CA1197202A - Antibiotic cl-1565 complex derivatives - Google Patents
Antibiotic cl-1565 complex derivativesInfo
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- CA1197202A CA1197202A CA000452122A CA452122A CA1197202A CA 1197202 A CA1197202 A CA 1197202A CA 000452122 A CA000452122 A CA 000452122A CA 452122 A CA452122 A CA 452122A CA 1197202 A CA1197202 A CA 1197202A
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Abstract
ABSTRACT
Novel pyranone compounds and related compounds, methods of preparing the compounds, and their use as cytotoxic and/or antileukemic agents or precursors, are provided.
Novel pyranone compounds and related compounds, methods of preparing the compounds, and their use as cytotoxic and/or antileukemic agents or precursors, are provided.
Description
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SUM~ARY AND DETAILED DESCRIPTION
The present invention in one preferred embodiment relates to novel pyranones, particularly 5,6-dihydro-2H-pyran-2-one compounds and related compounds, methods of preparing the compounds, and their use as cytotoxic and/or antileukemic agents or precursors. Thus, the invention in one aspect relates to compounds in substantially pure form, as follows:
a. Alcohols havi.ng the structural formulas la, lb and lc:
O ~ CH=CH-C-IH-CH2-TH-CH=CH-CH=CH-CH=CH-CH2R
HO OH
la, R1 = H; R2 = OH
lb, Rl = R2 = ~1 1C R1 = R2 = OH
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1~97~2 b. phosphates, preferably as their sodium salts or other salts, having the structural formulas 2a~
2b, and 2c:
~C02H
CH
R -CE~-CH-CH=CH-C CH-CH2-CH-CH=CH-CH=CH-CH=CH-CH2R
OH HO O~
2a, R = H; R2 = OH
2b, Rl = R2 _ H
2c, Rl = R2 = OH
c. lower alkyl or aryl esters of tne phosphdte function of said phosphates of b and of the phosphate function of the above mentioned CL 1565-A, CL 1565-B, and CL 1565-T, having the structural formulas 3a, 3b, and 3c, respectively:
~ Rl l I CIH3 OH 2 o ~ O ~ ~ CH=CH-7-CH-CH2-CH-CH=CH-CH=CH-CH=CH-C~2R
HO O~
3d, Rl = 1l; R2 = O~l 3b, Rl = R2 = H
3c, R = R2 = OH
d. acyl esters of said alcohols of a, said salts of said phosphate esters of b, sai; phosphate esters of c, and of the salts, preferably soclium salts, of compounds 3a, 3b, and 3c;
e. the cornpound designated CL 1565-PT-3; and
SUM~ARY AND DETAILED DESCRIPTION
The present invention in one preferred embodiment relates to novel pyranones, particularly 5,6-dihydro-2H-pyran-2-one compounds and related compounds, methods of preparing the compounds, and their use as cytotoxic and/or antileukemic agents or precursors. Thus, the invention in one aspect relates to compounds in substantially pure form, as follows:
a. Alcohols havi.ng the structural formulas la, lb and lc:
O ~ CH=CH-C-IH-CH2-TH-CH=CH-CH=CH-CH=CH-CH2R
HO OH
la, R1 = H; R2 = OH
lb, Rl = R2 = ~1 1C R1 = R2 = OH
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1~97~2 b. phosphates, preferably as their sodium salts or other salts, having the structural formulas 2a~
2b, and 2c:
~C02H
CH
R -CE~-CH-CH=CH-C CH-CH2-CH-CH=CH-CH=CH-CH=CH-CH2R
OH HO O~
2a, R = H; R2 = OH
2b, Rl = R2 _ H
2c, Rl = R2 = OH
c. lower alkyl or aryl esters of tne phosphdte function of said phosphates of b and of the phosphate function of the above mentioned CL 1565-A, CL 1565-B, and CL 1565-T, having the structural formulas 3a, 3b, and 3c, respectively:
~ Rl l I CIH3 OH 2 o ~ O ~ ~ CH=CH-7-CH-CH2-CH-CH=CH-CH=CH-CH=CH-C~2R
HO O~
3d, Rl = 1l; R2 = O~l 3b, Rl = R2 = H
3c, R = R2 = OH
d. acyl esters of said alcohols of a, said salts of said phosphate esters of b, sai; phosphate esters of c, and of the salts, preferably soclium salts, of compounds 3a, 3b, and 3c;
e. the cornpound designated CL 1565-PT-3; and
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f. pharmaceutically acceptable forms of said alcohols of a: said phosphates of b: said esters of c:
said acyl esters of d: and said CL 1565-PT-3 The term acyl as used herein refers to acyl groups of acids that may be straight chain, branch chain, substituted saturated, unsaturated, or aromatic acids such as, but not necessarily limited to, acetic, tri-fluoroacetic, propionic, n-butyric, isobutyric, valeric, caproic, pelargonic, enanthic, caprylic, lactic, acrylic, propargylic, palmitic, benzoic, phthalic, salicylic, C; nn~m; C and naphthoic acids. With respect to phosphate compounds of the invention, the substituted phosphoric acid function can be as a free acid or preferably as a salt form. Acceptable salts of the phosphate moiety can be selected from, but not necessarily limited to, a group consisting of alkali and alkaline earths, e.~., sodium, potassium, calcium, magnesium and lithium;
ammonium and substituted ammonium, including trialkyl-ammonium, dialkylammonium and alkylammonium, e.g., triethylammonium, trimethylammonium, diethylammonium, octylammonium and cetyltrimethylammonium; and cetylpyridinium. The term lower alkyl refers to Cl to C8 alkyl, preferably methyl. The term aryl refers to phenyl, benzyl, or substituted phenyl or benzyl.
Preferred compounds of the invention are:
1. 5,6-dihydro-6-(3,4,6,13-tetrahydroxy-3-methyl-1, 7,9,11-tridecatetraenyl)-2H-pyran-2-one (la).
2. 5,6-dihydro-6-(3,4,6-trihydroxy-3-methyl-1,7,~,11-tridecatetraenyl)-2H-pyran-2-one (lb).
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f. pharmaceutically acceptable forms of said alcohols of a: said phosphates of b: said esters of c:
said acyl esters of d: and said CL 1565-PT-3 The term acyl as used herein refers to acyl groups of acids that may be straight chain, branch chain, substituted saturated, unsaturated, or aromatic acids such as, but not necessarily limited to, acetic, tri-fluoroacetic, propionic, n-butyric, isobutyric, valeric, caproic, pelargonic, enanthic, caprylic, lactic, acrylic, propargylic, palmitic, benzoic, phthalic, salicylic, C; nn~m; C and naphthoic acids. With respect to phosphate compounds of the invention, the substituted phosphoric acid function can be as a free acid or preferably as a salt form. Acceptable salts of the phosphate moiety can be selected from, but not necessarily limited to, a group consisting of alkali and alkaline earths, e.~., sodium, potassium, calcium, magnesium and lithium;
ammonium and substituted ammonium, including trialkyl-ammonium, dialkylammonium and alkylammonium, e.g., triethylammonium, trimethylammonium, diethylammonium, octylammonium and cetyltrimethylammonium; and cetylpyridinium. The term lower alkyl refers to Cl to C8 alkyl, preferably methyl. The term aryl refers to phenyl, benzyl, or substituted phenyl or benzyl.
Preferred compounds of the invention are:
1. 5,6-dihydro-6-(3,4,6,13-tetrahydroxy-3-methyl-1, 7,9,11-tridecatetraenyl)-2H-pyran-2-one (la).
2. 5,6-dihydro-6-(3,4,6-trihydroxy-3-methyl-1,7,~,11-tridecatetraenyl)-2H-pyran-2-one (lb).
3. 5,6-dihydro-5-hydroxy-6-(3,4,6,13-tetrahydroxy-3-methyl-1,7,g,11-tridecatetraenyl)-2H-pyran-2-one (lc) .
4. CL 1565-PT-3, sodium salt mab/~
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5. ~,5,8,11,13-pentah~droxy-8-methyl-9-(phosphonooxy~-2,6,12,14,16-octadecapentaenoic acid, sodium salt (2c).
6. 5,8,11,18-tetrahydroxy-8-methyl-9-~phosphonooxy~-2,6,12,14,16-octadecapentaenoic acid, sodium salt (2a).
7. 5,8,11-trihydroxy-8-methyl~9-(phosphonooxy)-2,6,12,14,16-octadecapentaenoic acid, sodium salt.
8. 6-[6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one, sodium salt.
9. 6-[6-(acetyloxy)-3-hydroxy-3-methyl-4-(phosphono-oxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one, sodium salt.
10. 5-(acetyloxy)-6-[6,13-bis(acetyloxy)-3-h~-drox~-3-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one, sodium salt.
11. 5,6-dihydro-6-[4,6,13-tris(acetyloxy)-3-hydroxy-3-methyl-1,7,9,11-tridecatetraenyl~-2H-pyran-2-one.
12. 5,6-dihydro-6-[3,6,13-trihydroxy-3-methyl-4-(dimsthylphosphonooxy)-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one.
The invention in another aspect relates to a process for producing alcohols having the structural formulas la, lb, and lc, which comprises dephosphorylating pyranone phosphates having the structural formulas 3a, 3b, and 3c, respectively. The process is best carried out by reacting the pyranone phosphate with a phosphatase enzyme. The reaction is carried out in neutral or nearly neutral aqueous solution at moderate temperature, e.g., 37C., until the reaction is complete.
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The invention in another aspect rela-tes to a process for producing phospha-tes having the structural formulas 2a, 2b, and 2c, which comprises hydrolyzing pyranone phosphates having the structural formulas 3a, 3b, and 3c, respectively, under ring opening conditions.
The reaction can be carried out by treating the pyranone phosphate with a base such as an alkali metal hydroxide, preferably sodium hydroxlde, in an aqueous medium at ambient temperature.
The invention in another aspect relates to a process for producing acyl esters of alcohols of pyranone phosphates which comprises acylating the primary and secondary hydroxyl groups of alcohols having the str~ctural formulas la, lb, and lc ~ and pyranone phosphates having the structural formulas 3a, 3b, and 3c. The reaction can be carried out with a suitable acylating agent such as an acyl halide or acid anhydride, for example, p-bromobenzoyl chloride or acetic anhydride, preferably in the cold.
The invention in another aspect relates to a process for producing di- and tri- esters of phosphoric acid having the structural formulas 2a, 2b, and 2c and of pyranone phosphates having the structural formulas 3a, 3b, and 3c which comprises selectively esterif~ing the phosphate function of said phosphates and pyranone phos-~lates. The reaction can be carried out in a suitable solvent such as methanol with an esterifying agent such as diazomethane in the cold.
The invention in another aspect relates to a process for producing the compound CL 1565-PT-3 com-prisiny subjecting to chromatography a concentrate from beer containing the compound obtained by fermen-tation of a CL 1565-PT-3 producing strain of micro-organism employing an eluent tha-t is selected for the compound and isolating the compound from the eluate.
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For the process, one ernploys a concentrate of CL 1565-PT-3.
For the chrornatographic separation of CL 1565-PT-3, a system employing a reverse phase silica yel and a gradient elution usiny 0.05 M pH 7.2 phosphate buffer-acetonitrile is preferred.
Purification of compound or products obtained by the methods of the invention is accomplished in any suitable way, preferably by colu~n chromatography.
The invention in its composition aspect relates to pharmaceutical compositions comprising an alcohol compound having structural formula la, lb, or lc, and a pharmaceutically acceptable carrier.
The irvention in another aspect relates to pharmaceutical compositions comprising a phosphate compound having the structural formula 2a, 2b, or 2c, and a pharma-ceutically acceptable carrier.
The invention in another aspect relates to pharmaceutical compositions comprising a lower alkyl or aryl ester of the phosphate function of phosphates having structural formula 2a, 2b, or 2c, and further pyranone phosphate compounds having the structural formula 3a, 3b, or 3c, and a pharmaceutically acceptable carrier.
The invention in another aspect relates to pharmaceutical compositions comprising an acyl ester of an alcohol compound having structural formula la, lb, or lc, of a pyranone phosphate compound having structural formula 3a, 3b, or 3c, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
The invention in another aspect relates to pharmaceutical compositions comprising the compound CL 1565-PT-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
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PREPARI~TION OF. PHARM~CEUTICAL COMPOSIT:tONS
Pyranone compounds of the invention, particularl~
the alcohol compound having the structural formula la and the compounds designate~ CL 1565-PT-3 and CL 1565-A
diacetate in sodium salt form, have antitumor activity.
The compounds are active, for example, against P 388 lymphatic leukemia in vivo or either L1210 mouse leukemia cells or human colon adenocarcinoma cells in vitro.
Therefore, use of the compounds of the invention is con-templated for their antitumor activity as an active component of pharmaceutical compositions. When being utilized as cytotoxic or antileukemic agents, the compounds of the invention can be prepared and administered in various dosage forms, especially parenteral dosage forms. It will be clear to those skilled in the art that the dosage forms may comprise as the active component, one or more compounds of the invention.
The compounds are administered parenterally or intraperitoneally. Solutions of the ~ctive compound as either a salt or nonsalt form whichever is appropriate, can be prepared in an aqueous vehicle, optionally with a solubilizing agent or surfactant such as hydroxy-propylcelluloseO Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile injectable solutions or dispersions. In all cases the form must be sterile and must e fluid to the extent that easy syringeability exists. Xt must be stable under the conditions of manufacture and storage and must be preserved against the contamination action of mab/ `/~`~
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microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion Medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), N,N-dimethylacetamide, suitable mixtures thereof and vegetable oils. The 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 dispersion and by the use of surfactants. The prevention of the action of micro-organisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chloro-butanol, phenol, sorbic acid, thimerosal, and the like.
In many cases, it will be preferable to include isotonic agents, for e~:ample, sugars or sodium chlor:ide. Pro-longed absorption of the injectable compositions can be brought about by the use in the compositions o~ ayents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization accomplished by filtering. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of the sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingre~ient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
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~s used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agen-ts, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic com-positions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosage for the m~mm~l ian subiects tc be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitation inherent in the art of compound such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in unit dosage form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from about 10 mg to about 500 mg, with from about 25 mg to about 200 mg bein~
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preferred. Expressed in proportions, the active compound is generally present in from about 10 to about 500 my/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and the manner of administration of the said ingredients. The daily parenteral doses for mammalian subjects to be treated ranges from 0.01 mg/kg to 10 mg/kg. The preferred daily dosage range is 0.1 mg/kg to 1.0 mg/kg. In therapeutic use as cytotoxic or antitumor agents the compounds are administered at the initial dosage of about 0.01 mg to about 10 mg per kilogram. A dose range of about 0.1 mg to about 1.0 mg per kilogram is preferred.
