CA1098063A - Substances and process for their production - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Novel compounds are produced by the fermentation of a nutrient medium with the previously undescribed microorganism Streptomyces avermitilis.
They may be isolated by solvent extraction and chromatographic fractionation techniques. The compounds, which are described generically as C-076 have significant parasiticidal activity. The compounds may be included in compositions for the oral or parenteral administration to animals for the prevention and cure of parasitic infections.

Description

i3 SUMMARY OF THE INVENTION
.
This invention is concerned with novel chemical compounds. In particular, it is concerned with novel sompounds, collectively identified as C-076, which are pro-duced by the fermentation of a nutrient medium with a strain of the microorganism Streptomy~es ave~mitiZis. Thus, it is an object of this invention to provide compounds described generically as C-076 and a method for preparing such products.
It is a further object to provide novel compounds. It is a further object of this invention to provide a method for the recovery and purification of such compounds. These , "

f~

1 substances have significant and broad spectrum antiparasitic

2 activity and it is thus an additioIlal object of this

3 in~ention to pxovide novel antiparasitic compositions

4 containing one or more of the C-076 compounds.
S Furtiler objects of this invention will become apparent 6 :~rom the following description of this invention.
7 i~ESCRIPTION OF THE INVENTION
.. ... _ . .... __ .~. _ 8 In accordance witll this inventlon, a class 9 of substances descri~ed generically herein as C-076, i.s prepared bv gro~ing under controlled conditions, 11 ~)reviously undescribed strains of microorganis~.s. At 1~ least aight distinct ~ut closely related novel compounds 13 axe oroduced by Streptomyces avermitilis. They are 14 described herein as C-076 Ala, ~ , A2a, ~2b, Bla, ~1~, B2a, and ~2b. ~ll have significant antipaxasitlc 16 activity. They may be obtained by fermentation and 17 recovered in su~stantially pure form as described herein.
18 Based on taxonomic studies, the micro-lg organisms capable of producing these C-076 compounds are of a~new species of the genus Streptomyces, 21 which has been named StreptomYces avermitilis. One 22 such culture, isolated from soil is designated l~ 680 23 in the cultllre collection of ~erc~ & Co., lnc., Ra~lway, 24 New JerseyO A C-076 producing sample o~ t;lis culture has b~3en deposited in -the permanent culture collection o~
26 the Fermentation Sec~.ion o~ the Nortllern Utllization 27 Researcll Branch, U.S. Department of Agriculture at 28 Pe~oria, Illinois, and has ~een assi~nsd the accession 29 mlmber NR~I. 8165. ~ sample of NR~L 8165 has also been deposi~ed, without restriction as to availability, in the 1 permanent culture collection of the ~merican Type Culture 2 Collection at 12301 Parklawn Drive, Rockville, ~;aryland 3 20852, and has been assigned the accession number 4 ATCC 31,267.
The morphological and cultural characteristics 6 of Streptom~ces avermitilis are set forth below:
7 ~Iorphology; Sporophores form spirals AS side branches 8 on aerial mycelia. Spirals are compact but bec~me 9 more open as culkure ages. S~ores are in chains of more than 15 spores and are usuall~ spherical to oval 11 at 970 X magnification. Sporulation is obsexved on 12 oatmeal agar, glycerol-asparacJine ac3ar, saits-starch 13 agar and egg albumin agar. Spore surface is smooth as 14 seen ~y ele~tron microscopy.
Oatmeal agar 16 Vegetative growth: Reverse - very dar]~ brown 17 Aerial mycelium: "owdery, ~rownish gray (41i) 18 mixed with white.
19 501uble pigment: Brown C~ape~ Dox agar (sucrose nitrate agar) 21 Vegetative growth: Poor, colorless 22 Aerial mycelium: Scant, grayish 23 Soluble pigment: Light grayisll tan 24 Egg albumin agar Vegetative ~rowth: Tan 26 Aerial mycelium: ~Ioderate, light grayish-yellow-27 brown (3ge) mixed with white.
23 SQ1Ub1e pigment: Li~ht yellowish tan ~3~ 3 Glycerol asparagine agar 2 Vegetative growth: P~e~erse - yellowish brow 3 ~erial mycelium: T'owdery, brownish gray (41i) 4 mixed with wllite.
S Soluble pigment: Lig}lt, yellowish .brown 6 Inorganic salts-starch agar 7 Vegetative growth: Reverse - grayish yellowisi 8 }~rot~m.
9 ~erial mycelium: Potldery, light brownish gray (4ig) 3g edged with dar~-er 11 brownish gray (41i) .
12 Soluble pigment: Light yellowisll brown 13 veast extract-de2~trose + salts agar 14 Vegetative growth: r~everSe - dark brown Aerial mycelium: Lloderate r l~rownish white 16 Soluble pigment: Brown 17 ~east extract-malt extrast a~ar 18 Vegetative growth: ~everse - dar]~ brown 19 Aerial m~.tceliu~ oderate, brownish white Soluble pi.gment: Brown 21 :Peptone-iron-yeas~ extract agar 22 Vegetative growtll: Dar~; brown 2 3 A~rial mvcelium: None 24 Soluble pigment: Dar]; }~rown to black Melanin: Positive 26 H S production: 1:~05itive 2 7 Nutrient agar 2 8 Vegetative growth: Tan 2Y Aerial mycelium: Sparse, grayisl Soluble ~igment :. Light brown utrient starch agar 2 Vegetative growth: Tan 3 ~erial mycelium: Sparse, grayish w~lite 4 Soluble pigment: Light bro~m S Hydrolysis of starch: Good 6 Potato plug 7 Vegetative growth: Tan 8 Aerial mycelium: Brown mixed with grayish ~hite 9 Solu~le pigment: Grayish brown I,oef1er's Blood serum 11 Vegetative ~ro~th: Grayish tan 12 Aerial mycelium: None 13 Soluble pigment: Some browning of medium 14 Liqueaction: None Nutrient tyrosine agar 16 Vegetative growth: r~everse - dar]i brown to blac];
17 Aerial mycelium: Sparse, gra~,ish 18 Soluble pigment: Dar]; brown 19 Decomposition o~ tyrosine: None Carbon utilization 21 Pridham-Gottlieb basal medium + 1~ carbon source;
22 + = growth; no growth as compared ~o negati~e 23 control (no car~on source).
24 Glucose 25 Arabinose +
26 Cellulose 27 Fructose 28 Inositol +
29 ~actose +
30 Maltose +

i3 1 Mannitol +
2 Mannose +
3 Rafflnose +
4 Rhamnose f Sucrose +
6 Xylose +
7 ~utrient selatin agar 8 Vegetative growth: Tan 9 Aerial mycelium: Sparse~ gra~ish whike Soluble pigment: Light brown 11 Liquefaction of gelatin: Good 12 Gelatin stabs 13 Vegetative growth: Brown ring 14 Aerial mvcelium: None Soluble pigment: Greenish brown 16 Liquefaction of gelatin: Co:mplete 17 Skim milk agar 18 Vegetative gro~rth: Dar]; bro~n l9 Aerial mycelium: None Soluble pigment: Dark brown 21 Hydrolysis of casein: Good 22 Litmus milk 23 Vegetative growth: Dark bro~n growth ring 24 Aerial mycelium: ~one Color: Dark brown 26 Coagulation and/or peptoniza~ion: Complete 27 peptonization; becoming alkaline 28 (pH 8~1).

1 S]~im milk 2 Vegetative growth: Dark brown growth ring 3 Aerlal mycelium: None 4 Soluble pigment: Dark brown Coagulation and/or peptonization: Complete 6 peptonization; becoming al~aline 7 tpH 8.0).
8 Temperature range: (Veast extract-dextrose ~ salts agar) 9 28~C - Goo~ vegetative growth and aerial mycelia 37C - Good vegetatlve growth and aerial mycelia 11 50C - No grow-th 12 Oxygen requirement: (S~ab culture in yeast extract-13 dextrose ~ salts agar) 14 ~erobic ~11 readings ta~en after three weeks at 28C unless 16 noted otherwise. pH o all media ap~roximately neutral 17 (6.~ - 7.2) 18 C:olor number designations (~) taken from Color Harmony 19 ~lanual, 1958, 4th Edition Container Corporation of 20 America, Chicago, Illinois.
21 A careful comparison of the foregoing data 22 with pu~lished descriptions including Bergey's Manual 23 o Determinative Bacteriology (Eightll Edition) of 24 ~nown microorganisms reveals significant differences 25 tha~ indicate that the instant microorganism should 26 be classified as a new species. On this basis, it 27 was designated Streptomyces avermitilis.
28 The above description i5 illustrative 29 o~ a s~rain of Strep~omyces avermitilis which can be 30 employed in the production of the C 076 compounds ,3 l described herein. ~Iowever, the present invention also 2 emDraces mutants oL the above de~cribed microor~anism.
3 For example, tho~e C-076 producing mutants which are 4 obtained by natural selection or those producted by mutating agents includins X-ray irradiation, ultraviolet 6 irradiation, nltro~en mustard or like treatments are 7 also included within the ambit of this invention.
8 One example of such an organism is a strain of 9 Stre~omyces ave~mltil _ ~IA 4848 which was isolated after irradiation with ultraviolet light of Stre~tomvces ll avermitili.s ~ ~680. A lyoPhilized tube and a frozen vial ._.
12 o~ this culture has been depo3ited ln the permanent culture 13 collection of the ~merican Type Culture Collection, and 14 they have been assigned the accession num~ers 31272 and 31271 respectively. Sll~htly hiqller fermentation ~,~ield3 of 16 C-076 have been obtained using this frozen stoc~; as inoculum.

~-7 I'he C-076 compounds are produced during the 18 aerobic fermentation of suitable aclueous nutrient media l9 under conditions described hereinafter, wi~h a producin~

strain of Streptomyces avermitilis. A~qu~ous media such ~1 as those used for the production of man~ antibiotic 22 substances are suitable ~or use in this proces~ for the 23 preparation of C-076.

24 Such nutrient media contain sources of carbon cmd nitrogen assimilable by the microor~anism and 26 generally low levels of lnor~anic salts. In addition, 27 ~he fexmentation medla may contain traces of metals 28 necegsary for the ~rowth o~ the microor~an~sms. ~hese are 29 usually present in su~ficient concentration in the complex ~;ources of carbon and nitro~en whicll may be used as 31 nutrient sources, but can, of course, be added separatelv 32 to the medium if desired.

