U.S. patent application number 10/983710 was filed with the patent office on 2005-06-09 for tricyclo compounds, a process for their production and a pharmaceutical composition containing the same.
This patent application is currently assigned to FUJISAWA PHARMACEUTICAL CO., LTD.. Invention is credited to Goto, Toshio, Hatanaka, Hiroshi, Kino, Tohru, Okuhara, Masakuni, Tanaka, Hirokazu.
Application Number | 20050124646 10/983710 |
Document ID | / |
Family ID | 27262535 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124646 |
Kind Code |
A1 |
Okuhara, Masakuni ; et
al. |
June 9, 2005 |
Tricyclo compounds, a process for their production and a
pharmaceutical composition containing the same
Abstract
This invention relates to tricyclo compounds useful for
treatment and prevention of resistance by transplantation,
graft-versus-host diseases by medulla ossium transplantation,
autoimmune diseases, infectious diseases, and the like, which can
be represented by the following formula: 1 to a process for their
production, to a pharmaceutical composition containing the same and
to a use thereof.
Inventors: |
Okuhara, Masakuni; (Ibaraki,
JP) ; Tanaka, Hirokazu; (Ibaraki, JP) ; Goto,
Toshio; (Ibaraki, JP) ; Kino, Tohru;
(Tsuchiura-shi, JP) ; Hatanaka, Hiroshi; (Ibaraki,
JP) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
700 THIRTEENTH ST. NW
SUITE 300
WASHINGTON
DC
20005-3960
US
|
Assignee: |
FUJISAWA PHARMACEUTICAL CO.,
LTD.
Osaka
JP
|
Family ID: |
27262535 |
Appl. No.: |
10/983710 |
Filed: |
November 9, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10983710 |
Nov 9, 2004 |
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10426659 |
May 1, 2003 |
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10426659 |
May 1, 2003 |
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10247305 |
Sep 20, 2002 |
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10247305 |
Sep 20, 2002 |
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09721650 |
Nov 27, 2000 |
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6482845 |
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09721650 |
Nov 27, 2000 |
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09411630 |
Oct 1, 1999 |
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6201005 |
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09411630 |
Oct 1, 1999 |
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09084099 |
May 26, 1998 |
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6028097 |
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09084099 |
May 26, 1998 |
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08753950 |
Dec 3, 1996 |
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5830717 |
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08753950 |
Dec 3, 1996 |
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08450412 |
May 25, 1995 |
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5624842 |
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08450412 |
May 25, 1995 |
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07824384 |
Jan 23, 1992 |
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07824384 |
Jan 23, 1992 |
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07491205 |
Mar 9, 1990 |
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5110811 |
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07491205 |
Mar 9, 1990 |
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06868749 |
May 30, 1986 |
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4929611 |
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06868749 |
May 30, 1986 |
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06799855 |
Nov 20, 1985 |
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4894366 |
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Current U.S.
Class: |
514/291 ;
540/456; 540/457 |
Current CPC
Class: |
C07D 498/18 20130101;
A61P 31/04 20180101; C12P 19/44 20130101; C12N 1/205 20210501; A61P
37/06 20180101; A61P 37/00 20180101; C12P 17/188 20130101; Y10S
435/898 20130101; C07H 19/01 20130101; Y10S 435/886 20130101; A61P
31/00 20180101; C12R 2001/465 20210501; C12R 2001/55 20210501; Y02P
20/55 20151101 |
Class at
Publication: |
514/291 ;
540/456; 540/457 |
International
Class: |
A61K 031/4745; C07D
491/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 1984 |
GB |
8430455 |
Feb 5, 1985 |
GB |
8502869 |
Apr 1, 1985 |
GB |
8508420 |
Claims
1-19. (canceled)
20. A method for suppressing Mixed Lymphocyte Reaction by using a
compound of the formula: 16wherein R.sup.1 is hydroxy or
pharmaceutically acceptable protected hydroxy selected from the
group consisting of 1-(lower alkylthio)(lower)alkyloxy,
tri(lower)alkylsilyloxy, lower alkyl-diphenyl-siloxy,
pharmaceutically acceptable organic carboxylic acyloxy,
pharmaceutically acceptable organic sulfonic acyloxy, and
pharmaceutically acceptable organic carbamic acyloxy, R.sup.2 is
hydrogen or the same meanings as R.sup.1, R.sup.3 is methyl, ethyl,
propyl or allyl, n is an integer of 1 or 2, and the symbol of a
line and dotted line is a single bond or a double bond, or
pharmaceutically acceptable basic salt thereof.
21. The method of claim 20, wherein the compound is 17
22. The method of claim 20, wherein the compound is 18
23. The method of claim 20, wherein the compound is 19
Description
[0001] This invention relates to novel tricyclo compounds having
pharmacological activities, to a process for their production and
to a pharmaceutical composition containing the same.
[0002] More particularly, it relates to novel tricyclo compounds,
which have pharmacological activities such as immunosuppressive
activity, antimicrobial activity, and the like, to a process for
their production, to a pharmaceutical composition containing the
same and to a use thereof.
[0003] Accordingly, one object of this invention is to provide a
novel tricyclo compounds, which are useful for treatment and
prevention of resistance by transplantation, graft-versus-host
diseases by medulla ossium transplantation, autoimmune diseases,
infectious diseases, and the like.
[0004] Another object of this invention is to provide a process for
production of the tricyclo compounds by fermentation processes and
synthetic processes.
[0005] A further object of this invention is to provide a
pharmaceutical composition containing, as active ingredients, the
tricyclo compounds.
[0006] Still further object of this invention is to provide a use
of the tricyclo compounds for manufacturing a medicament for
treating and preventing resistance by transplantation,
graft-versus-host diseases by medulla ossium transplantation,
autoimmune diseases, infectious diseases, and the like.
[0007] With respect to the present invention, it is to be noted
that this invention is originated from and based on the first and
new discovery of new certain specific compounds, FR-900506,
FR-900520, FR-900523 and FR-900525 substances. In more detail, the
FR-900506, FR-900520, FR-900523 and FR-900525 substances were
firstly and newly isolated in pure form from culture broths
obtained by fermentation of new species belonging to genus
Streptomyces.
[0008] And, as a result of an extensive study for elucidation of
chemical structures of the FR-900506, FR-900520, FR-900523 and
FR-900525 substances, the inventors of this invention have
succeeded in determining the chemical structures thereof and in
producing the tricyclo compounds of this invention.
[0009] The new tricyclo compounds of this invention can be
represented by the following general formula: 2
[0010] wherein
[0011] R.sup.1 is hydroxy or protected hydroxy,
[0012] R.sup.2 is hydrogen, hydroxy or protected hydroxy,
[0013] R.sup.3 is methyl, ethyl, propyl or allyl,
[0014] n is an integer of 1 or 2, and
[0015] the symbol of a line and dotted line is a single bond or a
double bond,
[0016] and salts thereof.
[0017] Among the object compound (I), the following four specific
compounds were found to be produced by fermentation.
