CA1338140C - Antibiotic agents from streptomyces cyaneogriseus noncyanogenus and method of production thereof - Google Patents

Abstract

This invention relates to new agents designated LL-F28249.alpha., LL-F28249.beta., LL-F28249.gamma., LL-F28249.delta., LL-F28249.epsilon., LL-F28249?, LL-F28249?, LL-F28249.theta., LL-F28249?, LL-F28249?, LL-F28249.lambda., LL-F28249µ, LL-F28249?, and LL-F28249.omega., to their production by fermentation, to methods for their recovery and concentration from crude solutions, to processes for their purification and to pharmaceutically and pharmacologically-acceptable salts thereof. These substances have significant activity against helmintic, ectoparasitic and acaridal infections, and thus, are useful in so treating warmblooded animals afflicted with these infections, as well as treating plants inflicted with nematodes. The present invention includes within its scope the biologically pure culture which produces their agents, derived from a newly-discovered and previously uncultured microorganism, Streptomyces cyaneogriseus subsp. noncyanogenus, NRRL 15773.

Description

. I

~338i40 29,727 NOVEL AGENTS AND METHOD OF PRODUCTION THEREOF

BACKGROUND OF THE INVENTION

The present invention relates to new antibiotic compounds, collectively identified as LL-F28249, which are produced by the fermentation of a nutrient medium with the strain of the microorganism Streptomyces cy-aneo~riseus subsp. noncyano~enus LL-F28249, NRRL No.
15773 and to the pharmaceutically and pharmacologically-acceptable salts thereof.

~ - -2- 13381~0 SUMMARY OF THE INVENTION

The culture of Streptomyces cyaneogriseus subsp.
noncyanogenus (LL-F28249 and deposited under NRRL No.
15773 and hereinafter referred to as Streptomyces cy-aneogriseus noncyanogenus) which produces the agents~,~ ,y~ n ~ , vand~ components was isolated from mallee sand found in southern Australia.
- The structure and stereochemistry of LL-F28249, have not been fully defined, but the proposed structures 10are shown below. Component LL-F28249 was related to Hondamycin (Albimycin) which is disclosed in The Journal of Antibiotics, 22, No. 11, 521-526 (1969).
The strain is assigned to the genus Streptomyces based upon morphology and cell chemistry (content of the Lisomer of diaminopimelic acid). The strain's morphology and physiological data place it close to S. cyaneogriseus, as represented by ISP 5534 (ATCC 27426). Then, com-parisons of the formation of gray aerial mycelium soluble pigments on media (Table A) and coiled chains of smooth conidia (3-25 spores per chain) were made. The present strain is negative for blue soluble pigment wherein the comparison strain, ISR 5534, is positive. The strains have similar reactions in the ISP carbohydrate utili-zation tests indicating positive for arabinose, fructose, glucose, rhamnose and xylose, while indicating negative for inositol, mannitol, raffinose and sucrose (ISP 5534) slightly positive). However, the strains differ in sev-eral characters (Table B) out of 53 in the Gordon tests.
These differences support the creation of a subspecies of S. cyaneogenseus for the present microorganism.

Comparlson of F 28249 and ISP 5534 on ISP Morphology Test Medla (Numbers are from NBS-ISCC) Medlum F 28249 ISP 5534 Yeast-Malt A.m. Medium gray Light to medium gray (ISP 2) (265) (264-265) V.m. Light t~nn- Sh ( 75) Light t;lrlnl sh-white Deep yellow-brown to blackish-blue (188) S.p. Light brown Light brown Inorganic salts A.m. Light olive-gray ~112 Medium gray (265) Starch (ISP 4) to medium gray 265) V.m. Deep gray to black Gray-purplish-blue (266-267) (204) S.p. Grayish-yellowish- None brown Glycerol- A.m. 263 (white) to 263 (white) to Asparagine yellowish-gray (93) light gray (264) (ISP 5) V.m. Black ( 267) to light Gray-purplish-blue olive brown (96) (203-204) S.p. Slight brownish Light yellowish-gray Oatmeal A.m. Yellow-gray ( 93) None (ISP 3) V.m. Colorless Colorless S.p. Slight yellowish None 1 = A.m., aerial mycelium;
V.m. = vegetative mycelium;
S.p. = Soluble pigment . ~ ~.

1~38140 TABLE B
Comparison of Lederle F 28249 with ISP 5534 (Gordon Tests) Growth on/at Salicin + _ 45O +
Production of Urease +
DecaL~ylation of M~cate - +
Acid Production Raffinose - +
Sucrose - +

Both strains have the following reactions:
Positive Hydrolysis of casein, hy~ thine~ Y~n~ine~ tyrosine, adrenine, potato starch, gelatin, and esculin;
Production of ~ os~hA~ce Sensitivity to 1YSO~Æ
DecaLbo~ylation of acetate, citrate, lactate, malate, - oxalate and propionate Acid production from arabinose, cellobiose, dextrin, fructose, galactose, glucose, glycerol, lactose, maltose, ~nnose~ a{nethyl D-glucoside, rh~mnose, salicin, trehalose.
Negative Production of nitrate reductase D~cA-~ylation of benzoate and tartrate Acid from adonitol, dulcitol, erythritol, inositol, mannitol, sorbitol, ~-methyl-D-xyloside.
Growth on 5~ NaCl 13381~0 OH
CH3 ~ 4 ~_ ~,~ o ~ R~

0~0 OH A
- R6~ ~

R5 ~ \CH3 LL-F28249c~

-6- 13381~0 C~ 11 11 11 11 11 11 1 11 I~ 11 11 1~

S = I T T T T T T T T
= S S S S S 11 S S S S S

~ T I I T _ T T I T
+
O O O O O O O O O
~Y: ` I` I I I I I I

O
T T T

,rl T
S T

~J T T 2 T T T T S 2 T T T

S
C~
~ S = = = S S = S S S S

C~ S SS = =
1~ S S C~ C~ S = S S S C

SS S S S S S S
-- S S S S S S S S S S S S

~ I I I I I I I I I I I I
O 1 _1 J ~ ~ ~ ~ I ~

-7- 13381~0 QH

~ ~\CH
H3C ~ ~ , CH3 \CH3 OH

LL-F28 24 9v / ~ CH 3 OH - OH
H3C~I ~ CH3 ~ H3C--~CH3 --O
~ O=<

H3J 1~ CH3 H O ~ ~/ OH

LL-F28249w 13381~0 g It ls an ob~ect of thls lnventlon to provlde a, ~, y, ~ , e, ., ~, A, ~, v, and ~ compounds described and derlved from strePtomyces cYaneoqriseus noncYanoqenus~
NRRL 15773, and a method of thelr productlon by fermentatlon.
It ls a further ob~ect to provlde methods for thelr recovery and concentratlon from crude solutlons to purlfy sald compounds. These substances have slgnlflcant actlvlty agalnst helmlnthlc, ectoparasltlc and acarldal lnfectlons, and thus, are useful ln so treatlng warmblooded anlmals affllcted wlth these lnfectlons, as well as treatlng plants lnfllcted wlth nematodes. The utlllty of the presently dlsclosed fermentatlon broth and whole mash of the mlcroorganlsm Streptomyces cYaneoqrlseus noncYanoqenus NRRL
15773, and lts components a, ~, y, ~ , e, v, and ~ ls descrlbed elsewhere.
Accordlngly, the lnventlon hereln descrlbes a compound accordlng to the formula I, II and III, OH
~H3 ,~R~

~ ` \~o-l ~ "R
o~,o R6,-~A~B
Rs ~ CH3 - 1~38140 - 9a -whereln the radlcals Rl, R2, R3, R4, R5 and R6 and bonds A-B
and B-C are selected from the followlng groups:
a) when Rl ls CH(CH3)2, R2 ls H, R3 ls CH3, R4 ls CH3, R5 + R6 ls -O-CH2-, A-Bls a slngle bond and B-Cls a double bond;

~ 3) when Rl ls CH3, R2 ls H, R3 ls CH3, R4 ls CH3, R5 +
R61s-0-CH2-,A-Bls a slngle bond and B-Cls a double bond;

Y) when Rl ls CH3, R2 ls CH3, R3 ls CH3, R4 ls CH3, R5 +R61s-0-CH2-,A-Bls a slngle bond and B-Cls a double 10 bond;
~ ) when Rl ls CH3, R2 ls CH3, R3 ls CH3, R4 ls CH3, R5 ls OH, R6 ls CH2OH, A-Bls a slngle bond and B-Cls a double bond;
~ ) when Rl ls CH(CH3)2, R2 ls H, R3 ls H, R4 ls CH3, R5 +R61s-0-CH2-,A-Bls a slngle bond and B-Cls a double bond;

