable carriers for use in solid dosage forms include binding Ey agents (e.g. pregelatinised maize starch, polyvinylpyrrolidane or F : hydroxypropyl methylcellulose); fillers (e.g. lactose, 3 . micro-crystalline cellulase or calcium phosphate); lubricants (e.g. 3 ] 3 magnesium stearate, talc or silica); disintegrants (e.g. potato starch E or sodium starch glycollate); or wetting agents (e.g. sodium lauryl k 4 ' sulphate). Tablets may be coated by methods well known in the art. i £ in on

\ ~ 10 - ; y Examples of suitable pharmaceutically acceptable additives for use in liquid dosage forms include suspending agents (e.g. sorbitol syrup, b methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily , esters or ethyl alcohol); and preservatives (e.g. methyl or propyl : p-hydroxybenzoates or sorbic acid); stabilising and solubilising p agents may also be included. q

Pastes for oral administration may be formulated according to by methods well known in the art. Examples of suitable pharmaceutically b i 10 acceptable additives for use in paste formulations include suspending or gelling agents e.g. aluminium distearate or hydrogenated castor ii oil; dispersing agents e.g. palysorbates, non-aqueous vehicles e.g. oo i arachis oil or oily esters; stabilising and solubilising agents. The 3 compounds of the invention may also be administered in veterinary fs” medicine by incorporation thereof into animals daily solid or liquid E dietary intake, e.g. as part of the daily animal feed or drinking ! water. $

For buccal administration the composition may take the form of 3 tablets, pastes or lozenges formulated in conventional manner. ; k 20 The compounds of the invention may also be administered orally : in veterinary medicine in the form of a liquid drench in the form of, g ; for example, a solution,suspension or dispersion of the active ) ingredient together with a pharmaceutically acceptable carrier or 1 excipient. £ 25 The compounds of the invention may also, for example, be formulated as suppositories e.g. containing conventional suppository bases for use in veterinary or human medicine. a 12

BE

——

Co i —————— eee eer ee SS . 4 , 0

RETR LE Li Eat ise rd Libis sd EE Ai b,c co a 4 ge ; § ba § Na i - 11 - Es 4 24.314 i £4 4 Compounds according to the invention may be formulated for He : topical administration for use In veterinary and human medicine, 38 Ee ointments, creams, lotions, powders, pessaries, sprays, dips, aerosols Wy

F RA

1 or drops (e.g. eye OF nose drops) - gintments and creams May, for Rk * ps i us 8 i example, be formulated with an aqueous Of oily base with the addition id

BLES pI of suitable thickening and/or gelling agents. gintments for i \ oF J

L is 3: administration to the eye may be manufactured in a sterile manner Hi ’ using sterilised components. us J : par 30

A . i eS i Lotions may be formulated with an aqueous or oily base and will Fae ho in general also contain one Of more emulsifying agents, stabilising 25% 1 he FAS £3. agents, dispersing agents, suspending agents, thickening agents, OT by i . Ei colouring agents. LY or, : Evi

Powders may pe formed with the aid of any suitable powder base. $A

J y & -

Drops may be formulated with an agueous or non aqueous pase also ih

VF) §13 comprising one or more dispersing agents, stabilising agents, 2 4 . a solubilising agent or suspending agents. They may also contain 3 1H : i $ preservative. Hoy ' Fi : : Rt

J For topical administration by inhalation the compounds according By i Bo i to the invention may be delivered for use in veterinary Of human ia { 4 i 20 medicine in the form of an aerosol spray presentation or an 1] . Pes : . iY: ; ingufflator.. : Fy ’ The compounds of the invention may be administered in by ! i IN§ ! ; combination with other pharmaceutically active ingredients. The total ! 3 ¥ ] daily dosages of compounds of the invention employed in bath : 3 veterinary and human medicine will suitably be in the range 8 1-2000pg/%9 bodyweight, preferably from 10-1000 pg/kg MOTE preferably

Po &

b, from 100-500 pa/kg and these may be given in divided doses, e.g. 1-4 ¥ times per day. i t "The compounds according to the invention may be formulated in by

BET: i any convenient way for horticultural or agricultural use and the 0 invention therefore includes within its scope compasitions compr ising 3 a compound according to the invention adapted for horticultural or i: ‘ vig ; agricultural use. Such formulations include dry or liquid types, for Ie example dusts, including dust bases or concentrates, powders, 4 \ oH i including soluble or wettable powders, granulates, including of ho microgranules and dispersible granules, pellets, flowables, emulsions 3

E3nle i such as dilute emulsions or emulsifiable concentrates, dips such as ve root dips and seed dips, seed dressings, seed pellets, oil ‘ concentrates, oil solutions, injections e.g. stem injections, sprays, ul smokes and mists. of

Generally such formulations will include the compound in 5 association with a suitable carrier or diluent. Such carriers may be E liquid or solid and designed to aid the application of the compound 3 { either by way of dispersing it where it is to be applied or to provide 4 a formulation which can be made by the user into a dispersible x preparation. Such formulations are well known in the art and may be i d prepared by ..convent ional methods such as, for example by blending i : and/or grinding of the active ingredient(s) together with the carrier ; or diluent, e.q. solid carrier, solvent or surface active agent. & i . Suitable solid carriers, for use in formulations such as dusts, ] granul ates and powders may be selected from for example natural 1 mineral fillers, such as diatomite, talc, kaolinite, montmorillonite g pyrophyllite or attapulgite. Highly dispersed silicic acid or highly . ¥ a es Ce ————————————————————— \ - BT § : py: dispersed absorbent polymers may, if desired, be included in the 4 ; composition: Granulated adsorptive carriers which may be used may be ; pO TOUS (such as pumice, ground brick, sepiolite or bentonite) or b ' non-porous (such as calcite OT sand) - Suitable pregranulated ’

Es materials which may pe used and which may pe organic OF inorganic 2 include dolomite and ground plant residues. Fl

Ry suitable solvents for use as carriers OF diluents include = : aromatic hydrocarbons, aliphatic hydrocarbons, alcohols and glycols or ja. ethers thereof, ester, ketones, acid amides, strongly polar solvents, 3 optionally epoxidized vegetable oils and water. i conventional non-ionic, cationic OF anionic sur face-active 3) agents, e.g. ethoxylated alkyl phenols and alcohols, alkall metal or ’ i alkaline earth metal salts of alkyl benzene sulphonic acids, i

Hk ]ignosulphonic acids of sulphosuccinic acids or sulphonates of } 15 polymeric phenols which have good emulsifying, dispersing and/or i : i wetting properties may also pe used either alone or in combination in 4 k the compositions. H ) Stabilizers, anti-caking agents, anti-foaningd agents, viscosity k i regulators, pinders and adhesives, photostabilisers as well 38S : 20 fertilizers, feeding stimulants of other active substances May, if 2 i : desired, be: included in the compositions. The compounds of the 3 : | invention may also be formulated in admixture with other insecticides; acaricides and nemat icides. : in the formulations, the concentration of active material is i 25 generally from 0.01 to 99% and more preferably between 0.01% and 10% by weight . . .

Cd . 1 . i i w= p . ir £ - 1 - is ! A

Commercial products are generally provided as concentrated i . compositions to be diluted to an appropriate concentration of active fF = material for example from 0.001 to 0.0001% by weight far use. 1]

For use in horticulture and agriculture or for use in veterinary E medicine it may be desirable to use the whole fermentation broth, i without separation into Components or Factors, as a source of the 5 active compounds. It may be suitable to use dried broth (containing : mycelia) or to use lysed mycelia, live or dead mycelia separated from bE : the broth using solid/liquid separation or evaparation techniques or _ 10 to use the fermentation broth remaining after separation of the ; } mycelia. If desired the mycelia may be pasteurised or more & : preferably, dried e.g. by spray drying or roller drying. If desired 3 the broth or mycelia may be formulated into compositions including : , conventional inert carriers, excipients or diluents as described : 3 15 above. ;

It will be appreciated from the above that in general the k compounds of the invention may be used to combat infections or 3 } infestations by applying to the organism responsible for the infection : or infestation or a location thereof an effective amount of one or 1 more of said compounds. . According to a further aspect of the invention we provide a process for the production of Antibiotics S541 or a Component or. ]

Factor thereof as defined previously which comprises the step af cultivating an organism of the genus Streptomyces capable of producing ; 25 at least one of the compounds of the invention whereby at least one of : said compounds is produced, and if desired isolating said compound ! t !

a - 15 - ] ! : f

L therefrom. The organism is preferably one which principally produces one ar more compounds of the invention. ’ Based on taxonomic studies, 2a particular microorganism capable !