[0018] (1) The compound (I) wherein R.sup.1 and R.sup.2 are each
hydroxy, R.sup.3 is allyl, n is an integer of 2, and the symbol of
a line and dotted line is a single bond, which is entitled to the
FR-900506 substance;
[0019] (2) The compound (I) wherein R.sup.1 and R.sup.2 are each
hydroxy, R.sup.3 is ethyl, n is an integer of 2, and the symbol of
a line and dotted line is a single bond, which is entitled to the
FR-900520 substance (another name: the WS 7238A substance);
[0020] (3) The compound (I) wherein R.sup.1 and R.sup.2 are each
hydroxy, R.sup.3 is methyl, n is an integer of 2, and the symbol of
a line and dotted line is a single bond, which is entitled to the
FR-900523 substance (another name: the WS 7238B substance); and
[0021] (4) The compound (I) wherein R.sup.1 and R.sup.2 are each
hydroxy, R.sup.3 is allyl, n is an integer of 1, and the symbol of
a line and dotted line is a single bond, which is entitled to the
FR-900525 substance.
[0022] With respect to the tricyclo compounds (I) of this
invention, it is to be understood that there may be one or more
conformer(s) or stereoisomeric pairs such as optical and
geometrical isomers due to asymmetric carbon atom(s) and double
bond(s), and such isomers are also included within a scope of this
invention.
[0023] According to this invention, the object tricyclo compounds
(I) can be prepared by the following processes.
[0024] [I] Fermentation Processes: 3
[0025] [II] Synthetic Processes:
[0026] (1) Process 1 (Introduction of Hydroxy-Protective Group)
4
[0027] (2) Process 2 (Introduction of Hydroxy-Protective Group)
5
[0028] (3) Process 3 (Formation of Double Bond) 6
[0029] (4) Process 4 (Oxidation of Hydroxyethylene Group) 7
[0030] (5) Process 5 (Reduction of Allyl Group) 8
[0031] (6) Process 6 (Removal of the Carboxy-Protective Group)
9
[0032] in which
[0033] R.sup.1, R.sup.2, R.sup.3, n and the symbol of a line and
dotted line are each as defined above,
[0034] R.sub.a.sup.1 and R.sub.a.sup.2 are each protected
hydroxy,
[0035] R.sub.b.sup.1 is protected
carboxy(lower)alkylcarbamoyloxy,
[0036] R.sub.c.sup.1 is carboxy(lower)alkylcarbamoyloxy, and
[0037] R.sub.b.sup.2 is a leaving group.
[0038] Particulars of the above definitions and the preferred
embodiments thereof are explained in detail as follows.
[0039] The term "lower" used in the specification is intended to
mean 1 to 6 carbon atoms, unless otherwise indicated.
[0040] Suitable hydroxy-protective group in the "protected hydroxy"
may include:
[0041] 1-(lower alkylthio)(lower; alkyl such as lower
alkylthiomethyl (e.g. methythiomethyl, ethylthiomethyl,
propylthiomethyl, isopropylthiomethyl, butylthiomethyl,
isobutylthiomethyl, hexylthiomethyl, etc.), and the like, in which
the preferred one may be C.sub.1-C.sub.4alkylthiomethyl and the
most preferred one may be methylthiomethyl;
[0042] trisubstituted silyl such as tri(lower)alkylsilyl (e.g.
trimethylsilyl, triethylsilyl, tributylsilyl,
tert-butyl-dimethylsilyl, tri-tert-butylsilyl, etc.), lower
alkyl-diarylsilyl (e.g. methyl-diphenylsilyl, ethyl-diphenylsilyl,
propyl-diphehylsilyl, tert-butyl-diphenylsilyl, etc.), and the
like, in which the preferred one may be
tri(C.sub.1-C.sub.4)alkylsilyl and C.sub.1-C.sub.4alkyl-diphenylsi-
lyl, and the most preferred one may be tert-butyl-dimethylsilyl and
tert-butyl-diphenylsilyl;
[0043] acyl such as aliphatic acyl, aromatic acyl and aliphatic
acyl substituted with aromatic group, which are derived from
carboxylic, sulfonic and carbamic acids; and the like.
[0044] The aliphatic acyl may include lower alkanoyl which may have
one or more suitable substituent(s) such as carboxy (e.g. formyl,
acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl,
pivaloyl, hexanoyl, carboxvacetyl, carboxypropionyl,
carboxybutyryl, carboxyhexanoyl, etc.)
cyclo(lower)alkyloxy(lower)alkanoyl which may have one or more
suitable substituent(s) such as lower alkyl (e.g.
cyclopropyloxyacetyl, cyclobutyloxypropionyl,
cycloheptyloxybutyryl, menthyloxyacetyl, menthyloxypropionyl,
menthyloxybutyryl, menthyloxyheptanoyl, menthyloxyhexanoyl, etc.),
camphorsulfonyl, lower alkylcarbamoyl having one or more suitable
substituent(s) such as carboxy and a protected carboxy, for
example, carboxy(lower)alkylcarbamoyl (e.g. carboxymethylcarbamoyl,
carboxyethylcarbamoyl, carboxypropylcarbamoyl,
carboxybutylcarbamoyl, carboxypentylcarbamoyl,
carboxyhexylcarbamoyl, etc.), protected
carboxy(lower)alkylcarbamoyl such as
tri(lower)alkylsilyl(lower)alkoxycarbonyl(lower)alkylcarbamoyl
(e.g. trimethylsilylmethoxycarbonylethylcarbamoyl,
trimethylsilylethoxycarbonyl- propylcarbamoyl,
triethylsilylethoxycarbonylpropylcarbamoyl,
tert-butyldimethylsilylethoxycarbonylpropylcarbamoyl,
trimethylsilylpropoxycarbonylbutylcarbamoyl, etc.), and the
like.
[0045] The aromatic acyl may include aroyl which may have one or
more suitable substituent(s) such as nitro (e.g. benzoyl, toluoyl,
xyloyl, naphthoyl, nitrobenzoyl, dinitrobenzoyl, nitronaphthoyl,
etc.), arenesulfonyl which may have one or more suitable
substituent(s) such as halogen (e.g. benzenesulfonyl,
toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl,
fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl,
iodobenzenesulfonyl, etc.), and the like.
[0046] The aliphatic acyl substituted with aromatic group may
include ar(lower)alkanoyl which may hate one or more suitable
substituent(s) such as lower alkoxy and trihalo(lower)alkyl (e.g.
phenylacetyl, phenylpropionyl, phenylbutyryl,
2-trifluoromethyl-2-methoxy-2-phenylacety- l,
2-ethyl-2-trifluoromethyl-2-phenylacetyl,
2-trifluoromethyl-2-propoxy-2- -phenylacetyl, etc.), and the
like.