1 In general, carbohydrates such as sugars, for 2 example de~trose, sucrose, maltose, lactose, dextran, 3 cerelose and the li~e, and starches are suitable sources 4 of assimilable carbon in the nutrient mediaO The exact quantity of the carbc)n source wllich is utllized in the 6 medium will depend, in paxt, upon the other in~reclients 7 in the medium but it is usually found that an amount of 8 carbohydrate between about 0.5 and 5% by weight of the g medium is sa~is~actory. These car~on sources can ~e used individually or several suc~l carbon sources may b~
11 combi~ed in the same medium.
12 Various nitrogen sources sucll as yeast 13 hy~rolysates, yeast autoysate, soy~ean meal, casein 14 h~drolysates, yeast extracts, corn steep liquors, distillers solubles, cottonseed meal, meat e~tract and 16 t:he li~e, are readily assimilable by Streptomyces 17 dvarmitilis in the Production o~ the C-076 .. . _ 18 c:ompounds. The various sources of nitroqen can be used l9 alo~e or in combination in amounks ranging from about 0.2 to 6~ ~y weight of the medium.

21 ~mon~ the nutrient inorganlc salts whi~h can 22 ~e incorporate~ in the c1~1ture media are the customary 23 salts capable of yieldin~ sodium, ~otassiurn, magneslum, 24 ammoniuml calclum, phosphate, sulfate, chloride, carbonate and like ion5. ~lso incluc;ed are trace metals 26 such as cobalt, man~anese, iron and the li~ie.

27 It should be noted that the media described 28 hereirlbelow and in the E~amples are merely illustrative 29 of tlle wide variety of meclia which may be employed, and 30 are not intended to be limitative.

g_ ~aB~63 l The following are Examples of media suitable 2 for growing strains of Streptomyce3 avermltilis for 3 produc.in~ the C-075 compounds.
4 rIedium ~
~ .. . _

5 Corn meal 20.0 g

6 Distiller's solubles 10.0 ~

7 Soybean meal 15.0 g

8 Sodium citrate 4 0 g

9 CaC122H2O 0.5 g

10 Polyglycol P2000 2.5 rnl

11 gS 4.7H2O . 0.1 g

12 CCl2 6II2 0.01

13 FeSO4.7~2O 0.01

14 Distilled water 1000 ml L5 pEI 6.5 16 ~dium ~
17 Soluble starch 20.0 g.
18 Corn steep liquor 15.0 g.
l9 Cerelose 5 0 g 20 Soybean meal 4.0 g.
21 ( 4)2 S~4 4.0 g.
22 Corn meal 1.0 g.
23 Soybean oil 2.5 ml 24 ~;H2 PO4 o 3 g 25 CaCO3 6.0 g.
26 ~istilled water 1000 ml 27 pH 6.7 28 Medium C
29 Tomato p~ste 40.0 g.
30 Oat flour 15.0 g.
31 Distilled water 1000 ml 32 pH 6.0 1 Med 2 Oa~ flour 20.0 g.
3 Tomato paste 20.0 g.
4 Distilled water lO00 ml.
5 pH 5.5 6 Medium E
7 ]~extrose lO.0 g.
8 Peptone (Available from Difco 5.0 g.
9 Laboratories, Detroit, Michigan).
lO ~east autolysate (Available as Ardamine 3.0 g.
ll plI from Veast ~roducts Inc., 12 Paterson, New Jersey).
13 NaC1 12.7 g.
14 I~C1 0.72 g.
l5 FeSO4 (N~I4)2 SO4.6EI2O 0.035 g.
16 ~IgC12.6II2O 5.32 g.
17 CaCl2 2H2 0.73 gO
18 ~i5tilled water 1000 ml.
19 pH 7.4 20 The fermentation employing the C-076 21 producing microorganisms can be conducted at temperatures 22 ran~in~ rom about 20 to about 40C. For optimum 23 results, it is most convenienk to conduct these 24 ferme~tations at a temperature in the range of from a~out 24 to about 30C. ~emperatures of about 27-28C
26 are most preferred. Tlle pH of the Ilu~rient medium 27 suitable fox producing the C-076 compounds can 28 vary from about 5.0 to 9.0 with a preferred range of 29 from a~out ~.0 to 7.5 --11~

i63 1 Small scale fermentations are conveniently 2 carried out by placing suitable quantities of nutrient 3 medium in a flas~ emPloyin~ known sterile techniques, 4 inoculatin~ the flask with either spores or vegetative cellular growth of a C-076 producin~ s~rain of 6 Streptomyces avermitilis, loosely stoppering the necks 7 of the flas~; with cotton, and pe~mittin~ the fexmelltation 8 to proceed in a constant room temperature of abollt 9 28~C on a rotary sha}~er for about 3 to 10 days. For lar~er scale wcrk, it is preferable to c~nduct the 11 fermentation in suitable tanks provided witll an 12 agitator and a means of aerating th~ fel~nentation medium.
13 The rlutrient medium is made up in the tan~
14 and after .steriliz~tion is inoculated with a suitable ~ource of vegetative cellular growth of a C~076 16 producing s~rain of Streptomyces avermitilis. The _ _ 17 fermentation is allowed to continue for from 1 to 8 18 days while agitating and/or aerating tlle nu~rient 19 medium at a temperature in the ran~e of from about 24 to 37~C. The degree of aeration is dependent upon 21 several actors such as the size of the fermentor, 22 agitation speed and the like. Generally, the larger 23 scale fermenta~ions are agitated at about 95 to 24 150 RPM and about 2 to 20 cubic feet per minute of air.
The novel substances of this invention, 26 which are generically referred to herein as C-076, 27 are foun~ primarily ln the myceli~n on termination 28 o~ the Streptomyces avermitll s fermentation, and 29 may be recovered and separated from one another as described below. Four major and four minor components 1 of the C-076 as elaborated by Streptomyces avermitilis 2 llave been isolated. The eight different compounds are 3 identified herein as C-076 Ala, Alb, A2a, A2b, Bla, 4 Blb, B2a and B2b. The major components have been given the suffix "a" in our identification terminology and the 6 minor components the suffix "b~" The structural 7 dlfference between the "a" and "b" compounds is belived 8 to be the same ~or each of the four pairs.
9 As might be expected, even the major C-076 compounds are not produced in equal amounts by the 11 fermentations described herein. In general, it llas ~een 12 found that the ~1 compounds comprise about 20 to 30% bv 13 wei~ht of the total C-076 complex produced, the A2 14 compoun~s about 1-20% and the Bl and B2 compounds each about 25-35%. The weight ratio of the "a" serles o~
16 compounds to the "b" series is about 85:15 to 99:1.
17 The separation of the C-076 series of 18 compounds from the whole fermentation broth and the 19 recovery of the individual components is carried out by solvent extraction and application of chromatographic 21 fractionations with various chromatograpllic tec~niques 22 and solvent systems.
23 The C-076 compounds have sli~ht solubility 24 in water, but are soluble in organic solvents. Tllis property may be conviently employed to recover 26 them from the fermentation broth. Thus in one 27 recovery method, the whole fennentation broth is 28 iltered and the aqueous filtrate discarded. ~he wet 29 mycelial cake is then extracted with an appropriate organic solvent. While any organic solvent may be 31 employed, it is preferable to use a water miscible 1 solvent such as acetone, methanol, ethanol and the li];e.
2 Generally, several extractions are desirable to achieve 3 maximl~m recovery. The solvent removes the C-076 active 4 oomponents as well as other substances lacking the 5 antiparasitic activity of C-076. I~ the solvent is a 6 water miscible one, the water is also removed from the 7 wet mycelia. The extracted mycelia may ~e discarded.
8 The solvent extracts are evaporated to remove the or~anic 9 solvent and extracted several times ~Jith a second solvent.
10 When the first extraction employs a water miscible solvent, 11 the second extraction preferably employs a water immiscible 12 solvent such as chloroform, methylene chloride, carbon 13 tetrachloride, ethylace~ate, methylethyl ketone, 14 methylisobutyl ketone and the like. These latter extracts

15 are dried and concentrated usin~ known techniques to afford

16 a residue comprising C-076 admixed with other materials.

17 Tllis ~raction is then conveniently chromatographed in

18 order to separate the ac~ive C-076 compounds from other

19 material and also to separate and isolate the individual

20 C-076 compounds. The chromatographic techniquQs which may

21 be employed to purify the C-076 compounds are

22 generally ~nown to those skilled in this art. Examples

23 of such techniques are column chromatography,

24 using such media as silica gel, aluminium oxide, dextran

25 gels and the like, and elution of such columns with various

26 solvents, and/or a com~ination of two or more

27 solvents, in varying ratios. Liquid chromatography is

28 e~nployed for the detection of the C-076 compounds, and

29 hlgh pressure liquid chromatography may be employed to

30 isolate puriiied fractions containing one or more of such

31 compounds. Li~ewise, thin layer chromato~raphy may be l employed to detect the presence of, and to isolate the 2 individual C-076 compounds. The use of the foregoing 3 techniques as well as others known to those s.~illed in this 4 art, will afford purified compos~tions comprising the C-076 5 compounds as well as the individual C-076 compounds 6 themselves. The presence of the active C-076 compounds is 7 determined ~y analyzing the various chxomatograpllic fractions 8 for antiparasitic activity and also by the spectral g characteristics (such as ultraviolet and infrared) of said 10 compounds as described below.
ll Tlle spectral and other physical-chemic21 12 cllaractexistics of the individual C-076 compolmds are set 13 forth in tabular form in Table I. These compounds are very 14 soluble in most common organic solvents, and are of minimal 15 solubility in water.
16 The ultraviolet spectral data in Table T were 17 obtained on a Cary Model 15 Ultraviolet Spectrometer in 18 methanoi solutions in l cm. quartz cells. The concentration 19 Of the compound was approximately 2~ ~g/ml. The ultraviolet 20 absorption, while represented as that of a particular "a 21 series" compound, i9 actually the absorption of the "a 22 series" compound which contains a minor amount o~ a "b 23 series" compound. The "a" and "b" series dif~er only by a 24 ~CH2-moiety in a lowexalkyl substituent which dif~erence is 25 not associated with the chromopilore. Ultraviolet absorption 26 primarily characterizes the de~ree and nature of unsaturation 27 p:resent in the particular compo~md. The optical rotations 28 were determined using standard teci~iques with a ~arl 29 Zeiss polarimeter. The concentration factor (c) is given 30 as a percent of the compound in tl~e stated solvent.

