JPS606679A - Novel pyranone, related compounds and manufacture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel pyranone compounds, particularly K b, 6-dihydro-2H-bilan-2-one compounds and related compounds,
Streptomyces pulveraceus subsp. hostreus (
streptonyces pulveraceus
ubspecies fostreus) (ATCO 31906), a fermentation method for producing 5,6-cyhydro-2H-bilan-2-one compounds using this microbial strain, and pharmaceutical compositions containing pyranone compounds. and methods of using such pharmaceutical compositions.

The known microbial strain Streptomyces pulveraceus, deposited with the American Type Culture Collection and designated culture strain ATCC 13875, shares several characteristics in common with the 0L-1565 complex-producing strains of the present invention. have. However, the Streptomyces pulveracecus subsp. hostorex of the present invention is
OL-1565 is unique in several characteristics and its ability to produce complex antibiotics.

According to one aspect of the present invention, AT(j
A biologically pure strain of Streptomyces pulveraceus subsp. hostaceus, deposited as No. 031906, is provided.

According to another aspect of the present invention, compounds having the following structural formulas (1a)-(1c) (3a) to (3c) K are provided in substantially pure form.

(al M Alcohol having structural formula (ill), (1b) and (1c) (la) R1-HR2=OH (1t+) R1=R2-H (10) R1=R2±0H (b) Structural formula (2a) Bosphate (2a) preferably as a sodium salt or other salt with (2b) and (2c) R1=HR2-0H (21+) R1=R2=H (2c) R1-R2=OH (Q) said The phosphate functional content of the phosphate in (b) and the S formulas (3a-containing (6b) and (3c), respectively)
) with the compound 0L-1565-m, 0L-156
5-B and OL-156!1)-Lower alkyl or aryl ester of the bosphate functionality of -T (3a) OL-1565-A R1=HR,2=OH(
5b) OL-1565-B R1=R2=1 ((30)
OL-1565-T R1=R2=OH(d) Above (
the alcohol of a), the salt of the phosphate nitstool of (b), the phosphate ester of (C) and the compound (salts of (3b) and (3C) with 3a, preferably the acyl ester of the sodium salt, (e) CL-1565
- a compound designated as FT-3, and (f) said (a)
alcohol, the phosphate (b), the (cl)
, the acyl ester of (d) and the 0
Pharmaceutically acceptable forms of L-1565-FT-3.

The "acyl" used herein includes, but is not limited to, acetic acid, trifluoroacetic acid, propionic acid, n-butyric acid, isobutyric acid, -valeric acid, and caproic acid.
are linear or branched such as largonic acid, enanthic acid, caprylic acid, lactic acid, acrylic acid, pronulgylic acid, palmitic acid, benzoic acid, phthalic acid, tyricylic acid, cinnamic acid and naphthoic acid. refers to an acyl group of an acid that is substituted, saturated, unsaturated or aromatic. For the phosphate compounds of the present invention, the substituted phosphoric acid functionality may be present as the free acid or, preferably, as the IJium salt. Acceptable salts of the phosphate moiety include, but are not limited to, alkali and alkaline earth metals such as sodium.

potassium, calcium, magnesium and lithium,
Ammonium and trialkylammoniums, dialkylammoniums and alkylammoniums may be selected from the group consisting of substituted ammoniums and cetylviridinium, including triethylammonium, trimethylammonium, diethylammonium, octylammonium and cetyltrimethylammonium. By "L-lower alkyl" is meant c 1-OB alkyl, preferably methyl. The term "aryl" means phenyl or benzyl, substituted phenyl or benzyl.

Preferred compounds of the invention are as follows.

t5,6-:)hydro-6-(3,4,6,13-tetrahydroxy-6-meth-1.7,9.11-)
Ridecatetraenyl)-2H-bilan-2-one (1a
)2, 5.6-sihydro6-(3,4,6-trihydroxy-6-methyl-1,7,9,11-)lidecatetraenyl)-2H-vilan-2-one (1b)3 ,
5,6-hydro-5-hydroxy-6-(3,4,
6,13-tetrahydroxy-6-methyl-1,7,9
,11-)lidecatetraenyl)-2H-bilane-2-
on (1C) 4, 0L-1565-FT-3 sodium salt s, 4,
5. s, tt, 13--<phtahydroxy-8-methyl-9-(phosboxoxy)-2,6,12,14,1
6-octadecapentaene is the sodium salt (2C)6,
b, 8,11.18-tetrahydroxy-8-methyl-
9-(phosphonooxy) -2,6,12,14,16
7 Octadecabentaenoic acid sodium salt (2a) 7,s
, s, 11-trihydroxy-8-methyl-9-(phosphonooxine-2,6,12,14,16-octadecapentaenoic acid sodium salt a 6- (6,13-bis(acetyloxy)- 3-
Hydroxy-6-methyl-4-(phosphonooxy) -
1,7,9,11-)lidecatetraenyl]-5,6-
Sihydro 2H-bira/-2-one sodium salt 9.6-C6-(acetyloxy)-6-hydroxy-
3-methyl-4-(phosphonooxy)-1,7,9,1
1-)ridecatetraenyl) -b,s-dihydro-2H-bilan-2-one sodium salt 1α 5-(acetyloxy)-6-(6,13-bis(acetyloxy)-3-hydroxy-3 -Medel-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl)-5,6-:)hydro-2H-bilan-2-one sodium salt 1ts,s-:)hydro-6-( 4,6,13-)lis(acetyloxy)-3-hydroxy-3-methyl-1
,7,9,11-tridecatetraenyl)-7H-bilan-2-one 12,5,6-sihydro6-(3,6,13-trihydroxy-3-methyl-4-(dimethylphosphonooxy ) -1,7,9,11-tridecatetraenyl]
-2H-Pyranni-2-one In another aspect, the present invention provides the structural formula (3a
), (3b) and (3C) are dephosphorylated (dephosph6r71at
ion). The process is best carried out by reacting a pyranone phosphate with a phosphatase enzyme. The reaction is It is carried out in a neutral or nearly neutral aqueous solution at a suitable temperature, e.g. 37°C, until completion.

In another aspect, the invention comprises hydrolyzing pyranone phosphates having structures (2a), (2b) and (3c), respectively, under ring-opening conditions. The present invention relates to a method for producing a phosphate having (2c). The reaction can be carried out by treating the pyranone phosphate in an aqueous medium at ambient temperature with an alkali metal hydroxide, preferably a base such as sodium hydroxide.

In another aspect, the invention relates to structural formula (1aλ(1b)
and (1C) and alcohols with structural formula (3a
The present invention relates to a process for producing acyl esters of alcohols or pyranone phosphates, which comprises acylating the primary and secondary hydroxy groups of pyranone phosphates having λ(3b) and (3C). The reaction is suitably carried out cold using a suitable Zj acylating agent such as an acyl halide or acid anhydride such as p-bromobenzoyl chloride or acetic anhydride.

In another aspect, the present invention comprises selectively esterifying the phosphate functionalities of phosphates and pyranone phosphates. 3b) and (5C).The reaction may be carried out in a suitable solvent such as methanol using an esterification agent such as diazomethane when cold. can.

In another aspect, the present invention provides the use of an eluent that is selective for a compound to isolate 0L-1565-FT-
Production of compound 0L-1565-FT-3 consisting of subjecting the concentrate from the fermentation broth containing the compound obtained by fermentation of the 3-producing strain to chromatography and isolating the compound from the soluble nW solution. Regarding the method. For this method, a concentrate of 0L-1565-FT-3 is used. For the chromatographic separation of 0L-1565-FT-3, a reverse phase chromatography system utilizing silica gel and gradient elution using [105M (pH 7,2) phosphate buffer-acetonyl]IJ is preferred. be.

Purification of the compounds or products obtained by the method of the invention is accomplished by any suitable method, preferably column chromatography.