[0047] The more preferred acyl group thus defined may be
C.sub.1-C.sub.4alkanoyl which may have carboxy,
cyclo(C.sub.5-C.sub.6)alk- yloxy(C.sub.1-C.sub.4)alkanoyl having
two (C.sub.1-C.sub.4)alkyl groups on the cycloalkyl moiety,
camphorsulfonyl, carboxy(C.sub.1-C.sub.4)alkylcarb- amoyl,
tri(C.sub.1-C.sub.4)alkylsilyl(C.sub.1-C.sub.4)alkoxycarbonyl(C.sub-
.1-C.sub.4)alkylcarbamoyl, benzoyl which may have one or two nitro,
benzenesulfonyl having halogen, phenyl(C.sub.1-C.sub.4)alkanoyl
having C.sub.1-C.sub.4alkoxy and trihalo(C.sub.1-C.sub.4)alkyl, and
the most preferred one may be acetyl, carboxypropionyl,
menthyloxyacetyl, camphorsulfonyl, benzoyl, nitrobenzoyl,
dinitrobenzoyl, iodobenzenesulfonyl and
2-trifluoromethyl-2-methoxy-2-phenylacetyl.
[0048] Suitable "protected carboxy(lower)alkylcarbamoyl" and
"carboxy(lower)alkylcarbamoyl" moieties of the "protected
carboxy(lower)alkylcarbamoyloxy" and
"carboxy(lower)alkylcarbamoyloxy" groups may include the same as
those exemplified in the explanation of the hydroxy-protective
group mentioned above.
[0049] Suitable "leaving group" may include hydroxy, acyloxy in
which the acyl moiety may be those as exemplified above, and the
like.
[0050] The processes for production of the tricyclo compounds (I)
of this invention are explained in detail in the following.
[0051] [I] Fermentation Processes:
[0052] The FR-900506, FR-900520, FR-900523 and FR-900525 substances
of this invention can be produced by fermentation of FR-900506,
FR-900520, FR-900523 and/or FR-900525 substance(s)-producing
strains belonging to the genus Streptomyces such as Streptomyces
tsukubaensis No. 9993 and Streptomyces hygroscopicus subsp.
yakushimaensis No. 7238 in a nutrient medium.
[0053] Particulars of microorganisms used for the production of the
FR-900506, FR-900520, FR-900523 and FR-900525 substances are
explained in the following.
[0054] [A] The FR-900506, FR-900520 and FR-900525 substances of
this invention can be produced by fermentation of a FR-900506,
FR-900520 and/or FR-900525 substance(s)-producing strain belonging
to the genus Streptomyces such as
[0055] Streptomyces tsukubaensis No. 9993 in a nutrient medium.
The Microorganism
[0056] The microorganism which can be used for the production of
the FR-900506, FR-900520 and/or FR-900525 substances is FR-900506,
FR-900520 and/or FR-900525 substance(s)-producing strain belonging
to the genus Streptomyces, among which Streptomyces tsukubaensis
No. 9993 has been newly insulated from a soil sample collected at
Toyosato-cho, Tsukuba-gun, Ibaraki Prefecture, Japan.
[0057] A lyophilized sample of the newly isolated Streptomyces
tsukubaensis No. 9993 has been deposited with the Fermentation
Research Institute, Agency of Industrial Science and Technology
(No. 1-3, Higashi 1-chome, Yatabemachi Tsukuba-gun, Ibaraki
Prefecture, Japan) under the deposit number of FERM P-7886
(deposited date: Oct. 5th, 1984), and then converted to Budapest
Treaty route of the same depository on Oct. 19, 1985 under the new
deposit number of FERM BP-927.
[0058] It is to be understood that the production of the novel
FR-900506, FR-900520 and/or FR-900525 substance(s) is not limited
to the use of the particular organism described herein, which is
given for the illustrative purpose only. This invention also
includes the use of any mutants which are capable of producing the
FR-900506, FR-900520 and/or FR-900525 substances including natural
mutants as well as artificial mutants which can be produced from
the described organism by conventional means such as irradiation of
X-rays, ultra-violet radiation, treatment with
N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the
like.
[0059] The Streptomyces tsukubaensis No. 9993 has the following
morphological, cultural, biological and physiological
characteristics.
[0060] [1] Morphological Characteristics:
[0061] The methods described by Shirling and Gottlieb (Shirling, E.
B. and D. Gottlieb: Methods for characterization of Streptomyces
species. International Journal of Systematic Bacteriology, 16,
313-340, 1966) were employed principally for this taxonomic
study.
[0062] Morphological observations were made with light and electron
microscopes on cultures grown at 30.degree. C. for 14 days on
oatmeal agar, yeast-malt extract agar and inorganic salts-starch
agar. The mature sporophores formed Rectiflexibiles with 10 to 50
or more than 50 spores in each chain. The spores were oblong or
cylindrical, 0.5-0.7.times.0.7-0.8 .mu.m in size by electron
microscopic observation. Spore surfaces were smooth.
[0063] [2] Cultural Characteristics:
[0064] Cultural characteristics were observed on ten kinds of media
described by Shirling and Gottlieb as mentioned above, and by
Waksman (Waksman, S. A.: The actinomycetes, vol. 2: Classification,
identification and description of genera and species. The Williams
and Wilkins Co., Baltimore, 1961).
[0065] The incubation was made at 30.degree. C. for 14 days. The
color names used in this study were based on Guide to Color
Standard (manual published by Nippon Shikisai Kenkyusho, Tokyo).
Colonies belonged to the gray color series when grown on oatmeal
agar, yeast-malt extract agar and inorganic salts-starch agar.
Soluble pigment was produced in yeast-malt extract agar but not in
other media. The results are shown in Table 1.
1TABLE 1 Cultural Characteristics of Strain No. 9993 and
Streptomyces misakiensis IFO 12891 Cultural characteristics Medium
No. 9993 IFO 12891 Oatmeal Agar G Moderate Moderate A Gray Grayish
White R Pale Pink Colorless S None None Yeast-Malt G Moderate
Moderate Extract Agar A Light Gray Grayish White R Dull Reddish
Orange Light Brown S Dull Reddish Orange None Inorganic Salts- G
Moderate Moderate Starch Agar A Pale Yellow Orange to Grayish White
Light Gray R Dark Orange Pale Yellowish Brown S None None Glucose-
G Poor Moderate Asparagine A White Grayish White Agar R Pale Brown
Pale Yellowish Brown S None Pale Brown Glycerin- G Moderate
Moderate Asparagine A Pale Pink to White Grayish White Agar R Pale
Pink Pale Yellowish Brown S None Pale Brown Czapek Agar G Poor
Abundant A None Grayish White R Pale Pink Dark Orange to Dark Brown
S None None Nutrient Agar G Poor Poor A White, Poor White R
Colorless Colorless S None None Potato-Dextrose G Poor Moderate
Agar A None Yellowish Gray R Pale Pink Brown S None None Tyrosine
Agar G Moderate Moderate A White Grayish White to Light Gray R Dull
Reddish Orange Dark Orange to Black S None None Peptone-Yeast G
Poor Poor Extract-Iron A None None Agar R Colorless Colorless S
None None Abbreviation: G = Growth, A = Aerial Mass Color, R =
Reverse Side Color, S = Soluble Pigment,
[0066] The cell wall analysis was performed by the methods of
Becker et al. (Becker, B., M. P. Lechevalier, R. E. Gordon and H.