The dosage-s, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed.
Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For con-venience, the total daily dosage may be divided and administered in portions during the day if desired.
The compounds of the invention are also useful as intermediates or substrates for the chemical or bio-chemical synthesis or in situ delivery of pharmacologically active compounds.
The invention and the best mode of practicing the same are illustrated by the following examples of preferred embodiments of selected compounds and their preparation.
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5,6-Dihydro-6-(3,4,6,13-tetrah~droxy-3-methyl-1,7,9,11-tridecatetraenyl)-2f-l-pyran-2-one (CL 1565-A alcohol) (la) A solution of 1.4 g of the sodium salt of 5,6-dihyaro-6-(3,6,13-trihydroxy-3-methyl-4-(phosphono-oxy)-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one, (the sodium salt of CL 1565~A (3a)) and 1.0 g of alkaline phosphatase derived from bovine (calf) intestinal mucosa (Sigma Chemical Co., St. Louis, Missouri) in 140 ml of water was incubated at 37C for seven hours. The reaction mixture (pH 7.2) was then lyophilized and the resulting residue was triturated with methanol. The methanol-soluble product was chromatographed on C8-reverse phase silica gel. After a water wash, CL 1565-A-alcohol was eluted with water-acetonitrile (85:15). These latter fractions were combined, concentrated and lyophilized to yield 0.62 g of CL 1565-A-alcohol as a white solid.
CL 1565-A-alcohol can be detected in fermentation beers by using HPLC methods patterned after the HPLC procedure described in Example 2, below.
Properties of CL lg65-A alcohol Ultraviolet Absorption Spectrum in Methanol ~max 268 nm (al = 975) with inflections at 259 and 278 nm.
Elemental Analysis %C %H
Calcd. for ClgH2606-1/2H20: 63.51 7.52 Found: 63.41 7.51 Infrared Spectrum in CHC13 P~incipal absorptions at 1720, 1600, 1285, 30and 1060 reciprocal centimeters.
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Thin-layer Chromatography on Silica Gel-60 Solvent: chloroform-ethanol-0.5M pEI 5.5 sodium acetate buffer (40:70:20) Rf: 0.8 Solvent: chloroform-isopropanol l8:2) Rf: 0.19 Solvent: chloroform-methanol-water 175:25:1) Rf: 0.60 HPLC (see Example 2).
360 MHz Proton Magnetic Resonance Spectrum in D2O
Principal Signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 140 s(3H), 1.47 m(lH), 1.87 m(3H), 2.52-2.71 m(2H), 3.81 dd(lH), 4.27 d(2H), 4.95 t(lH), 5.13 m(lH), 5.65 ttlH), 5.95-6.15 m(4H), 6.20 t(lH), 6.44 t(lH), 6.61 t(lH), 6.89 dd(lH), 7.15 m(lH) parts per million downfield from sodium 2,2-dimethyl-2-silapentane~5-sulfonate (DSS).
! 90.4 MHz 13C-Nuclear Magnetic Resonance Spectrum in mab/J;
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Peak Number Chemical Shift Peak Number Chemical Shift 1 2~.8 11 127.0 2 31.9 12 128.8 3 41.4 13 129.8 4 64.9 14 133.6 67.3 15 137.3 6 76.4 16 137.4 7 77.9 17 140.4 8 81.5 18 151.9 1~ 9 122.4 19 170.5 126.8 *parts per million downfield from tetramethylsilane Cytotoxicity Against L1210 Cells ID50 = 2.5 ~g/ml 5,6-Dihydro-6-(3,4,6-trihydroxy-3-methyl-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one (CL 1565-B alcohol) (lb) CL 1565-B-alcohol was prepared in a manner similar to that used for the preparation of CL 1565-A-alcohol. CL 1565-B, sodium slat (10 mg) was dissolved in 5 ml water to which 10 mg of alkaline phosphatase (CalbiochemBehring Corp., San Diego, California) was added. The resulting solutio~- was stored at 37 for 14 hours. The reaction mixture was then lyophilized and the residual solid triturated with methanol. Concen-tration of the methanolic solution yielded 2 mg of CL 1565-B-alcohol _ 13 -mab/J~
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Properties of CL 1565-l~ alcohol Thin-layer Chromatography on E. Merck Silica Gel Solvent: chloroform-isopropanol (80:20) Rf: 0.38 High Pressure Liquid Chromatography Column: Lichrosorb~; RP-~> (Brownlee Labs, Berkeley, California) Solvent: water-acetonitrile (80:20) Flow-rate: 2 ml/min Retention time: 27.0 min Using the same HPLC conditions, the retention times of CL 1565-A-alcohol and CL 1565-B are 3.3 min and 1.0 min, respectively.
5,6-Dihydro-5-hydroxy-6-(3,4,6,l3-tetrahydroxy-3-methyl-1,7,9,11-tridecatetraenyl)-?H-pyran- -one (CL 1565-T alcohol) ( 1 c) ! 20 A solution of 10 mg of CL 1565-T, sodium salt and 7.5 mg of alkaline phosphatase in 1 ml of water was stored at 37 for 18 hours. The reaction mixture was then lyophilized and the residue was triturated with ethanol.
Removal of the ethanol in vac-uo afforded a residue containing CL 1565-T alcohol.
Properties of CL 1565-T alcohol:
Thin-layer Chromatography on Silica Gel G~ILF
(Analtech, Inc., Neward, Delaware) Solvent: chloroform-methanol-water (75:25:1) ~f: ~.44 Using the same TLC conditions, the observed Rf of CL 1565-T, sodium salt is 0.02.
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EXAMPLE
C~ 1565-PT-3. Sodium Salt Filtered fermentation beer (719 liters) prepared from a CL 1565-PT-3 producing microorganism was passed over 31 liters of Dowex~-l x 2 (chloride form) packed in a 30.5 cm (O.D.) column. The effluent and the sub-sequent water wash did no~ contain any detectable amounts of the CL 1565 components. The Dowex*-l resin was then eluted with lM sodium chloride-methanol (l:l) and the eluate was collected in two 10-liter and six 15-liter fractions. Most of the CL 1565-A, CL 1565-B, C~ 1565-T, and additional minor CL 1565 components appeared in eluates two through six. These fractions were combined and diluted with 246 liters of acetone. The resulting mlxture was stored at 5C overnight. The clear super-natant solution was removed and concentrated to 16 liters in vacuo. Lyophilization of this concentrate afforded 800 g of a solid. This product (740 g) was added to 552 g of a similar product isolated in the same manner and the combined solids were dissolved in 20 liters of water. The resulting solution (pH 6.0) was chromato-graphed on 50 liters of HP-20* resin contained in a 15 cm (O.D.) column. Elution of the HP-20~ column with 175 liters of water removed most ~f the CL 1565-T and all of the minor, more polar CL 1565 components, including CL 1565-PT-3 and CL 1565-C (2c). The fractions con-taining these components were combined and concentrated in vacuo~ The concentrate was chromatographed on 3 kg of 135 ~m C2-reverse phase silica gel (Merck RP-2, obtained from MCB, Inc., Indianapolis, Indiana).
Elution of this column with 0.05 M p~l 7.2 phosphate buffer yielded a fraction that contained CL 1565-T and several minor components as determined by HPLC anllysis.
This fraction was concentrated and rechromatographed _ 15 -*trade mark ~A
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The invention in another aspect relates to a process for producing alcohols having the structural formulas la, lb, and lc, which comprises dephosphorylating pyranone phosphates having the structural formulas 3a, 3b, and 3c, respectively. The process is best carried out by reacting the pyranone phosphate with a phosphatase enzyme. The reaction is carried out in neutral or nearly neutral aqueous solution at moderate temperature, e.g., 37C., until the reaction is complete.
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The invention in another aspect rela-tes to a process for producing phospha-tes having the structural formulas 2a, 2b, and 2c, which comprises hydrolyzing pyranone phosphates having the structural formulas 3a, 3b, and 3c, respectively, under ring opening conditions.
The reaction can be carried out by treating the pyranone phosphate with a base such as an alkali metal hydroxide, preferably sodium hydroxlde, in an aqueous medium at ambient temperature.
The invention in another aspect relates to a process for producing acyl esters of alcohols of pyranone phosphates which comprises acylating the primary and secondary hydroxyl groups of alcohols having the str~ctural formulas la, lb, and lc ~ and pyranone phosphates having the structural formulas 3a, 3b, and 3c. The reaction can be carried out with a suitable acylating agent such as an acyl halide or acid anhydride, for example, p-bromobenzoyl chloride or acetic anhydride, preferably in the cold.
The invention in another aspect relates to a process for producing di- and tri- esters of phosphoric acid having the structural formulas 2a, 2b, and 2c and of pyranone phosphates having the structural formulas 3a, 3b, and 3c which comprises selectively esterif~ing the phosphate function of said phosphates and pyranone phos-~lates. The reaction can be carried out in a suitable solvent such as methanol with an esterifying agent such as diazomethane in the cold.
The invention in another aspect relates to a process for producing the compound CL 1565-PT-3 com-prisiny subjecting to chromatography a concentrate from beer containing the compound obtained by fermen-tation of a CL 1565-PT-3 producing strain of micro-organism employing an eluent tha-t is selected for the compound and isolating the compound from the eluate.
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For the process, one ernploys a concentrate of CL 1565-PT-3.
For the chrornatographic separation of CL 1565-PT-3, a system employing a reverse phase silica yel and a gradient elution usiny 0.05 M pH 7.2 phosphate buffer-acetonitrile is preferred.
Purification of compound or products obtained by the methods of the invention is accomplished in any suitable way, preferably by colu~n chromatography.
The invention in its composition aspect relates to pharmaceutical compositions comprising an alcohol compound having structural formula la, lb, or lc, and a pharmaceutically acceptable carrier.
The irvention in another aspect relates to pharmaceutical compositions comprising a phosphate compound having the structural formula 2a, 2b, or 2c, and a pharma-ceutically acceptable carrier.
The invention in another aspect relates to pharmaceutical compositions comprising a lower alkyl or aryl ester of the phosphate function of phosphates having structural formula 2a, 2b, or 2c, and further pyranone phosphate compounds having the structural formula 3a, 3b, or 3c, and a pharmaceutically acceptable carrier.
The invention in another aspect relates to pharmaceutical compositions comprising an acyl ester of an alcohol compound having structural formula la, lb, or lc, of a pyranone phosphate compound having structural formula 3a, 3b, or 3c, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
The invention in another aspect relates to pharmaceutical compositions comprising the compound CL 1565-PT-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
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PREPARI~TION OF. PHARM~CEUTICAL COMPOSIT:tONS
Pyranone compounds of the invention, particularl~
the alcohol compound having the structural formula la and the compounds designate~ CL 1565-PT-3 and CL 1565-A
diacetate in sodium salt form, have antitumor activity.
The compounds are active, for example, against P 388 lymphatic leukemia in vivo or either L1210 mouse leukemia cells or human colon adenocarcinoma cells in vitro.
Therefore, use of the compounds of the invention is con-templated for their antitumor activity as an active component of pharmaceutical compositions. When being utilized as cytotoxic or antileukemic agents, the compounds of the invention can be prepared and administered in various dosage forms, especially parenteral dosage forms. It will be clear to those skilled in the art that the dosage forms may comprise as the active component, one or more compounds of the invention.
The compounds are administered parenterally or intraperitoneally. Solutions of the ~ctive compound as either a salt or nonsalt form whichever is appropriate, can be prepared in an aqueous vehicle, optionally with a solubilizing agent or surfactant such as hydroxy-propylcelluloseO Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile injectable solutions or dispersions. In all cases the form must be sterile and must e fluid to the extent that easy syringeability exists. Xt must be stable under the conditions of manufacture and storage and must be preserved against the contamination action of mab/ `/~`~
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microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion Medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), N,N-dimethylacetamide, suitable mixtures thereof and vegetable oils. The 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 dispersion and by the use of surfactants. The prevention of the action of micro-organisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chloro-butanol, phenol, sorbic acid, thimerosal, and the like.
In many cases, it will be preferable to include isotonic agents, for e~:ample, sugars or sodium chlor:ide. Pro-longed absorption of the injectable compositions can be brought about by the use in the compositions o~ ayents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization accomplished by filtering. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of the sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingre~ient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
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~s used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agen-ts, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic com-positions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosage for the m~mm~l ian subiects tc be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitation inherent in the art of compound such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in unit dosage form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from about 10 mg to about 500 mg, with from about 25 mg to about 200 mg bein~
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preferred. Expressed in proportions, the active compound is generally present in from about 10 to about 500 my/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and the manner of administration of the said ingredients. The daily parenteral doses for mammalian subjects to be treated ranges from 0.01 mg/kg to 10 mg/kg. The preferred daily dosage range is 0.1 mg/kg to 1.0 mg/kg. In therapeutic use as cytotoxic or antitumor agents the compounds are administered at the initial dosage of about 0.01 mg to about 10 mg per kilogram. A dose range of about 0.1 mg to about 1.0 mg per kilogram is preferred.
The dosage-s, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed.
Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For con-venience, the total daily dosage may be divided and administered in portions during the day if desired.
The compounds of the invention are also useful as intermediates or substrates for the chemical or bio-chemical synthesis or in situ delivery of pharmacologically active compounds.
The invention and the best mode of practicing the same are illustrated by the following examples of preferred embodiments of selected compounds and their preparation.
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5,6-Dihydro-6-(3,4,6,13-tetrah~droxy-3-methyl-1,7,9,11-tridecatetraenyl)-2f-l-pyran-2-one (CL 1565-A alcohol) (la) A solution of 1.4 g of the sodium salt of 5,6-dihyaro-6-(3,6,13-trihydroxy-3-methyl-4-(phosphono-oxy)-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one, (the sodium salt of CL 1565~A (3a)) and 1.0 g of alkaline phosphatase derived from bovine (calf) intestinal mucosa (Sigma Chemical Co., St. Louis, Missouri) in 140 ml of water was incubated at 37C for seven hours. The reaction mixture (pH 7.2) was then lyophilized and the resulting residue was triturated with methanol. The methanol-soluble product was chromatographed on C8-reverse phase silica gel. After a water wash, CL 1565-A-alcohol was eluted with water-acetonitrile (85:15). These latter fractions were combined, concentrated and lyophilized to yield 0.62 g of CL 1565-A-alcohol as a white solid.
CL 1565-A-alcohol can be detected in fermentation beers by using HPLC methods patterned after the HPLC procedure described in Example 2, below.
Properties of CL lg65-A alcohol Ultraviolet Absorption Spectrum in Methanol ~max 268 nm (al = 975) with inflections at 259 and 278 nm.