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t~ ~ o o ~r o co u~ o o 5co co ~ O ~ . o -- o~~ r ~ o o 0~ ~
r~
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:q C~ o~ CO
0~ +~o ~ t~ 11 o ~ o ~ ~ o ~
~1 co u~ O ~ ~ o L~
~r ~-- r~ ~ ~ . ~ ~ u~
m _l + co o~ _, _ _ ~CO o CO
o~ ~1 ~9 O ~ o ~ o ~ ~ a~ ~o c) e~' t~ CO ~ ~ ~
-- o ~; - ~ + ~n ~ C::l '* N -1 ~ O

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.
~i o ~ .
O + I l~ o ~ N O 1' . o u~ r ~ ul 5~ o ~: ~ In O ~ 1 ~ N N
(~5 Ct ~0 C) U~ N 0~ ~r N .~1 ~r O
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rd N CI ~ ~) O ~ X
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--16 ~

~3~3 15874 IA

1 13C nuclear ma~lletic resonance spectral data for C-076 Ala, A2a, Bla and B2~ are set fortll 3 in Ta~le II below. The spectra are obtained from a 4 Varian ~uclear ~lagnetic Spectrometer ~iodel CFT-20 in deuterated chloroform solutlon using tetramethylsilane 6 as an interrlal standard. The solution volum~ and the 7 concentration of sample are given or each case, ~ollowed ~ by the chemical shi~ts relative to tetrameth~l silane g for eacn compound glven i~ parts per million (ppm)O Tlle cbemical shifts are given for a single carbon atom unless 11 otherwise indicate~ in a parenthetical expr~ssion following 12 the chemical shift.

13 l'ABLE II
14 1 (0.6 ml._l 6 o ) lS 12.0, 13.0, 15.1, 16.4, 17.7, 18.4, 19.9, 20.3,27.5, 16 30.6, 34.3 (3C), 35.2, 36.6, 39.7, 40.5, 45.7, 56.4 17 (2C), 57.7, 67.3, 68.2 (2C), 68.4 (~C), 74.9, 76.1, 18 77.0, 77.5, 78.3, 79.4, 80.6 (2C), 32.0, 95.0, 95.8, 19 ~8.5, 118.4 (2C), 119.7, 124.9, 127.~, 135.2, 136.0, :~36.1, 137.6, 139.~, 173.8.
21 A,~,L~ UL~L_ 22 :Ll.8, 12.4, 13~8, lS.l, 17.7, 18.4, 19.9, 20.3, 27.3, 23 :34.3 (3C), 35.2, 35.8, 36.5, 39.8, 40.8, 41.2, 45.7, 24 'j6.4 (2C), 57.7, 67.3, 67.7, 68.2 (3C), 69.9, 70.g, J6.1, 77.0, 77.6, 78.3, 79-4t 80.6 (2C), 81.~, 94.9, 26 98.6, 99.7 r 117.7, 118.4, 119.7, 124.9, 135.7, 136.1, 27 137.6, 140.0, 173.7.

'' ii3 1 B1 (0.3 ml. 16%) 2 12~0, 12.9, 15.1, 16.4,17.7, 18.4, 19.9, 20.2, 27.5~
3 30.6, 3~.3 (3C~, 35.2, 36.6, 39.8, ~0.5, 45.7, 56.4 (2C), 4 67.3, 67.8, 68.2 ~2C), 68.4 (2C), 75.0, 76.1, 7~.3, 79.4 (2C), ~0.5 (2C), 82.0, 95.0, 95.8, 98.5, 118.1, 6 118.4, 120.4, 124.8, 127.9, 135.~, 136.2, 137.9 (2C), 7 139.7, 173.6.
8 B2 (0.6 ml. 16~) 9 11.8, 12.4, 13.8, 15.1, 1707, 18.4, 19.9, 20.2, 27.3, 34.4 (3C), 35.2, 35.8, 36.5, 39.8, 40.8, 41.2, 45.8, 11 56.4 (2C), 67.3, 67.7 (2C0, 68.3 (3C), 69.9, 70.9, 12 76.1, 7~.3, 79.4 (2C), 80.5 (2C), 81.8, 94.9, 98.6, 13 99.7, 117.7, 118.0, 120.4, 124.8, 135.7, 138.0 (~C), 14 :L~9.8, 173.5.

The characteristic mass spectral pea}~s of .
16 eight C-076 compounds are given i:n Table III. The 17 ~irst line of Table III represents the mass to charge 18 xatio (m/e) of the molecular ion of each respective 19 c:ompound, and the remaining numbers record the mass to charge ratio of the principal fragments o~ each compound.
21 ~ass to charge ratios found on the same horizontal row 22 indicate analagous fragments in each compound. The mass 23 to charge ratios which are given as whole numbers were 24 obtainea from an L~ Mass Spectrometer Model LK~-9000.
The mass to charge ratios which are given to the fouxth 26 decimal place were obtained from a High Resolution Varian 27 Mass Spectrometer l~Iodel ~T-731.

-18~

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C~ t~ L') L'l ~ ~ (~ 1 N N r~ r~
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a~ C5 r` L~) L~ '\1 ~ N ~Ir l r i r~

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a~ ~1 ~ ~1 1~ ,~1 ~ N ~ ~ L'~ O ~9 L") N ~ C~ ~ ~r t~ N ~~
C) H V~
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U~
_ _ . _ _ . , _ .. .... , _.. ~. _ _ ~ L'~ 3 C) ~'1 ~ ~1 !`
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r~ ~ o ~ c~ N G~ ~J CO O ~ ~ ")L") O ~O C' O ~J ~ r l ~ ~ ~ ~ ~)b'') Lr~r~l ~ CO C~ r- ~D
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r~ L~ L~ L~

~8~? ~3 15874 IA

1 The attached Figures 1 to 3 are accurate 2 reproductions of infrared and proton-nuclear magnetic 3 resonance spec.ra of four of tlle C-0~6 compounds.
4 Fi~ures l-a are infrared spectra for C-076 S .~la, A2a, Bla and B2a respectively. ~igures ~ to 3 are 6 proton-nuclear magnetic spectra for C~076 Ala, A2a, 7 Bla and B2a respectively. The infrared spectra wexe 8 obtained on a Per~in-Elmer Infrared S?ectrometer ~odel 9 421 in chloroform solution. mhe proton-nuclear magnetic resonance spectra were obtained in deuterated chloroform ll solution in a Varian Nuclear ~lagnetic Resonance 12 Spectrometer ~Iodel HA-100 and the s~ectra show chemical 13 shifts given in parts per million (p-,m) relative to 14 tetramethyl silane as an internal standard.
lS Based on experimental data, including tne 16 studies and measurements described herein, the C-076 17 compounds are believed to have the following planar 18 structural formula: Rl CH3 ~ ~ CH3 R-O ~ ~ ~ ~ ~ ~ R2 3 ~ ~
Il O ~

~9~ 3 15874 IA

1 wherein R is the cC -~-oleandrosyl-oC-L-oleandroside of 2 the structure:
CX3 ~ ~ C~ 0 HO ~ O ~ >
C~3O C~3O

3 and wherein the broken line indicates a single or a double 4 bond; Rl is hydroxy and is presen~ only wnen sald Dro~en line indicates a single bond.

6 R~ is propyl or butyl; and 7 P~3 is methoxy or hydro~.
8 In the foreaoing structural ~ormula, the g individual compounds are as set forth in Table IV.

T~BLE IV
11 Rl R2 R3 12 Ala Double bond butyl -OCH3 13 .~1~ Double bond prop~l -OC~3 14 A a -OE-I ~ut~l -OCH3 15 ~2b -OH propyl -OCEI3 16 Bla Double ~ond kutyl -OH
17 Blb Double bond propyl -OH
18 32a -OH butyl -OH
19 B2b -OH propyl -OEI

As mi~ht be expected, the compounds of this 21 invention wherein R~ is butyl (the "a seriesl' of compoundsj 22 and the corresponding compound wherein R2 is propyl (t:he ~3 l'b series"~, act similarly in most reco~ery procedures such 24 as those involving sol~ent extraction. In each ~air of "a" and "b" compounds the "a' compound is found in greater 26 amount and ~enerall~ make5 up about 85 to ~9~ of a mixture 27 of tlle "a" and "b" compoundsO The presence of the propyl -2~-i3 1 compounds is verified in the mass spectra of the 2 compounds wherein the mass peaks representing the 3 fragments containing the butyl group have companion 4 peaks witll a mass 14 units (or one-CH2 group) lower.
In addition, high pressure liquid chromatograph~ has 6 been employed to separate the Alb component from the 7 Ala component, and the mass spectrum of such Al~
8 compound has been verified by high resolution mass 9 spectrometry (see Table III).
The novel compounds o~ this invention llave 11 significant parasiticidal activity as anthelmintics, 12 insecticides and acaricides, in human and animal health 13 an~ in agriculture.
14 The disease or group of diseases descri~ed generally as helminthiasis is due to inEection of an 16 animal host ~itll parasitic worms known as helmintlls.
17 Helminthiasis is a prevale~t and serious economic problem 18 in domesticated animals such as swine, sheep, horses, 19 cattle, goats, dogs, cats and poultxy. ~nong t~e helminths, the group of worms described as nematodes causes widespread 21 and often times serious infection in various species o:E
22 animals. The most common genera oE nematodes in~ecting the 23 cmimals referred to above are ~Ia monchus, Trichostrongt~lus, 24 C?stertagia, Nematodirus, Cooperia, Ascaris, Bunostomum, oesopha~ostomum, Chabertia, Trichuris, Strong~lus, 26 ~lrichonema,Dic-tyocaulus, Capillaria, Hetera];is, -27 Toxocara, Ascaridia, Ox~uris, Ancylostoma, Uncinaria!

2~ ~~oxascaris and Parascarls. Cer~ain of these, such as 29 Nematodirus, Cooperia, and ~ ostomum attac~ primarily t.he intestinal tract while others, such as ~aemonchus and 31 Ostertagia, are more prevalent in the stomach while stlll others such as Dic-tvocaulus axe round in the lungs. S-till -2 other parasites may be located in other tissues and organs 3 of the body such as the heart arld blood vessels, sub-4 cutaneous and lymphatic tissue and the like. The parasitic S infections known as helminthiases lead to anarnia, 6 malm~ttrition, weakness, weight loss, severe damage to 7 the walls of the intestinal tract and other tissues 8 and organs and, if left untreated, may result 9 in death of the inected host. The C-076 compounds of 10 this invention have unexpec~edly high activity against 11 these parasites, and in addition are also active against 12 Dirof ilaria in d~gs, Nematospiroides, ~hzcia, 13 ~spiculuris in rodents, arthropod ec ~opctrasites of animals _ .
14 and birds such as tic}is, mites, lice, fleas, blowfl~, in 15 sheep Lucilia sp, bi.tin~ insects and such migrating 16 dipterous larvae as ~ sp in cattle, Gastrophilus 17 in horses, and Cuterebra sp. in rodents.