A. Lechevalier: Rapid differentiation between Nocardia and
Streptomyces by paper chromatography of whole cell hydrolysates:
Appl. Microbiol., 12, 421-423, 1964) and Yamaguchi (Yamaguchi, T.:
Comparison of the cell wall composition of morphologically distinct
actinomycetes: J. Bacteriol., 89, 444-453, 1965). Analysis of whole
cell hydrolysates of the strain No. 9993 showed the presence of
LL-diaminopimelic acid. Accordingly, the cell wall of this strain
is believed to be of type I.
[0067] [3] Biological and Physiological Properties:
[0068] Physiological properties of the strain No. 9993 were
determined according to the methods described by Shirling and
Gottlieb as mentioned above. The results are shown in Table 2.
Temperature range and optimum temperature for growth were
determined on yeast-malt extract agar using a temperature gradient
incubator (made by Toyo Kagaku Sangyo Co., Ltd.). Temperature range
for growth was from 18 to 35.degree. C. with optimum temperature at
28.degree. C. Milk peptonization and gelatin liquefaction were
positive. Melanoid pigment production was negative.
2TABLE 2 Physiological Properties of Strain No. 9993 and
Streptomyces misakiensis IFO 12891 Physiological properties No.
9993 IFO 12891 Temperature Range for Growth 18.degree.
C.-35.degree. C. 12.degree. C.-35.degree. C. Optimum Temperature
28.degree. C. 28.degree. C. Nitrate Reduction Negative Negative
Starch Hydrolysis Negative Positive Milk Coagulation Negative
Negative Milk Peptonization Positive Weakly Positive Melanin
Production Negative Negative Gelatin Liquefaction Positive Negative
H.sub.2S Production Negative Negative NaCl Tolerance (%) .ltoreq.3%
3%<, <5%
[0069] Utilization of carbon sources was examined according to the
methods of Pridham and Gottlieb (Pridham, T. G. and D. Gottlieb:
The utilization of carbon compounds by some Actinomycetales as an
aid for species determination: J. Bacteriol., 56, 107-114, 1948).
The growth was observed after 14 days incubation at 30.degree.
C.
[0070] Summarized carbon sources utilization of this strain is
shown in Table 3. Glycerin, maltose and sodium succinate could be
utilized by the strain No. 9993. Further, doubtful utilization of
D-glucose, sucrose, D-mannose and salicin was also observed.
3TABLE 3 Carbon Sources Utilization of Strain No. 9993 and
Streptomyces misakiensis IFO 12891 Carbon Sources No. 9993 IFO
12891 D-Glucose .+-. - Sucrose .+-. - Glycerin + - D-Xylose - -
D-Fructose - - Lactose - - Maltose + - Rhamnose - - Raffinose - -
D-Galactose - + L-Arabinose - - D-Mannose .+-. - D-Trehalose - -
Inositol - - D-Mannitol - - Inulin - + Cellulose - - Salicin .+-. -
Chitin - .+-. Sodium Citrate - - Sodium Succinate + - Sodium
Acetate - - Symbols: +: utilization .+-.: doubtful utilization -:
no utilization
[0071] Microscopic studies and cell wall composition analysis of
the strain No. 9993 indicate that this strain belongs to the genus
Streptomyces Waksman and Henrici 1943.
[0072] Accordingly, a comparison of this strain was made with
various Streptomyces species in the light of the published
descriptions (International Journal of Systematic Bacteriology, 18,
69 to 189, 279 to 392 (1968) and 19, 391 to 512 (1969), and Bergy's
Manual of Determinative Bacteriology 8th Edition (1974)].
[0073] As a result of the comparison, the strain No. 9993 is
considered to resemble Streptomyces aburaviensis Nishimura et. al.,
Streptomyces avellaneus Baldacci and Grein and Streptomyces
misakiensis Nakamura. Therefore, the cultural characteristics of
the strain No. 9993 were compared with the corresponding
Streptomyces aburaviensis IFO 12830, Streptomyces avellaneus IFO
13451 and Streptomyces misakiensis IFO 12891. As a result, the
strain No. 9993 was the most similar to Streptomyces misakiensis
IFO 12891. Therefore, the strain No. 9993 was further compared with
Streptomyces misakiensis IFO 12891 as shown in the above Tables 1,
2 and 3. From further comparison, the strain No. C9993 could be
differentiated from Streptomyces misakiensis IFO 12891 in the
following points, and therefore the strain No. 9993 is considered
to be a new species of Streptomyces and has been designated as
Streptomyces tsukubaensis sp. nov., referring to the soil collected
at Tsukuba-gun, from which the organism was isolated.
[0074] Difference from Streptomyces misakiensis IFO 12891
[0075] Cultural characteristics of the strain No. 9993 are
different from the Streptomyces misakiensis IFO 12891 on oatmeal
agar, yeast-malt extract agar, glucose-asparagine agar, Czapek agar
and potato-dextrose agar.
[0076] Starch hydrolysis of the strain No. 9993 is negative, but
that of the Streptomyces misakiensis IFO 12891 is positive.
[0077] Gelatin liquefaction of the strain No. 9993 is positive, but
that of the Streptomyces misakiensis IFO 12891 is negative.
[0078] In carbon sources utilization, the strain No. 9993 can
utilize glycerin, maltose and sodium succinate, but the
Streptomyces misakiensis IFO 12891 can not utilize them. And, the
strain No. 9993 can not utilize D-galactose and inulin, but the
Streptomyces misakiensis IFO 12891 can utilize them.
[0079] Production of FR-900506, FR-900520 and FR-900525
Substances
[0080] The novel FR-900506, FR-900520 and FR-900525 substances of
this invention can be produced by culturing a FR-900506, FR-900520
and/or FR-900525 substance(s)-producing strain belonging to the
genus Streptomyces (e.g. Streptomyces tsukubaensis No. 9993, FERM
BP-927) in a nutrient medium.
[0081] In general, the FR-900506, FR-900520 and/or FR-900525
substance(s) can be produced by culturing the FR-900506, FR-900520
and/or FR-900525 substance(s)-producing strain in an aqueous
nutrient medium containing sources of assimilable carbon and
nitrogen, preferably under aerobic conditions (e.g. shaking
culture, submerged culture, etc.).
[0082] The preferred sources of carbon in the nutrient medium are
carbohydrates such as glucose, xylose, galactose, glycerin, starch,
dextrin, and the like. Other sources which may be included are
maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium
succinate, and the like.
[0083] The preferred sources of nitrogen are yeast extract,
peptone, gluten meal, cottonseed meal, soybean meal, corn steep
liquor, dried yeast, wheat germ, feather meal, peanut powder etc.,
as well as inorganic and organic nitrogen compounds such as
ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium
phosphate, etc.), urea, amino acid, and the like.
[0084] The carbon and nitrogen sources, though advantageously
employed in combination, need not be used in their pure form,
because less pure materials which contain traces of growth factors
and considerable quantities of mineral nutrients, are also suitable
for use. When desired, there may be added to the medium mineral
salts such as sodium or calcium carbonate, sodium or potassium
phosphate, sodium or potassium chloride, sodium or potassium
iodide, magnesium salts, copper salts, cobalt salt and the like. If
necessary, especially when the culture medium foams seriously, a
defoaming agent, such as liquid paraffin, fatty oil, plant oil,
mineral oil or silicone may be added.