L of producing the above substances is of a new species of the genus : b 5 Streptomyces and has been named Streptomyces thermoarchaensis. A sample of this microorganism, which is 8 soil isolate, has been h nstiad sn ta semen couse oblestion SL IE br

Collections of [ndvete® and Marine Bacteria, Tory Rosser station, k

Aberdeen: United Kingdom, 3nd Nos been assigned the Accession numDEs :

NCB 12012. The morphological and cultural characteristics of b , Streptomyces thermoarchaensis NCIB 12015 are set forth hereinafter and : this organism, together with other antibiotics 5541 roducing strains : L of Streptomyces, provide another feature of this invention. In { particular, the invention extends to the new species of Streptomyces. [ the members of which possess the same essential morphological and cultural characteristics as Streptomyces : thermoarchaensis NCIB 12015.

The invention also extends to any compounds which are capable of being produced by Fermentation of S. thermoarchaensis NCIB 12015 and ’ 20 which are the optical isomers of the compounds of formula (1). . The organism of the genus Streptomyces will preferably be : Streptomyces thermoarchaensis NCIB 12015 or 2 mutant thereof.

Mutants of Streptomyces thermoarchaensis NCIB 12015 may arise spontaneously or may be produced by a variety of methods including those outlined in Techniques for the Development of Mic ro-organisms by : H.I.Adler in ‘Radiation and Radioisotopes for Industrial ‘

ks oo - i Arama i 18 ! 4, i 4

Ct li

Microorganisms’, proceedings of the Symposium, Vienna 1973, p241, by ; International Atomic Energy Authority. "Such methods include bi ionising radiation, chemical methods e.g. treatment with N-methyl-N'- 5 [RY hitro-N-nitrosoguanidine (NTG); heat; genetic techniques, such as i g recombination, transduction, transformation, lysogenisation and i } lysogenic conversion, and selective techniques for spontaneous ii mutants. Thus, for example we have obtained four mutant strains of 3)

Streptomyces thermoarchaensis NCIB 12015, and each of these has been ig! deposited in the permanent culture collection of the National 4

P Collections of Industrial and Marine Bacteria, Torry Research Station, i

Aberdeen, United Kingdom and has been assigned the Accession number er

NCIB 12111, NCIB 12112, NCIB 12113 and NCIB 12114. Streptomyces | E thermoarchaensis NCIB 12111, 12112, 12113 and 12114 and mutants thereof form a further aspect of the invention. 0

RA

Mutant strains NCIB 12111, 12112 and 12113 were derived by k treatment of spores of Streptomyces thermoarchaensis NCIB 12015 with “iE

NTG and then characterised by the one-step method of Holliday (R. er

Holliday (1956) Nature 178 987). 4

Mutant strain NCIB 12114 arose by spontaneous mutation of E

Streptomyces thermoarchaensis NCIB 12015 and was identified as being ; resistant’ to streptomycin after remaining viable following exposure to E 100pg/ml of streptomycin sulphate at 280C for 5 days. 4

Taxonomic studies indicate that Streptomyces thermoarchaensis

NCIB 12015 is a previously undisclosed microorganism of a novel species the characteristics of which are described hereinafter and are essentially those of the species as 8 whole. [It should be understood i b | : ; i .

gr —_— hicsdai Lai 2 RE it fhe state EA SS MR aR haan \pidigaintisd, shakin. - 17 - that the invention extends to all members of this species including any organism having substantially similar essential characteristics.

A On the preferred sporulation media, oatmeal agar, malt-yeast ] agar and inorganic galts-starch agar (Shirling, €.8B. and Gottlieb, D. 5

Eo (1966) Int. J. Syst. Bacteriol. 16, 313-340), Streptomyces i thermoarchaensis NCIB 12015 grows abundantly producing 3a stable i substrate mycelium and an aerial mycel ium bearing spores in open pl spiral chains as side branches off the main hyphae. On these media F. the reverse pigmentation is yellow/brown and the sporophores are qrey. d

At X100 magnification, sporaphores contain 2-5 turns per chain with Lt 5-10 spores within each turn of the spiral. On average, sporophores 2 contain between 20 and 50 spores. Scanning electron microscopy at a pr 3 magnification Xx12000 reveals the spores to be smooth walled and 9 ellipsoidal in shape with dimensions of g.7um x 1.4m at their widest Es points. Streptomyces thermoarchaensis NCIB 12015 is gram-positive ly

Streptamycts - = ——— Li and is able to grow and sporulate at temperatures between 209C and i 50°C. i

A comparison of the -faregoing data with published descriptions i in Bergey's Manual of Determinative Bacteriology (Eighth Edition) 4 indicates that the organism Streptomyces thermoarchaensis NCI8 12015 i ik ¢ belongs to the genus Streptomyces. i: : | Identification of Streptomyces thermoarchaensis NCIB 12015 to : species-group level was carried out using a computerised . 3 ; identification matrix reported by williams et al (3.Gen.Microbiol : (1983) 129, 1815-1830). The results of the 41 taxonomic tests : o described by the above authors are as follows for Streptomyces thermoarchaensis NCIB 12015: ; ; i E

[a

BIE da pron ifs fos LCS eae ANGE CK (ER 4 grind airy +2 Fn de i rc py , ans , Oi SE Rg REA AN ec ,

H ps ii i MPS le 5 3 Lk Adi fea oct A ko Eh RNC et Let atrs 3 . g %%

Re . fv - 18 - X

Ny

RR

CHARACTER RESULT bik oh

FEN fo uk = » » . amy!

Spore chain verticillati - J . LER . . . . AR

Spore chain retinaculiaperti - {ids

Bi

Spore chain rectiflexibiles - Bren : Co fi

Spore chain spirales + Et

Rl) ai

Fragmentation of mycelium - aed

Blak

Spore surface smoath + ix]

Void. be Dy ss.

Spore surface ruqose - © <3 f er 2X { bi lo Spore colour grey + FA af

SIREN

Spore colour red Co - iY)

Spore colour green ~ ili

I YR

Reverse yellow/brown + Xx i

Reverse red/orange - 100 i (5% { Melanin production - i . jeg 5 Mes st ; . YE) ; Use of adonitol - ne . el

Use of cellobiose + Ue + { ELH { be ocr ! Use of D-fructose + Batty rY, 23 hao - } Use of meso-inositol - bi SE . Rai ! Use of inulin + il . . gine)

Use of mannitol ~ Rt . . EY Oe . gs. vi

Use of raffinose + ARON val f:-¢ 40%

Use of rhamnose + y ye

Rs int, pl £ fee ! Use of D-xylose + bid r : br. eis i rae i . . . + SA

Use of DL-a-aminobutyric acid - Bil : pei b Use of L-histidine + ET : ve b Fut b Sid

Hg

Vila f wet

TA

2 itp,

Ci ] RINE

Tee ¢ yo vos

LO

—————————m———,,——— { - 19 -

CHARACTER RESULT

Use of L-hydroxyproline - . : i Degradation of allantoin +

Degradation of arbutin | +

Degradation of xanthine +

Degradation of pectin +

Degradation of lecithin -

Nitrate reduction +

Hydrogen sulphide production +

Tolerance of sodium azide (0.01%, w/v) -

Tolerance of sodium chloride (7% ,w/v) -

Tolerance of phenol (0.1%,w/v) +

Growth at 45°C +

Resistance to neomycin (50pg.ml- 1) -

Resistance to rifampicin (50ug.m1-1) + {

Antibiosis to Aspergillus niger LIV 131 +

Antibiosis to Bacillus subtilis NCiB 3610 -

Antibiosis to Streptomyces murinus Isp S509 + . 20 The organism was not identified as belonging to any of the 23 major : species groups (Williams, S.T. et al. (1983) J. Gen. Microbiol 129, : 1815-1830) or any of the minor species groups and single member clusters de fined by Williams and co-workers (1. Gen. Microbiol (1983) 129, 1743-1813). The characteristics of Streptomyces i 2s thermoarchaensis NCIB 12015 were also compared with descriptions of known Streptomyces species in Bergey's Manual of Determinative ;

F Bacteriology (Eigth €dition), in ISP reparts by Shirling and Gottlieb y

- 20 - is hi (Int. J. Syst. Bacteriol. (1968) 18, 69-189; Int. J. Syst. if

Bacteriol. (1968) 18, 279-392; Int. J. Syst. Bacteriol (1969). 19, b 391-512; Int. J. Syst Bacteriol (1972) 22, 265-394) and with new 5 species validly described in the International Journal of Systematic 1