The present invention in terms of compositions comprises structural formula (1a), (
1b) or (1C) and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides S structures (2a), (2b)
) or (2C) and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides structural formula (2a), (2
b) or (2C) and further a lower alkyl or aryl ester of the phosphate monofunctional pyranone phosphate compound having the structural formula <3a), (5b) or (3C) and a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

In another aspect, the present invention provides #g containing structural formula Ia (1b
) or (an alcohol compound having the structural formula (
5a), (5b) or (3C) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another aspect, the invention provides compounds 0L-1565-
A pharmaceutical composition also comprising PT-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The isolated microorganism is a novel streptomysade. This microorganism was sampled and deposited with the American Type Culture Collection and the ATO
It is preserved in the permanent culture strain collection as O31906.

This microorganism was also stored in cryotubes at the Warner-Lambert/Parke-Davis Culture Laboratory in Ann Arbor, Michigan.
It is stored as a dormant culture in cold vials and soil 1'a tubes.

The culture strain ATOC3190<S is capable of producing the 0L-1565 antibiotic jjIt complex in harvestable quantities by culturing in an aqueous nutrient medium containing assimilable nitrogen and carbon sources.

A new culture of Streptomyces (Streptomyces) 51906 showed different results when grown on a thin growth medium. .

It has morphological radioactivity and coloration. 31906 (isolate 426) on various media on which Streptomyces pulveraceus was grown side by side for comparison.
This is illustrated by the following table (Table 1) of I%. In this regard, colors are given for aerial hyphae (AMλ & surface color) for substrate hyphae and soluble pigments (BP). Spore types (
Spiral/8°M curvature/F and 1α muscle/-R) and their proportions of each spore type are also given. Color hoods (shown in parentheses) are R, lck@ratrom and O-1, Foes F, published by Container Corporation of 7) Rica, Chicago, Illinois, USA.
Edited by i1roOWRHARMONY MANLIALJ
ffr! According to the color code of the 4th edition (1958).

Second allo-Streptomyces pulveracecus subplant Hostreus 3
1906 and Streptomyces bulveraceus AT
Physiological properties of O013875 Skimmed milk - Coagulation Negative No soluble dye No soluble dye Nitrimonium salt reduction 10 Positive carbon availability (+) - Culture growth (-) - No growth d
- Glucose + + 1-1 Rabinose + - Cellulose - - d-Fructose + + 1-inositol - - Inulin 10 ← Maltose +.

d-mannitol − − melibiose + + raffinose + + rhamnose + + sorbitol − − siflorose − − d-pheasant rose + + d-galactose + + salicin + + comparative control (no legomene − − production of starting material (3a), (3b) and (3cXOL1565-A
The pyranone phosphate starting materials for the process of the invention, referred to as s-B and -T) are as follows: Examples A, B%C and D [Streptomyces open isolate AT
It can be produced by culturing a CL''1565 complex-producing strain of OO31906 under artificial conditions and isolating the material thus produced.

Example A: Seed culture and shake flask fermentation A culture designated as ATOO 31906 at the dormant stage was transferred to an M-26 agar slant and incubated at 28°C for 7 to 14 hours.
Incubate for days. A portion of the mimetic growth bacteria from this slant culture medium was used to prepare ARM 1550 seed medium containing 1
Inoculate 8X150m+ seed tubes.

This tube is shaken at 24° C. for 3 to 4 days.

C Engineering M 23 Slant culture medium Amidex corn starch powder 10f N-Z amine (type A) 21 Meat extract (Difco) 1r Yeast extract (Difco) 1r Cobaltous chloride 6H200,020? Agar 20
Distilled water 1000 m Pact yeast extract (Difco) 0.5 Glucose monohydrate 0.1 Camellia starch (Difco) 2.4 Bactodorypton (Difco) 0.5 Bacto meat extract (Difco) 0. 5 0a003 0.2 (Note) Before adding 0aCO5, adjust the pH to 7 with NaOH.
．． A part of the microbial growing bacteria (Irn1) from the congenital tube was adjusted to 5 and SM
Transfer 50 mA of the 64 production medium to a flj 300 mt6 baffled, shaken Erlenmeyer flask. The inoculated flask is cultured at 24°C for 5 days while shaking using a volute shaker (2" stroke) set in ORPM. After 5 days of fermentation, the #!1 fermentation liquid is yellow. The color is brown, the outside of the mycelium is granular, and the pH of the fermented liquid is about 5.5.

SM 64 Production Medium Whey (Kroger Dairy) 35.06%
Distilled water (Note) Adjust the pH to 6.5 with sodium hydroxide Example B: Protected fermentation in a 200 gallon (757 t) sea fermenter [&
Culture] A cryo-vial containing about 1 td of culture suspension is used as the inoculate source. The contents of this cryogenic vial are completely cured.
2 and containing 500 ml of 5D-05 culture medium.
sterilely transferred to a baffled Erlenmeyer flask. The inoculated flasks are incubated for 46-48 hours at 24°C on a vortex shaker at 15 ORPM speed.

5D-5Q seed medium Amperex 101110113 (A Labs)
0.5 Glucose monohydrate (Celerose) 0.1 Dextrin-Amidex B411 2.4 (Tama @ Millet product) N-Z case (Humko 5 heffield) 0
．． 5 Spray-dried meat soluble (Daylin Labe)
[1,3CacO50,2 Distilled water After 48 hours, the same content of the seed flask was 5D-054!
l! Transfer in a sterile manner to a 50 ton stainless steel fermenter containing 16A of child medium. Inoculated one machine -0 at 24℃ 18~2
Incubate for 4 hours, test at 500 RPM and iVV
Snow ξ-ge with air at speed M.

200 gallons (757 tons) using this microbial growth side
Production of tiy) Inoculate the fermenter.

[Production Ura Moromi]

200 gallons (7
Sterilize by heating with steam for 40 minutes at 121°C. The medium is cooled to 24° C. and then inoculated with approximately 16 Lk of microbial growth from 4 6OL+el m 4S. The inoculated medium was incubated at 24°C for 5~
Ferment for 7 days with 4iL3+ air at 190 RPM and s/mige with 1 VVM air.

Antifoam agents Dow Corning C and Polyglycol P-2
000 is used to lather-t i, lia section. 0L
-1565-A, 0L-1565-B and 0L-
Production of 1565-T is monitored throughout the fermentation cycle by recording fermentation parameters such as pH and % sedimentation or growth and by high pressure liquid chromatography sweeps as described below. 200 gallons (757t) r&1
An example of a b7+ filial profile in the pancreatic organ is the following 1".

06.0 0 - 12 5.8 5.6 - 24 5.1 13.3 - 36 5.15 14.7 - 48 5.55 19.5 - 725.45 22.0 3-6 96 5.95 24.7 10~20118 7.6
5 43. ．． 5 50-65152 7.80 59.
5 60~65142 7.90 40.0 60~7
0This one cap, the gas container, will be collected in 142 hours with 140 gallons (53 (1) of 1 tank),
ijp give.

Isolation example of 0L-1565-A 0 The harvested liquid from the above d fermentation was added to 54kji of Celite 545.
and filtered through a plate and frame pressure P machine (filter press).

P liquid (4751) with HP, -2011 fat (Gillis International product) 30.5 containing 120Li
Pass through an α(0,D, ) column. The resin was then treated with water (60 st) and 90:10 water/methanol (
170t). Most of 0L-1565-Au
From A11 +1, drain with 80:20 water/methanol. High pressure liquid chromatography analysis (HPLC) of the bath PiIL liquid carried out by the method shown below. The following elution profile is typically shown.

$1-3401 <2t #2-6401 11.5f #S-64017,Of Molten solutions #h2 and #3 were separately p-condensed and lyophilized to dark brown solids 90.2f and 7a7, respectively.
Get r money. These produce qOL and dissolve in 5tK of water. Obtained solution k J! Add to methanol 271 while stirring. After standing overnight at 5°C, the mixture is washed with 5 t of precipitated methanol. P
The liquid and washings are combined, concentrated in vacuo and lyophilized to yield solid 104.5f. Mix a portion (95f) of this product in 1.5 liters of water and gradually
Add to 7t. After 1 hour, filter the strained mixture and wash the precipitate with 2 tons of 1-propertool. The P solution and wash solution were combined, concentrated and lyophilized to form a solid 57?
get. This congener is typically 0L by HPLO analysis.
-1565-A has approximately 152.