[0085] As the conditions for the production of the FR-900506,
FR-900520 and FR-900525 substances in massive amounts, submerged
aerobic cultural conditions are preferred therefor. For the
production in small amounts, a shaking or surface culture in a
flask or bottle is employed. Furthermore, when the growth is
carried out in large tanks, it is preferable to use the vegetative
form of the organism for inoculation in the production tanks in
order to avoid growth lag in the process of production of the
FR-900506, FR-900520 and FR-900525 substances. Accordingly, it is
desirable first to produce a vegetative inoculum of the organism by
inoculating a relatively small quantity of culture medium with
spores or mycelia of the organism and culturing said inoculated
medium, and then to transfer the cultured vegetative inoculum
aseptically to large tanks. The medium, in which the vegetative
inoculum is produced, is substantially the same as or different
from the medium utilized for the production of the FR-900506,
FR-900520 and FR-900525 substances.
[0086] Agitation and aeration of the culture mixture may be
accomplished in a variety of ways. Agitation may be provided by a
propeller or similar mechanical agitation equipment, by revolving
or shaking the fermentor, by various pumping equipment or by the
passage of sterile air through the medium. Aeration may be effected
by passing sterile air through the fermentation mixture.
[0087] The fermentation is usually conducted at a temperature
between about 20.degree. C. and 40.degree. C., preferably
25-35.degree. C., for a period of about 50 hours to 150 hours,
which may be varied according to fermentation conditions and
scales.
[0088] Thus produced FR-900506,FR-900520 and/or FR-900525
substances can be recovered from the culture medium by conventional
means which are commonly used for the recovery of other known
biologically active substances. The FR-900506, FR-900520 and
FR-900525 substances produced are found in the cultured mycelium
and filtrate, and accordingly the FR-900506, FR-900520 and
FR-900525 substances can be isolated and purified from the mycelium
and the filtrate, which are obtained by filtering or centrifuging
the cultured broth, by a conventional method such as concentration
under reduced pressure, lyophilization, extraction with a
conventional solvent, pH adjustment, treatment with a conventional
resin (e.g. anion or cation exchange resin, non-ionic adsorption
resin, etc.), treatment with a conventional adsorbent (e.g.
activated charcoal, silicic acid, silica gel, cellulose, alumina,
etc.), crystallization, recrystallization, and the like.
Physical and Chemical Properties of FR-900506, FR-900520 and
FR-900525 Substances
[0089] The FR-900506, FR-900520 and FR-900525 substances produced
according to the aforementioned process possess the following
physical and chemical properties.
[0090] FR-900506 Substance
[0091] (1) Form and Color:
[0092] white powder
[0093] (2) Elemental Analysis:
4 C: 64.72%, H: 8.78%, N: 1.59% 64.59% 8.74% 1.62%
[0094] (3) Color Reaction:
[0095] Positive: cerium sulfate reaction, sulfuric acid reaction,
Ehrlich reaction, Dragendorff reaction and iodine vapor
reaction
[0096] Negative: ferric chloride reaction, ninhydrin reaction and
Molish reaction
[0097] (4) Solubility:
[0098] Soluble: methanol, ethanol, acetone, ethyl acetate,
chloroform, diethyl ether and benzene
[0099] Sparingly Soluble: hexane, petroleum ether
[0100] Insoluble: water
[0101] (5) Melting Point:
[0102] 85-90.degree. C.
[0103] (6) Specific Rotation:
[0104] [.alpha.].sub.D.sup.23: -73.degree. (c=0.8, CHCl.sub.3)
[0105] (7) Ultraviolet Absorption Spectrum:
[0106] end absorption
[0107] (8) Infrared Absorption Spectrum: 1 CHCl 3 v max : 3680 ,
3580 , 3520 , 2930 , 2870 , 2830 , 1745 , 1720 , 1700 , 1645 , 1450
, 1380 , 1350 , 1330 , 1310 , 1285 , 1170 , 1135 , 1090 , 1050 ,
1030 , 1000 , 990 , 960 ( sh ) , 918 cm - 1
[0108] (9) .sup.13C Nuclear Magnetic Resonance Spectrum: 2 ( ppm ,
C D Cl 3 ) : { 212.59 ( s ) 212.45 ( s ) , { 196.18 ( s ) 192.87 (
s ) , { 169.07 ( s ) 168.90 ( s ) , { 164.90 ( s ) 166.01 ( s ) , {
138.89 ( s ) 139.67 ( s ) , { 135.73 ( d ) 135.60 ( d ) , { 132.52
( s ) 131.99 ( s ) , { 130.27 ( d ) 130.21 ( d ) , { 122.87 ( d )
123.01 ( d ) , { 116.57 ( t ) 116.56 ( t ) , { 97.35 ( s ) 98.76 (
s ) , 84.41 ( d ) , { 77.79 ( d ) 78.22 ( d ) , { 75.54 ( d ) 76.97
( d ) , { 73.93 ( d ) 73.09 ( d ) , { 73.72 ( d ) 72.57 ( d ) , {
70.05 ( d ) 69.15 ( d ) , 56.75 ( d ) , { 53.03 ( d ) 53.13 ( d ) ,
{ 48.85 ( t ) 48.62 ( t ) , { 40.33 ( d ) 40.85 ( d ) , 39.40 ( t )
, 31.58 ( t ) , 30.79 ( t ) , { 27.27 ( t ) 26.34 ( t ) , 26.46 ( d
) , 24.65 ( t ) , { 20.45 ( q ) 19.73 ( q ) , { 14.06 ( q ) 14.23 (
q ) , { 9.69 ( q ) 9.98 ( q ) ,
[0109] the chart of which being shown in FIG. 1,
[0110] (10) .sup.1H Nuclear Magnetic Resonance Spectrum:
[0111] the chart of which being shown in FIG. 2,
[0112] (11) Thin Layer Chromatography:
5 Developing Stationary Phase Solvent Rf Values silica gel plate
chloroform:methanol 0.58 (10:1, v/v) ethyl acetate 0.52
[0113] (12) Property of the Substance:
[0114] neutral substance
[0115] With regard to the FR-900506 substance, it is to be noted
that in case of measurements of .sup.13C and .sup.1H nuclear
magnetic resonance spectra, this substance showed pairs of the
signals in various chemical shifts.
[0116] The FR-900506 substance thus characterized further possesses
the following properties.
[0117] (i) The measurements of .sup.13C Nuclear Magnetic Resonance
Spectra at 25.degree. C. and 60.degree. C. revealed the fact that
the intensities of each pair of the various signals therein were
changed.
[0118] (ii) The measurements of the thin layer chromatography and
the high performance liquid chromatography revealed that the
FR-900506 substance occurs as a single spot in the thin layer
chromatography and a single peak in the high performance liquid
chromatography, respectively.
[0119] This white powder of the FR-900506 substance could be
transformed into a form of crystals by recrystallization thereof
from acetonitrile, which possess the following physical and
chemical properties.
[0120] (1) Form and Color:
[0121] colorless prisms
[0122] (2) Elemental Analysis:
6 C: 64.30%, H: 8.92%, N: 1.77% 64.20%, 8.86%, 1.72%,
[0123] (3) Melting Point:
[0124] 127-129.degree. C.