Bacteriology since 1980. ol

No match could be made between Streptomyces thermoarchaensis j &

NCIB 12015 and a described species and on this basis we believe that ik

Streptomyces thermoarchaensis NCIB 12015 is the first known member of he a new species belonging to the genus Streptomyces. ola 0 Mutant strains NCIB 12111, 12112, 12113 and 12114, all have i substantially similar essential characteristics to Streptomyces ’ bo . thermoarchaensis. However, NCIB 12111 requires adenine for growth, 7 — iat]

NCIB 12112 requires serine for growth, NCIB 12113 requires histidine El ) for growth, and NCIB 12114 is resistant to streptomycin. Fy 2s The production of S541 by fermentation of a suitable i

Streptomyces organism may be effected by conventional means i.e. by # 4

Her culturing the Streptomyces organism in the presence af assimilable i sources of carbon, nitrogen and mineral salts. 53 : Assimilable sources of carbon, nitrogen and minerals may be id provided by either simple or complex nutrients. Sources of carbon I : will generally include glucose, maltose, starch, glycerol, molasses, i dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, he iy : glycerides, alcohols, alkanes and vegetable oils. Sources of carbon of ; will generally comprise from 0.5 to 10% by weight of the fermentation bel : 3 medium, Ha t fe t i i ir bo ‘ . pn

Ce — —

BE

J

- 21 - 1 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. 3

Sources of nitrogen will generally include soya bean meal, corn by steep liquors, distillers solubles, yeast extracts, cottonseed meal, 3 peptones, ground nut meal, malt extract, molasses, casein, amino ac id ke mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea ¥ and other amides may also be used. Sources of nitrogen will generally H comprise from 0.1 to 10% by weight of the fermentation medium.

Nutrient mineral salts which may be incorporated into the i ’ | culture medium include the generally used salts capable of yielding 3 : sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cabalt is manganese, vanadium, chromium, calcium, copper, molybdenum, boron, f { phosphate, sulphate, chloride and carbonate ions. i

An antifoam may be present to control excessive foaming and if added at intervals as required. Eb:

Cultivation of the Streptomyces organism will generally be f » effected at a temperature of from 20 to sg0c preferably from 25 to E : 40°c, especially around 340, and will desirably take place with 3 aeration and agitation e.g. by shaking or stirring. The medium may b initially be inoculated with a small quantity of a suspension of the i ¢ sporulated microorganism but in order to avoid a growth lag a 1 & vegetative inoculum of the organism may be prepared by inoculating a 3 small quantity of the culture medium with the spore form of the | i { organism, and the vegetative inoculum obtained may be transferred to Es fe 4 § “

Et

Te . F T .

RIN fi Ne, EERE tile 3 20H dct wt 0s g Pr STI gsi - 272 = the fermentation medium, or, more preferably to one or more seed ! stages where further growth takes place before transfer ta the principal fermentation mediun. The fermentation will generally be carried out in the pH range 5.5 to 6.5, preferably 5.5 to 7.5. y 5 . The fermentation may be carried out for a period of 2-10 days, 3 e.g. about 5 days. 0

Where it is desired to separate material containing Antibiotics §541 and any components or factors thereof from the whole fermentation i ; or to isolate any of the components or factors this may be carried out it by conventional isolation and separation techniques. Antibiotics 5541 | 3 , [7.4 according to the invention are predominantly contained in the mycelia 21 of the cells, but may also be found in the fermentation broth and, pi et the isolation techniques may also be applied to the fermentation broth i either before or after clarification. It will be appreciated that the be

PE choice of isolation techniques may be varied widely. A

Antibiotics 5541 may be isolated and separated by a variety of 2 fractionation techniques, for example adsorption-elution, bt precipitation, fractional crystallisation and solvent extraction which 0 may be combined in various ways. i is

Solvent extraction and chromatography and fractional bE crystallisation have been found to be most suitable for isolating and pi: separating the compounds of the invention. : a

Following the fermentation, the mycelia may be harvested using 1 conventional techniques, far example, filtration or centrifugation. ! 23 . Thereafter, for example, the material may be extracted from the \ mycelia with an appropriate organic solvent such as ketone. e.q. ‘ acetone, methylethyl ketone or methylisobutyl ketone; a hydrocarbon, . ] ‘

I

:

Ce ————————— A ——— rE 3 ease psa on I

L3 Ee I Eads Sha _ 23 ~- e.g. hexane; 3 halogenated hydrocarbon e.q- chloroform, _arbontetrachiorid® or methylene chlor ides an alcohol, €-9° methanol of ethanol; Of a diol, €-9- propane 1,2-diol; or an ester, €-9- i methyl acetate OF ethyl acetate. [t will be appreciated that if the mycelia contain significant amounts of water jp will be preferable Lo use a uater-soluble solvent.

Generally: more than ane extraction is desirable to achieve optimum recovery: preferably the first extraction is carried out using 2 water miscible solvent such 3S methanol of acetone. The antibiotics may be recovered as a crude extract by removal of the solvent. The solvent extracts may themselves be extracted, if desired after reduction of the solvent volume, for example by evaporation: AL this stage it is preferable to use 2 ater-inmiscible solvent such 38 : hexane, _nloroforms methylene chloride or ethyl acetate OF mixtures thereof sufficient water being added tO achieve satisfactory partition of the antibiotic compounds Removal of the

Jatec-inmiscible phase yields a material containing antibiotics 5541.

If desired Factor g may be separated by crystallisation from an appropriate solvent e.g. ; sopropanol. purification and/or separation of the active components and/or factors (completely or from other macrolide compounds present) may be effected by convent ional techniques such as for example: chromatography (including high per formance liquid _hromatography) on 2a 1 suitable support such 3S silica, @ on- functional acroreticular adsorption resin for example cross 1 inked polystyrene resins such 38S amberlite XAD-2 xAD-4 of xAD-1180 resins {Rohm & Haas Ltd), of an 5112 resin (kastell Ltd) or of an organic Lolvent-compat Pte

S

- 24 - { cross-linked dextran such as Sephadex LH20 (Pharmacia UK Ltd), or, in the case of hplc, reverse phase supports such as hydrocarbon linked silica e.g. Cj g~linked silica. The support may be in the form of a : bed, or more preferably packed in a column. In the case of non-functional macroreticular resins such as XAD-1180 or S112, . mixtures of organic solvents such as acetonitrile with water may be 4 i used for elution. 3

A solution of the compounds in a suitable solvent will generally i be loaded on to the silica or Sephadex columns, if desired after first 14 reducing the volume of solvent. The column may optionally be washed C: and then eluted with a solvent of suitable polarity. In the case of . :

Sephadex and silica, alcohols, such as methanol; hydrocarbans, such as fr hexane; acetonitrile; halogenated hydrocarbons, such as chloroform or 1 methylene chloride; or esters, such as ethyl acetate, may be used as Et

Ls solvents. Combinations of such solvents either alone or with water 2 may also be used. 3 4 Elution and separation/purification of the compounds of the k invention may be monitored by convent ional techniques such as 2 chromatography e.g. thin layer chramatography and high performance & ot » liquid chromatography or by utilising the properties of the compounds . b described previously. 3 : Chromatography over silica, preferably using an eluant such as 3 g | chloroform:ethyl acetate, readily separates Antibiotics $541 into

Components I and II, Component I being eluted first. Factors B, E and \

F can then readily be obtained from Component I using chromatography e.g. high performance liquid chromatography. Similarly Factors A, C and D may readily be isolated from Component [I. Alternatively, i ee —————————————— CE —————————— nS hi sR a pt gon SELON hii Ad as _ “1

Factor B can be separated from Factors £ and F by crystallisation from an alcohol such as methanal or iso-propanol. The mother liquors containing Factors E and F may, if desired, be subjected to further purification e.g. chromatography over silica and Factors E and F } $ isolated using high performance liquid chromatography. ;

Once obtained, the Factors may be further purified by crystallisation , ) e.g. from methanol, isa-propanol or a methanol/water mixture, and the ] invention extends to compounds according to the invention in : crystalline form. a

By a suitable combination of the foregoing procedures, the i J compounds according to the invention have been isolated as solids. It pl will be appreciated that the order in which the above purification : steps are carried out and the choice of those which are used may be 4 varied widely. i ps Thus, Factor B has been obtained as a crystalline solid having a ’ purity in excess of 90%. Similarly, Factors A, C, nD, £ and F have i also been obtained having a purity in excess of 90%. The Factors may, however, be used, as described above, at levels of purity appropriate - to their intended use. For use in human medicine, purities of at least 90%, preferably greater than 95%, are desirable. for veterinary ar agricultural or horticultural use, lower purities will suffice, for “example 50% or lower. :

The following Examples illustrate the invention. The following abbreviations are used . tle - thin layer chromatography (using Merck : 5735 silica 60 plates and developed with CHC14:ethyl acetate (3:1) unless otherwise specified); CCM - column chromatograpy using Merck 7734 silica 60 (200 x 4cm column unless otherwise specified) packed k a Teer fides TT | - , LL Py - 26 - and eluted with CHC1 j:ethyl acetate (3:1) unless otherwise specified; hplc - high per formance liquid chromatography; PE - petroleum ether (b.p. 60-800C unless otherwise specified); L - litre; EA - ethyl : acetate.