13 The instant compounds are also useful 19 against parasites which infect humans. - Tlle ntost common 20 genera of parasites o ~he gastro-intestinal tract 21 of man are Anc~lostoma, Necator, Ascaxis, Stron~yloides, 22 'rrichinella, CaPillaria, Trichuris, and Ente~obi.us.
. _ . _ . , . . _ . . .. .. _ . . . _ _ 23 Other medically important genera of parasites which are 2 4 found in the blood or other tissues and organs outside 25 t:he gas~ro-intestinal tract are the filiarial worms such as 26 Wuchereria, Brugia, Onchocerca and Loa, Dracuncuius 27 and extra intestinal stages of the intestinal worms 28 St..ongyloides and Trichine_1a. The compounds are also of 29 value against arthropods parasit:izing man, biting insects 30 and other dipterous pests causing annoyanca to man.

1 The compounds are also active against household 2 past5 such as the cockroach, slatella sp., clotAes moth, 3 Tlneola sp., carpet ~eetle, Attag~nus sp. and the housefly 4 ~lusca domestlca.
The compounds are also userul against insect 6 pests of stored grains sucll as Tribolium sp., ~enebrio 7 sp. and of agricultural plants such as splder mites, 8 (Tetranychus sp.), aphids, (Acyrthios~phon sp.); against 9 migratory orthopterans such as locusts and immature stages of insects living on plant tissue. The compounds are ll useful as a nematocide for the control of soil nematodes 12 and plant paxasites such as ~eloidogyne ~ which may be 13 of importance in agriculture.
14 These compounds may ~e ~dministered orally in a lS Imit dosage form such as a capsule, bolus or tablet, or as 16 a liquid drench where used as an anthelmintic in mammals.
17 The drench is normally a solution, suspension or dispersion 18 of the active ingredient usually in water together with a l9 suspending agent such as bentonite and a wetting agent or like excipient. Generally, the drenches also co~tain an 21 antifoaming agent. Drench formulations generally contains 22 ~rom about 0~001 to 0~5% by weight of the active compound.
23 Preferred drench ~ormulations may contain from 0.01 to 0.1 24 by weightO The capsules and boluses comprise the active ingredient admixed with a carrier vehicle such as starch, 26 talc, magnesium stearate, or di-calcium phosphate.
27 Where it is desired to administer the C-075 ~8 compounds in a dry, solid unit dosage form, capsules, 29 boluses or tablets containing the desired amount of active compound usuall~ are employed. These dosage forms are 31 prepared by intimately and uniformly mixing

32 ~he active ingredient witil suitable $inely divided l diluents, fillers, disinte~rating agents and/or binders 2 such as starch, lactose, talc, magnesium stearate, 3 vegetable gums and the like. Such unit dosage 4 formulations may be varied widely with respect to their total weight and content of the antiparasitic 6 a~ent depending upon factors such as the type of host 7 animal to be treated, the severity and type of infection 3 and the weight of the host.
9 When the active compound is to be administered via an animal feedstuff, it is intimately 11 dispersed in the feed or used as a top dressing or in 12 the fo.rm of pellets which may then be added to the 13 finished feed or optionally fed separately.
14 Alternatively, the antiparasitic compounds of our invention may be administered to animals 16 parenterally, for example, by intraruminal, intra-17 muscular, intratracheal, or su~cutaneous injection in 18 ~,rhich event the active in~redient is dissolved or dispersed l9 ln a liquid carrier vehicle. For parenteral administration, the active material is suitably a~mixed 21 with an acceptable vehicle, perferably o-f the vegetable 22 oil variety such as peanut oil, cotton seed oil and 23 the like. Other parenteral vehicles such as organic 24 preparation using solketal, glycerol, formal and aquecus parenteral formulations are also used. ~he active 26 C'-076 compound or compounds are dissolved or suspended 27 in the parenteral formulation for administration; such 28 formulations generally contain from 0.005 to 5%
29 ~y weigh~ of the ac~ive compound.

~ 63 15874 IA

1 .Although the antiparasitic agents of this 2 invention find their primary use in the treatment and/or 3 prevention of helminthiasis, they are also useful in 4 ~he prevention and treatment of diseases caused by other parasites, for example, arthropod parasites such 6 as tic~s, lice, fleas, mite5 and other bitin~ insects in 7 domes~icated animals and poultry. They are also efective 8 in treatment of parasitic diseases that occur in other 9 ani~als incLuding humans. The optimum amount to be employed for best results will, of course, depend upon the ll particular compound employed, the species of ani~al to be 12 trea~ed and the type and severity of parasitic infection or 13 infestation. Generally, good results are obta,ined with our 14 novel compounds by the oral administration of from about O.OOl to 10 mg. per kg. of animal body weigllt, sucll 16 ~otal dose being ~iven at one time or in divided doses 17 over a relatively short period of time such as 1-5 days.
18 With the preferred compounds o~ the invention, excellent 19 control o such parasites is obtained in animals by administering from about 0.025 to 0.5 mg. per kg. o~ body 21 weight in a single dose. Repaat treatments are given as 22 required to combat re-infections and are dependent upon 23 t,he species of parasite and the husbandry techni.ques 24 ~eing employed. The techniques for administering these matexials to animals are known to those skilled in the 26 veterinary field.
27 When the compounds described herein are 28 a.dministered as a componen-t of the feed oE the animals, or 29 dissolved or suspeIlded in the drin~ing water, compositions are provided in wllich the active compound 1 or compounds are intimately dispersed in an inert 2 carrier or diluent. By inert carrier is meant one that 3 will not react with the antiparasitic agent and one that 4 may be administered safely to animals. Preferably, a carrier for fee~ administration is one that is, or may 6 be, an ingredient of the animal ration.
7 Suitable compositions include feed premixes or 8 supplements in which the active ingredient is present in 9 relatively large amounts and which are suitable for direct feeding to the animal or for addition tto the feed either 11 directly or after an intermediate dilution or ~lending 12 step. Typical carriers or diluents suitable Eor such 13 composltions include, for example, dis~illersl dried grains, 14 corn meal, citrus meal, fermentation residues, ground oys~er shells, wheat shorts, molasses solubles, corn cob 16 meal, edible bean mill eed, soya grits, crushed limestone 17 and the like. ~he active C-076 compounds are intimattely 18 dLspersed throughout the caxrier by methods such as grinding, 19 s~irring, milling or tumbling. Compositions containing ~rom about 0.005 to 2.0~ ~y wei~ht oS the active compound 21 are particularly suitable as feed premixes. Feed 22 supplements, which are fed directly to the animal, contain 23 ~rom about 0.0002 to 0.3~ by weight of the active compounds.
24 Such supplements are added to the animal ~eed in an amount to give the finished feed the 26 concentration of active compound desired for the 27 tr~atment and control of parasitic diseases. Although 28 the desired concentration of active compound w.ill 29 vary depending upon the factors previously mentioned as well as upon the particular C-076 compound employed, 15874 I~

1 the compounds of this invention are usually fed at 2 concentrations of bet~,Jeen 0.00001 to 0.002% in the 3 feed in order to achieve the desired antiparasitic 4 result.
In using the compounds of this invention, the 6 lndividual C-076 components may be isolated and purified 7 and used in that form. Alternatively, mixtures of two or 8 more o the individual C-076 components may ~e used. It 9 is not nacessary to completely separate the various C-076 compounds obtained from the purification of the fermentatlon 11 broth. Generally, there is obtained a mixture containing 12 two or more of the C-076 compounds, but having other 13 unrelated compounds excluded ther.efrom, and such mixture 14 may be used for the prevention and treatment of parasitic diseases as described herein. Such a mixture generally 16 wlll contain unequal proportions of the C-076 compounds, 17 however, all of the compounds ha~e substantial activity 18 and the antiparasitic activity of the mixture can be 19 accuxately dete~minedO In particular i~ may not be necessary to separate the "b" components from the related "a'~
21 component. Such compounds differ only in the len~th of 22 the 25 side-chain. Separation of tilese closely related 23 compounds is generally not practiced since the "b"
2~ compound is present only in a very small percent by weight.
26 In addition, where the C-076 compounds are 27 to be added to an animal's feed, it is possible to 28 utilize tlle dried mycelial ca~e from the fermentation 29 ~roth. The mycelia contain a preponderance of the activity and since the level of the activity of the mycelia can be 31 determined, it can be added directly to the animal's feed.

-2~-1 The compounds of this invention have a broad 2 spectrum of activity agalnst many internal parasites at 3 low dosage levels and in many different animals. At 4 levels of about 2.5 mg. per ]iq. of animal body weight, 5 concentrated mixtures of C-076 compounds are fully 6 active in sheep against Haemonchus contortus, Ostert~
7 circumcincta, Trichostrongylus axei, ~richostrongylus . .
8 colubriformis, Coopexia s~ and Oesophagostomum 9 columbianum. Similarly in cattle at dosages as low as 0.0a3 mg./kg~ C-076 B2 is fully active aaainst 11 Ostertagia osterta~i, Trlchostrongylus axei, 12 ~richostrong_lus colubriformis, Oesophagostomum radiatum 13 and Dictyocaulus _lviparus. In addition, a horse infected 14 with bo~s (Gastrophilus intestinalis and Gastrophilus 15 ~ , large and small strongyles and ~ s 16 was successfully ~rea~ed with L0 mcJ./k~. (about 1~ active 17 compounds by weigh~) o~ a mixed concentrate of C-076 18 compounds, and a dog infected with the microfilarial stage 19 o~ heartworm (Diro~ilaria immitis) was successfully treated 20 With a single oral dose at 10 mg./k~. (about 1% active 21 compounds by weight) of a mixed concentrate of C-076 22 compounds. In rodents, such as mice, in~ections of 23 ~;yphacia, Nematosplroldes and Aspiculuris have been 24 ~;uccessfully treated by the oral administration of the 25 C-076 compounds or of the concentrates obtained from the 26 extraction of the mycelia.