[0125] (4) Specific Rotation:
[0126] [.alpha.].sub.D.sup.23: -84.4.degree. (c=1.02,
CHCl.sub.3)
[0127] (5) .sup.13C Nuclear Magnetic Resonance Spectrum: 3 ( ppm ,
C D Cl 3 ) : { 211.98 ( s ) 211.74 ( s ) , { 196.28 ( s ) 193.56 (
s ) , { 168.97 ( s ) 168.81 ( s ) , { 164.85 ( s ) 165.97 ( s ) , {
138.76 ( s ) 139.51 ( s ) , { 135.73 ( d ) 135.63 ( d ) , { 132.38
( s ) 131.90 ( s ) , { 130.39 ( d ) 130.17 ( d ) , { 122.82 ( d )
122.96 ( d ) , 116.43 ( t ) , { 97.19 ( s ) 98.63 ( s ) , 84.29 ( d
) , { 77.84 ( d ) 78.21 ( d ) , { 77.52 ( d ) 76.97 ( d ) , { 69.89
( d ) 69.00 ( d ) , { 56.63 ( d ) 54.87 ( d ) , { 52.97 ( d ) 52.82
( d ) , { 48.76 ( t ) 48.31 ( t ) , { 40.21 ( d ) 40.54 ( d ) ,
31.62 ( t ) , 30.72 ( t ) , 24.56 ( t ) , { 21.12 ( t ) 20.86 ( t )
, { 20.33 ( q ) 19.74 ( q ) , { 16.17 ( q ) 16.10 ( q ) , { 15.88 (
q ) 15.75 ( q ) , { 13.89 ( q ) 14.05 ( q ) , { 9.64 ( q ) 9.96 ( q
) ,
[0128] the chart of which being shown in FIG. 3,
[0129] (6) .sup.1H Nuclear Magnetic Resonance Spectrum:
[0130] the chart of which being shown in FIG. 4.
[0131] Other physical and chemical properties, that is, the color
reaction, solubility, ultraviolet absorption spectrum, infrared
absorption spectrum, thin layer chromatography and property of the
substance of the colorless prisms of the FR-900506 substance were
the same as those for the white powder of the same under the
identical conditions.
[0132] From the above physical and chemical properties and the
analysis of the X ray diffraction, the FR-900506 substance could be
determined to have the following chemical structure. 10
[0133]
17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-met-
hylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricycl-
o [22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
[0134] FR-900520 Substance
[0135] The physical and chemical properties are mentioned
later.
[0136] FR-900525 Substance
[0137] (1) Form and Color:
[0138] white powder
[0139] (2) Elemental Analysis:
[0140] C: 65.17%, H: 8.53%, N: 1.76%
[0141] (3) Color Reaction:
[0142] Positive: cerium sulfate reaction, sulfuric acid reaction,
Ehrlich reaction, Dragendorff reaction and iodine vapor
reaction
[0143] Negative: ferric chloride reaction, ninhydrin reaction and
Molish reaction
[0144] (4) Solubility:
[0145] Soluble: methanol, ethanol, acetone, ethyl acetate,
chloroform, diethyl ether and benzene
[0146] Sparingly Soluble: hexane, petroleum ether
[0147] Insoluble: water
[0148] (5) Melting Point:
[0149] 85-89.degree. C.
[0150] (6) Specific Rotation:
[0151] [.alpha.].sub.D.sup.23: -88.degree. (c=1.0, ChCl.sub.3)
[0152] (7) Ultraviolet Absorption Spectrum:
[0153] end absorption
[0154] (8) Infrared Absorption Spectrum: 4 CHCl 3 v max : 3680 ,
3580 , 3475 , 3340 , 2940 , 2880 , 2830 , 1755 , 1705 , 1635 , 1455
, 1382 , 1370 , 1330 , 1310 , 1273 , 1170 , 1135 , 1093 , 1050 ,
1020 , 995 , 970 , 920 , 867 cm - 1
[0155] (9) .sup.13C Nuclear Magnetic Resonance Spectrum: 5 ( ppm ,
C D Cl 3 ) : { 212.61 ( s ) 211.87 ( s ) , { 188.57 ( s ) 191.12 (
s ) , { 168.76 ( s ) 170.18 ( s ) , { 163.11 ( s ) 161.39 ( s ) , {
140.28 ( s ) 139.37 ( s ) , { 135.62 ( d ) 135.70 ( d ) , { 132.28
( s ) 131.34 ( s ) , { 130.39 ( d ) 130.00 ( d ) , { 122.50 ( d )
123.23 ( d ) , 116.48 ( t ) , { 99.16 ( s ) 99.11 ( s ) , { 84.42 (
d ) 84.48 ( d ) , { 78.60 ( d ) 79.86 ( d ) , { 76.73 ( d ) 77.33 (
d ) , { 59.97 ( d ) 60.45 ( d ) , 57.52 ( q ) , { 56.56 ( q ) 56.48
( q ) , { 56.14 ( q ) 55.97 ( q ) , { 53.45 ( d ) 53.26 ( d ) , {
49.15 ( t ) 49.73 ( t ) , { 48.46 ( t ) 47.62 ( t ) , { 44.47 ( t )
45.23 ( t ) , { 41.40 ( d ) 40.40 ( d ) , { 35.19 ( d ) 35.11 ( d )
, { 33.10 ( d ) 34.17 ( d ) , { 32.81 ( t ) 32.29 ( t ) , { 31.53 (
t ) 31.33 ( t ) , { 30.80 ( t ) 30.66 ( t ) , 28.60 ( t ) , { 26.03
( d ) 26.98 ( d ) , { 25.43 ( t ) 24.40 ( t ) , { 18.93 ( q ) 20.57
( q ) , { 14.09 ( q ) 13.95 ( q ) , { 9.85 ( q ) 10.00 ( q )
[0156] the chart of which being shown in FIG. 5,
[0157] (10) .sup.1H Nuclear Magnetic Resonance Spectrum:
[0158] the chart of which being shown in FIG. 6,
[0159] (11) Thin Layer Chromatography:
7 Developing Stationary Phase Solvent Rf Value silica gel plate
ethyl acetate 0.34
[0160] (12) Property of the Substance:
[0161] neutral substance
[0162] With regard to the FR-900525 substance, it is to be noted
that in case of measurements of .sup.13C and .sup.1H nuclear
magnetic resonance spectra, this substance showed pairs of the
signals in various chemical shifts, however, in case of
measurements of the thin layer chromatography and the high
Performance liquid chromatography, the FR-900525 substance showed a
single spot in the thin layer chromatography and a single peak in
the high performance liquid chromatography, respectively.
[0163] From the above physical and chemical properties and the
success of the determination of the chemical structure of the
FR-900506 substance, the FR-900525 substance could be determined to
have the following chemical structure. 11
[0164]
16-Allyl-1,13-dihydroxy-11-[2-(4-hydroxy-3-methoxycyclohexyl)-1-met-
hylvinyl]-1-22,24-dimethoxy-12,18,20,26-tetramethyl-10,27-dioxa-4-azatricy-
clo-[21.3.1.0.sup.4,8]heptacos-17-ene-2,3,9,15-tetraone
[0165] [B] The FR-900520 and FR-900523 substances of this invention
can be produced by fermentation of FR-900520 and/or FR-900523
substance(s)-producing strain belonging to the genus Streptomyces
such as Streptomyces hygroscopicus subsp. yakushimaensis No. 7238
in a nutrient medium.