~ ) when Rl ls CH2CH3, R2 ls H, R3 is CH3, R4 ls CH3, R5 +R61s-0-CH2,A-Bls a slngle bond and B-Cls a double bond;
Tl) when Rl ls CH(CH3)2, R2 ls H~ R3 ls CH3, R4 ls CH3, R5 + R6 ls -O-CH2-, A-Bls a double bond and B-Cls a slngle bond;

~) when Rl ls CH(CH3)2~ R2 ls H~ R3 1 3 4 CH2CH3, R5 + R6 ls -O-CH2-, A-Bls a slngle bond and B-Cls a double bond;

~) when Rl ls CH(CH3)2, R2 ls H, R3 ls CH2CH3, R4 ls CH3, R5 + R6 ls -O-CH2-, A-Bls a slngle bond and B-Cls a double bond5 13381~0 - 9b-1 CH3, R2 ls CH3, R3 ls CH3, R4 ls CH R
ls H, R6 ls CH3, A-B ls a single bond and B-C ls a double bond;
A ) when Rl ls CH(CH3)2, R2 ls CH3, R3 ls CH3, R4 ls CH3, R5 + R6 ls -O-CH2, A-B ls a slngle bond and B-C ls a double bond; and ~) when Rl ls CH(CH3)2, R2 ls CH3, R3 ls CH3, R4 ls CH3, R5 ls H, R6 ls CH3, A-B ls a slngle bond and B-C ls a double bond;

OH
C~H3 ~CH3 \0 J ~ ` CH/CH3 II

--`~CH
OH

and 6110g-7403 13381~0 -- gc H3C ~ CH3 O
m H3C ~ J O OH O ~
~ r OH
H~ CH3CE3 CH3 CH3 Another aspect of the lnventlon ls the compound deslgnated LL-F28249u accordlng to the formula OH

CH3~ GH3 \O ~"~CH/
H3C ~I ~ CH3 GH3 o ~ 3 OH

LL-F28249u whereln the compound has:
a) molecular welght: 592 (EI-MS);
b) molecular formula: C36H48O7;
.~ 61109-7403 ~L~

1338i~0 c) speclflc optlcal rotatlon: []D6 + 131 ~C .325, acetone);
d) Ultravlolet absorptlon spectrum: as shown ln Flgure LIII UV (CH30H) AMAX e 256 (E20, 500); 358 (E8, 830);
e) Infrared absorptlon spectrum: as shown ln Flgure LIV
(KBr dlsc);
f) Electron lmpact mass spectrum: as shown ln Flgure LV;
g) Proton nuclear magnetlc resonance spectrum (CDC13);
as shown ln Flgure LVI; and h) Carbon-13 nuclear magnetlc resonance spectrum (C-DCl3); as shown ln Flgure LVII, and descrlbed ln Table XI.
A further aspect of the lnventlon ls the compound deslgnated LL-F28249~ accordlng to the formula OH ~H3~oH
H3C ~ / ~CH3 ~H3C ~/\ CH3 o~
Y'J O OH O ~/ m HO~OH

LL-F28249~

,-- 9e - 1338140 whereln the compound has:
a) a molecular welght of 806 (FAB-MS);
b) a molecular formula, C45H7401z;
c) a speclflc optlcal rotatlon: [al26 = -49+4 (C 0.35, methanol);
d) a characterlstlc ultravlolet absorptlon spectrum as shown ln Flgure XV of the attached drawlngs;
e) a characterlstlc lnfrared absorptlon spectrum as shown ln Flgure XVI of the attached drawlngs;
f) a characterlstlc proton nuclear magnetlc resonance spectrum as shown ln Flgure XVII of the attached drawlngs;
g) a characterlstlc carbon-13 nuclear magnetlc resonance spectrum as shown ln Flgure XVIII of the attached drawlngs wlth slgnlflcant peaks at, 220.7;
219.6; 165.2; 148.7; 133.1; 132.3; 130.2; 122.3; 100.0;
82.9; 75.9; 73.0; 72.7; 72.6; 72.1; 69.0; 67.3; 63.6;
51.4; 46.2; 45.7; 42.2; 40.4; 38.3; 37.6; 36.1; 34.8;
33.5; 30.1; 26.6; 25.4; 24.5; 23.0; 21.1; 17.9; 14.3;
14.2; 12.1; 11.5; 10.9; 8.7; 8.3; 5.7; and h) a characterlstlc electron lmpact mass spectrum as shown ln Flgure XIX of the attached drawlngs wlth measured m/z values and proposed elemental composltlons as lndlcated below obtalned by hlgh resolutlon mass measurements, 462.3350 C28H4605 253.1797 C15H253 444.3237 C28H4404 235.1697 C15H232 425.2534 C23H377 224.1754 C14H242 13381~0 _ 9f _ 407.2439 C23H3506 209.1530 C13H2102 406.3046 C25H424 207.1744 C14H230 387.2895 C25H3903 184.1458 CllH2002 337.2010 Cl9H295 179.1048 CllH1502 297.2031 C17H2904 173.1205 C9H173 279.1944 C17H273 167.1051 ClOH152 261.1851 C17H252 155.1069 C9H152 These and further ob~ects wlll become apparent by the descriptlon of the drawlngs and detalled descrlptlon of the lnventlon which follow.

~; 61109-7403 .

-lO- 1338140 DE.sCRIPTION OF THE DRAWINGS
FIGURE 1: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 2: Characteristic infrared absorption spectrum of compound designated LL-F28249~, NRRL 15773. FIGURE 3: Characteristic proton nuclear magnetic reso-nance spectrum of compound~designated LL-F28249~, NRRL 15773, in CDC13 solution.
FIGURE 4: Characteristic carbon-13 nuclear magnetic resonance spectrum of compound designated LL-10F28249~, NRRL 15773, in CDC13 solution.
FIGURE 5: Characteristic electron impact mass spectrumof compound designated LL-F28249~, NRRL 15773.
FIGURE 6: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773. 5 FIGURE 7: Characteristic infrared absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 8: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 9: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 10: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249y, NRRL 15773.
FIGURE 11: Characteristic infrared absorption spectrum of 25compound LL-F28249y, NRRL 15773.
FIGURE 12: Characteristic proton nuclear magnetic reso-nance spectrum of compound LL-F28249y, NRRL
15773, in CDC13.
FIGURE 13: Characteristic carbon-13 nuclear magnetic 30- resonance spectrum of compound designated LL-F28249y, NRRL 15773, in CDC13.
FIGURE 14: Characteristic electron impact mass spectrum of compound designated LL-F28249y, NRRL 15773.
FIGURE 15: Characteristic ultraviolet absorption spectrum 35of compound designated LL-F28249~, NRRL 15773.
FIGURE 16: Characteristic infrared absorption spectrum of compound designated LL-F28249~, NRRL 15773.

13381~0 FIGURE 17: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 18: Characteristic nuclear magnetic resonance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
- FIGURE 19: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 20: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 21: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 22: Characteristic èlectron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 23: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249, NRRL 15773.
FIGURE 24: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 25: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 26: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 27: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 28: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 29: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249n, NRRL 15773.
FIGURE 30: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 31: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.

13381~0 FIGURE 32: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 33: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249a, NRRL 15773, in CDC13.
FIGURE 34: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 35: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 36: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LL-F28249~, NRRL 15773, in CDC13.
FIGURE 37: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 38: Characteristic carbon - 13 nuclear magnetic resonance spectrum of compound designated LL-F28249g, NRRL 15773, in CDC13 solution.
FIGURE 39: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 40: Characteristic infrared absorption spectrum of compound designated LL-F28249K, NRRL 15773.
FIGURE 41: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 42: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LLF28249 2S NRRL 15773.
FIGURE 43: Characteristic carbon - 13 nuclear magnetic resonance spectrum of compound designated LL-F28249 K, NRRL 15773.
FIGURE 44: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 45: Characteristic infrared absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 46: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773. 5 FIGURE 47: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LLF28249 , NRRL 15773.