Elemental Analysis %C %H
Calcd. for ClgH2606-1/2H20: 63.51 7.52 Found: 63.41 7.51 Infrared Spectrum in CHC13 P~incipal absorptions at 1720, 1600, 1285, 30and 1060 reciprocal centimeters.
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Thin-layer Chromatography on Silica Gel-60 Solvent: chloroform-ethanol-0.5M pEI 5.5 sodium acetate buffer (40:70:20) Rf: 0.8 Solvent: chloroform-isopropanol l8:2) Rf: 0.19 Solvent: chloroform-methanol-water 175:25:1) Rf: 0.60 HPLC (see Example 2).
360 MHz Proton Magnetic Resonance Spectrum in D2O
Principal Signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 140 s(3H), 1.47 m(lH), 1.87 m(3H), 2.52-2.71 m(2H), 3.81 dd(lH), 4.27 d(2H), 4.95 t(lH), 5.13 m(lH), 5.65 ttlH), 5.95-6.15 m(4H), 6.20 t(lH), 6.44 t(lH), 6.61 t(lH), 6.89 dd(lH), 7.15 m(lH) parts per million downfield from sodium 2,2-dimethyl-2-silapentane~5-sulfonate (DSS).
! 90.4 MHz 13C-Nuclear Magnetic Resonance Spectrum in mab/J;
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Peak Number Chemical Shift Peak Number Chemical Shift 1 2~.8 11 127.0 2 31.9 12 128.8 3 41.4 13 129.8 4 64.9 14 133.6 67.3 15 137.3 6 76.4 16 137.4 7 77.9 17 140.4 8 81.5 18 151.9 1~ 9 122.4 19 170.5 126.8 *parts per million downfield from tetramethylsilane Cytotoxicity Against L1210 Cells ID50 = 2.5 ~g/ml 5,6-Dihydro-6-(3,4,6-trihydroxy-3-methyl-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one (CL 1565-B alcohol) (lb) CL 1565-B-alcohol was prepared in a manner similar to that used for the preparation of CL 1565-A-alcohol. CL 1565-B, sodium slat (10 mg) was dissolved in 5 ml water to which 10 mg of alkaline phosphatase (CalbiochemBehring Corp., San Diego, California) was added. The resulting solutio~- was stored at 37 for 14 hours. The reaction mixture was then lyophilized and the residual solid triturated with methanol. Concen-tration of the methanolic solution yielded 2 mg of CL 1565-B-alcohol _ 13 -mab/J~
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Properties of CL 1565-l~ alcohol Thin-layer Chromatography on E. Merck Silica Gel Solvent: chloroform-isopropanol (80:20) Rf: 0.38 High Pressure Liquid Chromatography Column: Lichrosorb~; RP-~> (Brownlee Labs, Berkeley, California) Solvent: water-acetonitrile (80:20) Flow-rate: 2 ml/min Retention time: 27.0 min Using the same HPLC conditions, the retention times of CL 1565-A-alcohol and CL 1565-B are 3.3 min and 1.0 min, respectively.
5,6-Dihydro-5-hydroxy-6-(3,4,6,l3-tetrahydroxy-3-methyl-1,7,9,11-tridecatetraenyl)-?H-pyran- -one (CL 1565-T alcohol) ( 1 c) ! 20 A solution of 10 mg of CL 1565-T, sodium salt and 7.5 mg of alkaline phosphatase in 1 ml of water was stored at 37 for 18 hours. The reaction mixture was then lyophilized and the residue was triturated with ethanol.
Removal of the ethanol in vac-uo afforded a residue containing CL 1565-T alcohol.
Properties of CL 1565-T alcohol:
Thin-layer Chromatography on Silica Gel G~ILF
(Analtech, Inc., Neward, Delaware) Solvent: chloroform-methanol-water (75:25:1) ~f: ~.44 Using the same TLC conditions, the observed Rf of CL 1565-T, sodium salt is 0.02.
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EXAMPLE
C~ 1565-PT-3. Sodium Salt Filtered fermentation beer (719 liters) prepared from a CL 1565-PT-3 producing microorganism was passed over 31 liters of Dowex~-l x 2 (chloride form) packed in a 30.5 cm (O.D.) column. The effluent and the sub-sequent water wash did no~ contain any detectable amounts of the CL 1565 components. The Dowex*-l resin was then eluted with lM sodium chloride-methanol (l:l) and the eluate was collected in two 10-liter and six 15-liter fractions. Most of the CL 1565-A, CL 1565-B, C~ 1565-T, and additional minor CL 1565 components appeared in eluates two through six. These fractions were combined and diluted with 246 liters of acetone. The resulting mlxture was stored at 5C overnight. The clear super-natant solution was removed and concentrated to 16 liters in vacuo. Lyophilization of this concentrate afforded 800 g of a solid. This product (740 g) was added to 552 g of a similar product isolated in the same manner and the combined solids were dissolved in 20 liters of water. The resulting solution (pH 6.0) was chromato-graphed on 50 liters of HP-20* resin contained in a 15 cm (O.D.) column. Elution of the HP-20~ column with 175 liters of water removed most ~f the CL 1565-T and all of the minor, more polar CL 1565 components, including CL 1565-PT-3 and CL 1565-C (2c). The fractions con-taining these components were combined and concentrated in vacuo~ The concentrate was chromatographed on 3 kg of 135 ~m C2-reverse phase silica gel (Merck RP-2, obtained from MCB, Inc., Indianapolis, Indiana).
Elution of this column with 0.05 M p~l 7.2 phosphate buffer yielded a fraction that contained CL 1565-T and several minor components as determined by HPLC anllysis.
This fraction was concentrated and rechromatographed _ 15 -*trade mark ~A
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on 40 ~m C8-reverse pllase silica gel (Analytichem Internatlonal, Inc., Harbor City, California) ~sing a gradient elution system starting with 0.05 M pH 7.2 phosphate buffer and ending with 0.05 ~f p~l 7.2 phosphate buffer-acetonitrile (95:5). Before CL 1565-T was eluted, three minor CL 1565 components were eluted in separate groups of fractions. One of these fractions contains CL 1565-C, the isolation of which is described in Example 5. The component that was eluted in the last (the third) group of these fractions is called CL 1565-PT-3. This compound was isolated by concentration of the combined CL 1565-PT-3 fractions followed by the addition of ethanol. The inorganic salts that precipitated were filtered off and the filtrate was concentrated to d-;yness. The residue was dlssolved in ethanol and CL 1565-PT-3, sodlum salt was precipitated as a white solid by the addition of ethyl acetate.
Properties of CL 1565-PT-3. Sodium Salt Ultraviolet Absorption Spectrum in Methanol ~max 269 nm with inflectlons at 259 and 278 nm.
Infrared Spectrum in KBr principal absorptions at: 3400, 1750, 1640, 1175, 1060, and 980 reciprocal centimeters.
In Vivo Activity Against P388 Lymphatic I.eukemia in Mlce dose ~ 30 mg/kg; T/C x 100 - 150.
High Pressure Liquid Chromatography Column: Chromegabond* C-18, 4.6 mm I.D. x 30 cm (supplied by ES Industries, Marlton, NJ) Solvent: 0.1M pH 7.2 phosphate buffer-acetonitrile (88:12) Flowrate: 2 ml/min Detection: ultra~iolet absorption at 254 nm Retention Time: 1.69 min _16 *trade mark :~...... mnb / ! ~
1~9'7Z~2 4,5,8,11,18-Pentahydroxy-8-methyl-9-(phosphonoox~)-2,6,12,14,16-octadecapentaenoic acid. Sodium Salt (CL 1565-C, Sodium Salt) (2c, Sodium Salt) The CL 1565-C containing fraction desc~ibed in Example 4 was concentrated and chromatographed on 14Q g of 40 ~m C18-reverse phase silica gel using 0.05 M pH
7.0 phosphate buffer as the eluent. HPLC analysis of the ensuing fractions showed that CL 1565-C was rapidly eluted. The ~ractions that contained CL 1565-C were pooled (total volume, 220 ml), concentrated to 12 ml and rechromatographed on 140 g of C18-reverse phase silica gel using 0.05 M pH 7.1 phosphate buffer as the eluent. The eluates that contained CL 1565-C
as the only UV-absorbing material were combined and lyophilized. The product (3.55 g~ was dissolved in 7 ml of water and desalted on 140 g of C18-reverse phase silica gel using water as the eluent. The fractions containing CL 1565-C were combined and lyophilized to yield CL 1565-C, sodium salt as a white solid.
Properties of CL 1565-C, Sodium Salt Ultraviolet Absorption Spectrum in Methanol ~max 269 nm with inflections at 259 and 278 nm.
Infrared Spectrum in KBr Principal absorptions at: 3400, 1560, and ~50 reciprocal centimeters.
360 MHz H=~ ~ Spectrum in D2O
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.30 s(3H), 1.55-1.69 m(lH), 1.73 t(lH), 4.03-4.20 m(4EI), 4.68 t(lH), 4.94 t(lH), 5.55 t(lH), 5.65-5.85 m(3H), 5.85-5.95 m(2H), 6.16 t(lH), 6.36 t(lH), 6.56 t(lH), and 6.76 dd(lH) parts per million downfield from DSS.
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High Pressure Llquid Chromatography Column: Chromegabond* C-18, ~..h Tnm I.D. x 30 cm (supplied by ES Industries, Marlton, NJ) Solvent: O.lM pH 7.2 phosphate buffer-acetonitrile (88:l2) Flowrate: 2 mllmin Detection: ultraviolet absorption at 254 nm Retention Time: 1.69 min Using the same HPLC conditions, the following retention times were observed.
- CL 1565-PT-3 retention time = 2.20 min CL 1565-T retention time = 3.05 min CL 1565-~ retention time = 4.94 min Pr'èparation of CL 1565-C, Sodium Salt, from CL 1565-T
A solution of CL 1565-T, sodium salt ~22 mg) in 5 ml of water was adjusted to pH 11 with 0.1 N sodium hydroxide. After two hours the pH was readjusted to pH 11 and the reaction mixture was stored overnight at 5. The solution was adjusted to pH 7 and lyophilized to afford a white solid that contained CL 1565-C, sodium salt as shown by HPLC comparisons with a sample of CL 1565-C, sodium salt isolated using the procedure described in the previous example.
High Pressure 'Liquid Chromatography Column: ~IBondapak* C18-silica gel ~3.9 mm I.D. x 30 cm) Solvent: 0.05M pH 6.~ phosphate buffer-acetonitrile (92:8) Flowrate: 2 ml/min Detection: ultraviolet absorption at 254 nm Retention Time: 1.23 min ~sing the same conditions, the retention times of CL 1565-C isolated in tlle previous example and CL 1565-T
are 1.22 min and 2.30 min, respectively.
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5,8,11,13-Tetrahydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,l6-octadecapentaenoic acid, sodium salt (CL 1565-D, sodium salt) (2a, sodium salt).
CL 1565-A, sodium salt (100 mg) was dissolved ln 50 ml of water. The resulting solution was adjusted to pH 11 with dilute sodium hydroxide and stored at 5 overnight. The reaction mixture (pH 8.8) was again adjusted to pH 11 and stored at 5 overnight. After adjusting to pH 6, the solution was lyophilized to yield a white solid containing CL 1565-D, sodium salt.
Properties of CL 1565-D, Sodium Salt Ultraviolet Spectrum in Methanol ~max 268 nm with inflections at 259 and 278 nm.
Infrared Spectrum in KBr Principal absorptions at: 3400, 1650, 1560, 1435, 1350, 1090, and 970 reciprocal centimeters.
IIigh r~ess-lre Liquid Chromatography Column: ~Bondapak* C18-silica gel (4.6 mm I.D. x 30 cm) Solvent: 0.005M pH 7.3 phosphate buffer-acetonitrile (92:8) Flowrate: 2 ml/min Detection: UV absorption at 254 nm Retention Time: approximately 2~0 min.
Using the same conditions, the retention time of CL 1565-A is approximately 4.0 min.
5,8,11-Trihydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,16-octadecapentaenoic acid, sodium salt (CL 1565-E, sodium salt) (2b, sodium salt) In the same manner as Example 7 above, CL 1565-E, sodium salt~ the sodium salt of 2b, can be prepared starting with CL 1565-B, sodium salt (3b, sodium salt).
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5,8,9,11,18-~entahydroxy-8-meth~1_2,6,12,1~,16-octa-decapentaenoic acid, sodium salt A solution of CL 1565-A alcohol (la, 13 mg) in 1 ml of water was adjusted to pH 10.5 with lM sodium hydroxide. After standing for 30 minutes at room temperature, the reaction mixture (pH 8.2) was readjusted to pH 11.4 with lN sodium hydroxide. After four hours, the solution (pH 9.2) was lyophilized to yield a solid product containing 5,8,9,11,18-pentahydroxy-8-methyl-2,6~12;1~,l16-o~tadecapentaenoiCacid, sodium salt.
Properties:
Infrared Spectrum in KBr Principal absorptions at: 3400, 2920, 159D (CO2-), 1390, and 1060 reciprocal centimeters.
High Pressure Liquid Chromatography Column: Whatman Partisil* 10 ODS-3 (C-18 sllica gel) Solvent: water.acetonitrile (8:2) Flowrate: 2 ml/min Detection: UV absorptior at 268 nm Retention Time: 0.40 minutes.
Using the same conditions, the retention time of the starting material is 3.92 minutes.
Thin-layer Chromatography on Silica Gel 60 F254 (F. ~lerck) Solvent: chloroform-isopropanol (8:2) Detection: inspection under ultravlolet light and by spraying with a solution of 3% ceric sulfate in 3N sulfuric acld followed by heating at 110~ for ten minutes.
Rf: 0.0 Using the same system, the starting material is detected at an Rf of 0.2.
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In the same manner, 5,8,9,11-tetrahydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid, sodium salt and 4,5,8,9,11,18-hexahydroxy-8-methyl-2,6,l2,14,16-octadecapentaenoic acid, sodium salt can be prepared starting with compound lb and compound lc, respectively.
EXAMPLE 1 n 6-[6,13-Bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphon~-oxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-~-one, sodium salt (CL 1565-~ diacetate, sodium salt) CL 1565-A, sodium salt (30 mg) was acetylate~
by treatment with acetic anhydride (0.6 ml) in the presence of pyridine (0.3 ml) for 5 hours at 5. The volatile components were removed in vacuo and the residue was dissolved in 5% sodium bicarbonate solution and chromatographed over 20 ml of HP-20* resin. Eluti~n with methanol-water (70:30) yielded 21 mg of CL 1565-A
diacetate, sodium salt.