This product k 7.6 m(0,D,)'I in column
40 μm 018-silica gel coated with CR-541 (product of Analyte Chem International Co., Ltd.) 1.
2 is chromatographed above at about 1520 g.

The solution was [), 005M (pH 4,5) ammonium acetate buffer containing 5% acetonitrile.
05M (pH 4,5) ammonium acetate.

Fractions are collected and tested by HPLO.

The fractions containing 0L-1565-A 't'' were collected, concentrated and lyophilized. A portion of the filtrated product (570 mg) @ Prep-Pak soo/c
Prep LO/Elyetem with 18 columns
500 Fit (product of Waters Instrument Inc.) and 0.1 M (pH 6.5) phosphorus salt solution with 1 note of acetonitrile as a solubilizer. 0L-
The main fraction containing about 375 1565-A molecules is collected and vacuum-fed. u filled with aqueous solution in water
Pass through a column containing 200 11d of p-20 resin. Next, add resin to water 1400+17! CL 1565-A and IQ placed on the column.
i 50 methanol 3 50 t. Take a bath in f. A night in the bathhouse? True Nitrogen Shrink and Duckex 5 0X
2 (Nap') 5 through a column containing 5 ml. The resin effluent (pT 15.5) and the water wash were combined and lyophilized to produce 4-blue C! Obtain 180 tons of TJ-1565-A.

Analysis of this product typically indicates whether the product is CL-1
565-A contains about 1.5 mol of disodium per mol of free pHB. Enemy of μl theory (0IJ-1
565-A% (:'L 1565-B and CL 1ri65-T) is unstable/For a moment, this talented person's transformation is 1.0 in a good range. Sodium salt as a salt containing about 1.0 to 2.0 moles of disodium
Isolated in salt form such as l'ji'-.

Example D Filtrate (719t) produced in the same manner as described above
k 30.5 cmco, D,] Filled in tyra h t7 x 1x2 (chloride form) 3
1 pass above. Normal effluent and next water wash i is 0L-15651! i, does not contain any detectable amount of min. 0.1 as the entire fractionated total solvent system mentioned above.
M (pH 6, 8) 4141H salt EI solution CN&+)
/acetonitrile (88:12) and monitored by the HPLO method described below. Next, DOWEX 1
Eye Sebum'JkIM Sodium Chloride/Methanol (1:1
) and the eluent is collected in two 10t fractions and six 151 fractions. 0L-
1565-A%0L-1565-B, 0L-156
5-'r appears in Bath 14 liquids 2-6. These fractions are combined and diluted with 246 L of acetone.

The resulting mixture was stored at -5°C overnight. Tooru! Remove the above solution and concentrate in vacuo to 161.

Retention drying of this condensate gives a solids rating of 8002. This product C740t)? Approximately 550 tons of a similar product isolated in the same manner was added and the combined solids were added to 201 tons of water.
K#ji? do. The obtained #liquidCpH6,0) was adjusted to 15
G11CO, D,] HP-2 contained in the column
Chromatography on 0.501. water 175
The solubility of HP-20 column by t is mostly CL1
561-T'f: Remove. Most of the aL-1565-A component was eluted with 100 L of methanol/water (15:85), CL 1565-B and the remaining 9 OL-15
Is 65-A clean with 83L of methanol/water (50:50)? asanru. CL 1565-AKI;
t'11t: Combine the liquids, concentrate and freeze-dry to obtain a solid. By this solid-state FiHPLO analysis, CL
Contains approximately 110ft of 1565-A.

Part 752 of this product k O,05M (pH 6,8
) in 2 L of phosphate buffer and further 15 c+a (0
,D,) Purified by chromatography on a 100 μm C18 reverse phase column packed in 0.05 M (pH 6,8) phosphate buffer (Na+), Lycagel (product of Analytichem International Inc.) 52T (25 kP) do. The column is developed with a 0.05M phosphate buffer solution containing an increased ff (4.0-6.5%) of acetonitrile. The initial fraction contains all CL 1565-T. 0L-15
65-A is eluted in the next fraction. the only U
The fractions containing CL 1565-A v as the V absorption component are collected and concentrated in vacuo to 20t.

The concentrate is stored overnight at 5°C and the precipitated inorganic salts are filtered out. Next, the P solution was packed onto a 1511+111 [0, D,] column containing 28 tff of total HP-20 resin.

Wash the oil with water (66/,) and then (3L156
5-A is eluted with methanol/water (50:50) 42A. Most of 0L-1565-A contains r+41'
cL-15 containing some inorganic impurities was concentrated and dried for 1" h.
65-A is obtained. 1; 1 (machine impurities are product t methanol (50~100+'1g/me) K dissolved, all non-cd properties and suga removed, and actually transferred to the condensed area where it is vacuum concentrated. It can be removed by adding water and returning the P solution along the water.CL
A final 4ii preparation of 1-565-A is carried out by chromatography of the resulting aqueous ('AM) product on HP-20 resin.

(Properties of 3L 1565-A sodium salt UV absorption in Me'OH is 268 nm (a knee 805), 259 and 278 nm)
Main absorption of infrared absorption in KBr: 3400% 1710, 1630.14
20, 1387, 1260 ti155.1090
, 1060.975 s 920.820 and 775
(X-1 Optical rotation 3 [□n +2a26 (1.0% in 0-IM pH7mW@class buffer solution) Elemental analysis value (C19H2770I oN
&1.3P (for K) 0% H% Ha% P easy calculation value: 47. B4 5.86 6.27 6.49
Experimental value: 4B, 01 5.8B 6.05 6.3 Calculated value for 10 (by fast atom bombardment) (01gH25Na209P+H) -
'titkAij, -IQ/Z 455 experimental value for rr4/z475(01gH26Nao9P+H)+:
m/z 475.453 560 MHz proton magnetic resonance spectrum in D20 resulting signal (s = single line, d - double b'';, 1.'
e=Mie S, m=Multiple 49): 1.29s (5H)
, 1.5f3t (IH), 1.70m (IH).

2.49-2.58m (2H), 4.13-4.18
m (6H), 4.86t (IH), 5.19m (1H)
, 5.53t (IH), 5.9~6.0m (4H),
6.14t (IH), 6.32t (1H), 6.55t
(1)1), 6.75ad (1)1) and 7.09m
(IH) [ppm downfield from sodium 2.2-dimethyl-2-silasuntan-5-sulfonate (ms)] 1 in D20
30-Nuclear magnetic resonance main signal: 1 16a4 12 79.5 2 149.8 13 79.0 5 13a114 75.6 4135, b 15 64.4 5 134.4 16 62.7 6s 131.3 17 39.4 7 127.4 18 29.7 8 126.7 19 25.5 The 51p-core resonance in D20 is 85% bi
A doublet (J" 10 Hz) is shown at 0.504 ppm downfield from .

High pressure liquid chromatography column: fi BOndapakool B silica gel (3,9 craft, D, : UV absorption retention time at 254 nm: 2.8 min.
Careful chromatography of the concentrate obtained from the IL15651J# solution.
A fraction containing the L1565 component is given. (3
L1565-B and CL 1565-T are presented as essentially pure compounds. CL j565 components A, B and T are % 0, 05 M to 0 containing various proportions of acetonitrile at a flow rate of 1.5 g/min
．． μBondapak (
FDolB-Silica gel color A (!L9111 engineering, D,
X3Qcm) and detection by ultraviolet absorption at 254 nmK. Above H
Typical retention times for OI+ 1565-A%B and T using all PLO conditions are shown in the table below.

OL 1565-T 2.8 < 1.5, (3L15
65-A 4.3 <1.5 (3L 1565-B >
15 4.2 The solvent used in the crude fermentation liquid is a 1M
(pH 6,8) phosphate buffer/acetonitrile (88:
12) can be tested by the above method.