_~9_ 1 The C~076 compound3 of this invention are also 2 useful in combatting agricultural pests that inllict 3 damage upon crops while they are growing or while in 4 storage. The compounds ar2 applied using known techniques as sprays, dusts, emulsions and the like, to the 6 growing or stored crops to effect protection from such 7 agricultural pests.
~ The anthelmintic activity of C-076 may be g determined by orally administering vla the feed, a sample of C-076 individual compound, a mixture of C-076 11 compounds, a concent.rated extract, and the li]~e to a 12 mouse which had been infected 3 days earlier ~i~h 13 Nematos~iroides dubius. At 11, 12 and 13 days a~ter the 14 initiation of the medication, the feces of the mouse are examined for N.dubiu eggs, and on the next day the mouse 16 is sacri~iced and the n~ber of w0rm5 present in the 17 prox.~mal portion of the small intestine are determined.
18 An active compound is observed when there is a significant 19 3-eduction of eg~ and worm counts when compared to infected, 20 unmedicated controls.
21 The following examples are being provided in 22 order that the instant invention may be more fully understood.
23 Such examples are not to be construed as being limitative 24 of the invention.

EX~LE 1 26 The contents of a lyophilized tube of 27 Streptomyces avermitilis MA-4680 i9 transferred 28 aseptically to a 250 ml. Erlenmeyer flask containing 29 50 ml. of Medium 1. The inoculated flas~ is incubated .

1 for 3 days at 28C on a rotary shaking macIline at a 2 speed of 220 ~PM in a 2 inch radius circular orbit.
3 At the end of this time, a 250 ml. Erlenmeyer ,lask 4 containing 50 ml. of ~edium 2 is inoculated with a 2 ml. sample from the rirst flaskA This flask is 6 :incubated for 3 days at 28C on a rotary shaking 7 machine at a speed of 220 RPM in a 2 inch dia~eter 8 circular orbit. 50 Ml. of the resulting fer~entation 9 broth con-tainiIlg C-076 is effective against an N.dubius i.nfection in mice.

11 Composition of Media .
12 Medium 1 13 ~extrose 20 g.

14 Peptone 5 g.

15 ~leat Extract 5 g.

16 Primary Yeast 3 g.

17 NaCl 5 g.

18 CaCO3(after pH adjustment) 3 g.

l9 Distilled Water lO00 ml p~ 7.0 21 ~Iedium 2 22 Tomato Paste 20 g.

23 Modified Starch (CPC) 20 g.

24 ~rlmary Yeast 10 g.

25 CoC12 6H2O 0.00 26 DLstilled Water 1000 ml.

27 pH 7.2-7.4 EXA~IPLE 2 2 A lyophiliæed tube of Streptomyces 3 avermitilis iVL~-4680 is opened aseptically and the 4 contents suspended in 50 ml. of Medium l in a baffled 250 ml. Erlenmeyer flask. This flask is sha~en for 6 3 days at 28C on a rotary shaking machine 220 RPM
7 with a 2 inch diameter circular orbit. A 0.2 ml. portion 8 of this seed medium is used to inoculate a Slant of 9 Medium 3. The inoculated slant medium is incubated at l0 28C for l0 days and stored at 4C until used to 11 inoculate ~ more slants of Medium 3. These slants are 12 incubated in the dark for 8 days. One of ~hese slants is 13 used to incculate 3 baf~led 250 ml. Erlenmeyer flasks 14 containing 50 ml. of No. 4 Seed ~ledium. The seed flas]ss are 15 shaken for 2 days at 27 to 28C on a rotary shaking 16 machine at 220 RPM with a 2 inch diameter circular orbit.
17 rhe contents of these flasks are pooled and used to 18 inoculate (5% inoculum) baffled 250 ml. E:rlenmeyer flasks 19 containing 40 mlO of ~arious productioIl media. Flasks 20 containing media 2, 5 and 6 are incubated for 4 days 21 at 2~C on a rotary shaking machine at 220 ~ rith a 22 2 inch diameter circular or~it. The resulting broth 23 containing C-076 is then harvested and tested for 24 anthelmintic activity. In all cases 6.2 ml. of whole broth 25 and the solids obtained from centrifuging 25 ml. of whole 26 brot~l are fully active against N.dublus helminth inrections 27 iII mice~

~32-1 Medium 3 ~Slant Medium~
2 Dextrose 10.0 g.
3 Bacto Asparagine 0.5 g.
4 ~2~PO4 0,5 g.
5 Bat-to Agar 15.0 g.
6 Distilled Water 1000 ml.
7 pH 7.0 8 Medium 4 (Seed Medium) 9 Soluble Starch 10.0 g.
10 ~rdamine pH 5.0 g.
11 NZ Amine E 5.0 g-12 Beef Extract 3.0 g-13 MgSO4 7H2O 0.5 g.
14 Cerelose 1.0 g.
N~2H O4 0.190 g.
16 K;H PO 0~182 g.
17 CaCO3 0'5 g-18 Distilled Water 1000 ml.
19 pli 7.0-7.2 20 _edium 5 21 Toma~o Paste 40.0 g.
22 Oat ~lour 10.0 g.
23 Cerelose 10.0 g.
24 Corn Steep Liquor 5.0 g.
25 ~r.ace Elemen Mix 10.0 ml.
26 Dis~illed Water 1000 ml.
27 pH 6.8

-33-l Trace Element Mix 4.7H2O lO00 mg.
SO4.4~I2O lO00 mg.
4 CUCl2 2H2 25.0 mg.
5 CaC12 100.0 mg.
H2BO3 56.0 mg.
7 (NH~)2MoO4.4H2O 10.0 mg.
ZnS4 7H2 200.0 mg.
9 Distilled Water lO00 ml.
10 pH
11 ~iedium 6 . _ i 12 C~PC Inclustrial Starch 40.0 ~.
13 Mlodified (Available from CPC Corp.) 14 ~istiller 15 Solubles 7.0 g.
15 ~utolyzed Yeast (Ardamine pH 5.0 g.
16 availa~le from Yeast Producl:s Inc.
17 CoC12.6H2O 50.0 mg.
18 Distilled Water lO00 ml~
l~ pH 7.3 20 EX~MPLE 3 21 A 0.5 X l.0 cm. loop of one of the four slants 22 of Medium 3 prepared as in Example 2 is used to inoculate 23 a baffled 250 ml. Erlenmeyer flask containing 50 ml. of 24 Seed Medium No. 4. The seed flasX is qhaken for 1 day at 27 to 28C on a rotary shaking machine at 220 P~ with a 26 2 inch diameter circular orbit. The seed flask is then 27 stored stationary at 4C until it is ready to be used. The 2~o contents of this flask are then used to inoculate ~9 (5% inoculum) 20 un~affled 250 ml. Erlenmeyer ~lasks

-34-1 containing 40 ml. of Medium No. 2. After 4 days 2 incubation at 2gC on a rotary shaking machine at 3 220 RPM with a 2 inch diameter circular orbit, 19 of 4 the flasks are harvested and pooled. The combined fermentation broths containing C-076 are filtered 6 affording 500 ml. of filtrate and 84 g. of mycelia. 78 G.
7 of mycelia are extracted with 150 ml. of acstone for 8 1/2 hour with stirring and the mixture filtered. The filter 9 cake is washed with 50 ml. of acetone and the filtrate and wa,shings are combined and concentrated to 46.5 ml.
11 ~0 Ml. of the concentrate is adjusted to pH 4 with 12 dilute hydxochloric acid and extracted 3 time~ with 13 30 ml. portions of chloroform. The extracts are dried 14 by filtering through dry Infusorial Earth (Super-Cel) combined and concentrated ~o dryness in vacuo. The 16 oily residue of C-076 weighing 91.4 m~. is dissolved in 17 chloroform sufficient to make 3 ml. of solution which 18 riepresents 1% of broth volume. The c-076 obtained 19 in this recovery procedure is fully active again~t N.dublus infections in mice. In addition, the 21 cllloroform extraction achieved a 70 fold purification 22 o~ C-076 rom the t~hole brothO

23 EXA~LE 4 __ 24 A seed culture is prepared by inoculating 50 ml. of ~ledium 4 in a 250 ml. baffled Erlenmeyer 26 flask with a 0.5 X 1.0 cm. loop from one of the four 27 slants of ~Iedium 3 as prepared in Example 2. l~he flask 28 is incubated at 28C on a rotary shaking machine at 29 220 RPM with a 2 inch diameter circular orbit for 2 days.

-35-~ 3 15874 I~

1 The seed culture is used to inoculate a 2 liter Erlenmeyer 2 production flask containing 250 ml. o~ Medium 2. The 3 inoculum volume is 5.O ml. (2~). The production flask 4 is incubated at 28C on a rotary shaker at 220 RP~i for 4 days. At the end of this time the whole broth 6 contalning C-076 is harvested. 6 ~. or this whole broth 7 ~hen tested in a mouse infected with N.dubius is found to be 8 fully active.

9 EXA~IPL~ 5 . _ .
10 steP
11 A 250 ml. baffled Erlenmeyer flask containing 12 50 mlO of Medium 7 is inoculated with a frozen vial of 13 Streptom~ces avermitilis ~-4680. The flask is 14 incubated at 28C on a rotary shaking maclline at lS 160 RPM with a 2 inch diametar circular orbit at 160 r~P~I
16 ~or 24 hours.
17 i`ledium 7 18 ~ex~rose 1 gm.
19 CPC Industrial Starch Modified 10 gm.
20 l~iIeat Extract 3 gm.
21 NZ ~mine E 5 gm.
22 ~utoly2ed Yeast (Ardamine pH) 5 gm.
23 MgSO4.7H2O 0.05 ~m.
24 ~a4H~O4 0.19 gm.
~5 ~PO4 0.1~2 ~m.
26 C:aCO 0.5 gm.
27 Distilled Water 1000 ml.
28 p~ 7.0-7.2

-36-1 Step B
2 Two 2 liter baffled Erlenmeyer flasks 3 containing S00 ml. each of ~lediultt 7 are inoculated 4 with 10 ml . o~ the f lask contents of Step A. The 5 media are incubated at 2 8 C on a rotary shaking 6 machine at 160 ~PM with a 2 inch diameter circular or~it 7 for 24 hours.
8 _tep C
9 To a 756 liter stainless steel fermentor 10 containing 467 liters of Mediunt 8 is added 1 liter of 11 the whole fermerttation media from Step B. The 12 ~ermentor is stirred at 28C at 130 RPM for 96 hours 13 aIld with aeration at an air flow of 10 cubic feet per 14 minute.
15 Mediunt 8 _ _ 16 Tomato Pas te 2 0 gnt. /1 17 Primary Yeast N.F. 10 gm./l 18 Starch, modified, CPC 20 gnt./l 19 C'oC12 . 6H2o 5 mg. /1 20 Polyglycol 2000 0 . 321 ml ./1 21 ~is~tilled Water q. s.