I3381~0 FIGURE 48: Characteristic carbon - 13 nuclear magnetic resonance spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 49: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 50: Characteristic infrared absorption spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 51: Characteristic electron impact mass spectrum of compound designated LL-F28249~, NRRL 15773.
FIGURE 52: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LLF28249 , , NRRL 15773.
FIGURE 53: Characteristic ultraviolet absorption spectrum of compound designated LL-F28249v, NRRL 15773.
FIGURE 54: Characteristic infrared absorption spectrum of compound designated LL-F28249V, NRRL 15773.
FIGURE 55: Characteristic electron impact mass spectrum of compound designated LL-F28249V, NRRL 15773.
FIGURE 56: Characteristic proton nuclear magnetic reso-nance spectrum of compound designated LLF28249 , NRRL 15773.
FIGURE 57: Characteristic carbon - 13 nuclear magnetic resonance spectrum of compound designated LL-F28249v, NRRL 15773.

13381~0 DETAILED DESCRIPTION OF THE INVENTION
The physlochemlcal characterlstlcs for the a, ~, y, ~ , O, ~, K, A, ~, V and ~ components of LL-F2824g are described below:
LL-F28249a:
1) Molecular welght: 612 (FAB-MS);

2) Molecular formula: C36H52O8;

3) Speclflc optlcal rotatlon: [a]D6 c +133_3 ~C 0.3, acetone);

4) Ultravlolet absorptlon spectrum: as shown in Flgure I
UV (CH30H) AMAX = 244 nm (~ 28,000);

5) Infrared absorptlon spectrum: as shown ln Flgure II (KBr dlsc): 3439, 2960, 2925, 1714, 1454, 1374, 1338, 1171, 1120, 996, 967 cm~l;

6) Proton nuclear magnetlc resonance spectrum (CDC13): as shown ln Flgure III;

7) Carbon-13 nuclear magnetlc resonance spectrum (CDC13):
as shown ln Flgure IV and descrlbed ln Table I; and 0 8) Electron lmpact mass spectrum: as shown ln Flgure V
wlth accurate mass measurements and proposed elemental composltlons lndlcated ln Table II.
LL-F28249~:
1) Molecular welght: 584 (FAB-MS);
2) Molecular formula: C34H48O8;

3) Speclflc optlcal rotatlon: ~1D63+125 (C 0.30 acetone).
4) Ultravlolet absorptlon spectrum: as shown ln Flgure VI
UV (CH30H) AMAX = 244 nm (~ 25,600);

- 14a - 1338140 5) Infrared absorptlon spectrum: as shown ln Flgure VII
(KBr dlsc)~ 3520, 2910, 1735, 1717, 1450, 1375, 1335, 1180, 1170, 1119, 993, 727 cm~l;
6) Proton nuclear magnetlc resonance spectrum (CDC13): as shown ln Flgure VIII;
7) Carbon-13 nuclear magnetlc resonance spectrum (CDC13):
as shown ln Flgure XXXVIII and descrlbed ln Table II A;
and 8) Electron lmpact mass spectrum: as shown ln Flgure IX
wlth accurate mass measurements and proposed ele-I3381~0 mental compositions indicated in Table III.
LL-F28249r:
1) Molecular weight: 59~ (FAB-MS);
2) Molecular formula: C35H508;
3) Specific optical rotation: [~]26 = +150+4 (C 0.3, acetone);
4) Ultraviolet absorption spectrum: as shown in Figure X UVCHXOH = 244 nm (~ 27,100);
5) Infrared absorption spectrum: as shown in Figure XI
- (KBr disc): 3510, 2910, 1735, 1715, 1452, 1375, 1338, 1182, 1172, 1119, 995 cm~l;
6) Proton nuclear magnetic resonance spectrum (CDC13):
as shown in Figure XII;
7) Carbon-13 nuclear magnetic resonance spectrum (CDC13): as shown in Figure XIII and described ih Table IV; and 8) Electron impact mass spectrum: as shown in Figure XIV with accurate mass measurements and proposed elemental compositions indicated in Table V.
LL-F28249~:
20 1) Molecular weight: 806 (FAB-MS);
2) Molecular formula: C45H7412;
3) Specific optical rotation: [~]D6 = _49+3o (C 0.35, methanol);
4) Ultraviolet absorption spectrum: as shown in Figure XV UVCH30H = 225 nm ( ~27,400) 232 nm ( ~25,700);
5) Infrared absorption spectrum: as shown in Figure XVI
(KBr disc): 3480, 2965, 2935, 2880, 1703, 1647, 1458, 1380, 1292, 1223, 1135, 1098, 984 cm~l;
30 6) Proton nuclear magnetic resonance spectrum (CDC13):
as shown in Figure XVII;
7) Carbon-13 nuclear magnetic resonance spectrum (CDC13): as shown in Figure XVIII and described in Table VI; and 35 8) Electron impact mass spectrum: as shown in Figure XIX with accurate mass measurements and proposed elemental compositions indicated in Table VII.

LL-F28249~:
1) Molecular weight: 616 (EI-MS) 2) Molecular formula: C35H529 3) HPLC retention volume of 14.0 ml in the system in-dicated in Table VIII;
4) Ultraviolet absorption spectrum (methanol): as shown in Figure XX;
5) Proton nuclear magnetic resonance spectrum (CDCL3):
as shown in Figure XXI; and 6) Electron impact mass spectrum: as shown in Figure XXII.
LL-F28249~:
1) Molecular weight: 598 (EI-MS) 2) Molecular formula: C35 H50 8 3) HPLC retention volume of 14.8 ml in the system in-dicated in Table VIII;
4) Ultraviolet absorption spectrum (methanol): as shownin Figure XXIII;
5) Proton nuclear magnetic resonance spectrum (CDC13):
as shown in Figure XXIV; and 6) Electron impact mass spectrum: as shown in Figure XXV .
LL-F28249~:
1) Molecular weight: 598 (EI-MS) 2) Molecular formula: C35 H50 8 3) HPLC retention volume of 16.0 ml in tbe system in-dicated in Table VIII;
4) Ultraviolet absorption spectrum (methanol): as shown in Figuré XXVI;
5) Proton nuclear magnetic resonance spectrum (CDCL3):
as shown in Figure XXVII; and 6) Electron impact mass spectrum: as shown in Figure XXVIII.
LL-F28249n:
1) Molecular weight: 612 (EI-MS) 2) Molecular formula: C36 H52 8 3) HPLC retention volume of 23.5 ml in the system in-- dicated in Table VIII;

~ 17- 1~38140 4. Ultraviolet absorption spectrum (methanol): as shown in Figure XXIX;) 5) Proton nuclear magnetic resonance spectrum (CDC13):
as shown in Figure XXX; and 6) Electron impact mass spectrum: as shown in Figure XXXI.
LL-F28249~:
1) Molecular weight: 626 (EI-MS) 2) Molecular formula: C37Hs40g 3) HPLC retention volume of 24.5 ml in the system in-dicated in Table VIII;
4) Ultraviolet absorption spectrum (methanol): as s-hown in Figure XXXII;
5) Proton nuclear magnetic resonanace spectrum (CDC13):
as shown in Figure XXXIII; and 6) Electron impact mass spectrum: as shown in Figure XXXIV.
LL-F28249~-1) Molecular weight: 626 (EI-MS) 2) Molecular formula: C37 H54 8 3) HPLC retention volume of 26.0 ml in the system in-dicated in Table VIII;
4) Ultraviolet absorption spectrum (methanol): as sh-own in Figure XXXV;
5) Proton nuclear magnetic resonance spectrum (CDC13):
as shown in Figure XXXVI; and 6) Electron impact mass spectrum: as shown in Figure XXXVII.
LL-F28249K:
1) Molecular weight: 584 (EI-MS);
2) Molecular formula: C35 H52 7;
3) Specific optical rotation: [~]26D=+189-(C 0.165 acetone);
4) Ultraviolet absorption spectrum: as shown in Figure XXXIX UV ~ x3H=241nm (E20,400);
5) Infrared absorption spectrum: as shown in Figure XL
(KBr disc);