In this case, 2#+7! C at a flow rate of /min
L 1565-T% CL 1565-A and CL
The retention time of 1565-B is 2.7 minutes, 5.0 minutes.
minutes and >12 minutes.

CL 1565-T eluted faster than CL 1565-A from HP-20 resin and from reverse phase silica gel.

It can be isolated from the initial fraction of the 018-silica gel column described in the example above using % HP-20 resin.

(L 1565-B was gradually eluted from HHP-20m oil and reversed phase silica gel. CL 1566-A was gradually eluted from HHP-20m oil. -20 chromatography was eluted with 50% methanol during the run. This component was rechromatographed on HP-20 using essentially the same procedure as described for the 417 product of CL 1565-A. It can be isolated very well by chromatography followed by chromatography on 40 μma 1B-silica gel.

Properties of the CL 1565-T sodium salt UV absorption spectra in MeOH are almost identical to those for the CL 1565-A sodium salt.

λmax 268nm (a'-774), inflection at 260 and 278nm.

Infrared absorption spectrum main absorption in KBr: 5400.1715.1630, 13
80, 1260.1090, 970.81 and 7
70% mass spectrum (by fast atom bombardment) Total value for (019H25Na201 Qp+)1)+ = m7'z491 Experimental value: m/z 491 560 MHz proton magnetic resonance spectrum in D20 IH-NMR of aL1565-T The spectrum shows one proton signal of 1, which appears as deL at 4.34 ppm and DS
Downfield L7'c2.2~'5.21 from C3
) 1) % except for A where there are no n signals between m
The I H-NMR spectrum of CL 1565-A is very similar to that of Cutol.

Main signal of CL 1565-T sodium salt (8
- single doublet, d- doublet, seven- triplet, m- multiple ia)
: 1.30s (3H,), 1.55-1.64m (
IH), 1.73t (IH), 4.15-. 4.20n
(I H), 4.16d (2H), 4.34dd (I
H)% 4.94t(IH), 5.09dd(IT()
% 5.55t (IH), 5.89~6.06m (3H
), 6.16m (2H), 6.36t (IH), 6.
56t(IH), 6.76ticl(IH)%7.14
dcL (IH) (pp downfield from DBS
m).

90.4 Mn21SC4 magnetic resonance spectrum in D20 i 24.10 11 126.91 2 41.60 . 12 127.183 64.68
13 128.99 4 64.90 14 155.56 5 66.67 15 136.87 6 7&28 16 167.25 7 79.81 17 142.27 8 84.55' 18 149.469 124.4
0 19 169.6610 126.21 Shin Down fee from TM8 k l' L TapppmC
Properties of L 1565-B sodium salt Ultraviolet absorption spectrum in MeOH λmax 267 nm (a knee 805)% 259
Infrared absorption spectrum in KBr inflected at 277 nm and main absorption: 1720.1640.1385.120
360 MHz proton magnetic resonance spectrum in 0% 1060°970 and 820α-1 D20 Main signals (S-single line, d = doublet, t = triplet, m-multiffl+li!): 1. ! 128 (3H
), 1.58t (IH), 1.72t (IH), 1.7
9cl(3H), 2.45=268m(2H)%4.1
5t (IH), 4.89t (IH), 5.10m (IH)
), 5.49t (IH), 5ft 6.21m (6H),
6.50~6.64m (2H), 7.06~7.13
m (IHXDBS-7'-/ra downfield p
pm).

90.4 MHz j504 Magnetic Resonance Shaktor 1 in D20 20.70 11 127.24 2 25.06 12 129.47 3 31.91 13 129.90 4 41.85 14 134.66 5 66.85 15 135. 94 6 77.87 16 1.56.677 8α87
17 14α42 B 81.64 1B 152.01 9 122.41 19 170.5610 124.
45 Umbrella TM day to downfield L fc ppm5.6
-Sihydro6-(5,4,6,15-tetrabitroxy-6-methyl-i,7.9.ii-)ridecatetraxnyl)-2H-vilan-2-one (OL-1565
-A alcohol) (1a) 5,6-sihydro-6-(3,6,1
3-trihydroxy-3-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl)-
Sodium salt of 2H-pyran-2-one (CLl 56
5-Sodium salt of A(3a)) 1.4 f Oh! Alkaline phosphatase derived from bovine intestinal mucosa (
Sigma Chemical Company, Lowys, Missouri =-)1.
Incubate at -37°C for 7 hours. The reaction mixture (pH 7.2) is then lyophilized and the scraped residue obtained is triturated with methanol. The methanol soluble product is chromatographed on C8 reverse phase silica gel. After water washing, CL 1565-A-alcohol is eluted with water/acetonitrile (85:15). These latter fractions were combined, concentrated and lyophilized to give a white surface (1: LTE CL 1565-A-7 #Col A 6
Get 2-9. CL 1565-A-alcohol was prepared using HPI, O, which mimics the HPLO operation described in Example 2 below.
It can be detected in the fermentation liquid by using @.

CL 1565-A Alcohol properties Ultraviolet absorption spectrum in methanol λmaz' 268 nm (a knee 975), 259
and inflection elemental analysis value at 278 nm (C!19H260
0% H% calculated value: 6551 7.52 Experimental Ili: 63.41 7.51 Infrared spectrum main absorption in ouczB: 1720% 1600, 1285 and 1060 on silica gel-60 mIf of
1 Chromatography solvent: Chloroform/ethanol/, 0.5 M (pH 5,5) sodium acetate buffer (40 Nia 0:20) used, Rf: 0.8 Solvent: chloroform/isopropa/-/8:2) Used, Rf: 0.19 Solvent: Chloroform/methanol/water (75:25:1
) Use, %: 0.60 [IPLO (see Example 2) 360 MHz proton magnetic resonance spectrum main signal in 020 (B-single line, d-doublet, 1-triplet,
m2 multiplex #): 1.4os(a), 1.47m(
IH)% 1.87m(1), 252-2.71m(2
H), s, aiaat IH), 4.27a (2u)
, 4.95t (1H), 5.13m (IH), 5.65
t(1H), 5.95r-6,15m(4H)%6.2
0 t (IH)t 6.44 t (IH)a 6
．． 61t (IH), 6.89del (IH),
7.15m(IH) (sodium 2,2-dimethyl-
ppm downfield from 2-silapentane-5-sulfonate (DI3Ei)) 90.4 MHz 13C-Nuclear Magnetic Resonance Spectrum in D20 1 24.8 11 127.0 2 31.9 12 12 & 8 3 41.4 13 129. 8 4 64.9 14 13i6 5 67.3 15 157.5 6 76.4 16 137.4 7 77.9 17 140.4 8 B1.5 18 151.9 9 122.4 19 170.5 10 126. 8 (Note) *50-2.5 from #j cell toxicity against PTlm L 1210a cell downfield from tetramethylsilane
/jg/mt Example F 5.6-sihydro-6-(3,4,6-dolihydroxy-6-methyl-1.7,9.11-)ridecatetraenyl)-2H-vilan-2-one (CL 1565-
B-alcohol) (1b) OTJ 1565-B-alcohol is OL 1565
-A- Produced in a similar manner to that used for the production of alcohol. CL 1565-B sodium salt (10 my) is dissolved in 5 td of water and 10 η of this fLK alkaline phosphatase (Calbiochem Behring) is added. The resulting solution is stored at 37°C for 14 hours. The reaction mixture is then lyophilized and the remaining solid is triturated with methanol. Concentrate the methanol solution to obtain 211 g of CL 1565-B-alcohol.

OL 1565-B Alcohol Properties Merck Thin Layer Chromatography on Silica Gel Solvent: Chloroform/Isopropa/-M2O: 20) Rf: [L
38 High pressure liquid chromatography column: Liahrosorb RP-
8 (Prownley Laboratories product) Solvent: Water/acetonitrile (80:20) Flow rate: 2-7
min Retention time: 27.0 min Using the same HPLO conditions, the retention times for CL 1565-A-alcohol and CL 1565-B are 53 min and 1.0 min, respectively.

