Note: Descriptions are shown in the official language in which they were submitted.
1~?7~
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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b. phosphates, preferably as their sodium salts or
other salts, having the structural formulas 2a~
2b, and 2c:
~C02H
CH
CH CH3 OH
R -CE~-CH-CH=CH-C CH-CH2-CH-CH=CH-CH=CH-CH=CH-CH2R
OH HO O~
P3H2
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~
P3H2
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).
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
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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
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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~
m b/
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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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EXAMPLE 1
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
D2O
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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
EXAMPLE 2
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 -
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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.
EXAMPLE 3
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 -
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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
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EXAMPLE 5
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
EXAMPLE 6
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.
*trade mark
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EXAMPLE 7
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.
EXAMPLE 8
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).
-- 19 --
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RXAMPLE 9
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 -
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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 _
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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.
EXAMPLE 11
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 _
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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.
EXAMPLE 12
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).
mabt~ t
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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).
EXAMPLE 13
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).
- 24 -
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1~ ~72~;~
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
EXAMPLE 14
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
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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
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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.
_ ~9
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% 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
32 -
A *trade mark
m~b/ ~
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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
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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)
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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
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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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