Media A, B and C referred to in the Examples are :

Medium A al i D-Glucose 15.0 :

Glycerol 15.0 .

Soya Peptone 15.0

NaCl 3.0 } CaCO, 1.0

Distilled water to l litre, pH adjusted to pH 7.0 with aqueous ;

NaOH before autoclaving.

Medium B : a=!

D-Glucose 2.5 ) Malt dextrin MD 30E (Roquette (uk)Ltd) 25.0

Arkasoy 50 (British Arkady Co.Ltd) 12.5 : 20 Molasses 1.5

TK HPO, 0.125

Calcium carbonate 1.25 ; MOPS (3-{N-morphalino)propane- 21.0 sulphonic acid)

Distilled water to 1 litre, pH adjusted to 6.5 with SN NaOH before autoclaving.

ee ‘ —— la iiss TSS ER i / 4 - 21 -

Medium C

D-Glucose 2.5

Malt dextrin MD 30E (Roquette (UK) Ltd) 25.0

Arkasoy 20 12.5

Beet Molasses 1.5

K HPO, 0.125

CaC03 1.25 silicone 1520 (Dow Corning) 0.625

Distilled water to 1 litre, pH adjusted to 6.5 before sterilisation.

Example 1

Spores of Streptomyces thermoarchaensis NCIB 12015 were inoculated onto agar slants made up of the following ingredients: g-t : yeast extract (Oxoid L21) 0.5

Malt extract (Oxoid L39) 30.0

Mycological peptone (Oxoid L460) 5.0 agar No.3 (Oxoid L13) 15.0

Distilled water to 1 litre, pH approximately 5.4 “and incubated at 280c for 10 days. The mature slant was then covered with a 10% glycerol solution (6ml) and scraped with 3 sterile tool to loosen the spores and mycelium. g.aml aliquots of the resulting spore suspension were transferred to sterile polypropylene straws which were then heat-sealed and stored in liquid nitrogen vapour until required .

The contents of a single straw were used to inoculate 10ml of

Mediun A which was then incubated at 280¢ for 3 days on a shaker {

AL, | 2% LEIA : ‘ ER ha Bd faa tins spin i vce ge re i ol co spe a at atid: - 28 - rotating at 250rpm with a SOmm diameter orbital motion. This incubated medium was used to inoculate at a level of 2%, 15 tubes and two 250ml

Erlenmeyer flasks containing 10ml and 50ml respectively of Medium B.

The tubes and flasks were grown at 289C for 5 days, and the the cultures were then filtered separately under vacuum and the cells shaken for 30 minutes with a volume of methanol equal to that of culture filtrate. ; Activity against Caenorhabditis elegans was detected in extracts of cells grown in both tubes and flasks and these mycelial extracts 3 were bulked, evaporated to dryness and re-extracted with methanol to a concentrate (6ml) which was applied to a column of Sephadex LHZ20 1 (110 x 2.5cm) packed and eluted with methanol. 10ml Fractions were collected.

Fractions 21-28 were pooled and evaporated to yield an oily 1 1s residue (156mg) which was extracted with CHCl j:EA (3:1) to give an i extract (3ml) which was subjected to CCM (55x2.5cm column) 10ml X

Fractions were collected and analysed by tlc using plates containing 3 fluorescent indicator. Fractions 20 to 23 and Fractions 36 to 44 gave rise to two major areas which quenched the fluorescence and which we have identified as Component I (Rf 0.70) and Component II (RF 0.43).

Evaporation of fractions 20-23 yielded Component I as a solid (9mg) ;

Apax2380m, £13605 Anax265nm, E1350;and Apax2Sanm, . £4200. Evaporation of fractions 36 to 44 yielded Component 11 as a solid (11mg) A, 238mm, E1440;N,, 2650m, €1460;and

Amgx2S6nm, E! 280.

Co ——————— EE ——————————————— a A EERE Eins Heal ro nu aE ARE No hb / : f Cw

Example 2 \

Two 250ml Erlenmeyer flasks containing 5gml of Medium A were each inoculated with 0.2ml of a spore suspension of Streptomyces thermoarchaensis NCIB 12015 taken from a straw prepared as described in Example 1. The flasks were incubated at 280C for 3 days on a shaker rotating at 250rpm with a sQmm diameter orbital motion and the contents of both flasks were then used to inoculate 2a 20L fermenter vessel containing Medium B (12L). The culture was harvested after 5 days growth and processed as described in Example 3.

Example 3 :

Fermentation broth (12L) obtained as described in Example 2 was harvested after 5 days growth at 289C and centrifuged (4,200rpm at 3 10%C for 15 min). The cell pellet was mixed with methanol (5L) and allowed to stand for 20 hours at 4%. The mycelial extract was filtered, evaporated at 40°9C and sub jected to azeotropic distillation after addition of butan-1-ol (100ml). The extract was then treated with methanol (5%200ml) and the combined extracts were evaporated to 100ml and applied to 3 column of Sephadex LH20 (112 x Sem). The colunn was eluted with methanol and after a forerun of 200ml, 50ml fractions collected. fractions 40-90 were pooled and evaporated to “yield an bily residue (3.85q). The residue was extracted with 77ml of

CHC 3:EA (3:1), filtered and then subjected to CCM approximately 195ml fractions being col lected after 3 forerun of 200ml. fractions 124 to 142 containing Component I were pooled and evaporated to yield a solid (253mg) of which 216mg were purified by hple (Zorbax 00s, 25x2.1cm, 80% CH,CN/H 0) Fractions 250 to 320 cantaining Component 11 were pooled and evaporated to yield a solid }

(602mg) of which S40mg were purified by hnlc (as for fractions 124-142) and fractions from several runs were collected. ’ Material eluting from the hplc column was monitored by uv spectroscopy at 243nm. Peaks absorbing at this wavelength were dried down and i) tested for activity against Caenorhabditis elegans and ii) lk analysed by tlc. Four peaks which were active against Caenorhabditis , elegans also had an Rf value in the range 0.39 to 0.46 or 0.70 to i 0.75. gE gr:

Component I gave one peak with an Rf value of 0.70 to 0.75 and l this peak has been assigned as Factor B. Component [I gave three 5 peaks with an Rf value of 0.39 to 0.46 and these peaks have been a assigned as Factors A, C and D. jie

Factor A eluted from the hple column between 260 to 340ml after ah the injection of the sample and had an Rf value af 0.44 by tle. be

Factor B eluted from the hplc column between 270 to 310 ml after the py. injection of the sample and had an Rf value of 0.72 by tle. Factor C ie b eluted from the hplc column between 160 to 180 ml after the injection hn of the sample and had an Rf value of 0.4 by tlc. Factor D eluted from 18 iS the hplc column between 220 to 250ml after the injection of the sample a and had an Rf value of 0.42 by tlc. The further characteristics of on h¥

Factors A, 8, €C and D are described hereinafter. be

Example 4 ge 0.4ml of a spore suspension of organism Streptomyces x

Kasi 1 thermoarchaensis NCIB 12015 taken from a straw prepared as described h- 3 in Example 1 was used to inoculate a 250ml Erlenmyer flask containing i i

Medium A (S0ml). The flask was incubated at 289 ror 4 days on a hel shaker rotating at 250rpm with a 50mm diameter orbital motion. kid ia : Si ee on Co ———— A ——— eee ) ber mmm = ‘ws

Jag en ARR, hou B edi ASE RES mcmama "R EE - 31 - portions (Bml) were then used tO inoculate each of two 21 flat-bottomed flasks, each containing 400ml of the same medium, pefore : incubation under the same conditions for 3 days.