The Microorganism
[0166] The microorganism which can be used for the production of
the FR-900520 and/or FR-900523 substances is FR-900520 and/or
FR-900523 substance(s)-producing strain belonging to the genus
Streptomyces, among which Streptomyces hygroscopicus subsp.
yakushimaensis No. 7238 has been newly isolated from a soil sample
collected at Yakushima, Kagoshima Prefecture, Japan.
[0167] A lyophilized sample of the newly isolated Streptomyces
hygroscopicus subsp. yakushimaensis No. 7238 has been deposited
with the Fermentation Research Institute, Agency of Industrial
Science and Technology (No. 1-3, Higashi 1-chome, Yatabemachi,
Tsukuba-gun, Ibaraki Prefecture, Japan) under the number of FERM
P-8043 (deposited date: Jan. 12th, 1985), and then converted to
Budapest Treaty route of the same depository on Oct. 19, 1985 under
the new deposit number of FERM BP-928.
[0168] It is to be understood that the production of the novel
FR-900520 and FR-900523 substances is not limited to the use of the
particular organism described herein, which is given for the
illustrative purpose only. This invention also includes the use of
any mutants which are capable of producing the FR-900520 and/or
FR-900523 substance(s) including natural mutants as well as
artificial mutants which can be produced from the described
organism by conventional means such as irradiation of X-rays,
ultra-violet radiation, treatment with
N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the
like.
[0169] The Streptomyces hygroscopicus subsp. yakushimaensis No.
7238 has the following morphological, cultural, biological and
physiological characteristics.
[0170] [1] Morphological Characteristics:
[0171] The methods described by Shirling and Gottlieb (Shirling, E.
B. and D. Gottlieb: Methods for characterization of Streptomyces
species. International Journal of Systematic Bacteriology, 16,
313-340, 1966) were employed principally for this taxonomic
study.
[0172] Morphological observations were made with light and electron
microscopes on cultures grown at 30.degree. C. for 14 days on
oatmeal agar, yeast-malt extract agar and inorganic salts-starch
agar. The mature sporophores were moderately short and formed
Retinaculiaperti and Spirales with about 20 spores in each chain.
Hygroscopic spore mass were seen in the aerial mycelia on oatmeal
agar and inorganic salts-starch agar. Surface irregularities on
spores were intermediate between very short, thick spines and
warts.
[0173] [2] Cultural Characteristics:
[0174] Cultural characteristics were observed on ten kinds of media
described by Shirling and Gottlieb as mentioned above, and by
Waksman (Waksman, S. A.: The actinomycetes, vol. 2: Classification,
identification and description of genera and species. The Williams
and Wilkins Co., Baltimore, 1961).
[0175] The incubation was made at 30.degree. C. for 14 days. The
color names used in this study were based on Guide to Color
Standard (manual published by Nippon Shikisai Kenkyusho, Tokyo).
Colonies belonged to the gray color series when grown on oatmeal
agar, yeast-malt extract agar and inorganic salts-starch agar.
Soluble pigment was not produced in the examined media. The results
are shown in Table 4.
8TABLE 4 Cultural Characteristics of Strain No. 7238, Streptomyces
antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp.
glebosus IFO 13786 Cultural Characteristics Medium No. 7238 IFO
12839 IFO 13786 Oatmeal Agar G Poor Poor Poor A Grayish Yellow
Brown Grayish Yellow Brown Grayish Yellow Brown R Pale Yellow Pale
Yellow Pale Yellow S None None None Yeast-Malt G Moderate Abundant
Moderate Extract Agar A Grayish White Gray Gray R Pale Yellowish
Brown Pale Yellowish Brown Dark Orange S None None None Inorganic
Salts- G Moderate Moderate Moderate Starch Agar A Gray to Black
Gray Light Gray R Pale Yellow Orange Yellowish Gray Pale Yellow
Orange S None None None Glucose- G Moderate Moderate Moderate
Asparagine A Grayish White Gray White Agar R Pale Yellow Orange
Pale Yellow Orange Pale Yellow Orange S None None None Glycerin- G
Moderate Moderate Moderate Asparagine A White Gray Light Gray Agar
R Yellowish Gray Yellowish Gray Grayish Yellow Brown S None None
None Czapek Agar G Moderate Moderate Moderate A Grayish White
Grayish White White R Pale Yellowish Brown Pale Yellowish Brown
Pale Yellowish Brown S None None None Nutrient Agar G Moderate
Moderate Moderate A Grayish White Grayish White None R Pale Yellow
Pale Yellow Pale Yellow S None None None Potato-Dextrose G Moderate
Moderate Moderate Agar A White, Poor Pale Reddish Brown Pale Pink
to White R Pale Yellow Orange Pale Yellow Orange Pale Yellowish
Brown S None None None Tyrosine Agar G Moderate Moderate Moderate A
White Grayish White Gray to Black R Pale Yellowish Brown Brown Pale
Yellowish Brown S None Brown None Peptone-Yeast G Moderate Moderate
Moderate Extract-Iron A None Grayish White None Agar R Pale Yellow
Pale Yellow Colorless S None None None Abbreviation: G = Growth, A
= Aerial Mass Color, R = Reverse Side Color, S = Soluble
Pigment,
[0176] The cell wall analysis was performed by the methods of
Becker et al. (Becker, B., M. P. Lechevalier, R. E. Gordon and H.
A. Lechevalier: Rapid differentiation between Nocardia and
Streptomyces by paper chromatography of whole cell hydrolysates:
Appl. Microbiol., 12, 421-423, 1964) and Yamaguchi (Yamaguchi, T.:
Comparison of the cell wall composition of morphologically distinct
actinomycetes: J. Bacteriol., 89, 444-453, 1965). Analysis of whole
cell hydrolysates of the strain No. 7238 showed the presence of
LL-diaminopimelic acid. Accordingly, the cell wall of this strain
is believed to be of type I.
[0177] [3] Biological and Physiological Properties:
[0178] Physiological properties of the strain No. 7238 were
determined according to the methods described by Shirling and
Gottlieb as mentioned above. The results are shown in Table 5.
Temperature range and optimum temperature for growth were
determined on yeast-malt extract agar using a temperature gradient
incubator (made by Toyo Kagaku Sangyo Co., Ltd.). Temperature range
for growth was from 18 to 36.degree. C. with optimum temperature at
28.degree. C. Starch hydrolysis and gelatin liquefaction were
positive. No melanoid pigment was produced.