22 pH 7.2-7.4 23 ~ the end of this tinte 15 . 5 1. of the whole 24 broth is iltered and the mycelia containing C-076 washed 25 with water. The wet mycelia (2, 268 g~ ) are extracted with 26 3 liters o~ acetone with stirring. The ntixture is filtered 27 and the filtrate concentrated io 1550 ml. and adjusted to 28 pI~ 4. 0 with dilute HCl. This solu-tion is extracted 29 3 times wi~h equal volumes of chlorofo~m. The

-37-1 chloroform extracts are dried by filtering through 2 dry Infusorial Earth (Super-Cel), combined and 3 concentrated to dryness ln vacuo. The residual oil of 4 C-076 weighs 5.1~ g. 3.3 Mg. is fuliy active against N~dubius in mice.
6 4.69 G. of this oil is dissolved in 142 ml.
7 of chloroform and chromatographed on a column containing 8 go g. of silica gel pac~ed in chloroform. The column 9 is developed with 1400 ml. of chloroform. The column is then eluted with cllloroform/ethanol (49:1) collecting 11 145 cuts of 5 ml. each. Following this the column ls 12 eluted with chlorofoxm/ethanol (1~:1) collecting 13 fractions 146-226 of 5 ml. each. Fractions 49-72 are 14 combined and evaporated to dryness a~fording 200 mg.
of an oil (A). Fractions 79-184 are likewise combined 16 and evaporated affording 291 m~. of an oil (B). 400ug 17 of each fraction is fully active a~ainst N.dubius 13 in mice. These two fractions (A and 3) are analyzed 19 separately on silica gel thin layer chromatographic plates (Quanta/Gram QIF plat~s, available from 21 ~uanta/Gram Inc., Fairfield, New Jersey). The plates 22 are developed with chloroform/methanol (19:1). The 23 spots are analyæed for their ultraviolet activity and 24 one spot of each fraction has the characteristic ultraviolet absorption for the C 076 compounds 26 (see Table I). From fraction A, the spot with an 27 :~f of 0.83 and from fraction B the spot with an Rf of 28 0.57 have such absorption. These spots represent`the 29 C-076 ~ compounds and the C-076 B compounds respectively.

-38-i3 1 198 l~g~ of the oil (A) above is chromatographed 2 on 80 g. oiE silica gel packed in chloroform, elutlng 3 with chloroform/methanol (199:1) until 520 ml. is 4 collected, iEollowed by chloroi-o~m/methanol (99:1) collecting fractions oi- lO ml. each. The fraction 6 from 630 to 720 ml. affords 30.4 mg.; the fraction 7 from 730 to 950 ml. affords 78.4 mg. and the 8 fraction from 950 to 1040 ml. affords 20 m~. o an 9 oily matexial. Fractions l and 2 containing C-076 A
components when tested in mice against N.dubius at levels 11 of 1.0, 0.5 and 0.25 mg. are fully active.

12 ~XAMPL~ 6 13 Step A
.
14 A 250 ml. baffled Erler~eyer flask containing 50 ml. oE ~Iedium 8 is inoculated with a 16 fro~en vial of Streptomyces avermitilis ~ 4680.

17 The iElask is incu~ated at 28C on a rotary shaking 18 machine at 160 RP~I with a 2 inch diameter circular 19 orbit for 24 hours.

~S~ep B

21 A 2 liter baffled Erlenmeyer flask containing 22 500 mL. of Medium 8 is inoculated with lO ml. of the 23 i~las~ contents of Step A. The mediu~ is incubatPd at 24 28C on a rotary shaking machine at 160 ~PM with a ~' inch diame~er cixcular orbit for 24 hours.

39-.
.
- ~ , ' ': ' 1 Step C
__ 2 To a 189 liter stainless steel fermentor 3 containing 160 liters of Medium 9 is added 500 ml.
4 of inoculum from Step B. The fermentor is incubated S at 28C with stirring at 150 RPM for 24 hours aerating 6 at a rate of 3 cubic feet per minute.
7 ~Iedium 9 ~ . .
8 Dextrose 1 gm./l 9 Corn Starch 10 gm./l 10 Meat Extract 3 gm./l 11 Autolyzed Yeast (Ardamine pH) 5 gm./1 12 MgSO~.7H2O 0.05 gm./l 13 ~a2~IP4 0.10 gm./l 14 R~2PO4 0.182 g./l 15 CaC03 0.5 gm./l 16 Distilled Water q.s.

17 pH 7.0-7.2 19 To a 756 liter stainless steel fermentor containing 467 liters of ~edium 6 is added 43 lfters 21 of inoculum from Step C. The fermentor i~ incubated 22 at 28C with stirring at 130 ~PM for 144 hours and 23 ~lith aeration at an aix flow rate of 10 cubic feet per 24 minute.

C,tep_E

26 At the end of this time, the whole brotil 27 is filtered and the filter cake containing C-07Ç is washed 28 with water. The filter cake is slurried in 120 liter~ of 29 acetone for 30 minutes, filtered and the solids washed with 30 liters of acetone. The acetone washings are , -40- i 1 combined and evaporated under reduced pressure to a 2 volume of 40 liters. The concentrate is adjusted to 3 pH 4.0 with dilute hydrochloric aci~. The concentrate 4 is extxacted 3 times with equal volumes of chloro~onm.
The cllloroform extracts are dried by flltering through 6 a pad of dry Infusorial Earth (Super-Cel). The extracts 7 are passed through the Super-Cel, then combined. llhe 8 combined extrac~s are concentrated under reduced 9 pressure to a ~olume of 4 liters. The chloroform concentrate is filtered and passed through a column of 11 ;2.4 kg. of silica gel in chloro~orm. The column is 12 eluted with chloroform collecting ei~ht 3.5 liter 13 ~ractions. The column is then eluted with chloroform~
14 rnethanol (~9:1) collecting eight more 3.5 liter fractions (fractions 9-16). Fraction n~ er 3 is 16 concentrated to dryness afording 76 g. of an oily 17 ma~erial containing a preponderance of the C-076 materials.
18 97% o this material is dissolved in 685 ml.
19 o~ methylen~ chloride and chromaiographed through 800 g.
20 clf silicic acid (Mallinckrodt Chemical Co. 100 mesh 21 ~,eived again through an 80 mesh screen). The column 22 (7~62 cm. diameter, 36 cm. length) is developed with 23 methylene chloride/benzene (7:3), about 7.5 1, followed 24 by 5~ isopropanol in methylene chloride/benzene~7:3), 25 2.25 1. The fraction eluted with the 5~ isopxopanol 2~ in methylene chloride/ben~ene, which contains a 27 strongly colored band, contains virtually all of the 28 C-076 material, as determined by thin layer 29 chromatography (as described in Example 5). This 30 fraction (500 ml.) is evaporated and rechromatographed j -41-1 on 105 g. of silicic acid (column 3O7 cm. diameter, 2 18 cm. length) in methylene chloride. The column 3 is developed with 100 ml. portions of methylene 4 chloride containing 5, 10 and 20% ether. Furtller elution with 20go ether in methylene chloride 6 produces 2 colored bands. The fractions between the 7 two bands contained virtually all o the C-076 matexial 8 as determined by thin layer chromatograplly.
g The C-076 containins fraction is ci~omatographed on 59 ~. of silicic acid (column 11 3-7 cm. diameter, 11 cm. length) in methylPne chloride.
12 The column is developed with 10~ ether in methylene 13 chloride. After the first material begins to elute, a 14 :Eraction of 70 ml. is taken followad by 26 fractions of 5-6 ml. each. Fractions 3-26 are combined, 16 af~ording 1.35 g. of ma~erial, and analyzed by thin 17 layer chromatography (c~ilica gel plates-Ana].tech GF
18 254, developed with 5% isopropanol in methylene chloride).
19 The material with an R~ of 0.28 i.n this system is C-076 Al.
21 The column is then eluted with 20~ ether in 22 methylene chloride (200 ml.) followed by 50% ether in 23 methylene chloride (800 ml.). A small amount of 24 C'-076 Al/A2 mixture is eluted ~ollowed by all C~076 25 A2. The total residue of the C-076 A2 fxaction is 26 800 mg.
27 Further elution with 5~ isopropanol in 28 methylene chloride affords C-076 Bl (135 mg.). The 29 separation is followed by observing the ultraviolet j -42-1587~ IA

1 absorption of the eluent. C-076 Bl and A2 have very 2 similar Rf values on silica gel thin layer 3 chromatography plates (Analtech GF 254) in 4 5~ isopropanol in methylene chloride. However, the two componenis are clearly distinguishable on the 6 same plates developed with 15~ isopropanol in hexane.
7 The entire C-076 Al fraction is applied to 8 14 silica gel plates (Analtech HF 254, 20x20 cm. 500 9 thick). The plates are developed in 10~ isopropanol in hexane. The band containing the C-076 Al is 11 removed from the plates, extracted with ether, -12 evaporated and reapplied to 6 more plates and 13 developed 5 times with 5~ isopropanol in hexane. The 14 C-076 .~1 i5 removed from the pla~es and again chromatographed, developing ~ith pure ether affording 16 270 mg. of substantially pure C-076 ~1. The infrared 17 and nuclear magnetic resonance spectra for this sample 18 are reproduced as Figure 1 and 5 and Table II respectively.
19 The C-076 A2 fraction is chromatographed on 10 silica gel (Analtech HF 25~) plates, developing 21 5 times with 15% isopropanol in hexane affordinq 265 mg.
22 of substantially pure C-076 A2. The infrared and nuclear 23 magnetic resonance spectra for this sample are reproduced 24 in ~igures 2 and 6 respectively, and Table II.
The C-076 Bl fraction is chromatographed 26 on 2 plates (as above) in lS~ isopropanol in hexane 27 affording 55 mg~ of substantially pure C-076 B1. The 28 nuclear magnetic resonance spectrum of this sample is 29 reproduced in Figure 7, and 'rable II.