Properties of CL 1565-A Diacetate, Sodium Salt Ultraviolet Absorption Spectrum in Methanol ~max 268 nm (al = 650) with inflections at 259 and 278 nm Thin-layer Chromatography on Silica Gel 60 F254 (E. ~erck) Solvent: chloroform-isopropanol (8:2) Rf: 0.07 Solvent: chloroform-methanol-lN N~140~1 (25:30:4) Rf: 0.91 _ 21 _ *trade mark J~. mah/ I i~ ) :l~ g7Z~
Using this latter solvent, the Rf of CL 1565-A, sodLum salt is 0.27 Cytoto~icity Against L1210 Cells ID50 = approx. 6 ~g/ml High Pressure Liquid Chromatography Column: ~Bondapak7L C-18 (Waters Assoc., Inc.) Solvent: 0.05 M pH 6.8 phosphate buffer-acetonitrile (80:20) Flowrate: 1.5 ml/min Retention Time: 7.9 min Using the same HPLC conditions the retention time of CL 1565-A is 2.6 min.
360 MHz Proton Magnetic Resonance Spectrum in D20 Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.40 s (3H), 1.95 t (2H), 2.07 s (3H), 2.09 s (3H) 2.43-2.66 m (2H), 4.23 t (lH), 4.h5 d (2H), 5.10 m (lH), 5.51 t (lH), 5.74 m (lH), 5,87-6.08 m (4H), 6.19 t (lH), 6.39 t (lH), 6.64 t (lH), 6.81 d (lH), 7.10 m (lH) parts per million downfield from DSS.
5-(Acetyloxy)-6-~6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one, sodium salt (CL 1565-T ~:iacetate, sodium salt) A solution of CL 1565-T, sodium salt (30 mg) in acetic anhydride (0.6 ml) and pyridine (0.3 ml) was stored at 5 for 5 hours. After the volatile components were removed in vacuo, the residue was dissolved in 5%
(w/v) sodium bicarbonate and chromatographed over 20 ml of HP-20* resin. Elution wi~h methanolwater (30:30) and concentration of the eluate in vacuo yielded 12 mg of CL 1565-T triacetate, _ 22 _ *trade mark mab/ `t~
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Properties of CL 1565-~ Triacetate, Sodium Salt Ultraviolet Spectrum in Me-thanol ~max 268 nm wi-th inflections at 259 and 277 nm.
90 MHz Proton magnetic resonance spectrum in D2O
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.4 s(3H), 1.5-2.1 m(2H), 2.03 s(3H), 2.05 s(3H), 2.09 s(3H), 4.05-4.45 m(lH), 4.65 d(2H), 5.15-7.2 m(l2EI) parts per million downfield from DSS.
Thin-layer Chromatography on Silica Gel 60 F254 Solvent: chloroform-methanol (6:41 Rf: 0.37 In the same manner as above, 6-[6-acetyloxy-3-hydroxy-3-methy1-4-(phosphonooxy)-1,7,9,11-tridecatetra-enyl]-5,6-dihydro-2H-pyran-2-one, sodium salt (CL 1565-B acetate, sodium salt) can be prepared.
5~6-Dihydro-6-[4~6~13-tris(acetyloxy~)-3-hydroxy-3 methyl-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one CL 1565-A alcohol (30 mg) was ace-tylated by treatment with acetic anhydride (0.6 ml) in the presence of pyridine (0.3 ml) for 5 hours at 5. Following r~moval of the volatile components in vacuo, the reaction produce was chromatographed on silica gel (60-200 ~Im) r using chloroform followed by 5% methanol in chloroform.
Concentration of the 5~ methanol in chloroform eluate yielded CL 1565-A-a]cohol triacetate (35 mg).
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Properties of CL 1565-A-alcohol Triacetate Thin-layer Chromatography on Silica Gel 60 F254 (E. Merck) Solvent: chloroform-methanol (95:5) Rf: 0.43 Solvent: toluene-acetone (8:2) Rf: 0.21 360 MHz Proton Magnetic Resonance Spectrum in CDC13 Principal Signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.34 s(3H), 1.83-2.18 m ~2H), 2.07 s(3H), 2.13 s(3H), 2.14 s(3H), 2.45-2.55 m(2H), 4.69 d(2H), 4.98-5.09 m(2H), 5.47 t(lH), 5.75 m(lH), 5.85-6.03 m(3H), 6.11 d(lH), 6.17 t(lH), 6.44 t(lH), 6.57 t(lH), 6.77 dd(lH), 6.95 m(lH) parts per million down-field from tetramethylsilane.
Chemical Ionization (CH4~ Mass Spectrum m/z (% of base peak):
4.77 (M + H, 10), 459 (4), 417 (40), 399 (13), 373 (29), 357 (100), 339 (1~), 313 (79), 297 (64), 279 (12), 253 (20).
5,6-Dihydro-6-r4,6,13-tris(4-bromobenzoyloxy)-3-hydroxy-3-methyl-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one (CL 1565-A alcohol, tri-(4-bromobenzoate~) An excess of p-bromobenzoyl chloride was added to a solution of 20 mg of CL 1565-A alcohol in 1 ml of pyridine. After standing at room temperature for 48 hours, the pyridine was removed in vacuo and the residue partitioned between CH2C12 (10 ml) and saturated NaHCO3 (10 ml). The CH2C12 extract was washed with H2O (10 ml), and then evaporated to dryness.
The residue was chromatographed on silica gel to give CL 1565-A-alcohol, tri-(4-bromobenzoate) (19 mg).
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Properties of CL 1565-A alcohol, ~ri-(4-bromobenzoate) 90 M~lz Proton Magnetic Resonance Spectrum in CDC13:
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.36 s (3H), 1.9-2~4 m (411), 4.85 d t2H), 5.1-5.~ m (2H), 5.45-5.8 m (2~1), 5.82-6.12 m (5H), 6.186.9 m (4H), 7.35-8.0 m (12H) Thin-layer chromatography on Silica Gel Solvent: CHC13-isopropanol (4:1) Rf: 0.69 5,6-Dihydro-6-[3,6,13-trihydroxy-3-methyl-4-(dimethyl-phosphonooxy)-1,7,9sll-tridecatetraenyl]-2H-pyran-2-one CL 1565-A dimethyl ester CL 1565-A, sodium salt (25 mg) was dissolved in 1 ml of methanol and added with stirring at 0 to a mixture containing 1 ml Dowex* 50 x 2 (hydrogen form~ and 15 ml methanol. A solution of diazomethane in 20 ml of ether was added immediately and, after three minutes, the yellow solution was decanted from the Dowex* resin and concentrated to dryness in vacuo. The residual solid was triturated with chloroform and the chloroform-soluble material was purified by preparative layer chroma-tography on silica gel, using chloroform:methanol (8:2).
The ma~or UV absorbing band was removed from the silica gel plate to afford 9 mg of CL 1565-A dimethyl ester.
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Properties of CL 1565-A Dimeth~l Ester Thin-layer chromatography on Silica Gel 60 F254 (E. Merck) ~olvent: chloroform-methanoI (8:2) Rf: 0.49 Ultraviolet Spectrum in Methanol ~max 268 nm with inflections at 259 and 278 nm.
Infrared Spectrum in CHC13 Principal absorptions at: 3400, 1725, 1605, 1380, 1050, and 1020 reciprocal centimeters.
90 MHz Proton Magnetic Resonance Spectrum in CDC13 Principal Signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.3 s(3H), 1.55-1.85 m(2H), 2.35-2.55 m(2H), 3.78 s(3H), 3.91 s(3H), 4.23 d(2H), 4.35-5.10 m(3H), 5.5-7.0 m(lOH), parts per million down-field from tetramethylsilane.
~0 EXAMPLE 15 Preparation of Intravenous Formulations A solution of a compound prepared by any of the above examples is prepared in 1 liter of water for injection at room temperature with stirring. The solution is sterile filtered into 500 10 ml vials, each of which contains 5 ml of solution constituted to contain 75 mg of compound and is sealed under nitrogen.
Alternatively, after sterile filtration into vials, the water may be removed by lyophilization, and the vials then sealed aseptically, to provide a powder which is redissolved prior to injection.
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Preparation of Starting Materials Thc pyranone phosphate starting materials for the process of the invention designated as 3a, 3b and 3c (CL 1565~ B, and -T) can be made by cultivating a CL 1565 complex producing strain of a Streptomyces SD. isolate ATCC 31906 under artificial conditions and isolating the material.s thus produced as described in the following Examples, A, B, C and D.
EXAMPLE A
Seed development and shake flask fermentation The culture designated as ATCC 31906 in i~s dormant stage is transferred to a CIM-23 agar slant and incubated for 7 - 14 days at 28C. A portion of the microbial growth from the slant is used to inoculate an 18 x 150 mm seed tube containing 5 ml of ARM 1550 seed medlum. The seed tube is shaken at 24C fo~ 3 - 4 days.
CIM 23 agar slant Amidex* corn starch lOg N-Z amine, type A 2g Beef Extract (Difco) lg Yeast Extract (Difco) lg Cobaltous chloride-6 H20 . 0.020g Agar 20g Distilled water lOOOml ARM 1550 medium %
Bacto-Yeast Extract (Difco) 0.5 Glucose, Monohydrate 0.1 Soluble Starch (Difco) 2.4 Bacto-Tryptone (Difco) 0.5 Bacto-Beef Extract (Difco) 0.3 CaC03 0.2 NOTE: Adjust pH to 7.5 with NaOH
before adding CaC03 *trade mark ~"
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A portion (1 ml) of tl~e microbial grow~h from the seed tube is trarsferred to a 300 ml ErLenmeyer baffled shake flask containing 50 ml of SM 64 production medium.
The inoculated flask is incubated at 24C for 5 days with shaking using a gyratory shaker (2" thro~,T) set at 180 RPM. The fermentation beer after five days of fermentation is tan in color, the mycelia are granular in appearance, and the pH of the fermentation beer is about 5.5.
SM 64 Production Medium Whey (Kroger Dairy)35.0~ by volume Dextrin (Amidex* B411~
American Maize1.5~ by weight Pharmamedia (Traders Pro-tein) 4313071.5~ by welght Distilled water NOTE: Adjust pH to 6.5 with sodium hydroxide EXAMPLE B
Fermentation in 200-gallon fermentors.
Seed Development A cryogenic vial containing approximately 1 ml of culture suspension is used as the source of inocul~lm.
The contents of this cryogenic vial are thawed and aseptically transferred to a two liter, baffled Erlenmeyer flask containlng 500 ml of SD-05 seed medium.
The inoculated flask is incubated for 46-48 hours at 24C, on a gyratory shaker, at 130 RPM speed.
SD-05 Seed Medium %
Amberex* 1003 (Amber Labs) 0.5 Glucose Monohydrate (Cerelose) 0.1 Dextrin-Amidex* B411 (Corn Products) 2.4 N-Z case (Humko Sheffield) 0.5 Spray Dried Meat Solubles (Daylin Labs)0.3 3 ._ Distilled water _ 28 _ *trade mark ~` mab/ ~
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After 48 hours, the contents of the seed f:Lask are transferred aseptically to a 30-liter, stainless steel fermentor containing 16 liters of SD-05 seed medium. The inoculated fermentor is incubated for 18-24 hours at ~4C, stirred at 300 RPM, and sparged ~ith air at 1 W M rate. This microbial growth is used to inoculate the 200-gal production fermentor.
Production Fermentors A 200-gal fermentor which contains 160 gal of SM 64 is sterilized by heating with steam for 40 min.
at 121C. The medium is cooled to 24C and then inoculated with about 16 liters of the microbial growth from the 30-liter seed fermentor. The inoculated medium is allowed to ferment for five to seven days at 24C, 190 RPM
agitation, and sparged with 1 W M air. Antifoam agents, Dow Corning C and polyglycol P-2000, are used to control foaming.
The production of CL 1565-A, CL 1565-B and CL 1565-T is monitored throughout the fermentation cycle by recording fermentation parameters such as pH and percent sedimentation or growth and by a high pressure li~uid chromatographic procedure described below. An example of a fermentation profile in a 200-gal fermentor is shown in the following table.
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l~g7Z~2 % Sedimen- Micrograms Fermentation tation CL 1565-A/ml Time (hr) pH(growth) (IIPLC ~ssay) o 6.0 12 5.8 3.6 - 24 5.1 13.3 36 5.1514.7 48 5.3519.3 72 5.4522.0 3-6 96 5.9524.7 10-20 118 7.6543.3 50-65 132 7.~039.3 60-65 142 7.9040.0 60-70 This fermentor was harvested after 142 hours of fermentation with a harvest volume of 140 gal.
Isolation of CL 1565-A
Example C
The harvested beer from the above fermentation is mixed with 34 kg of Celite* 545 and filtered through a plate and frame filter press. The filtrate (473 liters) is percolated through a 30.5 cm (O.D.) column containing 120 liters of HP-20* resin (Gillies International, Inc., La Jolla, California). The resin is then washed with water (605 liters), and 90:10 water:methanol (170 liters).
Most of the CL 1565-A is then eluted from the resin with 80:20 water:methanol. High pressure liquid chromato-graphic analyses (HPLC), performed in the manner described below, of the ensuring eluates typically show the following elution profile.
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80:20 water:Methanol eluate grams o~ CL 1565-A
~1 = 340 liters <2 g #2 = 3~0 liters 11.5 g ~3 = 340 liters 7.0 g Eluates ~2 and #3 are separately concentrated and lyophilized to afford 90.2 g and 78.7 g, respectively, of dark brown solids. These products are combined and dissolved in 3 liters of water. The resulting solution is added to 27 liters of methanol with stirring. After standing overnight at 5C, the mixture is filtered and the precipitate is washed with 5 liters of methanol.
The filtrate and wash are combined, concentrated in vacuo, and lyophilized to yield 104.5 g of a solid. A
portion of this product (95 grams) in 1.5 liters of water is added slowly with mixing to 17 liters of l-propanol.
After one hour the resulting mixture is filtered and the precipitate is washed with 2 liters of l-propanol.
The filtrate and wash are comhined, concentrated, and lyophilized to afford 57 g of a solid which, by HPLC
analysis, typically contains about 15 g of CL 1565-A
This product is chromatographed, in approxi-mately 15 g lots, on 1.2 liters of 40 ~m C18-silica gel (Analytichem International, Inc., Harbor City, California) contained in a 7.6 cm (O.D.) column. The eluent is 0.005 M pH 4.5 ammonium acetate buffer followed by 0.005 M pH 4.5 ammonium acetate containing 5%
acetonitrile~ The fractions collected are assayed by HPLC. The fractions containing CL 1565-A are 30 pooled, concentrated, and lyophilized. A portion (570 mg) of the resulting product is rechromatographed using a Prep LC/System 500 apparatus fitted with a Prep~Pa}; ~00/C18 column (Waters Instruments, Inc., Milford, Massa-chusetts) and 0.1 M pH 6.5 phosphate buffer containing 10~ ace-tonitrile as the eluent. The major fractions, containing approximately 375 mg of mab/ Jl~
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CL-1565-A, are pooled and concentrated in vacuo. The aqueous soluti~n is passed throu~h a column containing 200 ml of ~IP-20 resin packed in water. The resin is then washed with 1400 ml of water and CL 1565-A 5 remaining on the column is eluted with 350 ml of 50%
methanol. The eluate is concentrated in vacuo and passed through a column containing 35 ml of Dowex-50X2 (Na ).