6) Electron impact mass spectrum: as shown in Figure XKI;
7) Proton nuclear magnetic resonance spectrum (CDC13);
as shown in Figure XLII; and 8) Carbon-13 nuclear magnetic resonance spectrum (CDC-13); as shown in Figure XLIII and described in Table IX.
LL-F28249~:
1) Molecular weight: 626 (FAB-MS);
2) Molecular formula: C37 Hs4 8;
10 3) Specific optical rotation: [~]D26 =+145 (C, 0.23 acetone);
4) Ultraviolet absorption spectrum: as shown in Figure XLIV UV ~ ~H=244nm (E30,000);
5) Infrared absorption spectrum: as shown in Figure XLV
(KBr disc);
6) Electron impact mass spectrum: as shown in Figure XLVI;
7) Proton nuclear magnetic resonance spectrum (CDC13);
as shown in Figure XLVII; and 20 8) Carbon-13 nuclear magnetic resonance spectrum (CDC-13); as shown in Figure XLVIII and described in Table X.
LL-F28249~:
1) Molecular weight: 612 (EI-MS);
25 2) Molecular formula: C37 H56 7;
3) Ultraviolet absorption spectrum: as shown in Figure XLIX UV ~ ~Q~41nm (E16,800);
4) Infrared absorption spectrum: as shown in Figure L
(KBr disc);
30 5) Electron impact mass spectrum: as shown in Figure LI;
6) Proton nuclear magnetic resonance spectrum (CDC13);
as shown in Figure LII.
LL-F28249v:
35 1) Molecular weight: 592 (EI-MS);
2) Molecular formula: C36 H48 7;
3) Specific optical rotation: [~]D26+136_(C.325, 13:~81~0 acetone);
4) Ultraviolet absorption spectrum: as shown in Figure LIII UV CH3H=256 (E20,500); 358(E 8,830);
5) Infrared absorption spectrum: as shown in Figure LIV
(KBr disc);
6) Electron impact mass spectrum: as shown in Figure LV;
7) Proton nuclear maagnetic resonance spectrum (CDC13);
as shown in Figure LVI; and 8) Carbon-13 nuclear magnetic resonance spectrum (CDC13);
as shown in Figure LVII, and described in Table XI.

~J
= X S ~ S' :~

U~
-~n .
.
_ _ ~ E
~r C

.

C
O

C~
~Y
m ~
~s: ~ C
E~ O
c Z O ~
~- x x - c l ~ u u c~ u c~ u u u u u --o :~ ~
.a ~ o u .

u ~ ~ c ~
~) o c ~c ~ ~ ~
~ ~ ~ ~ ~ r~ I~ ~` O ~ O ~ ~ C ~ ~ ~ a~
E
E O (~ ~
L~ C O
c ~ ~ U~
U aJ
J ~
~ C
c ~ G O
O C

_ 1~381~0 TABLE II
High Resolution Mass Measurements for LL-F28249~
m/zElemental Composition 612.3705 C36H52O8 594.3543 C36H507 576.3472 C36H48O6 484.3211 C30H44Os 482.2648 C29H38O6 466.3097 C30R42o4 448.2987 C30H40O3 442.2375 C26H34O6 425.2327 C26H33O5 354.2181 C23H30O3 314.1877 C20H26O3 278.1144 C15H18S
265.1786 C16H25O3 248.1405 ClsH20O3 247.1705 C16H23O2 237.1838 C15H252 219.1740 C15H23O
151.0753 C9H112 -22_ 1 3 3 8 1 ~ 0 TABLE IIa Carbon-13 NMR Data for LL-F28249~
Carbon Chemical Shift(ppm)* Carbon Chemical Shift(ppm) 1 173.3 18 68.3 2 142.6 19 67.8 3 139.5 20 67.7 4 137.7 21 48.4 137.3 22 45.7 6 133.9 23 41.0 7 123.8 24 40.8 8 123.4 25 36.1 9 120.3 26 35.9 **
120.2 27 34.7 11 118.0 28 22.3 12 99.7 29 19.8 13 80.2 30 15.5 14 79.4 31 13.8 76.7 32 13.1 16 69.2 33 10.8 17 68.6 * Downfield from TMS; CDC13 solution ** Two unresolved signals 1~381~10 TABLE III
Hiqh Resolution Mass Measurements for LL-F28249B
m/zElemental Composition 584.3388 C34H48O8 566.3306 C34H46O7 456.2864 C28840Os 442.2391 C26H34O6 438.2~80 C28~384 425;2331 C26~335 354.2187 c23830O3 314.1858 C20H26o3 278.1168 ClsHlgOs 237.1491 Cl4H21O3 219.1380 C14H19O2 209.1534 C13H2lO2 191.1418 C13HlgO
151.0750 CgH11O2 13381~0 TABLE IV
Carbon-13 NMR Data for LL-F28249y Chemical Shiftl Chemical Shift Carbon (ppm) Carbon (ppm) 1 173.6 19 68.3 2 142.4 20 67.9 3 139.9 21 57.7 4 137.3 22 48.5 S 136.0 23 45.8 6 134.0 24 41.2 7 123.8 2S ~ 40.8 8 123.6 26 36.2 9 120.4 27 36.1 119.6 28 36.0 11 118.5 29 34.8 12 99.8 30 22.3 13 80.5 31 19.9 14 77.8 32 15.5 77.0 33 13.8 16 76.8 34 13.1 17 69.3 35 10.8 18 68.6 Downfield from TMS; CDC13 solution.

` - -25~ 8 1~0 TABLE V
High Resolution Mass Measurements for LL-F28249y m/z Elemental Composition 598.3543 C3sH5008 580.3422 C35H4807 562.3292 c35H4606 496.2824 C30H4006 484.2440 C2gH3607 478.2687 C30H3805 456.2576 C27H3606 438.2772 C2gH3804 425.2341 C26H330S
420.26Sl C2gH3603 354.2199 C23H3003 314.1875 C20H26o3 292.1307 C16H2005 288.2075 ClgH2802 248.1397 C15H2003 237.1490 C14H2103 219.1382 C14H1902 209.1544 C13H212 191.1435 C13HlgO
151.0759 CgH112 -26- 1 3 ~ 8 1 ~ 0 TABLE VI
Carbon-13 NMR Data for LL-F28249~
Chemical Shiftl Chemical Shift Carbon (ppm) Carbon (ppm) 1 220.7 23 42.22 2 219.6 24 40.4 3 165.2 25 38.3 4 148.7 26 37.6 133.1 27 36.1 6 132.3 28 34.8 7 132.1 -29 33.5 8 130.2 30 30.1 9 122.3 31 26.6 100.0 32 25.4 11 82.9 33 24.5 12 75.9 34 23.0 13 73.0 35 21.1 14 72.7 36 17.9 72.6 37 14.3 16 72.1 38 14.2 17 69.0 39 12.1 18 67.3 40 11.5 19 63.6 41 10.9 51.4 42 8.7 21 46.2 43 8.3 22 45.7 44 5.7 lDownfield from TMS; CDC13 solution.
2Two unresolved signals.

TABLE VII
Hi~h Resolution Mass Measurements for LL-F28249~
m/z Elemental Composition 462.3350 C2gH4605 444.3237 C2gH4404 425.2534 C23H3707 407.2439 C23H3506 406.3046 C25H4204 387.2895 C2sH3903 337.2010 ClgH295 297.iO31 Cl7H29o4 279.1944 C17H273 261.1851 C17H2502 253.1797 ClsH2503 235.1697 C15H2302 224.1754 C14H2402 209.1530 C13H2102 207.1744 C14H230 184.1458 CllH2002 179.1048 CllH1502 173.1205 C9H173 167.1051 CloH1502 155.1069 C9H152 ` 13381~0 TABLE VIII
HPLC Retention Volumes for LL-F28249~ , n, ~ and ~
Compound Retention Volume*(ml) LL-F28249 ~ 19.8 LL-F28249 ~ 14.0 LL-F28249 ~ 14.8 LL-F28249~ 16.0 LL-F28249 n 23.5 LL-F28249~ 24.5 LL-F28249 ~ 26.0 ~System includes a column 3.9mm x 30cm packed with Clg reverse phase packing developed with methanol:water (80:20) at 1.0 ml/minute, detection was by absorbance at 254 nm.

1~8140 TABLE IX
Carbon-13 NMR Data for LL-F28249~
Carbon Chemical Shift(ppm)~ Carbon Chemical Shift(ppm) 1 173.9 19 56.7 2 140.7 20 48.4 3 138.3 21 47.7 4 136.6 22 41.1 136.5 23 40.6 6 133.8 14 37.1 7 124.7 25 36.3 8 124.4 26 36.0 9 123.8 27 35.9 120.1 28 34.6 11 118.5 29 22.0 12 99.7 30 19.3 13 77.2 31 16.0 14 76.6~* 32 13.8 76.5 33 13.3 16 69.3 34 13.1 17 6~.6 35 10.7 18 67.3 Downfield from TMS; CDC13 solution.
~* Coincident with CDC13 signals.