53~ 3 . .
2 The fermentation described in Example 6 3 is repeated twice and the whole broths are combined.
4 The fermentation broth is worked up as describe~ in Example 6 recovering 3.3 1 of an initial chloroform 6 extract which contains 60 mg./ml. of total solids 7 and is estimated to be 0.5~ C-076 by thin layer 8 chromatographic analysis.
9 3 Liters of this chloroform solution i5 chromatogxaphed on 2400 g. of silica gel (Davidson 11 Grade 6~ packed in chloroform. The column 12 (9.5x 122 cm.) is developed with eight 3800 ml.
13 portions of chloroform (fractions 1-8) followed by 14 eight 3800 ml. portions of chloroform/methanol (49:1 ~ractions 9-16). The individual fractions are 16 analyzed by thin layer chromatography (silica gel 17 plates, Quanta/Gram QIF) develop~d with chloroform/
L8 methanol 19:1. Fractions 9~ 13 and 14 are each 19 evaporated to dryness affording 6.63 g. of solids containing the C-0~6 A components in fractions 9-11, 21 24.91 g. of solids containing C~076 B components in 22 fractions 12-13, and 4.71 g. of ~olids also containing 23 the C-076 B components in fraction 14.
24 The material from fractions 12-14 are combined (29.62 g.~, dissolved in 100 ml. of 26 ~etllylene chloride and chromatographed on 400 g. of 27 silica gel ~Davidson Grade 62) in methylene chloride.
28 The column is eluted with 1500 mlu of methylene 29 chloride/2-propanol (99:1); 1500 ml~ of methylene chlor de/2-propanol (49:1); 2000 ml. of methylene 31 chloride/2-propanol (19:1); and 1000 ml. of methylene 1 chloride/2-propanol (9:1). The eluent volumes 2 between 5500-6000 ml. (2.56 ~.) and 6000-G500 ml.
3 (5.03 g.) are combined in 25 ml. of methylene 4 chloride and chromatographed on 60 g. of silica gel in hexane. A forerun of 70 ml. of hexane and 100 ml.
6 of hexane/diethyl ether (4:1) are ta~en and the 7 column then developed with 600 ml. of hexane/diethyl 8 ether (1:4) taking 200 ml. cuts, and finally eluting 9 T~ith 700 ml. of ether tallng 100 ml. cuts. Column eluent volumes from 400 to 600 ml. affords 2O035 g.
11 of solids containing C-076 Bl components; volumeq 12 600-1100 contained 0.881 g. of solids containing 13 mixed C-076 Bl/B2 components; and volumes 14 1100-1500 ml. contained 0.381 g. of solids containing ~'-076 B2 components.
16 The mixed C-076 Bl/B2 components are then 17 cLissolved in 4.2 ml. of methyl alcohol/water (4:1) and 18 chromatograplled on C18 Rorasil (Bondapa~-37-75 micron 19 size) in the same solvent~ The reverse phase high pressure column (more polar components eluted first) 21 is 1.2 meters by 16 mm. and is eluted at a rate of 22 800 ml. per hour taking 21.3 ml. fractions. The 23 presence of C-076 components is monitored by observing 24 the ultraviolet absorption of the fractions. C-076 B2 is recovered in fractions 24 to 37 and C-076 Bl is 26 recovered in ~ractions 51-70 recovering 195.4 mg. of 27 C-076 B2 and 137 mg. of C-076 Bl.
2~ Each sample is then separately 29 chromatographed on 4 g. columns of silica gel (Davidson Grade 62) in methylene chloride. The --a~S_ ~8Q~3 1 columns are eluted with 35 ml. of methylene chloride/
2 ~et~lanol (9:1). The last 20 ml. of eluent from each 3 column is collected and evaporated to dryness affording 4 155.3 mg. of C-076 B2 and 90 mg. of C-076 Bl respectively.
6 Then 50 mg. of C-076 Bl and 100 mg. of 7 C-076 B2 are chromatographed on preparative silica 3 gel plates (Analtech HF 254), developed with 12~
9 isopropanol in hexane followed by development with ether, recovering C-076 Bl and C-076 B2 which are 11 ubstantially pure. The infrared absorption 12 spectrum of the thus recovered C 076 Bl and B2 is as 13 shown in Figures 3 and 4 respectively. The nuclear 14 m~agnetic spectrum of the thus recovered C-076 B2 is as sho~n in Figure 8.

-~6-~81~i3 1 EX~IPLE 8 2 ~ 250 ml. baffled Erlenmeyer flask containing 3 50 ml. of the following medium:
4 Lactose 2.0 Distiller's solubles 1.5 6 Autolyzed yeast, Ardamine pH 0.5 7 pH - before sterilizatlon 7.0 8 i.s inoculated with the contents of one frozen vial of 9 Streptomyces avermitilis MA 4848 and incubated on a rotary sha~er at 28C for 24 hours at 150 ~M.
11 10 Ml. of the abo~te fermentation medLa is 12 employed to inoculate 500 ml. of the same medium as 13 above in a 2 liter baffled Erlenmeyer flask. The 14 fermentation media is incubated a~ 150 R~M on a rotary shaker at 28C fox 24 hours.
16 All of the foregoin~ media is employed ~o 17 inoculate 467 liters oE the following media in a 756 18 liter stainless steel fermentor:
19 Lactose 2.0 Distiller's solubles 1~5 21 Autolyzed yeast,Ardamine pH 0.5~
22 Polyglycol 2000 0.32 ml./liter 23 p~ - before sterilization 7.0 24 T~Ie fermentation media is incubated at 28C for 40 hours w:Lth an air flow 10 cublc feet per minute and an 26 agi~ation rate 130 RPM.
27 230 Liters of the above media is employed to 28 inoculate 4,310 liters of the following medium in a 29 5,670 liter stainless steel fermentor:

1 Dextrose ? Peptonized milk 2.4 3 Autolyzed yaast,Ardamine pH 0.25~
4 Polyglycol 2000 2.5 ml./liter pH - before sterilization 7.0 6 The fermentation conkinues for 144 hours at 26C with 7 2ln air flow rate of 54.3 cubic feet per minute and 8 ~l~it~tion o~ 120 RPM.
g The fermentation media are filtered and the mycelial filter cake washed with about 550 liters of 11 water, the filtrate and washings are discarded. The 12 filter caXe is agitated with about 1500 liters of acetone 13 for about one hour and filtered. The filter cake is 14 ~ashed with a mixture of about 150 liters of acetone and

40 liters of deionized water affording about 2000 liters 16 of extract.
17 The foregoing fermentation and extraction is 18 r~epeated on the same scale affording a further 2000 19 liters of acetone extract which is combined with the first extract and evaporated to a volume of about 800 liters.
~1 The pH of the concentrate is adjusted to about 4.~ with 22 concentrated hydrochloric acid and combined with about 23 800 lit~rs of methylene chloride. The combined solvents ar.e ~gitated for about 4 hours and separated. The aclueous layer is combined with an additlonal 800 liters 26 ol. methylene chloride and agitated for about 4 hours~ The 27 layers are 5eparated and each methylene chloride extract 28 s~!parately treated with about 10 kilograms of Super-Cel 29 and filtered. Both extracts are evaporated to a combined volume of about 60 liters~

1 EX~LE 9 .... _ 2 The 60 liter solution of C-076 in methylene 3 chloride of the previous example is concentrated to 4 dryness in vacuo and the residue is combined 3 ti~es S with 60 liter portions of methanol and evaporated to 6 dryness to remove any re~idual methylene chloride. The 7 final methanol concentrate volume is approximately 36 8 :Liters. The methanol solution is stored overnight and 9 filtered. The filter cake is washed with 40 liters o~
~resh methanol and the methanol filtrates and washings 11 are combined. The methanol solution is combined with 12 ~5 liters of eth~lene glycol and 130 liters of heptane.
13 The 2 layer solution is agitated for 5 minutes and the 14 lower layer (e-~hylene glycol and methanol) is separated.
I'he heptane solution is washed with a mixture of 20 16 liters of ethylene gLycol and 6.:3 liters methanol. After 17 ~ive minutes of agitation, the lower layer is separated 18 and com~ined with the first ethylene glycol/methanol 19 extract. An equal volume of water (approximately 150 liters) containing 79 g. of salt per liter is added to 21 the e~hylene glycol/me~hanol extracts. This solution is 22 extracted wi~h 150 liters of etllyl ether with agi~ation 23 for 5 minutes. The ether layer is washed with 75 liters 24 of water (1/2 volume) and agitated for 5 minutes and the layers separated. ~his procedure is repeated an 26 additional 2 times (the final water was contains 20 g. of 27 Clllt per liter) af~ording a ~inal ether layer volume of 28 l:L0 liters. The ether layer is concentrated in vacuo, to 29 a mimimum volume, keeping the te~perature less than 25C. 40 Liters of methylene chloride is added to the 31 residue and the solution is evaporated to dryness. This 1 proceduxe is repeated and the final residue concentrated 2 in vacuo at 50C to dryness.

3 EXA~LE 10 4 A 30 centimeter diameter column is prepared with a layer of 34 kilograms of activated alumina 6 ~ollowed by a layer of 34 kilograms o~ activated 7 ,-arbon in a solution of methylene chloride. The residue 8 ~rom the previous example is dissolved in methylene g chloride to a volume of 34 liters and applied to the column ancl eluted with 34 liters of methylene chloride.
11 t:hese fractions are discarded. A 3~ solution o 12 isopropanol and methylene chloricle ~20.8 liters of 13 i~5opropanol and 660 liters of met:hylene chloride) is 14 applied to the column and eluted in approximately 200 l,iter fractions. The combined ic;opropanol and methylene 16 chloride fractions are evaporatecl ln vacuG at a batll 17 temperature of about 60C to a volume of ahout 2Q liters.
18 ~he bath temperature is reduced t.o about 45C and the 19 extract is evaporated to dryness in_vacuo. Th residue is dissolved in 10 parts methylene chloride, 10 parts 21 hexana and one part methanol to a final volume of 15 22 liters. This solution i5 a~plied directly to the Sephadex LH-20 column of the next example.

-5~-Ei3 2 ~ 30 centimeter diameter column is prepared in 3 methanol with 36 Xilograms of Sephadex LX-20 (available 4 from Pharmacia Fine Chemicals, 800 Centennial Avenue, Piscataway, New Jersey 08854) and washed with a solvent 6 consistlng of 10 parts methylene chloride, 10 parts 7 hexane and one part methanol. One-fourth of the C-076 8 solution of Example 10 is applied to the column and the 9 column elu~ed a~ a rate of 250 ml. per minute. Two 20 liter forecuts are collected and discarded followed by 11 20 two liter rich cuts (identified as fractions 1-20), 12 followed by a single 20 liter ta.il cut, which is discarded.
13 Fractions 1-8 are found to contain the C-076 A compounds 14 and fractions 9-20 are found to contain the C-076 B compounds.