The effluent (pH 5.5) and a water wash of the resin are combined and lyo?hilized to yield 180 mg of purified CL 1565-A, isolated as a sodium salt.
Analysis of this product shows typically that the product contains approximately 1.3 moles of sodium per l.0 mole of parent CL 1565-A free acid. Because the free acids (CL 1565-A, CL 1565-B, and CL 1565-T) are labile, they preferably are isolated in the salt form such as the sodium salt form, preferably as the salts having about l.0 to about 2.0 moles of sodium per 1.0 mole of free acid.
Example D
Filtered beer (719 liters), prepared in the same manner as described above, are passed over 31 liters of Dowex -1 x 2 ~chloride for-m) packed in a 30.5 cm [O.D.
column. The effluent and the subsequent water ~ash usually do not contain any detectable amounts of the CL
1565 components. The entire fractionation described herein i8, monitored by the HPLC method described below usin~ 0.1 pH 6.ô phosphate buffer (Na+)-acetonitrile t88:12) as the solvent system. The Dowex-l resin is then eluted with lM
sodium chloride-methanol (1:1) and the eluate is collected in two 10-liter and six 15-liter fractions. The CL 1565-A, CL 1565-B, CL 1565-T appear in eluates two through six.
These fractions are combined and diluted with 246 liters of acetone. The resulting mixture is stored at 5C over-night. The clear supernatant solution is removed A *trade mark m~b/ ~
z and concentrated to 16 liters in vacuo. Lyophilization of this concentrate affords about 800 g of a solid. This product (740 g) is added to about 550 g of a similar product isolated in the same manner and the combined solids are dissolved in 20 liters of water. The resulting solution (pH 6.0) is chromatographed on 50 liters of *
HP-20 resin contained in a 15 cm [O.D.] column. Elution of the HP-20 column with 175 liters of water removes most of the CL 1565-T. ~lost of the CL 1565-A component is eluted with 100 liters of methanol-water (15:85);
CL 1565-B and the remaining amount of CL 1565-A are eluted with B3 liters of methanol-water (50:50). The eluates richest in CL 1565-A are combined, concentrated, and lyophilized to afford a solid which, by HPLC analysis, contains about 110 g of CL 1565-A.
A 75-gram portion of this product is dissolved in two liters of 0.05 M pH 6.8 phosphate buffer and further purified by chromatography on 52 liters (25 kg) ~0 of 100 ~m C18 reverse phase silica gel (Analytichem International, Inc., Harbor City, California) packed in 0.05 M pH 6.8 phosphate buffer (Na ) in a 15 cm [O.D.]
column. The column is developed with 0.05M phosphate buffer containing increasing amounts (4.0-6.5%) of - acetonitrile. The early fractions contain CL 1565-T.
CL 1565-A is eluted in subsequent fractions. The fractions containing CL 1565-A as the only UV-absorbing component are pooled and concentrated in vacuo to 20 liters. This concentrate is stored overnight at 5C and the inorganic salt that precipitates is filtered off. The filtrate is then charged on a 15 cm [O.D.] column containing 28 liters of HP-20 resin. The resin is washed with water (66 liters), and CL 1565-A is then eluted with 42 liters of methanol-water (50:50). The eluates that contain the majority of the CL 1565-A are combined (26 liters), *trade mark mab/ l 1~7Z~Z
concentrated, and lyophllized to yield CL 1565-A c~ntaining some inorganic impurities. The inorganic impuriti~s can be removed by dissolving the product in methanol tat 50 to 100 mg/ml), filtering off any insoluble material, and converting the filtrate to an aqueous solution by con-tinually adding water to the filtrate as it i~ being concentrated in vacuo. Final purification of CL 1565-A
is effected by chromatography of the resulti~g aqueous concentrate on HP-20 resin.
Properties of CL 1565-A. Sodium Salt Ultraviolet Absorption Spectrum in MeOH
~max 268 nm (al = 805) with inflections at 25g and 278 nm Infrared Absorption Spectrum in KBr Principal absorptions at: 3400, 1710, 1630, 1420, 1387, 1260, 1155, 1090, 1060, 975, 920, 820, and 775 reciprocal centimeters.
Optical Rotation [a]D3 + 28.2 (1.0% in 0.1 M pH 7 phosphate buEfer) Elemental Analysis %C _ZH ZNa ZP
Calcd. for ClgH27.7lo al.3 5.86 6.27 6.49 Found: - 48.01 5.88 6.05 6.3 Mass Spectrum Svia fast atom bombardment) Calcd- for [C19~25Na29P+H] = m/z 475 [ClgH26NaOgP+~] = m¦z 453 Found: m/z 475, 453 _ 34 -t~trade mark mab/ ~
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360 MHz Proton Magnetic Resonance Spectrum in D2O
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.29 s(3H), 1.58 t~lH), 1.70 m(lH), 2.49-2.58 m(2H), 4.13-4.]8 m(3H), 4.86 t(lH), 5.09 m(lH~, 5.53 t(lH), 5.9-6.~ (4H), 6.14 t(lH), 6.32 t(lH), 6.55 t~lH), 6.75 c H), and 7.09 m(lH) parts per million downfield from sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS).
C-Nuclear Magnetic Resonance Spectrum in D2O
Principal signals at:
peak number peak number 1 168.4 12 79.5 2 149.8 13 79.0 3 138.1 14 75.6 4 135.0 15 64.4 134.4 16 62~7 6 131.3 17 39.4 7 127.4 18 29.7 8 126.7 19 23.5 parts per 9 124.9 million down-124.8 field from 11 120.1 tetramethyl-silane ~TMS).
The P-Nuclear Magnetic Resonance Spectrum in D2O
exhibits a doublet (J = 10 Hz) at 0.504 ppm downfield from 8S% phosphoric acid.
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Hlgh Pressure Liquid Chromatography Column: ~Bondapak C18 silica gel (3.9 mm I.D.
x 30 cm) Solvent: 0.005M pH 7.3 sodium phosphate buffer-acetonitrile (90:10) Flowrate: 2 ml/min Detection: ultraviolet absorption at 254 nm Retention time: 2.8 min Isolation of Additional CL 1565 Components Careful chromatography of the concentrates obtained from CL 1565-beers on C18-silica gel or HP-20 resin affords fractions that contain CL 1565 components other than CL 1565-A. CL 1565-B and CL 1565-1 are isolated as essentially pure compounds. CL 1565 components A, B and T can be readily distinguished by HPLC on a ~Bondapak C18-silica gel column (3.9 mm I.D. x 30 cm) using 0.05M - O.lOM phosphate buffers containing varying proportions of acetonitrile at a flowrate of 1.5 ml/min and detection by ultraviolet absorption at 254 nm.
Typical retention times of CL 1565-A, B and T using the above HPLC conditions are given in the following table.
Retention time (min) in:
Solvent ~# So].vent B##
CL 1565-T2.8 <1.5 CL 1565-A4.3 <1.5 CL 1565-B>15 4.2 # 0.05M pH 7.4 phosphate buffer-acetonitrile (87:13) ## 0.05M pH 7.4 phosphate buffer-acetonitrile (78:22) *trade mark mab / 1~, ~
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Crude beers can be ass~yed in the above manner except the solvent used is 0.1 M pH 6.8 phosphate buffer-acetonitrile (88:12). In this case, at a flo~rate of 2 ml/min, the retention times of CL 1565-T, CL 1565-A
and CL 1565-B are approximately 2.7, 5.0 and >12 minutes, respectively.
CL 1565-T is eluted earlier than CL 1565-A
from HP-20 resin and from reverse phase silica gel. It can be isolated from the early fractions of the C18-silica gel column described in example D, above, using HP-20 resin.
CL 1565-B is eluted more slowly than CL 1565-A
*
from HP-20 resin and from reverse phase silica gel.
CL 1565-B is eluted with 50% methanol during the HP-20 chromatography of the crude Dowex-l product described in example D, above. This component can best be isolated *
by rechromatography on HP-20 followed by chromatography on 40 ~m C18-silica gel using essentially the same procedure described for the purification of CL 1565-A.
Properties of CL 1565-T. Sodium Salt Ultraviolet Absorption Spectrum in MeOH
Nearly identical to that for CL 1565-A, sodium salt with al = 774 at ~max 268 nm and inflections at 260 and 278 nm.
Infrared Absorption Spectrum in KBr Principal absorptions at: 3400, 1715, 1630, 1380, 1260, 1090, 970, 830 and 770 reciprocal centimeters.
Mass Spectrum (via fast atom b-ombardment) Calcd. for [ClgH25Na2O10P+H] = m/z 491 Pound: m/z 491 - 37 ~
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360 MHz Proton Magnetic Resonance Spectrum in D2O
The H-NMR spectrum of CL 1565-T is very similar to the H-NMR spectrum of CL 1565-A with the exception that the formex spectrum e~hibits a characteristic one proton signal appearing as a doublet of doublets at 4.34 ppm and is devoid of any signals between 2.2 - 3.2 ppm downfield from DSS.
Principal Signals oE CL 1565-T, sodium salt are at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.30 s(3H), 1.55-1.64 m~lH), 1.73 t(lH), 4.13-4.20 m(lH), 4.16 d(2H), 4.34 dd(lH), 4.94 t(lH), 5.09 dd(lH), 5.55 t(lH), 5.89 -6.06 m~3H), 6.16 m(2H), 6.36 t(lH), 6.56 t (lH), 6.76 dd(lH), 7.14 dd(lH) parts per million downfield from DSS
90.4 MHz 13C-Nuclear Magnetic Resonance Spectrum in D2O:
Peak Number Chemical Shift Peak Number Chemical Shift 1 24.10 11 126.91 2 41.60 12 127.18 3 64.68 13 128.99 4 64.90 14 133.36 66.67 15 136.87 6 78.28 16 137.23 7 79.81 17 142.27 8 84.33 18 14g.46 9 124.40 19 169.66 126.21 *parts per million downfield from TMS
_ 38 -mab/~
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Properties of CL 1565-r,. So~iulll Salt Ultraviolet ~b~;orption Spec~rum in MeOH
~max 267 nm (al - 805) with inflections at 259 and 277 nm Infrared Absorption Spectrum in KBr Principa] absorptions at: 1720, 1640, 1385, L200, 1060, 970 and 820 reciprocal centimeters.
360 MHz Proton Magnetic Resonance Spectrum in D2O
Principal Signals at:
(s=singlet, d=doublet, t=triplet, m-multiplet) 1.32 s (3H), 1.58 t (lH), 1.72 t (lH~, 1.79 d ~3H~, 2.45-2.68 m (2H), 4.15 t (11A~) ~ 4.89 t (lH), 5.10 m (lH), 5.49 t (lH), 5.83-6.21 m (6H), 6.50-6.64 n (2H), 7.06-7.13 m (lH) parts per million downfleld from DSS.
90.4 MH~ 13C-Nuclear Magnetic Resonance Spectrum in D2O:
Peak Number Chemical Shift* Peak ~umber Chemical Shift*
1 20.70 11 127.24 2 25.06 12 129 47 3 31.91 13 129.90 4 41.85 14 134.66 66.85 15 135.94 6 77.87 16 136.67 7 80.X7 17 14~.42 8 81.64 18 152.~1 9 122.41 19 170.56 124.45 *parts per million downfield from TMS
39 ~
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on 40 ~m C8-reverse pllase silica gel (Analytichem Internatlonal, Inc., Harbor City, California) ~sing a gradient elution system starting with 0.05 M pH 7.2 phosphate buffer and ending with 0.05 ~f p~l 7.2 phosphate buffer-acetonitrile (95:5). Before CL 1565-T was eluted, three minor CL 1565 components were eluted in separate groups of fractions. One of these fractions contains CL 1565-C, the isolation of which is described in Example 5. The component that was eluted in the last (the third) group of these fractions is called CL 1565-PT-3. This compound was isolated by concentration of the combined CL 1565-PT-3 fractions followed by the addition of ethanol. The inorganic salts that precipitated were filtered off and the filtrate was concentrated to d-;yness. The residue was dlssolved in ethanol and CL 1565-PT-3, sodlum salt was precipitated as a white solid by the addition of ethyl acetate.
Properties of CL 1565-PT-3. Sodium Salt Ultraviolet Absorption Spectrum in Methanol ~max 269 nm with inflectlons at 259 and 278 nm.
Infrared Spectrum in KBr principal absorptions at: 3400, 1750, 1640, 1175, 1060, and 980 reciprocal centimeters.
In Vivo Activity Against P388 Lymphatic I.eukemia in Mlce dose ~ 30 mg/kg; T/C x 100 - 150.
High Pressure Liquid Chromatography Column: Chromegabond* C-18, 4.6 mm I.D. x 30 cm (supplied by ES Industries, Marlton, NJ) Solvent: 0.1M pH 7.2 phosphate buffer-acetonitrile (88:12) Flowrate: 2 ml/min Detection: ultra~iolet absorption at 254 nm Retention Time: 1.69 min _16 *trade mark :~...... mnb / ! ~
1~9'7Z~2 4,5,8,11,18-Pentahydroxy-8-methyl-9-(phosphonoox~)-2,6,12,14,16-octadecapentaenoic acid. Sodium Salt (CL 1565-C, Sodium Salt) (2c, Sodium Salt) The CL 1565-C containing fraction desc~ibed in Example 4 was concentrated and chromatographed on 14Q g of 40 ~m C18-reverse phase silica gel using 0.05 M pH
7.0 phosphate buffer as the eluent. HPLC analysis of the ensuing fractions showed that CL 1565-C was rapidly eluted. The ~ractions that contained CL 1565-C were pooled (total volume, 220 ml), concentrated to 12 ml and rechromatographed on 140 g of C18-reverse phase silica gel using 0.05 M pH 7.1 phosphate buffer as the eluent. The eluates that contained CL 1565-C
as the only UV-absorbing material were combined and lyophilized. The product (3.55 g~ was dissolved in 7 ml of water and desalted on 140 g of C18-reverse phase silica gel using water as the eluent. The fractions containing CL 1565-C were combined and lyophilized to yield CL 1565-C, sodium salt as a white solid.