` - 13~81~

TABLE X
Carbon-13 NMR Data for LL-F28249~
Carbon Chemical shift(ppm)- Carbon Chemical Shift(ppm) 1 ' 173.6 19 68.3 2 142.5 20 67.9 3 139.8 21 57.8 4 137.4 22 48.6 137.2 23 45.8 6 136.0 24 41.2 7 130.7 25 40.9 8 123.6 26 36.1 9 120.3 27 36.0 119.7 28 34.9 11 118.6 29 26.9 12 99.8 30 23.0 13 80.5 31 22.4 14 77.7 32 20.0 77.6 33 15.7 16 76.7 34 14.0 17 69.3 35 11.1 18 68.6 * Downfield from TMS; CDC13 solution.
*~ Two unresolved signals.

`` ` 13~8140 .

TABLE XI
Carbon-13 NMR Data for LL-F28249~
Carbon hemical Shift(ppm)* Carbon Chemical Shift(ppmJ
1 167.4 18 69.4 2 150.5 19 68.7 3 142.9 20 68.3 4 142.0 21 48.4 137.2 ~* 22 41.0 **
6 132.1 23 35.9 7 130.7 24 35.6 8 125.8 25 35.5 9 125.5 26 34.4 124.2 27 29.7 11 123.7 28 26.8 12 123.2 29 22.9 13 121.3 30 22.8 14 118.0 31 22.1 100.0 32 15.3 16 76.7 33 13.9 17 74.6 34 11.0 * Downfield from TMS; CDC13 solution.
** Two unresolved signals.

13381~o TABLE XII
Chromatographic Data TLC * HPLC **
Component Relative Rf Retention Time(minutes) 1.00 13.8 ~ .797 9.3 r 1.42 12.6 .758 10.4 1.06 10.9 1.12 11.5 1.03 16.2 1.27 17.3 1.27 18.2 1.83 24.7 1.56 19.1 1.92 38.0 1.95 42.3 ~ ~ .212 7.1 * Analtech Silica Gel GHLF250~developed with ethyl acetate:methylene chloride (1:3), detection by : charring with H2S04.
~, ** Altex Ultrasphere~ODS 5~ 4.6mmx25cm developed with 85% methanol in water at 1.0 ml/minute, detection by absorbance at 254 nm.

The new agents designated LL-F28249~ y ,~ ,~
, ~,n, ~"~K, A,~,vand~are formed during the cultivation under controlled conditions of Streptomyces cyaneo~riseus noncyano~enus, NRRL 15773.
This organism is maintained in the culture col-lection of the Medical Research Division, American Cya-namid Company, Pearl River, New York as culture number LL-F28249. A viable culture of this new microorganism has been deposited with the Patent Culture Collection Labora-tory, Northern Regional Research Center, U. S. Department of Agriculture, Peoria, Illinois 61604, and has been added to its permanent collection. It is freely available to the public in this depository under its accession number NRRL
15773.
For the production of these new agents the pres-ent invention is not limited to this particular organism.
In fact, it is desired and intended to include the use of naturally-occurring mutants of this organism, as well as induced mutants produced from this organism by various mutagenic means known to those skilled in the art, such as exposure to nitrogen mustard, X-ray radiation, ultra-violet radiation, N'-methyl-N'nitro-N-nitrosoguanidine, actinophages and the like. It is also desired and intended to include inter- and intraspecific genetic recombinants produced by genetic techniques known to those skilled in the art such as for example, conjugation, transduction and genetic engineering techniques.
Cultivation of Streptomyces cyaneo~riseus non-cyaneogenus, NRRL 15773 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of agents LL-F28249~,~ ,y, ~ , n,~
~ ,A,~,v and~include an assimilable source of carbon, such as dextrin, sucrose, molasses, glycerol, etc.; an as-similable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep li-quor, etc.; and inorganic anions and cations, such as potassium, `. 13381~0 -sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc., are supplied as impurities of other consti-tuents of the media. Aeration in tanks and bottles is supplied by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoam agent such as silicone oil may be added as needed.
Example 1 Inoculum Preparation A typical medium used to grow the various stages of inoculum was prepared according to the following for-mula:
Dextrose.................... 1.0%
Dextrin..................... 2.0%
Yeast extract............... 0.5%
NZ amine.................... 0.5%
Calcium carbonate........... 0.1%
Water........ qs................. 100%
This medium was sterilized. A 100 ml portion of this sterile medium, in a flask, was inoculated with my-celial scrapings from an agar slant of Streptomyces cyaneo~riseus noncyano~enus NRRL 15773. The medium was then agitated vigorously on a rotary shaker for 48-72 hours at 28C providing primary inoculum. This primary inoculum was then used to inoculate one liter of the above sterile medium, which was then grown aerobically at 28C
for 48 hours providing secondary inoculum.
Example 2 Fermentation A fermentation medium of the following formu-lation was prepared.
Dextrin..................... 1.0%
Soya peptone................ 1.0%
Molasses.................... 2.0%
Calcium carbonate........... 0.1%
Water........ qs................. 100%

This medium was sterilized and then a 30 liter portion was inoculated with one liter of secondary inocu-lum prepared as described in Example 1. The fermentation was conducted at 30C, with a sterile air flow of 30 liters per minute, backpressure of 8 psig and agitation by an impeller operated at 500 rpm for 91 hours at which time the mash was harvested.
Example 3 Isolation of LL-F28249~, B andr A total of 26 liters of whole harvest mash, prepared as described in Example 2 was mixed with 1500 g of diatomaceous earth and filtered. The mycelial cake was washed with 5 liters of water and the filtrate and wash discarded. The mycelial cake was mixed with 10 liters of methanol for one hour, then filtered and washed with 5 liters of methanol. The methanol extract and methanol wash were combined and evaporated to an aqueous residue of about 1-2 liters. This aqueous residue was mixed with twice its volume of methylene chloride and mixed for 1/2 hour. The methylene chloride phase was separated and then concentrated to a syrup giving 27 g of crude material.
This-27 g of crude material was dissolved in a mixture of methylene chloride and methanol, filtered throu-gh cotton and anhydrous sodium sulfate and then evap-orated, giving 7.0 g of an oil.
A 170 g portion of silica gel was slurried in 12.5% ethyl acetate in methylene chloride and poured to form a column 2.5x58 cm. The oil was dissolved in 12.5%
ethyl acetate in methylene chloride and applied to the column. The column was developed with the same solvent mixture. The mobile phase was run at 1.3 ml/minute ini-tially and 15 minute fractions were collected. The flow rate slowed to about 0.5 ml/minute after 10 fractions, so fractions 1-10 were 20 ml decreasing to about 10 ml uni-formly and fractions 11-98 were about 7 ml. At fraction 99 the flow rate was increased to give 25 ml fractions in 10 minutes. A total of 105 fractions were coliected.

-36- 13381~0 These fractions were tested by thin layer chromatography in ethyl acetate:methylene chloride (1:1).
Fractions 30-54 were combinet and evaporated giving 1.08 g of an oil containing LL-F28249y.
Fractions 55-62 were combined and evaporated giving 150 mg of solid containing LL-F28249~ and ~.
The 150 mg of solid containing LL-F28249 ~and was chromatographed by preparative HPLC using a reverse-phase column (Wbatman C8, 2.2x50 cm) developed with 80%
(v/v) methanol in water. The flow rate was about 10 ml/-minute and 2 minute fractions were collected.
Fractions S8-69 were combined, the methanol was evaporated, t-butanol was added and the mixture was lyo-philized, giving 60 mg of pure LL-F2824~ .
Fractions 40-43 were combined, the methanol was evaporated and the resitual aqueous suspension was ex-tracted with methylene chloride which, upon evaporation, gave 10 mg of pure LL-F28249 ~
The 1.08 g of oil containing LL-F28249y was dis-solved in 10% ethyl acetate in methylene chloride and applied to a column (2.5x50 cm) pac~ed with silica gel.
The column was developed with lOZ ethyl acetate in meth-ylene chloride, eluting at a flow rate of 2 ml/minute and collecting 12 minute fractions. Fractions 19-29 were combined and evaporated to a residue. This residue was purified by preparative reverse-phase chromatography as described for the andB components. Fractions 55-62 were combined, the methanol was evaporated in vacuo, t-bu-tanol was added and the mixture was lyophilized giving 60 mg of pure LL-F28249y.
Example 4 Large Scale Fermentation An inoculum of Streptomyces cyaneogriseus non-cyanogenus, NRRL 15773 was prepared as described in Ex-ample 1, using 100 ml of primary inoculum to produce 10 liters of secondary inoculum.