The contents of both flasks were then used tO inoculate 3 fermenteC vessel (70L) containing Medium B (40L) supplemented with silicone 525 {Dow-Cornings 0.0625% (v/v)1. The fermentation was carried out: with agitation and aeration sufficient to maintain 2 dissolved oxygen level of greater than 20% of saturation, with gilicone antifoam added 3S required. The fermentation was harvested after 10 days, and the broth (40L) was clarified by centrifugation (15000 r.p.m). The residual supernatant was displaced with water } : (sL), and the recovered cells (1.4kg) were frozen at -200.

After 2 week the frozen cells were thawed, suspended in methanol (15L) and stirred gently for 15h. The suspension was then filtered and the solid residue was re-extracted with methanol (10L) . The combined filtrate (25L) was diluted with water (12L) and extracted with PE (250). after 30 min the phases were separated by : centrifugation.

The lower, methanol phase was re-extracted three times with 0 PE (250, 15L and 15L)- The combined pg phases (80L) were concentrated by three passes through 23 pfaudler g.8-12v-27 wiped- film evaporator {Vapour pressure 0.1 bar, vapour temperature 209, steam temperature 1279), and the concentrate (BL) was dried with sodium sulphate (1%q) and further Concentrated under reduced pressure at 40? in 3 rotary film evaporator. The oily residue (15ml) was dissolved in a mixture of CHCl, and EA (70ml, 3.1 v/v) and subjected to ccM, fractions of approx imately 40m] being collected after a foreruft of 1,400ml.

PER ui EL ns pm . co } - 32 -

Fractions 45 - 65 were combined and evaporated to yield Factor B ; (940mg; as defined in Example 3), which was crystallised twice from methanol and finally from nitromethane. The crystals were submitted for single crystal X-ray diffraction analysis, which showed that they ! were orthorhombic, clear prisms with a = 10.171(3), b = 13.317(5), ¢ = 25.032(7)A, ¥ = 3391A3, Z = 4, space group P2242), De = 1.18gem- 3, !

R = 0.053 for 2169 independent observed reflections (0 <589) measured on a diffractometer with Cu-Ka radiation (A = 1.54178A). The structure as determined by X-Tay crystallography is shown in Figure 5. )

Example 5 :

An inoculum of Streptomyces thermoarchaensis NCIB 12015 was 1 prepared as described in Example 4 with the growth period being two i days, and used to inoculate a fermenter vessel (70L) containing Medium 8 (40L) supplemented with polypropylene 2000 (0.06% v/v) instead of

Silicone 525. Polypropylene 2000 was added as required throughout the fermentation to control foaming. The fermentation was carried out at 280C, with agitation and aeration sufficient to maintain a dissolved oxygen level of greater than 30% saturation. After 24 hours of fermentation, a portion of broth (9L) was transferred to 2 fermenter (700L) containing medium (450L) made up as follows:

—————————oe—————— i” Co ———————————————————————— ee eee ST ys - 33 - Ke art

D-glucose 2.8 ’ Malt Dextrin (MD30E) 27.8 arkasoy 50 13.9 p

Molasses 1.7 3

K HPO, 0.14 i

By caC03 1.39 i silicone 525 (Dow Corning) 0.06% (v/v) of

Adjusted to pH 6.2 pefore sterilisation. oh 19 The fermentation was carried out at 280C with agitation and 2 aeration suf ficient ko maintain 3 dissolved oxygen level of greater i than 20% saturation. polypropylene 2000 antifoam was added as 3 required. After 2 days the pH was controlled to 7.2 with the 5 addition of H,50 The fermentation Was harvested after 5 days. i

The broth (450L) was clarified by centrifugation and the .

HER residual supernatant was displaced with water (200). The recovered oH cells (25.5kg) were stirred for 1 hour in sufficient methanol to give gs: a total volume of 75L. The suspension Was Filtered and the solid by

REY residue was re-extracted with methanol (35L) and filtered. The Er combined filtrate (871) was diluted with water (40U) and extracted pi with PE. After 30 min. the phases were separated by centrifugation by and the lower methanol phase was re-extracted with PE (300) after the E addition of water (s0L). After separation the lower phase was again extracted with pe (300). The combined PE phases (85L) were 5 2 concentrated bY three passes through 3 pfandler g.8-12v-27 wiped- film evaporator (vapourl pressure 0.1 bar,’ vapour temperature 200, steam 3

P: temperature 1279). The concentrate (9L) was dried with sod ium : y io: - a - hie sulphate (2kg) and further concentrated under reduced pressure at 409 E in a rotary film evaporator. The oily residue (130g) was dissolved in y

CHCl3 to give 190ml and this was subjected to CCM [column packed and j washed (500ml) in CHCl,] fractions of approximately 40ml being 5 i $ collected after a forerun of 1,400ml. k by

Fractions 32-46 were combined and evaporated to yield an oil i:

Prat

Ls (21.2a). Fractions 47-95 were combined and evaporated to give an oil 58 (20.1g) which was dissolved in CHCl 3:EA (3:1) to 50ml, and subjected Ni

ER to CCM, fractions of approximately 40m] being collected after a RY. hy] nae forerun of 1,400 ml. fractions 22-36 were combined and evaporated to BE give an oil (3.1g) which was added to the oil obtained from fractions iE fh: 32-46 from the first column. The combined oils were dissolved in A prey boiling methanol (4ml) which was then added to hot propan-2-ol (20ml) i . 8 iE to yield on standing crystalline Factor 8 (2.579). a

Mother liquor after crystallisation of Factor B was evaporated i

RI to yield an oil which was dissolved in an equal volume of CHCl, and i

Sa © loaded onto a column (30x2.2cm) of Merck Kieselgel 60 (70-230 mesh -q

ASTM, Art. No. 7734) packed in CH,CL,. The bed was washed with id 1).

Fest

CH,Cl, (2 bed volumes) and eluted with CHCL3:EA (3:1) (2 bed volumes). be : BN

Evaporation of the eluate yielded an oil which was dissolved in poy

Ri methanol and subjected to preparative hplc on Spherisorb S5 005-2 5 . (250mmx20mm, Phase Sep.Ltd.). The sample (Sml) was pumped onto the I column over a period of 1 minute and the calunn was eluted with i:

A acetonitrile:water (7:3) under the following conditions: i} 3 . 3 i oe } iN ob i ex : : 0? pd . 2 © ’ ) yy

Py

J giv Co —————————————————————— eee a ~- 35 -

Time (mins) Flow (ml/min) 0.00 0.00) injection 1.00 0.00 ) time 1.10 30.00 ; 39.90 30.00 40.00 35.00 75.00 35.00

Material eluting from the hplc column was monitored by uv spectroscopy at 238nm. gvaporation of the combined fractions with peaks eluting at 26.3 minutes yielded factor £ as 2 solid. fvaporation of the combined fractions with peaks eluting at 14.4 minutes yielded Factor F as 2 solid. The further characteristics of Factors £ and F are described hereinafter. 1

Example 6

Fermentation broth (similar to that prepared in Example 2) harvested after 117hr was autoclaved (121°C, the), cooled to room \ temperature and stirred on 2 magnetic stirrer to give 2 homogeneous suspension of cells. Two portions (2ml) were centrifuged (12,0009; 2 min., room temperature) the supernatants were decanted and the residual cells were suspended in water (2ml), thoroughly mixed and “sub jected to centrifugation again (12,0009. , 2 min., roow temperature). After decantation of the supernatants, the cells were . washed twice more with distilled water (2m} portions). The washed cells were then thoroughly mixed with either water (2ml) or methanol (2m) and left at room temperature with occasional shaking for 1.5hr.

The suspensions were again centrifuged (12,00049, 7 mins, room b ion!” / - 36 - temperature) and the supernatants were sequentially diluted in water. The cells from the aqueous suspension were re-suspended in water and immediately sequentially diluted in water. Portions (10 ul) of each of the dilutions were added to a suspension (200yul) of the nematode Caenorhabditis elegans in a buffer solution containing

Na, HPO, (6g/L), K, HPO, (3g/L), NaCl (5g/L) and MgSO,.7H J (0.25g/L) and adjusted to pH 7.0. After 4hr the nematode suspensions were examined to find which dilutions of test mixture caused total inhibition of motility in greater than 98% of the nematodes in the assay suspension. [It was found that 1 in 5, 1 in 25, 1 in 250 and 1 : in 500 dilutions of the methanol extract, 1 in 5, 1 in 25, 1 in 250, 1 in 500 and 1 in 1000 dilutions of the cell suspension and 1 in 2, 1 in ! : 4 and 1 in 8 dilutions of the aqueous extract caused such inhibition of the nematodes when 10pl were added to 200ul of nematode suspensions.