Properties of CL 1565-C, Sodium Salt Ultraviolet Absorption Spectrum in Methanol ~max 269 nm with inflections at 259 and 278 nm.
Infrared Spectrum in KBr Principal absorptions at: 3400, 1560, and ~50 reciprocal centimeters.
360 MHz H=~ ~ Spectrum in D2O
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.30 s(3H), 1.55-1.69 m(lH), 1.73 t(lH), 4.03-4.20 m(4EI), 4.68 t(lH), 4.94 t(lH), 5.55 t(lH), 5.65-5.85 m(3H), 5.85-5.95 m(2H), 6.16 t(lH), 6.36 t(lH), 6.56 t(lH), and 6.76 dd(lH) parts per million downfield from DSS.
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High Pressure Llquid Chromatography Column: Chromegabond* C-18, ~..h Tnm I.D. x 30 cm (supplied by ES Industries, Marlton, NJ) Solvent: O.lM pH 7.2 phosphate buffer-acetonitrile (88:l2) Flowrate: 2 mllmin Detection: ultraviolet absorption at 254 nm Retention Time: 1.69 min Using the same HPLC conditions, the following retention times were observed.
- CL 1565-PT-3 retention time = 2.20 min CL 1565-T retention time = 3.05 min CL 1565-~ retention time = 4.94 min Pr'èparation of CL 1565-C, Sodium Salt, from CL 1565-T
A solution of CL 1565-T, sodium salt ~22 mg) in 5 ml of water was adjusted to pH 11 with 0.1 N sodium hydroxide. After two hours the pH was readjusted to pH 11 and the reaction mixture was stored overnight at 5. The solution was adjusted to pH 7 and lyophilized to afford a white solid that contained CL 1565-C, sodium salt as shown by HPLC comparisons with a sample of CL 1565-C, sodium salt isolated using the procedure described in the previous example.
High Pressure 'Liquid Chromatography Column: ~IBondapak* C18-silica gel ~3.9 mm I.D. x 30 cm) Solvent: 0.05M pH 6.~ phosphate buffer-acetonitrile (92:8) Flowrate: 2 ml/min Detection: ultraviolet absorption at 254 nm Retention Time: 1.23 min ~sing the same conditions, the retention times of CL 1565-C isolated in tlle previous example and CL 1565-T
are 1.22 min and 2.30 min, respectively.
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5,8,11,13-Tetrahydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,l6-octadecapentaenoic acid, sodium salt (CL 1565-D, sodium salt) (2a, sodium salt).
CL 1565-A, sodium salt (100 mg) was dissolved ln 50 ml of water. The resulting solution was adjusted to pH 11 with dilute sodium hydroxide and stored at 5 overnight. The reaction mixture (pH 8.8) was again adjusted to pH 11 and stored at 5 overnight. After adjusting to pH 6, the solution was lyophilized to yield a white solid containing CL 1565-D, sodium salt.
Properties of CL 1565-D, Sodium Salt Ultraviolet Spectrum in Methanol ~max 268 nm with inflections at 259 and 278 nm.
Infrared Spectrum in KBr Principal absorptions at: 3400, 1650, 1560, 1435, 1350, 1090, and 970 reciprocal centimeters.
IIigh r~ess-lre Liquid Chromatography Column: ~Bondapak* C18-silica gel (4.6 mm I.D. x 30 cm) Solvent: 0.005M pH 7.3 phosphate buffer-acetonitrile (92:8) Flowrate: 2 ml/min Detection: UV absorption at 254 nm Retention Time: approximately 2~0 min.
Using the same conditions, the retention time of CL 1565-A is approximately 4.0 min.
5,8,11-Trihydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,16-octadecapentaenoic acid, sodium salt (CL 1565-E, sodium salt) (2b, sodium salt) In the same manner as Example 7 above, CL 1565-E, sodium salt~ the sodium salt of 2b, can be prepared starting with CL 1565-B, sodium salt (3b, sodium salt).
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5,8,9,11,18-~entahydroxy-8-meth~1_2,6,12,1~,16-octa-decapentaenoic acid, sodium salt A solution of CL 1565-A alcohol (la, 13 mg) in 1 ml of water was adjusted to pH 10.5 with lM sodium hydroxide. After standing for 30 minutes at room temperature, the reaction mixture (pH 8.2) was readjusted to pH 11.4 with lN sodium hydroxide. After four hours, the solution (pH 9.2) was lyophilized to yield a solid product containing 5,8,9,11,18-pentahydroxy-8-methyl-2,6~12;1~,l16-o~tadecapentaenoiCacid, sodium salt.
Properties:
Infrared Spectrum in KBr Principal absorptions at: 3400, 2920, 159D (CO2-), 1390, and 1060 reciprocal centimeters.
High Pressure Liquid Chromatography Column: Whatman Partisil* 10 ODS-3 (C-18 sllica gel) Solvent: water.acetonitrile (8:2) Flowrate: 2 ml/min Detection: UV absorptior at 268 nm Retention Time: 0.40 minutes.
Using the same conditions, the retention time of the starting material is 3.92 minutes.
Thin-layer Chromatography on Silica Gel 60 F254 (F. ~lerck) Solvent: chloroform-isopropanol (8:2) Detection: inspection under ultravlolet light and by spraying with a solution of 3% ceric sulfate in 3N sulfuric acld followed by heating at 110~ for ten minutes.
Rf: 0.0 Using the same system, the starting material is detected at an Rf of 0.2.
_ 20 -*trade mark mab/ ~
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In the same manner, 5,8,9,11-tetrahydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid, sodium salt and 4,5,8,9,11,18-hexahydroxy-8-methyl-2,6,l2,14,16-octadecapentaenoic acid, sodium salt can be prepared starting with compound lb and compound lc, respectively.
EXAMPLE 1 n 6-[6,13-Bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphon~-oxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-~-one, sodium salt (CL 1565-~ diacetate, sodium salt) CL 1565-A, sodium salt (30 mg) was acetylate~
by treatment with acetic anhydride (0.6 ml) in the presence of pyridine (0.3 ml) for 5 hours at 5. The volatile components were removed in vacuo and the residue was dissolved in 5% sodium bicarbonate solution and chromatographed over 20 ml of HP-20* resin. Eluti~n with methanol-water (70:30) yielded 21 mg of CL 1565-A
diacetate, sodium salt.
Properties of CL 1565-A Diacetate, Sodium Salt Ultraviolet Absorption Spectrum in Methanol ~max 268 nm (al = 650) with inflections at 259 and 278 nm Thin-layer Chromatography on Silica Gel 60 F254 (E. ~erck) Solvent: chloroform-isopropanol (8:2) Rf: 0.07 Solvent: chloroform-methanol-lN N~140~1 (25:30:4) Rf: 0.91 _ 21 _ *trade mark J~. mah/ I i~ ) :l~ g7Z~
Using this latter solvent, the Rf of CL 1565-A, sodLum salt is 0.27 Cytoto~icity Against L1210 Cells ID50 = approx. 6 ~g/ml High Pressure Liquid Chromatography Column: ~Bondapak7L C-18 (Waters Assoc., Inc.) Solvent: 0.05 M pH 6.8 phosphate buffer-acetonitrile (80:20) Flowrate: 1.5 ml/min Retention Time: 7.9 min Using the same HPLC conditions the retention time of CL 1565-A is 2.6 min.
360 MHz Proton Magnetic Resonance Spectrum in D20 Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.40 s (3H), 1.95 t (2H), 2.07 s (3H), 2.09 s (3H) 2.43-2.66 m (2H), 4.23 t (lH), 4.h5 d (2H), 5.10 m (lH), 5.51 t (lH), 5.74 m (lH), 5,87-6.08 m (4H), 6.19 t (lH), 6.39 t (lH), 6.64 t (lH), 6.81 d (lH), 7.10 m (lH) parts per million downfield from DSS.
5-(Acetyloxy)-6-~6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one, sodium salt (CL 1565-T ~:iacetate, sodium salt) A solution of CL 1565-T, sodium salt (30 mg) in acetic anhydride (0.6 ml) and pyridine (0.3 ml) was stored at 5 for 5 hours. After the volatile components were removed in vacuo, the residue was dissolved in 5%
(w/v) sodium bicarbonate and chromatographed over 20 ml of HP-20* resin. Elution wi~h methanolwater (30:30) and concentration of the eluate in vacuo yielded 12 mg of CL 1565-T triacetate, _ 22 _ *trade mark mab/ `t~
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Properties of CL 1565-~ Triacetate, Sodium Salt Ultraviolet Spectrum in Me-thanol ~max 268 nm wi-th inflections at 259 and 277 nm.
90 MHz Proton magnetic resonance spectrum in D2O
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.4 s(3H), 1.5-2.1 m(2H), 2.03 s(3H), 2.05 s(3H), 2.09 s(3H), 4.05-4.45 m(lH), 4.65 d(2H), 5.15-7.2 m(l2EI) parts per million downfield from DSS.
Thin-layer Chromatography on Silica Gel 60 F254 Solvent: chloroform-methanol (6:41 Rf: 0.37 In the same manner as above, 6-[6-acetyloxy-3-hydroxy-3-methy1-4-(phosphonooxy)-1,7,9,11-tridecatetra-enyl]-5,6-dihydro-2H-pyran-2-one, sodium salt (CL 1565-B acetate, sodium salt) can be prepared.
5~6-Dihydro-6-[4~6~13-tris(acetyloxy~)-3-hydroxy-3 methyl-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one CL 1565-A alcohol (30 mg) was ace-tylated by treatment with acetic anhydride (0.6 ml) in the presence of pyridine (0.3 ml) for 5 hours at 5. Following r~moval of the volatile components in vacuo, the reaction produce was chromatographed on silica gel (60-200 ~Im) r using chloroform followed by 5% methanol in chloroform.
Concentration of the 5~ methanol in chloroform eluate yielded CL 1565-A-a]cohol triacetate (35 mg).
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Properties of CL 1565-A-alcohol Triacetate Thin-layer Chromatography on Silica Gel 60 F254 (E. Merck) Solvent: chloroform-methanol (95:5) Rf: 0.43 Solvent: toluene-acetone (8:2) Rf: 0.21 360 MHz Proton Magnetic Resonance Spectrum in CDC13 Principal Signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.34 s(3H), 1.83-2.18 m ~2H), 2.07 s(3H), 2.13 s(3H), 2.14 s(3H), 2.45-2.55 m(2H), 4.69 d(2H), 4.98-5.09 m(2H), 5.47 t(lH), 5.75 m(lH), 5.85-6.03 m(3H), 6.11 d(lH), 6.17 t(lH), 6.44 t(lH), 6.57 t(lH), 6.77 dd(lH), 6.95 m(lH) parts per million down-field from tetramethylsilane.
Chemical Ionization (CH4~ Mass Spectrum m/z (% of base peak):
4.77 (M + H, 10), 459 (4), 417 (40), 399 (13), 373 (29), 357 (100), 339 (1~), 313 (79), 297 (64), 279 (12), 253 (20).
5,6-Dihydro-6-r4,6,13-tris(4-bromobenzoyloxy)-3-hydroxy-3-methyl-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one (CL 1565-A alcohol, tri-(4-bromobenzoate~) An excess of p-bromobenzoyl chloride was added to a solution of 20 mg of CL 1565-A alcohol in 1 ml of pyridine. After standing at room temperature for 48 hours, the pyridine was removed in vacuo and the residue partitioned between CH2C12 (10 ml) and saturated NaHCO3 (10 ml). The CH2C12 extract was washed with H2O (10 ml), and then evaporated to dryness.
The residue was chromatographed on silica gel to give CL 1565-A-alcohol, tri-(4-bromobenzoate) (19 mg).
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Properties of CL 1565-A alcohol, ~ri-(4-bromobenzoate) 90 M~lz Proton Magnetic Resonance Spectrum in CDC13:
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.36 s (3H), 1.9-2~4 m (411), 4.85 d t2H), 5.1-5.~ m (2H), 5.45-5.8 m (2~1), 5.82-6.12 m (5H), 6.186.9 m (4H), 7.35-8.0 m (12H) Thin-layer chromatography on Silica Gel Solvent: CHC13-isopropanol (4:1) Rf: 0.69 5,6-Dihydro-6-[3,6,13-trihydroxy-3-methyl-4-(dimethyl-phosphonooxy)-1,7,9sll-tridecatetraenyl]-2H-pyran-2-one CL 1565-A dimethyl ester CL 1565-A, sodium salt (25 mg) was dissolved in 1 ml of methanol and added with stirring at 0 to a mixture containing 1 ml Dowex* 50 x 2 (hydrogen form~ and 15 ml methanol. A solution of diazomethane in 20 ml of ether was added immediately and, after three minutes, the yellow solution was decanted from the Dowex* resin and concentrated to dryness in vacuo. The residual solid was triturated with chloroform and the chloroform-soluble material was purified by preparative layer chroma-tography on silica gel, using chloroform:methanol (8:2).
The ma~or UV absorbing band was removed from the silica gel plate to afford 9 mg of CL 1565-A dimethyl ester.
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Properties of CL 1565-A Dimeth~l Ester Thin-layer chromatography on Silica Gel 60 F254 (E. Merck) ~olvent: chloroform-methanoI (8:2) Rf: 0.49 Ultraviolet Spectrum in Methanol ~max 268 nm with inflections at 259 and 278 nm.
Infrared Spectrum in CHC13 Principal absorptions at: 3400, 1725, 1605, 1380, 1050, and 1020 reciprocal centimeters.
90 MHz Proton Magnetic Resonance Spectrum in CDC13 Principal Signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.3 s(3H), 1.55-1.85 m(2H), 2.35-2.55 m(2H), 3.78 s(3H), 3.91 s(3H), 4.23 d(2H), 4.35-5.10 m(3H), 5.5-7.0 m(lOH), parts per million down-field from tetramethylsilane.
~0 EXAMPLE 15 Preparation of Intravenous Formulations A solution of a compound prepared by any of the above examples is prepared in 1 liter of water for injection at room temperature with stirring. The solution is sterile filtered into 500 10 ml vials, each of which contains 5 ml of solution constituted to contain 75 mg of compound and is sealed under nitrogen.
Alternatively, after sterile filtration into vials, the water may be removed by lyophilization, and the vials then sealed aseptically, to provide a powder which is redissolved prior to injection.
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Preparation of Starting Materials Thc pyranone phosphate starting materials for the process of the invention designated as 3a, 3b and 3c (CL 1565~ B, and -T) can be made by cultivating a CL 1565 complex producing strain of a Streptomyces SD. isolate ATCC 31906 under artificial conditions and isolating the material.s thus produced as described in the following Examples, A, B, C and D.