Two 300 llter fermentatlons were conducted as descrlbed ln Example 2 uslng 10 llters of the above secondary lnoculum for each 300 llters of fermentatlon medlum. At the end of 118 hours the mashes were harvested.
ExamPle 5 Isolatlon of LL-F28249~
A total of 450 llters of harvest mash from the two 300 llter fermentatlons descrlbed ln Example 4 was treated as descrlbed ln the flrst portlon of Example 3 glvlng crude materlal as a syrup.
Thls syrupy resldue was washed wlth hexane to remove non-polar materlals and the remalnlng 9 g of lnsoluble materlal was sub~ected to Sephadex LH-20 partltlon chromatography.
The chromatographlc column was prepared wlth 9 llters of Sephadex LH-20~, prevlously swelled ln methanol, to form a column lOxllO cm. The column was equlllbrated by passlng about 4800 ml of moblle phase [methylene chlorlde:hexane:methanol (10:10:1)] through lt at a flow rate of 5ml/mlnute. The 9 g of lnsoluble materlal was charged onto the column ln 50 ml of the moblle phase. An lnltlal forerun of 2150 ml was obtalned at a flow rate of 5 ml/mlnute. The flow rate was then lncreased to 8 ml/mlnute and fractlons were collected every 45 mlnutes. Fractlons 9-12 were comblned and the solvents evaporated ln vacuo glvlng 4.9 g of resldue.
Thls resldue was dlssolved ln a lsl mlxture of cyclohexane and ethyl acetate and allowed to evaporate slowly at room temperature. The addltlon of n-hexane gave a , - 37a - 1~38140 preclpltate whlch was collected, glvlng 3.1 g of solld.
A 3.0 g portlon of thls solld was further purlfled by preclpltatlon from 25 ml of methylene chlorlde uslng 50 ml of n-hexane.
The preclpltate thus obtalned was redlssolved ln 15 ml of methylene chlorlde and preclpltated wlth 25 ml of n-hexane, glvlng 510 mg of pure LL-F28249~.

-r 61109-7403 , ~

Example 6 Isolation of LL-F28249~ , n, ~ and~
Fractions 4-7 from the Sephadex LH-20 column described in Example 5 were combined and the solvents evaporated in vacuo to give 1.9 g of residue.
This residue was chromatographed on a 200 g silica gel column (2.5cm x 83cm) using 10% ethyl acetate in methylene chloride as the eluant. The flow rate was approximately 2 ml/minute and fractions were collected every 12 minutes.
Fractions 65-67 and 73-79 were combined together and the solvents were evaporated in vacuo to yield 250 mg of residue.
This 250 mg of residue was subjected to pre-parative reverse-phase chromatography as described in Example 3 except using 75% methanol in water as the mobile phase. The flow rate was about 10 ml/minute. The first 2000 ml portion of eluate was diverted to waste then 72 fractions were collected at 2.0 minute intervals. After diverting another portion of eluate to waste (between 300-400 ml) fractions were collected again but at 2.5 minuteintervals.
Fractions were combined as indicated below. The combined fractions were allowed to evaporate in a fume hood overnight, then the components were extracted into methylene chloride. Follwing evaporation of the solvent about 1 mg each of the pure components were obtained.
Fractions Combined Compound 30 28-31 LL-F28249~
81-83 LL-F28249 n 93_95 LL-F28249 Example 7 Isolation of LL-F28249~ andv A total of 390 liters of fermentation mash, harvested from fermentations conducted as described in Example 2, was processed essentially as described in the first paragraph of Example 3, giving 120 ml of methylene chloride concentrate. This concentrate was diluted with 200 ml of hexane and chilled overnight at 4C. The resulting precipitate was removed by filtration and discarded. The filtrate was diluted with 300 ml of hexane. The resulting precipitate (A) was collected by filtration and saved. This filtrate was evaporated to dryness and the oily residue was then dissolved in 200 ml of methylene chloride and diluted with 1700 ml of hexane.
The resulting precipitate (B) was collected by filtration and saved. This filtrate was concentrated to an oily residue which was then redissolved in 50 ml of methylene chloride, 950 ml of methanol was added and this solution was stored at 4C for 3 days. The resulting precipitate was removed by filtration and discarded. The filtrate was evaporated to dryness and the residue (C) combined with (A) and (B) and subjected to chromatography as follows:
The 5.0xlO9cm column was slurry-packed with Woelm TSC
silica gel in ethyl acetate:methylene chloride (1:9). The column was developed with the same solvent mixture at a rate of 25 ml/minute. The first 2 liters of effluent were discarded, then sixteen 400 ml fractions were collected.
Fractions 2 and 3 were combined and evaporated giving 3.9 g of oily material (D).
Fractions 4 through 7 were combined and evaporated giving 9.5 g of oily material which was dissolved in hexane and chromatograpbed on a 2.5xllOcm column slurry-packed with 300 g of Woelm silica gel in ethyl acetate:hexane (1:4). The column was developed with the same solvent system at a rate of 4 ml/minute, collecting fractions at 7 minute intervals.

Fractlons 45-54 were combined and evaporated, givlng 0.3 g of materlal (E).
Fractlons 63-135 were comblned, evaporated to dryness, then redlssolved ln t-butanol and lyophlllzed glvlng 4.6 g of off-whlte solld (F).
LL-F28249~ and ~
Materlal (D) and (E) were comblned and chromatographed on a 2.5xllOcm column packed wlth 300 g of Woelm slllca gel, developlng wlth ethyl acetate:hexane (1:9).
The flow rate was malntalned at 4ml/mlnute and fractlons were collected at 7 mlnute lntervals.
Fractlons 67-115 were comblned and evaporated to dryness, glvlng 920 mg of resldue (G).
Thls resldue (G) was chromatographed by preparatlve HPLC uslng a reverse phase column (Whatman~ C8, 2.2x50cm) and developlng wlth 85% (v/v) methanol ln water. The flow rate was about lOml/mlnute and fractlons were collected at 2.5 mlnute lntervals.
Fractions 33-40 were combined, concentrated to remove the methanol, then extracted wlth methylene chlorlde.
The resldue obtalned upon evaporatlon was dlssolved ln t-butanol and then lyophlllzed, glvlng 60 mg of LL-F28249~.
Fractlons 52-58 were slmllarly processed glvlng a small quantlty of LL-F28249~.

A one gram portlon of materlal (F) was chromatographed by reverse phase HPLC, as descrlbed above, except that 80% (v/v) methanol ln water was used as eluent.

.~ , Fractlons 61-75 were comblned and processed as above, glvlng 100 mg of LL-F28249A.
LL-F28249~
A 396 g portlon of material essentlally the same as materlal ~D) above, was dlssolved ln 500 ml of methanol and then chllled at 40 for several hours. The resultlng preci-pltate was removed by filtratlon, washed wlth cold methanol and dlscarded. The comblned flltrate and wash was evapor-ated. The resldual oll was dlssolved ln hexane and charged on a Sx50 cm dry-packed slllca gel column (Malllnkrodt SlllcARD cc-7). The column was eluted wlth ethyl acetate:
hexane (1.5:8.5) at a rate of about 50 ml/mlnute.
Four fractlons were collected.
Fractlon Volume (llters) Fraction 3 was evaporated, glvlng 5.0 g of resldue whlch was purifled by preparatlve reverse phase HPLC (Waters C18, 5x60cm). The column was lnltlally developed wlth 16 llters of 80% methanol ln water (v/v) at 100 ml/mlnute, then wlth 6.4 llters of 84% methanol ln water (v/v). The flrst llter of effluent was dlscarded and then fractlons of 400 ml were collected.
Fractlons 44-47 were comblned and processed as described above, glvlng 390 mg of LL-F29249v as a pale yellow solld.