Example G 5.6-sihydro-5-hydroxy-6-(3,4,6
゜15-tetrahydroxy-5-methyl-1,7,9,
(11-tridecatetraenyl)-2H-vilan-2-one (CL 1565-T alcohol) (1c) A solution of 10 mg of CL 1565-T sodium salt and 7.59 g of alkaline phosphatase in 1-1 water at 67°C for 18 Store time. The reaction mixture is then lyophilized and the residue is triturated with ethanol. The ethanol is removed in vacuo to yield a residue containing OL' 1565-T-alcohol.

9L 1565-T Alcohol properties Thin layer chromatography on silica gel GHLF (Analtech product) Solvent: Chloroform/methanol/water (75:25=1
) Rf: 0.44 Using the same TLO conditions, the observed Rf of CL 1565-T sodium salt is α02.

Example H OL 1565-FT-3 Sodium Salt CL 1565
-FT-Fffl produced from 5-producing microorganisms
Ferment? '[(7191)'t 30.5 cm (0, D,
) Dowex 1×2 (chloride form) 51 packed in a column is passed over. The effluent and the next wash are CL 1
It does not have any detectable amounts of the 565 components. Next, DOWEX-14! The H fat was eluted with 1M sodium chloride/methanol (1:1) and the eluent was diluted with two 10
Collect in the t fraction and six 154 fractions. Most CL 1565-A, CL 1
565-B, OL' 1565-T and additional small amounts of CL 1565 components appear in the 2nd to 6th eluents. Combine these fractions and add 24 ml of acetone.
Dilute with 6L. The resulting mixture is stored at 5°C overnight. The clear supernatant solution is removed and concentrated in vacuo to 16t. This concentrate was lyophilized to a solid of 800 t'fl.
:obtain.

To this product C740t)' is added 552t of a similar product separated from jP in the same way and the combined solid is dissolved in 20t of water. The resulting solution (pH 6,0)'e 15t!
HP-20 resin contained in the (0, D,) column 5
0 is chromatographed above.

HP-20 column was eluted with 175t of water and most of the 0L
-1565-T and CL1565-FT=3 and C
Remove all small amounts of highly polar CL 1565 components, including TJ1!565-0 (2C). The 7 lactations containing these components are combined and concentrated in vacuo.

This concentrate was transferred to 135 μm C2-reversed phase silica gel (MC
Chromatography on Merck RP-2) 31y, manufactured by Company B. This column was 0.05M (pH 7,
2) Elution with phosphate buffer to obtain a fraction containing CL 1565-T and some minor components as determined by HPLC analysis. This fraction was concentrated to 40 μm cB using a gradient elution system starting with 0.05 M (pH 7,2) phosphate buffer and ending with 0.05 M (pH 7,2) phosphate buffer/acetonitrile (95:5). - Rechromatography on reverse phase silica gel (Analytechem International product). Before CL 1565-T is eluted, three minor 1565 components are eluted in separate groups of 7-ractions. One of these hook johns is CL 1565-
C! ' Contains. Its isolation was described in Example 1. The component eluting in the last (third) group of these fractions is designated CL 1565-PT-5.

This compound is isolated by concentrating the combined CL 1565-PT-3 fractions and then immersing them in ethanol.

The precipitated inorganic salts are removed and the p solution is concentrated to dryness. The residue was dissolved in ethanol and (L1565-PT-
! The sodium salt is precipitated as a white solid by addition of ethyl acetate.

Ultraviolet milk yield spectrum of CL 1565-PT-5 sodium salt in methanol λmaz 269 n
Infrared spectrum resulting in absorption in KEr inflected at m%, 259 and 278 nm: 3400% 1750% 1640
, 1175, 1060 and 983 cm-1 In vivo activity against P588 lymphocytic leukemia in mice Use 1p% T10X 100-
1 s.

High pressure liquid chromatography column Nichrome Megabond C-18,4,6 Fighter, D,
×30cIn(NJ%r-Luton(D Be i:y
Solvent: 0. IM pH 7,2 phosphate buffer - acetonitrile (88:12) Flow rate: 2-7 minutes Detection: Ultraviolet absorption at 254 nm Retention time: 1.69 minutes Example: 4.5, 8.11.18-Pentahydroxy- 8-methyl-9-(phosphonooxy)-2,6,12,14,
16-octadecapentaenoic acid sodium salt (CL 1
565-C Sodium @) (2c Sodium Salt) Example 4
The shoulder-containing fraction of OL 1565-0 described in 1 was concentrated and purified to 40 μm 01B-reversed phase silica gel 1 using Q, 05M (pH 7,0) phosphate buffer as eluent.
Chromatograph on 40v. HPLO analysis of the collected fractions shows that CL1565-0 is easily eluted. Fractions containing CL1565-0 't' were collected (total volume 220 d) and concentrated to 1
2d and 0.05M (pH 7,1
) Using phosphate buffer't-018-reversed phase silica gel 1
Re-chromatograph on 401. the only UV
% g of solution containing CL 1565-0 as absorption material
! Sweat 41 and lyophilize. Product (6,55F
)' in 7 d of water and desalted on 018-reversed phase silica gel 1402 using water as eluent. C.L.
Fractions containing all 1565-0 'ff' are combined and lyophilized to yield CL 1565-0 sodium as a white solid.

Properties of CL 1565-0 Sodium Salt Ultraviolet absorption in methanol λmax 269 nln s 259 and 278
Infrared spectrum principal absorption in KBr inflected in nm: 3400.1560 and 1650°-
Of 1D20! 160 MH2IE-NMR main signal (6-single line, d-triplet, a-triplet, m=multiplet): 1.30s (3H), 1.55
~1.69m(IH)% 1.73t(IH), 4.0
3-4.20m (4H)% 4.68t (IH), 4.
94t (IH), 5.55t (1H)%5.65~5
．． 85m (3H), 5.85-5.95m (2H), 6
．． 16t (IH), 6.36t (tH), 6.56t (
IH) and 6.76 da (1H) (ppm downfield from DiB), high pressure liquid chromatography column: Chromebond C-18,4,6 Fighter, D,X
3QCm (supplied by ES Industries, Marlton, NJ) Solvent 2Q, 1M pH 7, 2 phosphate buffer-acetonitrile (88:12) Flow rate: 2g/min Detection: UV absorbance at 254nm Retention time: 1.69 min Same HPLO Using the following conditions, the retention times were observed.

CL 1565-FT-3 retention time is 2.20 minutes C! L
1565-T Retention time - 3.05 minutes CL 1565
-A Retention time = 4.94 min Example Preparation of CL 1565-0 sodium salt from J CL 1565-T CL 1565-T sodium salt (22J1
Adjust the solution in g) to pH 11 with 0.1N sodium hydroxide. After 2 hours the pH is readjusted to pH 11 and the reaction mixture is stored at 5° C. overnight. The bath solution is adjusted to pH 7 and lyophilized to obtain a white solid containing the OL' 1565-0 sodium salt. This was isolated using the procedure described in the previous example. As seen by HPLO comparison with a sample of L 1565-0 sodium salt.

High Pressure Liquid Chromatography Column: 11 Bonclapak 01 B-Silica Gel (6,9 Fighter, D, UV absorption retention time: 1.23 min Using the same sample, CL1565-
The retention times of 0 and 0L-1565-T were 1.
22 minutes and 2.50 minutes.

Example L 5.8.11. i3-tetrahydroxy-8-methyl-
9-(phosphonooxy) -2,6,12,14,16
- Octadecapentaenoic acid sodium salt (CL 156
5-D sodium salt A) (2a sodium salt) CI, 1
565-A sodium salt (100al?)'e water 50
H solution to 1Bt. Did you get it? : Makes the solution diluted) I
Adjust the pH to 11 with Jum and store at 5°C overnight.

The reaction mixture (pH 8.8) is again adjusted to I)Hll and stored at 5°C overnight. I) After adjusting to H6, the solution is lyophilized to obtain a white solid containing CL 1565-D sodium salt.