EXAMPLE A
Seed development and shake flask fermentation The culture designated as ATCC 31906 in i~s dormant stage is transferred to a CIM-23 agar slant and incubated for 7 - 14 days at 28C. A portion of the microbial growth from the slant is used to inoculate an 18 x 150 mm seed tube containing 5 ml of ARM 1550 seed medlum. The seed tube is shaken at 24C fo~ 3 - 4 days.
CIM 23 agar slant Amidex* corn starch lOg N-Z amine, type A 2g Beef Extract (Difco) lg Yeast Extract (Difco) lg Cobaltous chloride-6 H20 . 0.020g Agar 20g Distilled water lOOOml ARM 1550 medium %
Bacto-Yeast Extract (Difco) 0.5 Glucose, Monohydrate 0.1 Soluble Starch (Difco) 2.4 Bacto-Tryptone (Difco) 0.5 Bacto-Beef Extract (Difco) 0.3 CaC03 0.2 NOTE: Adjust pH to 7.5 with NaOH
before adding CaC03 *trade mark ~"
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A portion (1 ml) of tl~e microbial grow~h from the seed tube is trarsferred to a 300 ml ErLenmeyer baffled shake flask containing 50 ml of SM 64 production medium.
The inoculated flask is incubated at 24C for 5 days with shaking using a gyratory shaker (2" thro~,T) set at 180 RPM. The fermentation beer after five days of fermentation is tan in color, the mycelia are granular in appearance, and the pH of the fermentation beer is about 5.5.
SM 64 Production Medium Whey (Kroger Dairy)35.0~ by volume Dextrin (Amidex* B411~
American Maize1.5~ by weight Pharmamedia (Traders Pro-tein) 4313071.5~ by welght Distilled water NOTE: Adjust pH to 6.5 with sodium hydroxide EXAMPLE B
Fermentation in 200-gallon fermentors.
Seed Development A cryogenic vial containing approximately 1 ml of culture suspension is used as the source of inocul~lm.
The contents of this cryogenic vial are thawed and aseptically transferred to a two liter, baffled Erlenmeyer flask containlng 500 ml of SD-05 seed medium.
The inoculated flask is incubated for 46-48 hours at 24C, on a gyratory shaker, at 130 RPM speed.
SD-05 Seed Medium %
Amberex* 1003 (Amber Labs) 0.5 Glucose Monohydrate (Cerelose) 0.1 Dextrin-Amidex* B411 (Corn Products) 2.4 N-Z case (Humko Sheffield) 0.5 Spray Dried Meat Solubles (Daylin Labs)0.3 3 ._ Distilled water _ 28 _ *trade mark ~` mab/ ~
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After 48 hours, the contents of the seed f:Lask are transferred aseptically to a 30-liter, stainless steel fermentor containing 16 liters of SD-05 seed medium. The inoculated fermentor is incubated for 18-24 hours at ~4C, stirred at 300 RPM, and sparged ~ith air at 1 W M rate. This microbial growth is used to inoculate the 200-gal production fermentor.
Production Fermentors A 200-gal fermentor which contains 160 gal of SM 64 is sterilized by heating with steam for 40 min.
at 121C. The medium is cooled to 24C and then inoculated with about 16 liters of the microbial growth from the 30-liter seed fermentor. The inoculated medium is allowed to ferment for five to seven days at 24C, 190 RPM
agitation, and sparged with 1 W M air. Antifoam agents, Dow Corning C and polyglycol P-2000, are used to control foaming.
The production of CL 1565-A, CL 1565-B and CL 1565-T is monitored throughout the fermentation cycle by recording fermentation parameters such as pH and percent sedimentation or growth and by a high pressure li~uid chromatographic procedure described below. An example of a fermentation profile in a 200-gal fermentor is shown in the following table.
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l~g7Z~2 % Sedimen- Micrograms Fermentation tation CL 1565-A/ml Time (hr) pH(growth) (IIPLC ~ssay) o 6.0 12 5.8 3.6 - 24 5.1 13.3 36 5.1514.7 48 5.3519.3 72 5.4522.0 3-6 96 5.9524.7 10-20 118 7.6543.3 50-65 132 7.~039.3 60-65 142 7.9040.0 60-70 This fermentor was harvested after 142 hours of fermentation with a harvest volume of 140 gal.
Isolation of CL 1565-A
Example C
The harvested beer from the above fermentation is mixed with 34 kg of Celite* 545 and filtered through a plate and frame filter press. The filtrate (473 liters) is percolated through a 30.5 cm (O.D.) column containing 120 liters of HP-20* resin (Gillies International, Inc., La Jolla, California). The resin is then washed with water (605 liters), and 90:10 water:methanol (170 liters).
Most of the CL 1565-A is then eluted from the resin with 80:20 water:methanol. High pressure liquid chromato-graphic analyses (HPLC), performed in the manner described below, of the ensuring eluates typically show the following elution profile.
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80:20 water:Methanol eluate grams o~ CL 1565-A
~1 = 340 liters <2 g #2 = 3~0 liters 11.5 g ~3 = 340 liters 7.0 g Eluates ~2 and #3 are separately concentrated and lyophilized to afford 90.2 g and 78.7 g, respectively, of dark brown solids. These products are combined and dissolved in 3 liters of water. The resulting solution is added to 27 liters of methanol with stirring. After standing overnight at 5C, the mixture is filtered and the precipitate is washed with 5 liters of methanol.
The filtrate and wash are combined, concentrated in vacuo, and lyophilized to yield 104.5 g of a solid. A
portion of this product (95 grams) in 1.5 liters of water is added slowly with mixing to 17 liters of l-propanol.
After one hour the resulting mixture is filtered and the precipitate is washed with 2 liters of l-propanol.
The filtrate and wash are comhined, concentrated, and lyophilized to afford 57 g of a solid which, by HPLC
analysis, typically contains about 15 g of CL 1565-A
This product is chromatographed, in approxi-mately 15 g lots, on 1.2 liters of 40 ~m C18-silica gel (Analytichem International, Inc., Harbor City, California) contained in a 7.6 cm (O.D.) column. The eluent is 0.005 M pH 4.5 ammonium acetate buffer followed by 0.005 M pH 4.5 ammonium acetate containing 5%
acetonitrile~ The fractions collected are assayed by HPLC. The fractions containing CL 1565-A are 30 pooled, concentrated, and lyophilized. A portion (570 mg) of the resulting product is rechromatographed using a Prep LC/System 500 apparatus fitted with a Prep~Pa}; ~00/C18 column (Waters Instruments, Inc., Milford, Massa-chusetts) and 0.1 M pH 6.5 phosphate buffer containing 10~ ace-tonitrile as the eluent. The major fractions, containing approximately 375 mg of mab/ Jl~
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CL-1565-A, are pooled and concentrated in vacuo. The aqueous soluti~n is passed throu~h a column containing 200 ml of ~IP-20 resin packed in water. The resin is then washed with 1400 ml of water and CL 1565-A 5 remaining on the column is eluted with 350 ml of 50%
methanol. The eluate is concentrated in vacuo and passed through a column containing 35 ml of Dowex-50X2 (Na ).
The effluent (pH 5.5) and a water wash of the resin are combined and lyo?hilized to yield 180 mg of purified CL 1565-A, isolated as a sodium salt.
Analysis of this product shows typically that the product contains approximately 1.3 moles of sodium per l.0 mole of parent CL 1565-A free acid. Because the free acids (CL 1565-A, CL 1565-B, and CL 1565-T) are labile, they preferably are isolated in the salt form such as the sodium salt form, preferably as the salts having about l.0 to about 2.0 moles of sodium per 1.0 mole of free acid.
Example D
Filtered beer (719 liters), prepared in the same manner as described above, are passed over 31 liters of Dowex -1 x 2 ~chloride for-m) packed in a 30.5 cm [O.D.
column. The effluent and the subsequent water ~ash usually do not contain any detectable amounts of the CL
1565 components. The entire fractionation described herein i8, monitored by the HPLC method described below usin~ 0.1 pH 6.ô phosphate buffer (Na+)-acetonitrile t88:12) as the solvent system. The Dowex-l resin is then eluted with lM
sodium chloride-methanol (1:1) and the eluate is collected in two 10-liter and six 15-liter fractions. The CL 1565-A, CL 1565-B, CL 1565-T appear in eluates two through six.
These fractions are combined and diluted with 246 liters of acetone. The resulting mixture is stored at 5C over-night. The clear supernatant solution is removed A *trade mark m~b/ ~
z and concentrated to 16 liters in vacuo. Lyophilization of this concentrate affords about 800 g of a solid. This product (740 g) is added to about 550 g of a similar product isolated in the same manner and the combined solids are dissolved in 20 liters of water. The resulting solution (pH 6.0) is chromatographed on 50 liters of *
HP-20 resin contained in a 15 cm [O.D.] column. Elution of the HP-20 column with 175 liters of water removes most of the CL 1565-T. ~lost of the CL 1565-A component is eluted with 100 liters of methanol-water (15:85);
CL 1565-B and the remaining amount of CL 1565-A are eluted with B3 liters of methanol-water (50:50). The eluates richest in CL 1565-A are combined, concentrated, and lyophilized to afford a solid which, by HPLC analysis, contains about 110 g of CL 1565-A.
A 75-gram portion of this product is dissolved in two liters of 0.05 M pH 6.8 phosphate buffer and further purified by chromatography on 52 liters (25 kg) ~0 of 100 ~m C18 reverse phase silica gel (Analytichem International, Inc., Harbor City, California) packed in 0.05 M pH 6.8 phosphate buffer (Na ) in a 15 cm [O.D.]
column. The column is developed with 0.05M phosphate buffer containing increasing amounts (4.0-6.5%) of - acetonitrile. The early fractions contain CL 1565-T.
CL 1565-A is eluted in subsequent fractions. The fractions containing CL 1565-A as the only UV-absorbing component are pooled and concentrated in vacuo to 20 liters. This concentrate is stored overnight at 5C and the inorganic salt that precipitates is filtered off. The filtrate is then charged on a 15 cm [O.D.] column containing 28 liters of HP-20 resin. The resin is washed with water (66 liters), and CL 1565-A is then eluted with 42 liters of methanol-water (50:50). The eluates that contain the majority of the CL 1565-A are combined (26 liters), *trade mark mab/ l 1~7Z~Z
concentrated, and lyophllized to yield CL 1565-A c~ntaining some inorganic impurities. The inorganic impuriti~s can be removed by dissolving the product in methanol tat 50 to 100 mg/ml), filtering off any insoluble material, and converting the filtrate to an aqueous solution by con-tinually adding water to the filtrate as it i~ being concentrated in vacuo. Final purification of CL 1565-A
is effected by chromatography of the resulti~g aqueous concentrate on HP-20 resin.
Properties of CL 1565-A. Sodium Salt Ultraviolet Absorption Spectrum in MeOH
~max 268 nm (al = 805) with inflections at 25g and 278 nm Infrared Absorption Spectrum in KBr Principal absorptions at: 3400, 1710, 1630, 1420, 1387, 1260, 1155, 1090, 1060, 975, 920, 820, and 775 reciprocal centimeters.
Optical Rotation [a]D3 + 28.2 (1.0% in 0.1 M pH 7 phosphate buEfer) Elemental Analysis %C _ZH ZNa ZP
Calcd. for ClgH27.7lo al.3 5.86 6.27 6.49 Found: - 48.01 5.88 6.05 6.3 Mass Spectrum Svia fast atom bombardment) Calcd- for [C19~25Na29P+H] = m/z 475 [ClgH26NaOgP+~] = m¦z 453 Found: m/z 475, 453 _ 34 -t~trade mark mab/ ~
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360 MHz Proton Magnetic Resonance Spectrum in D2O
Principal signals at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.29 s(3H), 1.58 t~lH), 1.70 m(lH), 2.49-2.58 m(2H), 4.13-4.]8 m(3H), 4.86 t(lH), 5.09 m(lH~, 5.53 t(lH), 5.9-6.~ (4H), 6.14 t(lH), 6.32 t(lH), 6.55 t~lH), 6.75 c H), and 7.09 m(lH) parts per million downfield from sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS).
C-Nuclear Magnetic Resonance Spectrum in D2O
Principal signals at:
peak number peak number 1 168.4 12 79.5 2 149.8 13 79.0 3 138.1 14 75.6 4 135.0 15 64.4 134.4 16 62~7 6 131.3 17 39.4 7 127.4 18 29.7 8 126.7 19 23.5 parts per 9 124.9 million down-124.8 field from 11 120.1 tetramethyl-silane ~TMS).
The P-Nuclear Magnetic Resonance Spectrum in D2O
exhibits a doublet (J = 10 Hz) at 0.504 ppm downfield from 8S% phosphoric acid.
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Hlgh Pressure Liquid Chromatography Column: ~Bondapak C18 silica gel (3.9 mm I.D.
x 30 cm) Solvent: 0.005M pH 7.3 sodium phosphate buffer-acetonitrile (90:10) Flowrate: 2 ml/min Detection: ultraviolet absorption at 254 nm Retention time: 2.8 min Isolation of Additional CL 1565 Components Careful chromatography of the concentrates obtained from CL 1565-beers on C18-silica gel or HP-20 resin affords fractions that contain CL 1565 components other than CL 1565-A. CL 1565-B and CL 1565-1 are isolated as essentially pure compounds. CL 1565 components A, B and T can be readily distinguished by HPLC on a ~Bondapak C18-silica gel column (3.9 mm I.D. x 30 cm) using 0.05M - O.lOM phosphate buffers containing varying proportions of acetonitrile at a flowrate of 1.5 ml/min and detection by ultraviolet absorption at 254 nm.
Typical retention times of CL 1565-A, B and T using the above HPLC conditions are given in the following table.
Retention time (min) in:
Solvent ~# So].vent B##
CL 1565-T2.8 <1.5 CL 1565-A4.3 <1.5 CL 1565-B>15 4.2 # 0.05M pH 7.4 phosphate buffer-acetonitrile (87:13) ## 0.05M pH 7.4 phosphate buffer-acetonitrile (78:22) *trade mark mab / 1~, ~
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Crude beers can be ass~yed in the above manner except the solvent used is 0.1 M pH 6.8 phosphate buffer-acetonitrile (88:12). In this case, at a flo~rate of 2 ml/min, the retention times of CL 1565-T, CL 1565-A
and CL 1565-B are approximately 2.7, 5.0 and >12 minutes, respectively.
CL 1565-T is eluted earlier than CL 1565-A
from HP-20 resin and from reverse phase silica gel. It can be isolated from the early fractions of the C18-silica gel column described in example D, above, using HP-20 resin.
CL 1565-B is eluted more slowly than CL 1565-A
*
from HP-20 resin and from reverse phase silica gel.