Claims (18)

1. A LL-F28249 compound selected from the group according to formula I, II and III, I

wherein the radicals R1, R2, R3, R4, R5 and R6 and bonds A-B
and B-C are selected from the following groups:
.alpha.) when R1 is CH(CH3)2, R2 is H, R3 is CH3, R4 is CH3, R5 + R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;
.beta.) when R1 is CH3, R2 is H, R3 is CH3, R4 is CH3, R5 +
R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;
?) CH3, R2 is CH3, R3 is CH3, R3 is CH3, R4 is CH3, R5 + R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;

?) when R1 is CH3, R2 is CH3, R3 is CH3, R4 is CH3, R5 is OH, R6 is CH2OH, A-B is a single bond and B-C is a double - 42a -bond;

.epsilon.) when R1 is CH(CH3)2, R2 is H, R3 is H, R4 is CH3, R5 + R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;

?) when R1 is CH2CH3, R2 is H, R3 is CH3, R4 is CH3, R5 + R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;
?) when R1 is CH(CH3)2, R2 is H, R3 is CH3, R4 is CH3, R5 + R6 is -O-CH2-, A-B is a double bond and B-C is a single bond;
.theta.) when R1 is CH(CH3)2, R2 is H, R3 is CH3, R4 is CH2CH3, R5 + R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;
?) when R1 is CH(CH3)2, R2 is H, R3 is CH2CH3, R4 is CH3, R5 + R6 is -O-CH2-, A-B is a single bond and B-C is a double bond;
?) when R1 is CH3, R2 is CH3, R3 is CH3, R4 is CH3, R5 is H, R6 is CH3, A-B is a single bond and B-C is a double bond;
.lambda.) when R1 is CH(CH3)2, R2 is CH3, R3 is CH3, R4 is CH3, R5 + R6 is -O-CH2, A-B is a single bond and B-C is a double bond; and µ) when R1 is CH(CH3)2, R2 is CH3, R3 is CH3, R4 is CH3, R5 is H, R6 is CH3, A-B is a single bond and B-C is a double bond, II

and III

- 43a -

2. The compound designated LL-F28249.alpha. accord-ing to claim 1 wherein the compound has:
a) a molecular weight of 612 (FAB-MS);
b) a molecular formula, C36H52O8;
c) a specific optical rotation, [.alpha.]?6 = +1333° (C 0.3, acetone);
d) a characteristic ultraviolet absorption spectrum as shown in Figure I of the attached drawings;
e) a characteristic infrared absorption spectrum as shown in Figure II of the attached drawings;
f) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure III of the attached draw-ings;
g) a characteristic carbon-13 nuclear magnetic resonance spectrum as shown in Figure IV of the attached draw-ings with significant peaks at, 173.4; 142.8; 139.4;
137.7; 137.3; 137.2; 130.6; 123.3; 120.3; 118.0; 99.7;
80.2; 79.3; 76.7; 69.3; 68.5; 68.4; 67.8; 67.7; 48.4;
45.7; 41.1; 40.7; 36.1; 36.0; 35.9; 34.7; 26.8; 22.8;
22.2; 19.9; 15.5; 13.9; 11.0; and h) a characteristic electron impact mass spectrum as shown in Figure V of the attached drawings with mea-sured m/z values and proposed elemental compositions as indicated below obtained by high resolution mass measurements, 612.3705 C36H52O8 3S4.2181 C23H30O3 594.3543 C36H50O7 314.1877 C20H26O3 576.3472 C36H48O6 278.1144 C15H18O5 484.3211 C30H44O5 265.1786 C16H25O3 482.2648 C29H38O6 248.1405 C15H20O3 466.3097 C30H42O4 247.1705 C16H23O2 448.2987 C30H40O3 237.i838 C15H25O2 442.2375 C26H34O6 219.1740 C15H23O
425.2327 C26H33O5 151.0753 C9H11O2.

3. The compound designated LL-F28249.beta. accord-ing to claim 1 wherein the compound has:
a) a molecular weight of 584 (FAB-MS);
b) a molecular formula, C34H48O8;
c) specific optical rotation: [.alpha.] ?6 =+125° (C 0.30, ace-tone).
d) a characteristic ultraviolet absorption spectrum as shown in Figure VI of the attached drawings;
e) a characteristic infrared absorption spectrum as shown in Figure VII of the attached drawings;
f) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure VIII of the attached draw-ings;
g) a characteristic carbon-13 nuclear magnetic resonance spectrum as shown in Figure XXXVIII of the attached drawings, with significant peaks at 173.3; 142.6;
139.5; 137.7; 137.3; 133.9; 123.8; 123.4; 120.3; 120.2;
118.0; 99.7; 80.2; 79.4; 76.7; 69.2; 68.6; 68.3; 67.8;
67.7; 48.4; 45.7; 41.0; 40.8; 36.1; 35.9; 34.7; 22.3;
19.8; 15.5; 13.8; 13.1; 10.8; and h) a characteristic electron impact mass spectrum as shown in Figure IX of the attached drawings with mea-sured m/z values and proposed elemental compositions as indicated below obtained by high resolution mass measurements, 584.3388 C34H48O8 314.1858 C20H26O3 566.3306 C34H46O7 278.1168 C15H18O5 456.2864 C28H40O5 237.1491 C14H21O3 442.2391 C26H34O6 219.1380 C14H19O2 438.2780 C28H38O4 209.1534 C13H21O2 425.2331 C26H33O5 191.1418 C13H19O
354.2187 C23H30O3 151.0750 C9H11O2.

4. The compound designated LL-F28249.gamma. accord-ing to claim 1 wherein the compound has:
a) a molecular weight of 598 (FAB-MS);
b) a molecular formula, C35H50O8;
c) a specific optical rotation: [.alpha.]?6 = +1504? (C 0.3, acetone);

d) a characteristic ultraviolet absorption spectrum as shown in Figure X of the attached drawings;
e) a characteristic infrared absorption spectrum as shown in Figure XI of the attached drawings;
f) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XII of the attached draw-ings;
g) a characteristic carbon-13 nuclear magnetic resonance spectrum as shown in Figure XIII of the attached draw-ings with significant peaks at, 173.6; 142.4; 139.9;
137.3; 136.0; 134.0; 123.8; 123.6; 120.4; 119.6;
118.5; 99.8; 80.5; 77.8; 77.0; 76.8; 69.3; 68.6;
68.3; 67.9; 57.7; 48.5; 45.8; 41.2; 40.8; 36.2; 36.1;
36.0; 34.8; 22.3; 19.9; 15.5; 13.8; 13.1; 10.8; and h) a characteristic electron impact mass spectrum as shown in Figure XIV of the attached drawings with measured m/z values and proposed elemental composi-tions as indicated below obtained by high resolution mass measurements.
598.3543 C35H50O8 354.2199 C23H30O3 580.3422 C35H48O7 314.1875 C20H26O3 562.3292 C35H46O6 292.1307 C16H20O5 496.2824 C30H40O6 288.2075 C19H28O2 484.2440 C28H36O7 248.13g7 C15H20O3 478.2687 C30H38O5 237.1490 C14H21O3 456.2576 C27H36O6 219.1382 C14H19O2 438.2772 C28H38O4 209.1544 C13H21O2 425.2341 C26H33O5 191.1435 C13H19O
420.2651 C28H36O3 151.0759 C9H11O2.

5. The compound designated LL-F282496 according to claim 1 wherein the compound has:
a) a molecular weight of 616 (EI-MS);
b) a molecular formula, C35H52O9;
c) a HPLC retention volume of 14.0 ml;
d) a characteristic ultraviolet absorption spectrum shown in Figure XX of the attached drawings;
e) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XXI of the attached drawings;
f) a characteristic electron impact mass spectrum as shown in Figure XXII of the attached drawings.

6. The compound deslgnated LL-F28249.epsilon. according to claim 1 wherein the compound has:
a) a molecular weight of 598 (EI-MS);
b) molecular formula, C35H50O8;
c) a HPLC retention volume of 14.8 ml;
d) a characteristic ultravlolet absorption spectrum as shown in Figure XXIII of the attached drawings;
e) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XXIV of the attached drawings; and f) a characteristic electron impact mass spectrum as shown in Figure XXV of the attached drawings.

7. The compound according to claim 1 and designated as LL-F28249? whereln the compound has:

a) a molecular weight of 598 (EI-MS);
b) a molecular formula, C35H50O8;
c) a HPLC retention volume of 16.0 ml;
d) a characteristic ultraviolet absorption spectrum as shown in Figure XXVI of the attached drawings;
e) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XXVII of the attached drawings; and f) a characteristic electron impact mass spectrum as shown in Figure XXVIII of the attached drawings.