EX~`~LE 12 16 The process of Example 11 is repeated on the 17 ~ame scale three more times and all of the fractions 18 containing the C-076 B components (fractions 9-20) are 19 combined and evaporated to dryness, a~fording 818 g.
of crude mixed C-076 B components. The sample is found to 21 c:ontain 55~ C-076 Bl and 39~ of C-076 32. 680.5 G. of 22 t:his sample is dissolved in 2 liters of methylene chloride 23 and placed in a 22 liter three neck round bottom flas~
Z4 ~ollowed by the addltion of 13.6 liters of methanol. 'l'he ~lethylene chloxide is removed by distillation. 13.6 Liters 26 of ethylene glycol is added as the methanol is being 27 dis~illed under reduced pressure. The rate of distillatio 28 is main~ained such that the temperatùre of the solution ~51-1 did not go below 65C. When the addition of the ethylene 2 glycol is complete, the solution is allowed to cool at 3 5C for sixteen hours. The crystals are filtered and 4 washed with 1 liter of cold ethylene glycol. The S crystals are then redissolved in 2 liters of methylene 6 chloride the solution placed in a 22 liter three necked 7 round bottom flas]c. The procedure described above is 8 repeated twice. The first time 12.5 liters each of 9 methanol and ethylene glycol is employed and the second ~ime 13.6 liters each of methanol and ethylene glycol is 11 employed. The final crystals are washed with 1 liter of 12 cold ethylene glycol and 1 liter of water. The crystals 13 are dissolved in 4 liters of water and dried by filtering 14 ~hrough sodium sulfate. The benzene solution is concentrated to a volume of 2 lit.ers and lyophilized 16 affording 241.2gm. of a white powder consisting of 98 17 ~-076 Bl and 1~ of C-076 B2.
18 The mother liquors (22 liters) from the first 19 two crystallizatiolls above are combined and diluted with 22 liters of water. The aqueous solution is extracted 21 with 60 liters of toluene and again with 15 liters of 22 t:oluene. The toluene extract is then washed with a8 23 ].iters of water. The organic phase is filtered through 24 Super-Cel to remove any residual water and evaporated affording 336 gm. of solid material consisting of 79 26 C-076 B2 and 16~ C-076 Bl compounds.

2 In the four Sephadex L~-20 columns o~ the 3 procedure of Example 11, fractions 1~8 contain the C-076 ~ A compounds and are combined. By HPLC analysis the mixture is found to contain 252 g. of C-076 A2a, 16 g. of 6 A2b, 94 g. of Ala and 24 g. of Alb. The material is 7 dissolved in a solvent system consisting of hexane:
8 toluene:methanol in the proportion of 6:1:1 and applied g to the Sephadex LH-20 column of the same dimensions as the one used in ~xample 11 in the above solvent.
11 Fractions are collected at the rate of 250 ml. per minute 12 and a 20 liter forecut is collected and discarded. Furthex 13 elution af~ords 2 additional 20 li~er forecuts which are 14 also discarded and 50 four liter rich cuts which contain lS ~-076 A compounds. Fractions 3-8 are found to contain 16 predominately C 076 Al component:s (40.2 g. Ala and 6.7 g.
17 Alb), and fractions 29-36 are found to contain C-076 A2 18 compounds (117~2 g. A2a and 7.35 g. of A2b). Fractions 19 9-28 contain a mixture of C-076 Al and A2 compounds.

E ~ ~LE 14 21 A sample of 150 g. of C-076 Bl from Example 12 22 is dissolved in 3 liters of a solvent mixture of hexane:
23 toluene:methanol in the ratio of 3:1:1. The solution is 24 passed throu~h a column of Sephadex LH-20 (of the same 25 dimensions as ~he one used in ExamPle ll) in the above 26 solvent taking fractions at the rate of 250 ml. per 27 minutes. After two 20 liter portions of the solvent 28 mixture are collected and discarded, forecut of 10 liters 29 is taken and discarded. Then 30 richcuts of 2 liters each 1 are taken. Fractions 1-13 and 25-30 are discarded.
2 Fractions 14-16 are combined and contain 80 g. of 3 predominately C-076 Bla. Fractions 22-24 are combined 4 and contain 6.7 g. of predominately C-076 Blb. Fractions 17-21 contain a mixture of C-07G Bla and slb.
6 Fractions 17-21 above are combined and 7 concentrated and passed through a Sephadex LH-20 column 8 with the same solvent system as above. Three 20 liter 9 forecuts are taken and discaxded. Richcuts are then taken as follows: 5 cuts of 2 liters each (fractions 1-5);
11 20 cu~s of 1 liter each (fractions 6-25); and 10 cuts 12 of 2 li~ers each (fractlons 26-35). ~ractions 1 15 are 13 discarded; fractions 16-21 conta:in 13.5 g. of C~076 14 3~a and 0.4 g. of C-076 Blb; fractions 22-26 con~ain 44 g. of C-076 Bla and 0.13 g. oi- C-076 Blb; fractions 16 27-30 contain 10.~ g. of C-076 Bla and 0.8 g. o~ C-076 Blb.

17 EXA~PLE_15 18 ` A mixture of all 8 C-076 components are 19 chromatographed on a high pressure liquid chromatography column 4mm. X 30 cm. packed with 10 micron ~ Bondapak 21 C'18 silica gel (available from Waters Associates Inc., 22 ~aple Street, Milford, ~lassachusetts 01757) eluting with 23 85:15 (v/v~ methanol:water at a constant 40C. At a 24 flow rate o~ 1.2 ml. per minute all eight compounds are separated and the elution volumes, which under the ~ore~oing constant conditions are characteristic of 27 the individual compounds are as follows:

~5~-6~

1 Elution Volume (Ve) Ml 2 C-076 B2b 5 9O
3 C-076 B2a 6.52 4 C-076 A2b 7.12 5 C-076 A2a 7.88 6 C-076 Blb 8.36 7 C-076 Bla 9.60 8 C-076 Alb 10.24 9 C~076 Ala 11.88 10The separation of C-076 "b" components from 11 the respective "a" components is accomplished using 12 techniques such as high pressure liquid chromatography.
13 An absolute methanol solution of 30 microliters of a 14 mixture of C-076 Ala and Alb, estimated to contain 30 micrograms of C-076 Alb is placed on a 3x250 mm. hi~h 16 pressure liquid chromato~xaphy column containing 17 Spherisorb 5 micron ODS (availab:Le from Spectra Physics) 18 as pac~ing. The column is eluted with 85 :15 methanol-1~ water at a rate of 0~15 ml./min. The elution of the products are followed by observing the ultraviolet 21 absorption of the eluent and collecting the lndividual 22 component~ at the outlet of the W monitor. 30 Micrograms 23 of C-076 Alb is recovered and analyzed in a mass 24 spectrometer. The mass spectrum of this sample is recorded in the second column of Table III.

Claims (23)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A process for the preparation of a compound of the formula:

wherein R is:

and wherein the broken line indicates a single or a double bond; Rl is hydroxy and is present only when said broken line indicates a single bond;
R2 is propyl or butyl; and R3 is methoxy or hydroxy, which comprises fermenting with a strain of Streptomyces avermitilis, an aqueous nutrient medium containing an assimi-lable source of carbon, an assimilable source of nitrogen and inorganic salts under aerobic conditions and recovering said compound from said fermentation broth.

2. The process of Claim 1, wherein the strain is Streptomyces avermitilis NRRL 8165 (ATTC 31267).

3. The process of Claim 1, wherein the fermentation is conducted at a temperature of from about 20 to 40°C and a pH of from about 5.0 to 9Ø

4. The process of Claim 3, wherein the temperature is from about 24 to 30°C and the pH is from about 6.0 to 7.5.

5. The process of Claim 4, wherein the nutrient medium contains about 0.5 to 5% by weight of carbon sources and about 0.2 to 6% by weight of nitrogen sources.

6. The process of Claim 5, wherein the fermentation is carried out for about 1 to 8 days.

7. The process of Claim 1, wherein in the isolated compound, the broken line indicates a double bond, R2 is propyl and R3 is methoxy.

8. The process of Claim 1, wherein in the isolated compound, the broken line indicates a double bond, R2 is propyl and R3 is hydroxy.

9. The process of Claim 1, wherein in the isolated compound, the broken line indicates a double bond, R2 is butyl and R3 is methoxy.

10. The process of Claim 1, wherein in the isolated compound, the broken line indicates a double bond, R2 is butyl and R3 is hydroxy.

11. The process of Claim 1, wherein in the isolated compound, the broken line indicates a single bond, Rl is hydroxy, R2 is propyl and R3 is methoxy.

12. The process of Claim l, wherein in the isolated compound, the broken line indicates a single bond, Rl is hydroxy, R2 is propyl and R3 is hydroxy.

13. The process of Claim l, wherein in the isolated compound, the broken line indicates a single bond, Rl is hydroxy, R2 is butyl and R3 is methoxy.

14. The process of Claim l, wherein in the isolated compound, the broken line indicates a single bond, Rl is hydroxy, R2 is butyl and R3 is hydroxy.

15. A compound having the formula:

wherein R is:

and wherein the broken line indicates a single or a double bond; Rl is hydroxy and is present only when said broken line indicates a single bond;
R2 is propyl or butyl; and R3 is methoxy or hydroxy, when prepared by the process defined in Claim 1 or 2 or by an obvious chemical equivalent.

16. The compound of Claim 15, wherein the broken line indicates a double bond, R2 is propyl and R3 is methoxy, when prepared by the process defined in Claim 7 or by an obvious chemical equivalent.

17. The compound of Claim 15, wherein the broken line indicates a double bond, R2 is propyl and R3 is hydroxy, when prepared by the process defined in Claim 8 or by an obvious chemical equivalent.

18. The compound of Claim 15, wherein the broken line indicates a double bond, R2 is butyl and R3 is methoxy, when prepared by the process defined in Claim 9 or by an obvious chemical equivalent.

19. The compound of Claim 15, wherein the broken line indicates a double bond, R2 is butyl and R3 is hydroxy, when prepared by the process defined in Claim 10 or by an obvious chemical equivalent.

20. The compound of Claim 15, wherein the broken line indicates a single bond, R1 is hydroxy, R2 is propyl and R3 is methoxy, when prepared by the process defined in Claim 11 or by an obvious chemical equivalent.

21. The compound of Claim 15, wherein the broken line indicates a single bond, R1 is hydroxy, R2 is propyl and R3 is hydroxy, when prepared by the process defined in Claim 12 or by an obvious chemical equivalent.

22. The compound of Claim 15, wherein the broken line indicates a single bond, R1 is hydroxy, R2 is butyl and R3 is methoxy, when prepared by the process defined in Claim 13 or by an obvious chemical equivalent.

23. The compound of Claim 15, wherein the broken line indicates a single bond, R1 is hydroxy, R2 is butyl and R3 is hydroxy, when prepared by the process defined in Claim 14 or by an obvious chemical equivalent.