Example 7 250ml Erlenmyer flasks containing either 50ml of Medium A or 50ml of

Mediun B were inoculated with 0.4ml of a spore suspension of

Streptomyces thermoarchaensis NCIB 12015 taken from a straw prepared as described in Example 1. The flasks containing Medium A or Medium B , were incubated at 28% for 2 days on a rotary shaker operating at “250rev/min with a 50mm diam. throw. Portions (8ml) from each medium were then used to inoculate 2 litre flat-bottomed flasks containing 400ml of the same medium (A or B respectively). These flasks were incubated under the same conditions for two days.

ee hY

I

’ Two 70L fermenters were each inoculated with 2 flasks of Medium

A and one other 70L fermenter was inoculated with two flasks of Medium g. Each Fermenter contained 40L of Medium C.

The fermentations were carried out at 340, with agitation and aeration sufficient to maintain a dissolved oxygen level greater than 30% of saturation. After approximately 26h of fermentation the pH ws controlled to 7.2 with the addition of aqueous H SO. polypropylene glycol 2000 antifoam was added 3s required. After 5 days, these fermentations were harvested and bulked.

One other 70L fermenter, which was also inoculated with two flasks containing Medium B, contained Medium B supplemented with i silicone 1520 (0.06%) . The fermentation was carried out at 289% with agitation and aeration suf ficient to maintain 2a dissolved oxygen level of greater than 30% of saturation. polypropylene glycol 2000 was added as required to control foaming. After 24 hours, a 9 L portion was transferred to a 700L fermenter containing 450 L of Medium C.

The fermentation was carried out at 340C with agitation and aeration sufficient to maintain a dissolved oxygen level of greater . than 30% of gaturation. Foaming was controlled by the addition of polypropylene glycol 2000 and after approximately 24 hours the pH was controlled to 7.2 with the addition of aqueous H SO. The fermentation was harvested after 4 days and bulked with the three aL fermentions described above.

The bulked harvest broths were centrifuged through 2 sharples

AS16PY at about 120L/h. The residual supernatant in the centrifugal bowl was displaced with water. \

SE

- 38 -

The recovered cells (11.65kg) were emulsified in methanol (33L) " with a Silverson mixer. After 60 min the suspension was filtered through a twill cloth and the residue was once again emulsified in methanol (361). After 40 min the suspension was again filtered. The filtrates from the two methanol extractions were combined.

The combined extracts (53.5L) were mixed with water (271) and PE (27L). After stirring for 20 min the two phases were separated on a

Westfalia MEM 1256 centrifuge. The lower aqueous methanal phase (70L) was mixed with water (37L) and PE (27L) and stirred and separated as pefore. The interfacial emulsion in the PE phase was broken with acetone (4L). The lower aqueous methanol phase (108L) was then mixed with water (40L) and PE (27L) for a third time, and stirred and i separated as before, with acetone (41) being used to clear the interfacial emulsion. The three hexane extracts were then combined.

The combined PE extract (BSL) was concentrated with a wiped film evaporator (vapour pressure 0.15 bar, vapour temperature 269). The concentrate (3L) was dried with sodium sulphate (2kg) and then Further evaporated under reduced oressure at 409. The resultant oil (6399) was dissolved in 300ml of a mixture of chloroform and EA (3:1 v/v) and filtered and washed through glass fibre paper. The filtrate and washings (1060ml) were subjectd to CCM (1500mm x 100mm diam) with “elution at a flow rate of 6L/h.

The fraction eluting between 8.8 and 13.1L was bulked and ‘ evaporated at low pressure to an oil (56.3g), while that eluting between 13.1L and 24.6L was similarly reduced at low pressure to a pale yellow solid (153.4q). The early fraction was shown to contain largely factor 8 while the later fraction contained a mixture of . d ee

Co Co ————————————————— re eee TET a \ factors A, B, ¢c and D- The Factor B in this later fraction was progressively removed by repeating the chromatography CCM 3s described above, twice - the last time 00 fresh silica - under similar conditions except that the flow rate was reduced tO L/h. 3 The peaks containing factors a, C and DO from the second of these columns eluted between g.8 and 17.6L, the residual Factor B which it contained being separated in the third column from which Factors A, C and DO eluted petween 14 and 28L. This final bulked eluate was reduced at low pressure to 3 solid (1149) - The peaks containing factor B from the two columns (7.5-8.8L and 10.3-13.06L respectively) were evaporated vo oils (10.79 and 109 respectively) and were combined with the oil i obtained from the first of the three columns. :

The oils containing factor B were dissolved in boiling methanol (25m) and mixed with boiling propan-2-01 (100mL) . gn cooling to 4°

Factor B crystallised. it was filtered off, washed with methanol (200mL) , cooled to _209, and dried under yacuun to give 25.39 of g

Factor B. ’ The solid from the third silica column which contained factors

A, C and D was dried under vacuum to constant weight (879) - Samples 1 (20q) of this solid were dissolved in methanol (190mL) and made UP to 230ml with 7:3 (v/v) acetonitrile:water: portions (smL) of the j solution were then chromatographed on a column (250mm x 21 .2mm diam) : of spherisord 005-2 (5 um particle diam), with 733 acetonitrile water as the eluting solvent. The flow rate was held at 20mL/min for about 10 sec; jt was then steadily increased over 3 22min period to 3am / min, and was held at this rate for 2 further 3 min. The eluting factors were detected at 238nm. factor C eluted petween 11.0 and 13.4 f gi ad iil kd col pets cag rae Cisians ania - . — | : /. - 40 - ! min, Factor D between 13.4 and 17.4 min and Factor A between 17.4 and 23.0min.

The fractions containing Factor C from each chromatographic "separation were bulked and reduced at low pressure to a solid.

Fractions containing Factor A were similarly reduced to a solid.

Fractions containing Factor D were also bulked and reduced to an impure solid (7g). This was redissolved in methanol (65mL), mixed with 7:3 acetonitrile water and rechromatographed on the spherisorb 00S2 column as already described except that the flow was kept constant at 20mL/min throughout. The Factor D now eluted between 16 and 20 min, and this fraction was bulked from each chromatographic run. The bulked eluate was reduced to a solid. The three salids containing Factors A, C and D were dried over P,0¢ under vacuum to constant weight (55g, 7.0g and 1.21g respectively).

The four solids isolated from this process were each shown to be similar to authentic samples of Factors A, 8, C and D.

Example 8 250ml Erlenmyer flasks containing 50mL of medium B were inoculated

Y with 0.5ml of a spore suspension of each of Streptomyces thermoarchaensis NCIB 12111, 12112, 12113 and 12114 taken from straws prepared as described in Example 1. - Flasks containing Streptomyces thermoarchaensis NCIB 12111, NCIB 12112 and NCIB 12113 were incubated at 319C on a rotary shaker. The flask containing Streptomyces thermoarchaensis NCIB 12114 was . incubated at 289C for 2 days and then lmL of broth was transferred to another 250ml Erlenmyer flask containing 50mL of medium 8. This flask

HE a ~ - 41 was incubated at 310C on a rotary shaker. All flasks were shaken at 250rev/min with 2 50mm diameter throw. after 6 days incubation, a 10mL sample of each broth was centrifuged at 1,250qg for 45 minutes, and processed as follows. The supernatant was discarded and the pellet resuspended to 10mL in i methanol. The suspension was shaken vigorously and left for 1 hour with occasional mixing. The suspension was then centrifuged at 10,000q for 5 minutes and the supernatant analysed DY hplc (S5 QDS-2, 10cm x 4.6mm, 70% CH4CN/O. 1M NH HPO) - Peaks were monitored at 2660m.

Analysis by hplc showed the presence of Factors A, g, C and D § in each case. ' ]

Example 9 ’ ‘ 3 factors A, g, C, D, £ and F have peen found to have the following : t characteristics: i) They contain carbon, hydrogen and oxygen only. ii) Electron Impact (€.1.) mass spectroscopy of Factors A, 8, C, Dy E and F gave the following results : : !