8. The compound designated LL-F28249? according to claim 1 wherein the compound has:
a) a molecular weight of 612 (EI-MS);
b) a molecular formula, C36H52O8;
c) a HPLC retention value of 23.5 ml;
d) a characteristic ultraviolet absorption spectrum as shown in Figure XXIX of the attached drawings;
e) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XXX of the attached drawings; and f) a characteristic electron impact mass spectrum as shown in Figure XXXI of the attached drawings.

9. The compound designated LL-F28249.THETA. according to claim 1 wherein the compound has:
a) a molecular weight of 626 (EI-MS);
b) a molecular formula, C37H54O8;

c) a HPLC retention value of 24.5 ml;
d) a characteristic ultraviolet absorption spectrum as shown in Figure XXXII of the attached drawings;
e) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XXXIII of the attached drawings; and f) a characteristic electron impact mass spectrum as shown in Figure XXXIV of the attached drawings.

10. The compound designated LL-F28249? according to claim 1 wherein the compound has:
a) a molecular weight of 626 (EI-MS);
b) a molecular formula, C37H54O8;
c) a HPLC retention value of 26.0 ml d) a characteristic ultraviolet absorption spectrum as shown in Figure XXXV of the attached drawings;
e) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XXXVI of the attached drawings; and f) a characteristic electron impact mass spectrum as shown in Figure XXXVII of the attached drawings.

11. The compound designated LL-F28249K according to claim 1 wherein the compound has:
a) molecular weight 584 (EI-MS);
b) molecular formula: C35H52O7;
c) Specific optical notation: [.alpha.]?6=+189°- (C 0.165, acetone);

d) Ultraviolet absorption spectrum: as shown in Figure XXXIX UV (CH30H) .lambda.MAX = 241 nm (E20,400);
e) Infrared absorption spectrum: as shown in Figure XL
(KBr disc);
f) Electron impact mass spectrum: as shown in Figure XLI;
g) Proton nuclear magnetic resonance spectrum (CDCl3);
as shown in Figure XLII; and h) Carbon-13 nuclear magnetic resonance spectrum (CDCl3); as shown in Figure XLIII and described in Table IX.

12. The compound designated LL-F28249A according to claim 1 wherein the compound has:
a) molecular weight: 626 (FAB-MS);
b) Molecular formula: C37H54O8;
c) specific optical notation: [.alpha.]?=+145° (C, 0.23 acetone);
Ultraviolet absorption spectrum: as shown in Figure XLIV UV (CH3OH) .lambda.MAX = 244 nm (E30,000);
e) Infrared absorption spectrum: as shown in Figure XLV (KBr disc);
f) Electron impact mass spectrum: as shown in Figure XLVI;
g) Proton nuclear magnetic resonance spectrum (CDCl3);
as shown in Figure XLVII; and h) Carbon-13 nuclear magnetic resonance spectrum (CD-Cl3); as shown in Figure XLVIII and described in Table X.

13. The compound designated LL-F28249µ according to claim 1 wherein the compound has:
a) molecular weight: 612 (EI-MS);
b) molecular formula: C37H56O7;
c) Ultraviolet absorption spectrum: as shown in Figure XLIX UV (CH3OH) .lambda.MAX = 241 nm (E16,800);
d) Infrared absorption spectrum: as shown in Figure L
(KBr disc);
e) Electron impact mass spectrum: as shown in Figure LI;
f) Proton nuclear magnetic resonance spectrum (CDCl3);
as shown in Figure LII.

14. The compound designated LL-F28249v according to the formula II

LL-F28249v wherein the compound has:
a) molecular weight: 592 (EI-MS);
b) molecular formula: C36H48O7;
c) specific optical notation: [.alpha.]?6 + 131° (C .325, acetone);
d) Ultraviolet absorption spectrum: as shown in Figure LIII UV (CH3OH) .lambda.MAX = 256 (E20, 500); 358 (E8,830);
e) Infrared absorption spectrum: as shown in Figure LIV
(KBr disc);
f) Electron impact mass spectrum as shown in Figure LV;
g) Proton nuclear magnetic resonance spectrum (CDCl3);
as shown in Figure LVI; and h) Carbon-13 nuclear magnetic resonance spectrum (CDCl3); as shown in Figure LVII, and described in Table XI.

15. The compound designated LL-F28249.omega. according to the formula III

wherein the compound has:
a) a molecular weight of 806 (FAB-MS);
b) a molecular formula, C45H74O12;
c) a specific optical rotation: [.alpha.]?6 = -494°

(C 0.35, methanol);
d) a characteristic ultraviolet absorption spectrum as shown in Figure XV of the attached drawings;
e) a characteristic infrared absorption spectrum as shown in Figure XVI of the attached drawings;
f) a characteristic proton nuclear magnetic resonance spectrum as shown in Figure XVII of the attached drawings;
g) a characteristic carbon-13 nuclear magnetic resonance spectrum as shown in Figure XVIII of the attached drawings with significant peaks at, 220.7;

219.6; 165.2; 148.7; 133.1; 132.3; 130.2; 122.3; 100.0;
82.9; 75.9; 73.0; 72.7; 72.6; 72.1; 69.0; 67.3; 63.6;
51.4; 46.2; 45.7; 42.2; 40.4; 38.3; 37.6; 36.1; 34.8;
33.5; 30.1; 26.6; 25.4; 24.5; 23.0; 21.1; 17.9; 14.3;
14.2; 12.1; 11.5; 10.9; 8.7; 8.3; 5.7; and h) a characteristic electron impact mass spectrum as shown in Figure XIX of the attached drawings with measured m/z values and proposed elemental compositions as indicated below obtained by high resolution mass measurements, 462.3350 C28H46O5 253.1797 C15H25O3 444.3237 C28H44O4 235.1697 C15H23O2 425.2534 C23H37O7 224.1754 C14H24O2 407.2439 C23H35O6 209.1530 C13H21O2 406.3046 C25H42O4 207.1744 C14H23O
387.2895 C25H39O3 184.1458 C11H20O2 337.2010 C19H29O5 179.1048 C11H15O2 297.2031 C17H29O4 173.1205 C9H17O3 279.1944 C17H27O3 167.1051 C10H15O2 261.1851 C17H25O2 155.1069 C9H15O2

16. A process for producing agents LL-F28249.alpha., LL-F28249.beta., LL-F28249.gamma., LL-F28249.sigma., LL-F28249.epsilon., LL-F28249?, LL-F28249?, LL-F282498, LL-F28249?, LL-F28249K LL-F28249.lambda., LL-F28249µ, LL-F28249v, and LL-F28249.omega. which comprises:
aerobically fermenting the organism streptomyces cyaneogriseus noncyanoqenus, NRRL 15773 mutant or an LL-F28249 .alpha., .beta., .gamma., .sigma., .epsilon., ?, ?, .THETA., ?, K, .lambda., µ, ? and .omega. producing mutant thereof, in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic anions and cations, until a substantial amount of LL-F28249 .alpha., .beta., .gamma., .sigma., .epsilon., ?, ?, .THETA., ?, K, .lambda., µ, ? and .omega. are produced in said medium; and then recovering the agents therefrom.

17. A process for producing agents LL-F28249.alpha., LL-F28249.beta., LL-F28249.gamma., LL-F2824g.sigma., LL-F28249.epsilon., LL-F28249?, LL-F28249?, LL-F28249.THETA., LL-F28249?, LL-F28249K LL-F28249.lambda., LL-F28249µ, LL-F28249v, and LL-F28249.omega. which comprises:
aerobically fermenting a liquid medium containing assimilable sources of carbon, nitrogen and inorganic anions and cations, which medium has been inoculated with a viable culture of the organism Streptomyces cyaneogriseus noncyanoqenus, NRRL 15773 or an LL-F28249 .alpha., .beta., .gamma., .sigma., .epsilon., ?, ?, .THETA., ?, K, .lambda., µ, v and .omega.
producing mutant thereof; maintaining said fermentation culture with sterile aeration and agitation at a temperature of 24°-32°C for a period of 80-200 hours; harvesting the mash; and extracting the agents.

18. A biologically pure culture of the microorganism Streptomyces cyaneogriseus noncyanoqenus, NRRL 15773, said culture being capable of producing agents LL-F28249.alpha., LL-F28249.beta., LL-F28249.gamma., LL-F28249.sigma., LL-F28249.epsilon., LL-F28249?, LL-F28249?, LL-F28249.THETA., LL-F28249?, LL-F28249K LL-F28249.lambda., LL-F28249µ, LL-F28249v, and LL-F28249.omega. in recoverable quantities upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic anions and cations.