Properties of CL 1565-D sodium salt Ultraviolet spectrum in methanol λwax 268 nm % 259 and 278 n
Infrared spectrum principal absorption in KBr inflected at m: 3400% 1650.1560.1
435% 1650.1090 and 97 [1 clL-1 High pressure liquid chromatography column: μBondapalc 01B-silica gel (4,6w engineering, D, 8) Flow rate: 2d/min detection"UV absorption retention time at 254nm [about 2.0 minutes Using the same conditions, the retention time of 1.OL 11ss-h is about 4.0 minutes.

Example M s,a,ii-)dihydroxy-8-methyl-9-(
CL 1565-B sodium salt (51) sodium Departing with salt) and CL
1565-]! monosodium salt (sodium salt of 2b) can be produced.

Example N 5.8,9. it, i8-bophthahydroxy-8-methyl-2,6,12114116-octazocapene sodium citrate CL 1565-A alcohol (1t113
mg) solution i1N sodium hydroxide at pH 10.5
Adjust to. After standing for 30 minutes at room temperature, the reaction mixture C
pHa2) Re-adjust to pH 11.4 with e1N sodium hydroxide. After 4 hours, the solution (pH 92) was completely lyophilized to give 5,8,9,11.18-pentahydroxy-8-methyl-2,6t12. A solid product containing F4.16-octadecabontaenoic acid sodium salt is obtained.

Infrared spectrum principal absorption in KBr' 3400 s 2920.1590 (0
02, 1590 and 1060cIIL-1 High pressure liquid chromatography column: Whatman Verticil 10oDEJ-5 (0
-18 silica gel) Solvent: Water/acetonitrile CB: 2) Flow rate = 2-/min Detection: UV absorption at 268 nm Retention time: 0.90 min Using the same conditions, the retention time of the starting material is 592 min.

Thin chromatography on silica gel 60 F254 (E, Merck) Solvent: chloroform/isopropanol (8:2) Detection: under ultraviolet light and sprayed with a solution of 5% sulfuric acid tBltA in 6N sulfuric acid and at 110° Observe Rf by heating for 10 min at α0. Using the same system, the starting material is detected at R of α2.

In the same method, starting with compound (1b) and compound (18)'e respectively, 5, 8, 9. ti-tetrahydroxy-8-methyl-2,6,12,14,1
6-octadecapentaenoic acid sodium salt and 4.5
, 8,9,11,16-hexahydroxy-8-methyl-2,6,12,14,16-octadeca can prepare citrate sodium salt.

Example 0 6-[6,13-bis(acetyloxy)-6-hydroxy-3-methyl-4-(phosphonooxy)-1-7
t91it-)lidecatetraenyl)-516-dihydro-2H-bilan-2-one sodium salt (cL1
s4s-A diacetate sodium salt) pyridine (α3
-) for 5 hours at 5°C in the presence of acetic anhydride (0,6
OL 1565-A sodium salt (30 µ) is acetylated by treatment with m). The volatile components are removed in vacuo and the residue is dissolved in 50% bicarbonate solution and chromatographed on HP-20 resin 20d. CL 1565-A diacetate sodium salt 21 was eluted with methanol/water (70:30).
Get wg.

Properties of CL 1565-A diacetate sodium salt Ultraviolet absorption spectrum in methanol λmaX 268 nm (a:=650) % 25
Thin layer chromatography on silica gel 60 F254 (1, Merck) inflected at 9 and 278nn+ Solvent: Chloroform/isopropanol (8:2) Rf
:0.07 Solvent: Chloroform/methanol/iN NH40H (
25:50:4) Rf: 1lL91 Using the latter solvent, the Rf of CL 1565-A sodium salt is (L27).

50"" Approximately 6 μg/mL from cytotoxicity to It1210 cells High-pressure liquid chromatography column: μEondapak 0-18 (Waters Assoc) Solvent: (105M (pH 6,8 fan salt buffer/acetonitrile (80:20)) Flow rate : 1.5 wt/min Retention time: 19 minutes Using the same HPLO conditions, the retention time for CL 1565-A is 2.6 minutes.

560 MHz proton magnetic resonance spectrum main signals in D20 (8 = single line, d - doublet, 1 - triple i
, Bm multiplex iiM): 1.40g (3H), 1
．． 95t (2H), 2.07s (3H), , 2.09
g(3H)%2.43-2.66m(2H), 4.25
t(1)1), 4.65d(2H), 5.10m(
IH), 5.51t (IH), 5.74m (1H), 5
．． 87~6.08m (4H), 6.19t(1)1)
, 6.39t (IT(), 6.64tC1H), 6.
81dd (1H), 7.10m (1H) (DI38 down; feet+l.

Example P 5-(acetyloxy)-6-(6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(
Phosphonooxy) -1,7,9,11-)lidecatetraenyl) -s,b-dihydro-2H-pyran-2-
O'L1565-T sodium salt (CL 1565-T diacetate sodium salt) A solution of O'L1565-T sodium salt (30*y) in acetic anhydride (0,6d) and pyridine (o,5d) was dissolved in 5" After removing the volatile components, the residue 't5% (W/L) is dissolved in sodium bicarbonate and chromatographed on up-20*fat 20+d. Methanol/water (50:30 ) to elute g and concentrate in full vacuo to give CL 1565-T ) lyacetate 1289.

CL 1565-T) Properties of lyacetate sodium salt Ultraviolet spectrum in methanol λmax 268 nm s 259 and 277 n
90 MHz proton magnetic resonance spectrum main signal in inflection D20 at m (e - single line, d = doublet, t =
triplet, m-multiplet): 1.4s (3H), 1.
5-2.1m(2H)%2.03+3(3H)%2.
05s (3H), 2.09s (3H), 4.05~4
．． 45i (IH), 4.65d (2H), 5.15-7
．． 2m (121) (1) Pm downfield from DSS) Thin layer chromatography on silica gel 60 F254 Solvent: Chloroform/methanol (6:4) Rf: 0.
57 In the same method as described above, 6-(6-acetyloxy-5-hydroxy-6-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl)
-5,6-hydro-2H-bilan-2-one sodium #7ACCL 1565-B acetate sodium salt) can be prepared.

Example Q 5.6-sihydro6- (4,6,13-)lis(acetyloxy)-3-hydroxy-5-methyl-1,7
,9,11-)lidecatetraenyl]-2H-bilane-
OL by treatment with acetic anhydride (0,6Int) for 5 h at 56 in the presence of 2-one pyridine (0,3d).
1565-A al:=y-ru(3Q ag) f
7 Cetylate. The reaction product, free of volatile components, is then chromatographed on silica gel (60-200 μm) using 5% methanol in chloroform.

Concentrate the 5% methanol in chloroform eluent to CL
1565-A-alcohol triacetate (35II
g) is obtained.

1565-A-Alcohol triacetate properties Thin layer chromatography on silica gel 60 F254 (E, Merck) Solvent: Chloroform/methanol (95:5) R (:0
．． 43 Solvent: toluene/acetone (8:2) Rf: 0,21 The 360 MHz-j roton magnetic resonance in cDct5 is the main signal of the cuttle (e = single-rJl, d-211, 13 weight hrn = poly-TJffjJ ): 1.54s
(3H), 1.83-2.18m (2H), 2.07s
(3H), 2.15s (3H), 2.14s (3H),
2.45-2.55m (2H), 4.69d (2H),
4.98-5.09m (2H), 5.47t (IH)
, 5.75m (IH), 5.85~6.05m (5H
), 41ta (1H), 6.17t (IH), 6.44
t(1H), 6.57t(IH), 6.77tid(I
H), 6.95m<1H) ('Pl'm downfield from tetramethylsilane) chemical ionization (OH
4) Mass spectrum m/Z (% of base peak) 477(M-)-H, 10), 459(4), 417(
40), 399(13), 373(29),! 157 (
100), 359(19), 513(79), 297(
64), 279(12), 253(20) Example R 5,6-sihydro6- (4,6,13-tris(4
-bromobenzoyloxy)-5-hydroxy-5-methyl-i e 7 e 9111-tridecatetraenyl-2H-bilan-2-one [0L1565-A°alcoholtri(4-bromobenzoate)] Excess p
-bromobenzoylchloride F"t is added to a solution of 20xy of OL 1565-A alcohol in 1 me of pyridine. After standing at room temperature for 48 hours, the pyridine is removed in vacuo and the residue @oa2at2(10-) and saturated NaH
Allocate between OO3 (10Nt).