Factor molecular ion | corresponding to molecular formula

A 612.37 CiHs Ds 8 598.35 Castsals c . 584.34 : Cy Hyele

Db 598.35 Csstsals £ 612.3638 Cots Oe 3 626.3807 C37Hsu08 pro—— | ; ’ - 42 -

Fast Atom Bombardment (FAB) mass spectroscopy gave the following results:

Factor +ve FAB -ve FAB mol. wt. * A M/Z 635[M+Nal* M/Z 611[M-H]- 612 ; M/Z 613(MaH T+

B M/Z 691[M+H+qglycerol J+ 598

M/Z 599[M+H J+

M/Z 581[MH-H 0+

M/Z 563[MH-2H,0]*

C M/Z 607(M+Nal+ M/Z 583(M-H]- 584

D M/Z 621(M+Nal+ M/Z 597(M-H]}~- 598

Field desorption mass spectroscopy of Factor E gave the following result M/Z 612 M+, and of Factor F gave the result M/Z 626 M*,

An E.I.spectrum of Factor A with accurate mass measurement gave ions at 612.37 Cy Hc 0g; 466.31 CoH, 0; 448.30 C4 H, 055 425.23 C,ygH3304; - 354.22 Cy3H3404; 297.22 Cy H, 40; 278.11 Cy gH g0s; 247.17 C Ho40,; 219.18 CisHo305 95.05 CgH70. . | An E.I.Spectrum of Factor B with accurate mass measurement gave ions at 598.35 C3gHg0q; 438.28 CogHyg0y; 420.26 CogH 3605; 316.19 CoqH,03; 248.14 C cH 0035. 151:08 CH yy 0,. ‘An E.I. spectrum of Factor C with accurate mass measurement gave ions at : 584.34 Cy,H,g0g; 566.33 C3 Hyc07; 438.28 C,gH 340, : An E.I. spectrum of Factor D with accurate mass measurement gave ions at: 598.35 CyeHo0gi 452.29 CogHyg0,: 634.28 CogHq05.

ee rr ee ————————————————— eer y “ ay

An accurate mass measurement of Factor £ in the €.1- jonisation mode gave an ion at: 452.2908 CogHudd us and for Factor f an jon at 566.3067 Caottaudu: iii) factors A, By c, 0, £ and F have characteristic IR spectra in promo form including the following peaks * for Factor A at about 3510 (oH) , 1712 (ester) and 998 cm (Cc-0)s for Factor g at about 3510 (OH), 1710 (ester) and 996 cm! (c-0)3 for Factor ¢ including peaks at about 3510 (oH), 172 (ester) and 996 cmt (c-0);3

For factor p including peaks at about 3508 (oH), 1711 (ester) and 996 cmt (C-0);

For factor £ including pe aks at about 3500 (pH), 1708 (ester) and 994cm* (c-0)3 / and for Factor F including pands at about 3500 (oH), 1708 (ester), and 997cm-1 (C-0).

The full spectra for Factors A, By c, Oy f£ and F are shown 1n figures 1,2,3,4,6 and 7 respectively of the accompanying drawings. : iv) factors A, By c, 0, £ and F have 3a Uv spectrum in methanol (c = 0.002%) showing the following (where © = inflexion and M = maximum) 0 factor Mom el Fagor Mom) el

A 252 (1) 318 D 252 (1) 263 . L264.5 (M) 468 2064.5 (M) 393 ,39 (1) 830 Cae (1) 362 : 8 52 (1) 302 * E 5p (1) 266 2044.5 (M) 426 244 (M) 402 239 (1 394 238 (M) 313

C 252 (n 316 * F 252 (1D 285 y ly ‘ ¥ . k } 44 _ ki 264.5 (M) 470 264.5 (M) 421 239 (1) 432 239 (M) 389 (* methanol ¢ = 0.001%)

It should be noted that while the Ajay values above are characteristic of each Factor, the el values reflect the purity of the material as it has been obtained. However, the ratios of the gl values are characteristic of the compound per Se. i v) A 200 Miz proton mmr spectrum of solution of each Factor in deutero-chloroform includes signals [t values with multiplicities, coupling constants (Hz) and integration values in parentheses] centred at about: ¥

Factor A : 4.1 to 4.4(m,2H);6.61 (broad s,1H);4.6 to 4.75(m,2H); : 4.81(d,9,1H); 5.05(m,1H);5.34(s,2H);5.69(3,5,1H); 6.06(d,5, 1H); 6.17(m, 1H); 6.26(d,11,1H);6.37(m, 1H); 6.66(d,10,1H); 6.74(q,2,1H); 7.42(m,1H);7.7 to 7.9(m,SH); 8.14(s;3H); 8.40(s,3H); 8.47(s,3H); : 8.61(t, 11,10); 8.96(d,7,3H); 9.06(d,7,3H); 9.02(d,7,3H); 9.13(q,11,1H); 9.21(d,7,3H).

Factor B : 4.2 to 4.4(m,2H); 4.55(a,7,1H); 4.65(broad,s,1H); 4.6 to 4.8(m,2H); 5.06(m,1H); 5.3 to 5.5(m,2H); 6.01(dS, 1H); 6.07(d,5,1H); 6.12(s,1H); 6.26(d,11,1H); 6.24(m,1H); 6.3 to 6.5(m,2H); 6.53(s,3H); 6.73(q,2,1H); 7.62(m, 1H); 7.6-8.0(m,4H); 8.22(s,3H); 8.35(d,7,3H); © 8.41(s,3H); 8.49(s,3H); B.62(t,11,1H); 9.03(d,6,3H); 9.12(q,11,1H); 9.22(d,7,3H).

Factor C : 4.29(d,11,t,2,1H);4.4 to 4.6(m,3H);4.56(broad s,1H); 5.14(dd,15,10,1H); 5.23(m,1H); 5.65(broad 5,2H); 5.72(d,6,1H); ; 5.95(d,10,1H); 5.99(d,6,1H); 6.08(broad s,1H); 6.1 to 6.4(m,3H); 6.62(q,3,1H); 7.7 to 8.1(m,calt);8.18(s,3H); 8.33(s,31);

Br ee a Co —————————— A ————— ee TE

Bi, con DER AT [SETI y

A) - tan psatame ee g.00(d,7,34)3 9.08(d,7,3H); 9.12(q,12,1H)

Factor D : 4.18 to 4.4 (m, 2H) 4.87 to 4.81 (m,8H); 5.04 (m, 1H); 5.3% (s,2H)3 5.72 (d,7,1H); 6.07 (d,7,1H)3 6.15 to 6.05 (m,4H)s 6.74 (q,b,1H)7 4 - 8.1 (m,BH)3 8.16 (s,3H)38.41 (s,3H)38.49 (s,3M);8.62 (t,L11,1H)3 8.92 - 9.05 (m,6M);9-21 (d,7,31)-

Factor E*: 4.1 to 4.3 (m, 2H); 4.5 to 4.8 (m,aH total); 5.00 CRE il 5.2 to 5.5 (m,2H)3 6.01 (d,5,1H)36:0 (d,5,1H)3 6.11 (s,1H)3 6.1 to 6.6 (m,3H)36-4 (d,10, 1H); 6.51 (s,34)36.70 (q,2,1H); 7-60 (m,1H);8.20 (5,31); B.u1 (s,3H)3 8.07 (s,3M); 8.60 (t,11,11)39.00 (v,7,3H)s 9.02 (d,6,31)3911 (q,11,11)59.20 (d,7,3H)-

Factor fF: 4.2 to 4.4 (m,2H)s 4.62 (s,1H); c2 4.70 (m,2H); 480 (d,9,1)5 5:04 (mH); 5:2 £0 3-2 (m,2H); 5-99 (4,5,11); 6:0° (d,5,1H);6:1 (s,1H); 6.1 to 6. (m,2H); €8 6.36 (m,1H)36.82 (d,10,1H)3 6.51 (s,3H); 6.70 (q,2,1H)37-42 (m,H);7.58 (m, 1H); 8.19 I (s,3H);8.40 (s,3H);8.87 (s,3H);8-60 (£,11,14)3 8.95 (d,7,3H)39:0% (d,7,30):9.01 (d,7,3H); 9.10 (q,11,1H)3 9.2 (d,6,3H) vi) A oise-decoupled 25.05 MHz carbon-13 nmr spectryu® of a solution ; 1 of each factor in geutero-chloro fort include pe aks (6 values with multiplicities of signals in off- resonance gpectrum in parentheses] at about: ; ) Factor A: 173.2(s)3 142 .6(d)5 139.2(s); 137.6(s)3 137.108) ’ 137.0(d); 130.4(s); 123.104); 120.1(d);117.8(d)3 99.5(s); 80.0(s); 79.0(d); 76.5(d)3 69.0(d); 68.3%; g7.4(d); 48.2(L)5 45.5(d)s 40.9003 : 40.5(t)s 35.8%; 34.5(t)3 22.1(a)3 34.5(t);5 26.6(d)3 272.6(a); 22.0(al)s 19.7); 15-3(a)s 13.70q);10.8(a)

F

A a i ih id i i da a a i A Cee

Factor B: 173.4(s); 162.1(d); 139.5(s); 137.1(s); 135.7(s); 133.7(s); 123.6(d); 123.3(d);120.0(d);119.3(d); 118.2(d); 99.5(s); 80.1(s); 77.3(d);76.6(d);76.4(d); 69.0(d);68.3(d);67.9 *;67.6 © #;57.5(q);68.2(t); 45.4(d); 40.7(t); 40.5(t); 35.8%; 34.5(t); 22.1(q); 19.6(q); 15.3(q);13.6(q); 12.9(q); 10.5(q).