CL 1565-B is eluted with 50% methanol during the HP-20 chromatography of the crude Dowex-l product described in example D, above. This component can best be isolated *
by rechromatography on HP-20 followed by chromatography on 40 ~m C18-silica gel using essentially the same procedure described for the purification of CL 1565-A.
Properties of CL 1565-T. Sodium Salt Ultraviolet Absorption Spectrum in MeOH
Nearly identical to that for CL 1565-A, sodium salt with al = 774 at ~max 268 nm and inflections at 260 and 278 nm.
Infrared Absorption Spectrum in KBr Principal absorptions at: 3400, 1715, 1630, 1380, 1260, 1090, 970, 830 and 770 reciprocal centimeters.
Mass Spectrum (via fast atom b-ombardment) Calcd. for [ClgH25Na2O10P+H] = m/z 491 Pound: m/z 491 - 37 ~
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360 MHz Proton Magnetic Resonance Spectrum in D2O
The H-NMR spectrum of CL 1565-T is very similar to the H-NMR spectrum of CL 1565-A with the exception that the formex spectrum e~hibits a characteristic one proton signal appearing as a doublet of doublets at 4.34 ppm and is devoid of any signals between 2.2 - 3.2 ppm downfield from DSS.
Principal Signals oE CL 1565-T, sodium salt are at:
(s=singlet, d=doublet, t=triplet, m=multiplet) 1.30 s(3H), 1.55-1.64 m~lH), 1.73 t(lH), 4.13-4.20 m(lH), 4.16 d(2H), 4.34 dd(lH), 4.94 t(lH), 5.09 dd(lH), 5.55 t(lH), 5.89 -6.06 m~3H), 6.16 m(2H), 6.36 t(lH), 6.56 t (lH), 6.76 dd(lH), 7.14 dd(lH) parts per million downfield from DSS
90.4 MHz 13C-Nuclear Magnetic Resonance Spectrum in D2O:
Peak Number Chemical Shift Peak Number Chemical Shift 1 24.10 11 126.91 2 41.60 12 127.18 3 64.68 13 128.99 4 64.90 14 133.36 66.67 15 136.87 6 78.28 16 137.23 7 79.81 17 142.27 8 84.33 18 14g.46 9 124.40 19 169.66 126.21 *parts per million downfield from TMS
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Properties of CL 1565-r,. So~iulll Salt Ultraviolet ~b~;orption Spec~rum in MeOH
~max 267 nm (al - 805) with inflections at 259 and 277 nm Infrared Absorption Spectrum in KBr Principa] absorptions at: 1720, 1640, 1385, L200, 1060, 970 and 820 reciprocal centimeters.
360 MHz Proton Magnetic Resonance Spectrum in D2O
Principal Signals at:
(s=singlet, d=doublet, t=triplet, m-multiplet) 1.32 s (3H), 1.58 t (lH), 1.72 t (lH~, 1.79 d ~3H~, 2.45-2.68 m (2H), 4.15 t (11A~) ~ 4.89 t (lH), 5.10 m (lH), 5.49 t (lH), 5.83-6.21 m (6H), 6.50-6.64 n (2H), 7.06-7.13 m (lH) parts per million downfleld from DSS.
90.4 MH~ 13C-Nuclear Magnetic Resonance Spectrum in D2O:
Peak Number Chemical Shift* Peak ~umber Chemical Shift*
1 20.70 11 127.24 2 25.06 12 129 47 3 31.91 13 129.90 4 41.85 14 134.66 66.85 15 135.94 6 77.87 16 136.67 7 80.X7 17 14~.42 8 81.64 18 152.~1 9 122.41 19 170.56 124.45 *parts per million downfield from TMS
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Claims (58)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing a compound of general formula wherein for the compound of formula (3a), R1 represents H and R2 represents -OH;
for the compound of formula (3b), R1 and R2 represent H; and for the compound of formula (3c), R1 and R2 represents -OH;
said process comprising:
cultivating a CL 1565 complex producing strain of a Streptomyces SD. isolate ATCC 31906 to produce the desired compounds of formula (3a) to (3c); and isolating the desired compounds of formula (3a) to (3b).
for the compound of formula (3b), R1 and R2 represent H; and for the compound of formula (3c), R1 and R2 represents -OH;
said process comprising:
cultivating a CL 1565 complex producing strain of a Streptomyces SD. isolate ATCC 31906 to produce the desired compounds of formula (3a) to (3c); and isolating the desired compounds of formula (3a) to (3b).
2. A compound of general formula wherein R1 and R2 are as defined in claim 1; and wherein:
the compound of formula (3a) is designated CL 1565-A, the compound of formula (3b) is designated CL 1565-B, and the compound of formula (3c) is designated CL 1565-T;
when prepared by the process defined in claim 1.
the compound of formula (3a) is designated CL 1565-A, the compound of formula (3b) is designated CL 1565-B, and the compound of formula (3c) is designated CL 1565-T;
when prepared by the process defined in claim 1.
3. The process defined in claim 1, and further comprising acylating at least one primary or secondary -OH
group of the compounds of formula (3a) to (3c).
group of the compounds of formula (3a) to (3c).
4. An acyl ester of the compounds of formula (3a) co (3c) as defined in claim 2, when prepared by the process defined in claim 3.
5. The process defined in claim 1, and further comprising preparing a pharmaceutically acceptable salt of the compounds of formula (3a) to (3c).
6. A pharmaceutically acceptable salt of the compounds of formula (3a) to (3c) as defined in claim 2, when prepared by the process defined in claim 5.
7. The process defined in claim 3, and further comprising preparing a pharmaceutically acceptable salt of an acyl ester of the compounds of formula (3a) to (3c).
8. A pharmaceutically acceptable salt of an acyl ester of compounds (3a) to (3c) as defined in claim 2, when prepared by the process defined in claim 7.
9. The process defined in claim 1, and further comprising preparing a loweralkyl or aryl mono- or diester of the phosphate function of the compounds of formula (3a) to (3c).
10. A loweralkyl or aryl phosphate mono- or diester of the compounds of formula (3a) to (3c) as defined in claim 2, when prepared by the process defined in claim 9.
11. Tile process defined in claim 7, comprising acetylating the 6- and 13-OH groups of the compound of formula (3a), and preparing the Na salt of the product.
12. 6-[6,13-Bis(acetyloxy)-3-hydroxy-3-methyl-4-phosphonooxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one (sodium salt), when prepared by the process defined in claim 11.
13. The process defined in claim 7, comprising acetylating the 6-OH group of the compound of formula (b), and preparing the Na salt of the product.
14. 6-[6-(Acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy) 1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one (sodium salt), when prepared by the process defined in claim 13.
15. The process defined in claim 7, comprising acetylating the 5-, 6- and 13-OH groups of the compound of formula (3c), and preparing the Na salt of the product.
16. 5-Acetyloxy-6-[6,13-bis(acetyloxy)-3-hydroxy-3-methyl-4-(phosphonooxy)-1,7,9,11-tridecatetraenyl]-5,6-dihydro-2H-pyran-2-one (sodium salt), when prepared by the process defined in claim 15.
17. The process defined in claim 9, comprising preparing the dimethyl phosphate ester of the compound of formula (3a).
18. 5,6-Dihydro-6-[3,6,13-trihydroxy-3-methyl-4-dimethylphosphonooxy-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one, when prepared by the process defined in claim 17.
19. The process defined in claim 1, and further comprising dephosphorylating the compounds of formula (3a) to (3c).
20. A compound of general formula wherein:
for the compound of formula (1a), R1 represents H and R2 represents -OH;
for the compound of formula (1b), R1 and R2 represent H, and for the compound of formula (1c), R1 and R2 represent -OH;
when prepared by the process defined in claim 19.
for the compound of formula (1a), R1 represents H and R2 represents -OH;
for the compound of formula (1b), R1 and R2 represent H, and for the compound of formula (1c), R1 and R2 represent -OH;
when prepared by the process defined in claim 19.
21. The process defined in claim 19, and further comprising acylating at least one primary or secondary -OH
group of the compounds of formula (1a) to (1c).
group of the compounds of formula (1a) to (1c).
22. An acyl ester of the compounds of formula (1a) to (1c) as defined in claim 29, when prepared by the process defined in claim 21.
23. The process defined in claim 19, comprising dephosphorylating the compound of formula (3a).
24. 5,6-Dihydro-6-(3,4,6,13-tetrahydroxy-3-methyl-1,7,9,11-tridecatetraenyl)- 2H-pyran-2-one, when prepared by the process defined in claim 23.
25. The process defined in claim 19, comprising dephosphorylating the compound of formula (3b).
26. 5,6-Dihydro-6-(3,4,6-trihydroxy-3-methyl-1,7,9,11-tridecatetraenyl)- 2H-pyran-2-one, when prepared by the process defined in claim 25.
27. The process defined in claim 19, comprising dephosphorylating the compound of formula (3c).
28. 5,6-Dihydro-5-hydroxy-6-(3,4,6,13-tetrahydroxy-3-methyl-1,7,9,11-tridecatetraenyl)-2H-pyran-2-one, when prepared by the process defined in claim 27.
29. The process defined in claim 21, comprising acetylating the 4-, 6- and 13-OH groups of the compound of formula (1a).
30. 5,6-Dihydro-6-[4,6,13-tris(acetyloxy)-3-hydroxy-3-methyl-1,7,9,11-tridecatetraenyl]-2H-pyran-2-one, when prepared by the process defined in claim 29.
31. The process defined in claim 1, and further comprising hydrolyzing, under ring opening conditions, the compounds of formula (3a) to (3c).
32. A compound of general formula wherein:
for the compound of formula (2a), R1 represents H and R2 represents -OH, for the compound of formula (2b), R1 and R2 represent H, and for the compound of formula (2c), R1 and R2 represent -OH;
when prepdred by the process defined in claim 31.
for the compound of formula (2a), R1 represents H and R2 represents -OH, for the compound of formula (2b), R1 and R2 represent H, and for the compound of formula (2c), R1 and R2 represent -OH;
when prepdred by the process defined in claim 31.
33. The process defined in claim 31, and further comprising preparing a loweralkyl or aryl mono- or diester of the phosphate function of the compounds of formula (2a) to (2c).
34. A loweralkyl or aryl phosphate mono- or di-ester of the compounds of formula (2a) to (2c) as defined in claim 32, when prepared by the process defined in claim 33.
35. The process defined in claim 31, and further comprising preparing a pharmaceutically acceptable salt of the compounds of formula (2a) to (2c).
36. A pharmaceutically acceptable salt of the compounds of formula (2a) to (2c) as defined in claim 32, when prepared by the process defined in claim 35.
37. The process defined in claim 35, comprising hydrolyzing, under ring opening conditions, the compound of formula (3a), and preparing the Na salt of the product.
38. 5,8,11,18-Tetrahydroxy-8-methyl-9-(phosphono-oxy)-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 37.
39. The process defined in claim 35, comprising hydrolyzing, under ring opening conditions, the compound of formula (3b), and preparing the Na salt of the product.
40. 5,8,11-Trihydroxy-8-methyl-9-(phosphonooxy)-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 39.
41. The process defined in claim 35, comprising hydrolyzing, under ring opening conditions, the compound of formula (3c), and preparing the Na salt of the product.
42. 4,5,8,11,18-pentahydroxy-8-methyl-9-(phosphono-oxy)-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 41.
43. The process defined in claim 31, and further comprising dephosphorylating the compounds of formula (2a) to (2c), and preparing a pharmaceutically acceptable salt of the products.
44. The process defined in claim 43 t comprising dephosphorylating the compound of formula (2a), and preparing the Na salt of the product.
45. 5,8,9,11,18-pentahydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 44.
46. The process defined in claim 43, comprising dephosphorylating the compound of formula (2b), and preparing the Na salt of the product.
47. 5,8,9,11-Tetrahydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 46.
48. The process defined in claim 43, comprising dephosphorylating the compound of formula (2c), and preparing the Na salt of the product.
49. 4,5,8,9,11,18-hexahydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 48.
50. The process defined in claim 31, and further comprising dephosphorylating the compounds of formula (2a) to (2c), acylating at least one primary or secondary -OH
group of the productl and preparing a pharmaceutically acceptable salt thereof.
group of the productl and preparing a pharmaceutically acceptable salt thereof.
51. The process defined in claim 50, comprising dephosphorylating the compound of formula (2a), acetylating the 5-, 9-, 11- and 18-OH groups, and preparing the Na salt of the product.
52. 5,9,11,18-Tetra(acetyloxy)-8-hydroxy-8-methyl-2,6,12,14,16-octadecapentaenoic acid (sodium salt), when prepared by the process defined in claim 51.
53. A process for preparing a compound designated CL 1565-PT-3, wherein said compound is characterized by:
(i) an ultraviolet absorption spectrum in methanol of Amax 269 nm with inflections at 259 and 278 nm; and (ii) principal infrared absorption in KBr of 3,400, 1,750, 1,640, 1,175, 1,060 and 980 cm-1;
said process comprising:
cultivating a CL 1565-PT-3 producing microorganism to produce the desired compound; and isolating the desired CL 1565-PT-3.
(i) an ultraviolet absorption spectrum in methanol of Amax 269 nm with inflections at 259 and 278 nm; and (ii) principal infrared absorption in KBr of 3,400, 1,750, 1,640, 1,175, 1,060 and 980 cm-1;
said process comprising:
cultivating a CL 1565-PT-3 producing microorganism to produce the desired compound; and isolating the desired CL 1565-PT-3.
54. A compound designated CL 1565-PT-3 as defined in claim 53, when prepared by the process defined in claim 53.
55. The process defined in claim 53, and further comprising preparing a pharmaceutically acceptable salt of the compound designated CL 1565-PT-3.
56. A pharmaceutically acceptable salt of a compound designated CL 1565-PT-3 as defined in claim 54, when prepared by the process defined in claim 55.
57. The process of claim 55, comprising preparing the Na salt.
58. The Na salt of a compound designated CL 1565-PT-3 as defined in claim 54, when prepared by the process defined in claim 57.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US49388883A | 1983-05-12 | 1983-05-12 | |
| US493,888 | 1983-05-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1197202A true CA1197202A (en) | 1985-11-26 |
Family
ID=23962119
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000452122A Expired CA1197202A (en) | 1983-05-12 | 1984-04-16 | Antibiotic cl-1565 complex derivatives |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS606679A (en) |
| CA (1) | CA1197202A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101550162B (en) * | 2008-04-03 | 2015-11-25 | 北京华昊中天生物技术有限公司 | Fostriecin derivant and pharmaceutical usage thereof |
-
1984
- 1984-04-16 CA CA000452122A patent/CA1197202A/en not_active Expired
- 1984-05-11 JP JP9308784A patent/JPS606679A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPS606679A (en) | 1985-01-14 |
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