The an2cz2 extract is washed with H2O (10 m) and then evaporated to dryness. Chromatography of the residue on silica gel yields CL 1565-A-alcoholtri(4-bromobenzoate) (19).

Properties of CL 1565-A alcohol tri(4-bromobenzoate) 9 Q MHz proton magnetic resonance spectrum in CDCl2 Main signal (s-singlete, d-doublet, a-triplet, m=multiplet) : 1.56s (3H), 1.9
~2.4m (4H), 4.85 (1 (2H), 5.1~
5.4m (21)% 5.45-5.8m (2H), 5.
82-6.12m (5H), 6.1E) 4.9m (4H
), 7.35~arm(12H) Thin layer chromatography on silica gel Solvent: 0HO13/Isopronozonol (4:1) Rf: 0.69 Example 5.6-Sihydro6- (3,6,13- Trihydroxy-6-methyl-4-(dimethylphosphonooxy)
-1,7,9,11-)lidecatetraenyl]-2H-
Bilan-2-one (CL 1565-A dimethyl ester) CL 1565-A f ) lium salt (25Q) dissolved in t-methanol 1 d and with stirring at 0'' Dowex 50X 2 (hydrogen form) 1 d and methanol 15
-Add a mixture containing. A solution of diazomethane in ether was prepared directly and after 3 minutes the yellow solution was decanted from the Dowex resin and concentrated to dryness in vacuo. The remaining solids were triturated with chloroform and the chloroform-soluble material was dissolved in chloroform/methanol (8
:2) Elimination by preparative chromatography on silica gel using t. Main tff
The absorption band was removed from the silica gel plate to Or, +1
565-A dimethyl ester 9.1 is obtained.

Properties of OL 1565-A dimethyl ester Thin layer chromatography on silica gel 607254 (Et, Merck) g agent: / loloform / methanol (8:2) Rf: 0
．． 49 UV radiation in methanol has an inflection a at 268 nm, 259 and 278 nm.
x Main absorption of infrared rays in oaazB: 5400% 1725, 1605
% 9Q MHz proton magnetic resonance spectrum in 110% 105D and 1020 cIL-1 aDatB Main signals (s = singlet, d-doublet, a-triplet, m611 multiplet): 13s (3H), 1.55
~1.85m (2H), 235~2.55m (2)I)
, &78a (5H), &91a (5H), 4.23cL
(2H), 4.35-5.10m (3H), 5.5-7
．． 0m (10H) (ppm downfield from tetramethylsilane > Pharmacological A, Animal Tumor Activity CL 1565-A natrivime salt used in the National Cancer Institute's Cancer Therapy IIP screening program) It exhibits antitumor activity in animal tumor models.The results are shown in Table 3.

CtL 1565-A sodium salt is used for murine leukemia L
1210 significantly prolonged the survival time of mice inoculated with 1210 and is also therapeutic when evaluated in this tumor mouse model as seen from the activity data summarized in Tables 4 and 5.

Table 4 Mouse leukemia P3888 X Mouse leukemia L1210 XX (curable) Mouse MI58

In case of mouse mammary CDQFI, treatment is 64 days after tumor transplantation.
Treatment started on ffllc in 1 day, which means the tumor is at an advanced stage. The symbols in the table are explained as follows.

-X- Shows borderline activity.

×- shows activity. Early endpoints are not significant and show statistical significance.

XX = exhibits significant activity and produces a certain percentage of "healing". A strong endpoint indicates sufficient anti-tumor selectivity to warrant consideration for development into a clinical trial.

N〇- shows no activity.

Table 5 1 6.25 1 day x9 207 0/6°2 12.
50 1 day x 9 337 3/66.25 1 day x 9
194 1/6 3L12 1 day x9 191 0/6 3 12.50 1 day x9 246 0/6f6.25
1 day x9 216 Q/6 &12 1 day x9 195 0/6 1.56 1 day x9 150 0/6 4 12.50 1 day x9 268 o/'/) g6.
25 1 day X 9 236 1/6 512 1 day Surviving Animals at the Completion of the Observation Period d) Tumor Reduction - Evaluation of the reduction in tumor cells that can grow below the tumor present immediately before treatment.

If the Log1o cell death value is positive, fewer cells are present at the end of treatment than at the beginning. If negative, the tumor will grow under treatment.

e) Animals surviving 30 days are considered treated. f) Observation period B of 45 days. The active KDso (50% inhibitory concentration in tissue culture) in a cyst culture is determined for different single loci. The data are shown below.

Lewis Lung Cancer 1.3 L1210 Leukemia 0.15 HOT-8 Human Colon 1fIIt 6 M0A-518 Mouse Tumor & Cancer 1.1 Pyranone Compounds of the Invention, Particularly Alcohol Compounds Having Structural Formula (1a) and CL 1565-FT-5 c+IJ15 in the form of the IJium salt
65-A diacetate has anti-lung tumor activity. For example, these compounds are active in vivo against P3881J/sensitivity leukemia or in vitro against L1, 210 murine leukemia cells or human colon adenocarcinoma cells. The use of the compounds of the invention is therefore contemplated for anti-malignant activity as active ingredients in pharmaceutical compositions. When utilized as cytotoxic or anti-leukemic agents, the compounds of the present invention can be prepared and administered in a variety of use forms, particularly parenteral use forms. As will be clear to those skilled in the art, the use forms may have one or more compounds of the invention as active ingredients.

Compounds of the invention may be administered parenterally or intraperitoneally. Solutions of the active compounds in suitable salt or non-salt forms can be prepared in aqueous vehicles optionally using solubilizing agents or surfactants such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile injectable solutions or dispersions.
include. In all cases, this form is sterile
It must be liquid and must be liquid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene gelyl and liquid polyethylene glycol), N,N-dimethylacetamide, suitable mixtures thereof and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, rubic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of the injectable compositions is brought about by the use in the compositions of additives that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by mixing the active compounds as required in the appropriate solvent with various of the other ingredients enumerated above and then sterilizing by filtration.
Ru. Generally, dispersions are prepared by incorporating the sterile active ingredient into a sterile vehicle containing the basic dispersion medium and the required other ingredients as described above. In the case of sterile powders for the production of sterile injectable solutions, preferred manufacturing methods include vacuum drying and freeze drying techniques which yield powders of the active ingredients and other desired ingredients forming sterile supersolutions as described above. be.

Pharmaceutically compatible carriers as used herein include any solvents, dispersion media, coatings, anti- and anti-fungal agents, isotonic agents and absorption delaying agents. The use of such media and agents for pharmaceutically active substances is known in the art. Its use in therapeutic compositions is contemplated, except where the underlying medium or agent is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate all parenteral compositions in unit dosage form for ease of administration and uniformity of use.

As used herein, the unit usage form means a physically discrete unit suitable as a unit usage shield for the mammalian subject to be treated. Each unit of L represents a total amount of active substance calculated to provide the desired therapeutic effect in association with the required pharmaceutical carrier. Specifications for the novel unit dosage form of the present invention are: - the %Ag% of the active substance in contact with the active substance; and the specific therapeutic effect achieved; The use of compounds such as -M as active substances for the treatment of leprosy in organisms with the diseased state is of great benefit.

The main active ingredient may be formulated in effective amounts with a suitable pharmaceutically acceptable carrier in unit dosage forms as described above for convenient and effective administration. Unit usage forms contain, for example, amounts of the principal active compound ranging from about 10 to 500 cm.