Factor C : 173.3(s); 142.2(d); 140.3(s); 138.5(s); 137.0(s); 134.9(s); 123.9(d); 121.1(d); 120.6(d);118.1(d); 100.2(s); 80.6(s); 80.1(d); 77.4(d);69.2(d);69.0(d); 68.3 *;68.0(d); 67.9(d);48.6(t); 46.3(d);61.4(t); 36.5 *;36.3 *;36.1(d);35.0(t); 22.6(q);20.0(q); 15.4(q);14.3(q);13.1(q);10.8(q).

Factor D: 173.2 (s); 142.5 (d); 139.1 (s); 137.5 (s); 137.1 (s); 132.1 (s); 131.4 (d); 123.1 (d);120.1 (d);117.8 (d); 99.5 (s); 79.9 } (s); 79.2 (d); 76.5 (d);69.0 (d); 68B.3%;68.1%;67.6%; 67.4 *; 48.2 (t);45.5 (d);40.8 (t); 640.5 (t);35.7 *;34.5 (t);22.0 (q);20.6 (t);19.6 (gq); 15.3q);13.7 (q);13.6 (q);10.7 (a). : * multiplicity uncertain. vii) Circular dichroism curves for factors A, B, C and D (ca. 0.1%

Co solutions in methanol) are shown in Figure 8. The curves are closely \ comparable in the region 230 to 260 nm associated with absorption of the diene chromophore. This indicates that the absolute configurations at C,, C;, Cy; and C4 are the same in all four Factors. ’ } ot ee . —_————

CN

47 -

The following are examples af formulations according to the invention.

The term ‘Active Ingredient’ as used hereinafter means a compound of the invention and may be for example ONE of Factors A, 8, GC p, kL oor

F. ’ hy

Multidose parenteral injection I w/v ~~ Rende active Ingredient "4.0 1 = 5% w/v

Benzyl alcohol 2.0 ;

Glyceryl triacetate 30.0 propylene glycol to 100.0 ;

Dissolve the active ingredient in the penzyl alcohol and glyceryl! : triacetate. Add propylene glycol and make UP to volume. Filter the 3 solution tO remove any particulate contamination. pspectically Fill 3 the product into injection vials and close with rubber seals Of plugs . held in position by aluminium overseals. ferminally sterilise the ; product by heating in an autoclave. perosol spray active Ingredient 0.1 0.01 - 0.50% w/v

Trichloroethane : 29.9

Ir ichlorofluoromethane 35.0

Dichlorodifluoromethant 35.0 my

_ : 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 1

Method of manufacture - web granulation ;; ma

Active Ingredient 250.0 \

Magnesium stearate 1% w/w 4.5 : i

Maize starch 5% w/w 22.5 a

Sodium starch glycolate 2% w/w 9.0 ‘ 2

Sodium lauryl sulphate 1% w/w 4.5 {

Microcrystalline cellulose to tablet core weight of 450mg

Add sufficient quantity of a 10% starch paste to the active ingredient : to produce a suitable wet mass for granulation. Prepare the granules y . and dry using a tray or fFluid-bed drier. Sift through a seive, 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 citable colour may be included in the film-coating solution.

—————— ise WE i SRE ERE MAI go : \ i k y wid - 4a

Veterinary tablet for smal l/domestic animal use

Method of manufactur - dry granulation ma

Active Ingredient 50.0

Magnesium stearate 7.5

Microcrystalline cellulose to tablet core weight of 75.0

Blend the active ingredient with the magnesium stearate and microcrystallise cellulose. Compact the blend into slugs. Break down the slugs by passing through 3 rotary granulator to produce free-flowing Tablets. Compress into tablets.

The tablet cores can then be film-coated, if desired, 8S “re described above. yeterinary jntrammary injection mg/ dase Range

Active Ingredient 150mg 150-500mg ' polysorbate 60 3.0% w/w) ta 3 or 59

White Beeswax 6.0% w/w) to 3g to 3 or 59 arachis oil 91.0% w/w) to 3 of 59 . "Heat the arachis oil, white beeswsX and polysorbate ¢0 to 160% with stirring. Maintain at 160% for two hours and then cool to room temperature with stirring. aseptically add the active ingredient to the vehicle and disperse ysina 8 nigh gpeed mixer. Refine by passing through a colloid mill. aseptically Fill the product into sterile plastic syringes.

» di _ 50 -

Veterinary oral drench active Ingredient 0.35 0.05-0.50% 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 puffer, if necessary. Make up to final volume with the water. Fill the product into the drench container.

Veterinary oral paste

A Range

Active Ingredient 7.5 1-10% w/w

Saccharin 25.0

Polysorbate 85 3.0

Aluminium distearate 5.0 fractionated coconut oil to 100.0

Disperse the aluminium distearate in the fractionated coconut 0il and ) polysorbate 85 by heating. Cool to room temperature and disperse the saccharin in the oily vehicle. Dispense the active ingredient in the base. Fill into plastic syringes. i

DD

RS ks Ebi PEE oo *

Granules for veterinary in-feed administration

Active Ingredient 2.5 0.05-5% w/w imestone flour to 100.0

Blend the Active Ingredient with the limestone flour. Prepare the granules using 3 web granulation process. Dry using a tray or fluid-bed drier. Fill into the appropriate container . fmulsifiable Concentrate

Active ingredient 50g

Anionic emulsifier 409g : (e.g. Phenyl sulphonate CALX) :

Nen- ionic emulsifier 60g (e.q. gyperonic NP13) aromatic solvent (e.g. Solvesso 100) to 1 litre.

Mix all ingredients, stir until dissolved. ; Granules (a) Active ingredient 50g

Wood resin 40g ” Gypsum granules (20-60 mesh) to 1k3d (e.q- pgsorb 100A) (b) Active ingredient s0q

Syperonic NP13 40q : Gypsum granules (20-60 mesh) to 1kg-

Dissolve all ingredients in a volatile solvent e.g. methylene } chloride, add to granules tumbling in mixer. Ory to remove solvent.

The activity of Factors A, B, C, D, £ and F was determined using a variety of pests and their hosts including the following:

Tetranychus urticae (French bean and Myrobalan B plum), Myzus persicae (Chinese cabbage and radish), Heliothis virescens (cotton), Chilo partellus (Rape bean) Meloidogyne incognita (Mung bean), Panonchus ulmi (Myrobalan B plum), Phorodon humuli (hop), Aulacorthum circumflexum : (cyclamen). E

The product was used in the Form of a liquid preparation. The preparations were made by dissolving the product in acetone. The solutions were then diluted with water containing 0.1% or 0.01% by weight of a wetting agent until the liquid preparations contained the required concentration of the product. .

The test procedure adopted with regard to each pest comprised supporting a number of the pests on a medium which was usually a host plant and treating the medium with the preparation (residual test). In the case of Tetranychus urticae both the pests and the medium were treated with the preparation (contact test).

Following this procedure Factors A to F were found to be effective at concentrations (by weight of product) of 500 parts per million or less.

Claims (2)

_ .— CLAIMS:

1. A method for combating parasites or fungi in aariculture, horticulture or forestry, which comprises applying to plants or other vegetation or a celected location thereof one or more compounds of the aeneral formula 111. OH CHj 2 ot Hog oS 1 ~~ 2 CH3 thy si T 3 K | 0 ] LH Rl x ” (II) OH | _H

Lo . 0 z CHy ¥ OR a

2. A method as claimed in claim 1 in which said parasites are insect, acarine or nematode nests.

ABSTRACT Compounds are described having the formula OH { = C “is H aw Hs = 0 \ AN = .- A CH 0 = H CH Ww Pon rH rl (111) 0 0 Ne on [a Ca y = CN 0 A Cex) EI iN Co . = CH, Co, “oo } H OR’ in which rl is a methyl, ethyl or isopropyl group and R? is a hydrogen atom or a methyl group.

The compounds may be used in agriculture or medicine as antiparasitics, und may be prepared by culturing certain Streptomyces strains, in particular Streptomyces thermoarchaensis NCIB 12015.