Approximately 25-2001 g is preferred. In terms of proportions, the active compound is generally about 10 to 500
Present in IIg. In the case of compositions containing supplementary active ingredients, the amounts used will be determined by the customary usage and method of administration of said ingredients. The daily parenteral use f for the mammalian subject being treated is 0.01 Qlk
g ~10 Qlkg (in the D range).

The preferred daily usage range is 0.11ifl/kt~
It is 1.0 mg/kt. In therapeutic use as a cytotoxic or antineoplastic agent, the compound is approximately 0.0.
Administered at an initial dosage of 0.01-10 mg. Approximately 0.1 to 1 per 1. OQ is preferred. However, the usage may vary depending on the requirements of the recipient, the severity of the condition to be treated and the compound used. Determination of the appropriate amount to use for a particular situation is within the skill of one in the art. Generally, treatment involves subtotal amounts of less than the optimal use of the compound. Therefore, the dosage should be filled in small portions of 1 and 2 to reach the optimal effect under the circumstances.For good practice, the total daily dosage should be divided if necessary and spread throughout the day. It can be administered in small doses.

The compounds of the present invention are useful as intermediates or substrates for chemical synthesis or as introductions to chemically active compounds.

Patent applicant Warner-Lambert Kompany Continued from page 1 Priority claim @ May 25, 1983 Φ United States (US) [
497875 0 Inventor Richard H Bunge 2520 Traver Road, Ann Arbor, Michigan, USA (48 105) @l! Inventor: Timothy Earl Hurley 3735 Green Prior Drive, Ann Arbor, Michigan, USA (48 105) 0 Inventor: James C. French Ramsey, Ann Arbor, Michigan, USA (48 105) 3 50 0 Inventor: Joseph Ino B Tanatsuk 5284 Troy Collington Drive, Michigan, USA (48 098) 0 Inventor William E. Dobson 1275 Braddy Boulevard, Windsor, Ontario, Canada N8 Nis 3 K2 0 Inventor Planche・Dee Graham Michigan, USA (48 239) Redford Livend 15978

Claims (1)

[Claims] 1) The following (a) structure, iti formula (Ia included (1b) and (1c)
Alcohols (Ia) with R1=HR2=OR (1b) R1=HR2=OR (1c) R1=R2=OH (b) Phosphates K) with structural formulas (2a), (2b) and (2c) 5H2 ( 2a) R1=HR2=oH (211) R1-R2SeH (20) R1-R2=OH (0) The phosphate of (b) above and further 0L having the structural formula <3a (3b) and (3c), respectively −
1565m, 0L-1565-B and 0L-156
Ester of the phosphate functional group of pyranone phosphate designated as 5-T\ POI! 1H2 (3a) R1-HR2=OH (3b) R1 precipitate R2-11 <5a) H1 ■ R2 OH (phrase acyl ester (s) of alcohol of above (a) and pyranone phosphate of above (0) 0L- A compound called 15ss-pr-s and (f) the phosphate of (b) above, (0) above.
the pyranone phosphate ester of (d), the pyranone acyl ester of (d) and the 0L-1565-PT-3
A substantially pure form of the compound consisting of a pharmaceutically acceptable salt of. An alcohol according to claim 1 having the structural formulas (1a), (1b) and (1c). 3) A phosphate according to claim 1 having the structural formula (2a, (2b) and (2c)) and a pharmaceutically acceptable salt thereof. 4) A structure according to claim 1. Formula (2a included (
2b) and (2c) and furthermore the structural formula (3a) according to claim 1, (
Lower alkyl or aryl esters of the phosphate functionality of pyranone phosphates with sb) and (3c). 5) Structural formula (Ia) according to claim 1. (1b) and <'IQ) and the structural formula (3a) according to claim 1. Acyl esters of pyranone phosphates having (3b) and (3o) and pharmaceutically acceptable salts of pyranone phosphate acyl esters. 6) 0L-1565-FT-3 compound and its pharmaceutically acceptable salts. 7) 5.6-:) Hydro-6-(3,4,6,13-tetrahydroxy-6-methyl-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one. 5.6- :)Hydro-6-(3,4,6-dolihydroxy-3-methyl-1,7,9,11-)lidecatetraenyl)-2H-pyran-2-one, 5.6 -Sihydro-5-hydroxy-6-(3,4,6,13-tetrahydroxy-3-methyl-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one, 5.8, 9. it, 18-pentahydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid sodium salt, b, 8,9.11-tetrahydroxy-8-methyl-2
, 6,12,14,16-octadecanic acid sodium salt. 4.5,8,9,11,18-hexahydroxy-8-
Methyl-2,6,12,14,16-octadecapentaenoic acid sodium salt, 0L-1565-FT-3 sodium salt, 4.5,8,
11,18-pentahydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,16 6-octadecabentaenoic acid sodium salt. 5.8,11.18-tetrahydroxy-8-methyl-
9-(phosphonooxy) -2,6,12,14,16
- Octadecapentaenoic acid sodium salt. 5.8.11-)dihydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,16-octadecapentaenoic acid sodium salt. 6- (6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy) -1,
7,9,it-)lidecatetraeninol-5,6-:
) Hydro-2H-pyran-2-one sodium salt, 6-[6-(acetyloxy)-3-hydroxy-3-
Methyl-4-(phosphonooxy)-1,7,9,11-
tridecatetraenyl) -5,6-dihydro-2H-
Pyran-2-one sodium salt, 5-acetyloxy-6-(6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy)-1-7e9e 11-Fridecatetraenyl ]-5,6-sihydro 2H-pyran-2-one sodium salt. 5.9,11.18-tetra(acetyloxy)-8-
Hydroxy-8-methyl-2,6,12,14,16-
Octadecapentaenoic acid sodium salt. 5.6-Sihydro6-(4,6,13-tris(acetyloxy)-6-hydroxy-3-methyl-1,7
,9,11-)lidecatetocenyl]-2H-bilane-
2-one and 5,6-sihydro-6-(3,6,13-)dihydroxy-3-methyl-4-dimethylphosphonooxy-1,
7,9,11-)lidecatetraenyl]-2H-bilan-2-one. 8) Structural formula (3a) according to claim 1,
Structural formula (Ia) according to item 1, which comprises dephosphorylating a pyranone phosphate having (3b) and (3C), respectively. A method for producing an alcohol having (1b) and (10). 9) The above tri if detection range PI under ring-opening conditions
Structural formula described in item 1 (3a containing (3b) and (3C)
A process for producing phosphates having the structural formulas (2a-containing (2b) and (2c)) according to claim P11, which comprises hydrolyzing pyranone phosphates having the following, respectively. Structural formula (3a) described in item 1
. A process for preparing acyl esters of alcohols and pyranone phosphates according to claim 1, which comprises acylating the primary and secondary hydroxy groups of the alcohol with (6b) and (3c). 11 phosphates having structural formulas (2a-containing (2b) and (2c) and structural formulas of (5a), (3b) and (3C). 12) Preparing the concentrate from the fermentation broth containing the compound CL 1565-FT-3 in a manner selective for the compound. Compound 0L-1565 consisting of chromatography using an eluent to isolate the compound from the eluent.
-Production method of FT-3. 13) Structural formula (1a) according to claim 2
A pharmaceutical composition comprising an alcohol compound having b (1b) or (1C) and a pharmaceutically acceptable carrier. 14) Structural formula (2a) according to claim 3
, (2b) or (2C) and a pharmaceutically acceptable carrier. 15) G or toy according to claim 4
A pharmaceutical composition comprising an -o-substituted phosphonooxy-containing compound and a pharmaceutically acceptable carrier. 16) A pharmaceutical composition comprising the acyl ester compound according to claim 5 and a pharmaceutically acceptable carrier. 17) OL-1565 described in claim 6
- A pharmaceutical composition consisting of a FT-3 compound and a pharmaceutically acceptable carrier. 18) 0L-156 in amounts that can be harvested by culturing in an aqueous nutrient medium containing assimilable crabmeat and carbon sources.
5 Microorganisms capable of producing antibiotic complexes Streptomyces pulveraceus subsp. Hostreus ATac 3190
6 biologically pure cultures.