9TABLE 5 Physiological Properties of Strain No. 7238, Streptomyces
antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp.
glebosus IFO 13786 Physiological properties No. 7238 IFO 12839 IFO
13786 Temperature Range 18.degree. C.-36.degree. C. 18.degree.
C.-38.degree. C. 16.degree. C.-35.degree. C. for Growth Optimum
Temperature 28.degree. C. 28.degree. C. 27.degree. C. Nitrate
Reduction Negative Negative Negative Starch Hydrolysis Positive
Positive Positive Milk Coagulation Negative Negative Negative Milk
Peptonization Negative Negative Positive Melanin Production
Negative Negative Negative Gelatin Liquefaction Positive Positive
Positive H.sub.2S Production Negative Negative Negative Urease
Activity Negative Negative Negative NaCl Tolerance (%) 7%<,
<10% 7%<, <10% 5%<, <7%
[0179] Utilization of carbon sources was examined according to the
methods of Pridham and Gottlieb (Pridham, T. G. and D. Gottlieb:
The utilization of carbon compounds by some Actinomycetales as an
aid for species determination: J. Bacteriol., 56, 107-114, 1948).
The growth was observed after 14 days incubation at 30.degree.
C.
[0180] Summarized carbon sources utilization of this strain is
shown in Table 6. D-Glucose, sucrose, lactose, maltose,
D-trehalose, inositol, inulin and salicin could be utilized by the
strain No. 7238.
10TABLE 6 Carbon Sources Utilization of Strain No. 7238,
Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus
subsp. glebosus IFO 13786 Carbon Sources No. 7238 IFO 12839 IFO
13786 D-Glucose + + + Sucrose + + + Glycerin - + + D-Xylose - .+-.
+ D-Fructose - + + Lactose + + - Maltose + - + Rhamnose - + -
Raffinose - + + D-Galactose - + + L-Arabinose - .+-. .+-. D-Mannose
- + + D-Trehalose + .+-. + Inositol + + + D-Mannitol - + + Inulin +
+ - Cellulose .+-. - - Salicin + + - Chitin .+-. - - Sodium Citrate
- - .+-. Sodium Succinate - + + Sodium Acetate - - - Symbols: +:
utilization .+-.: doubtful utilization -: no utilization
[0181] Microscopic studies and cell wall composition analysis of
the strain No. 7238 indicate that this strain belongs to the genus
Streptomyces Waksman and Henrici 1943.
[0182] Accordingly, a comparison of this strain was made with
various Streptomyces species in the light of the published
descriptions [International Journal of Systematic Bacteriology, 18,
69 to 189, 279 to 392 (1968) and 19, 391 to 512 (1969), and Bergy's
Manual of Determinative Bacteriology 8th Edition (1974)].
[0183] As a result of the comparison, the strain No. 7238 is
considered to resemble Streptomyces antimycoticus Waksman 1957 and
Streptomyces hygroscoicus subsp. glebosus Ohmori, et. al. 1962.
Therefore, the cultural characteristics of the strain No. 7238 were
further compared with the corresponding Streptomyces antimycoticus
IFC 12839 and Streptomyces hygroscopicus subsp. glebosus IFO 13786
as shown in the above Tables 4, 5 and 6. From further comparison,
the strain No. 7238 could be differentiated from Streptomyces
antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp.
glebosus IFO 13786 in the following points.
[0184] (i) Difference from Streptomyces antimycoticus IFO 12839
[0185] Cultural characteristics of the strain No. 7238 are
different from the Streptomyces antimycoticus IFO 12839 on
yeast-malt extract agar, glucose-asparagine agar,
glycerin-asparagine agar, potato-dextrose agar and tyrosine
agar.
[0186] In carbon sources utilization, the strain No. 7238 can
utilize maltose, but the Streptomyces antimycoticus IFO 12839 can
not utilize it. And, the strain No. 7238 can not utilize glycerin,
D-fructose, rhamnose, raffinose, D-galactose, D-mannose, mannitol
and sodium succinate, but the Streptomyces antimycoticus IFO 12839
can utilize them.
[0187] (ii) Difference from Streptomyces hygroscopicus subsp.
glebosus IFO 13786
[0188] Cultural characteristics of the strain No. 7238 are
different from the Streptomyces hygroscopicus subsp. glebosus IFO
13786 on yeast-malt extract agar, potato-dextrose agar and tyrosine
agar.
[0189] Milk peptonization of the strain No. 7238 is negative, but
that of the Streptomyces hygaroscopicus subsp. glebosus IFO 13786
is positive. The strain No. 7238 can grow in the presence of 7%
NaCl, but the Streptomyces hygroscopicus subsp. glebosus IFO 13786
can not grow under the same condition.
[0190] In carbon sources utilization, the strain No. 7238 can
utilize lactose, inulin and salicin, but the Streptomyces
hygroscopicus subsp. glebosus IFO 13786 can not utilize them. And,
the strain No. 7238 can not utilize glycerin, D-xylose, D-fructose,
raffinose, D-galactose, D-mannose, mannitol and sodium succinate,
but the Streptomyces hygroscopicus subsp. glebosus IFO 13786 can
utilize them.
[0191] However, the strain No. 7238 forms hygroscopic spore mass in
the aerial mycelia on oatmeal agar and inorganic salts-starch agar,
and further morphological and cultural characteristics of the
strain No. 7238 are similar to the Streptomyces hygroscopicus
subsp. glebosus IFO 13786. Therefore, the strain No. 7238 is
considered to belong to Streptomyces hygroscopicus, but the strain
No. 7238 is different from the Streptomyces hygroscopicus subsp.
glebosus IFO 13786, though this known strain is the most similar to
the strain No. 7238 in Streptomyces hygroscopicus subspecies. From
the above facts, the strain No. 7238 is considered to be a new
subspecies of Streptomyces hygroscopicus and has been designated as
Streptomyces hygroscopicus subsp. yakushimaensis subsp. nov.,
referring to the soil collected at Yakushima, from which the
organism was isolated.
Production of FR-900520 and FR-900523 Substances
[0192] The novel FR-900520 and/or FR-900523 substance(s) can be
produced by culturing FR-900520 and/or FR-900523
substance(s)-producing strain belonging to the genus Streptomyces
(e.g. Streptomyces hygroscopicus subsp. yakushimaensis No. 7238,
FERM BP-928) in a nutrient medium.
[0193] In general, the FR-900520 and/or FR-900523 substance(s) can
be produced by culturing the FR-900520 and/or FR-900523
substance(s)-producing strain in an aqueous nutrient medium
containing sources of assimilable carbon and nitrogen, preferably
under aerobic conditions (e.g. shaking culture, submerged culture,
etc.).
[0194] The preferred sources of carbon in the nutrient medium are
carbohydrates such as glucose, sucrose, lactose, glycerin, starch,
dextrin, and the like. Other sources which may be included are
maltose, D-trehalose, inositol, inulin, salicin, and the like.
[0195] The preferred sources of nitrogen are yeast extract,
peptone, gluten meal, cottonseed meal, soybean meal, corn steep
liquor, dried yeast, wheat germ, feather meal, peanut powder etc.,
as well as inorganic and organic nitrogen compounds such as
ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium
phosphate, etc.), urea, amino acid, and the like.
[0196] The carbon and nitrogen sources, though advantageously
employed in combination, need not be used in their pure form,
because less pure materials which contain traces of growth factors
and considerable quantities of mineral nutrients, are also suitable
for use. When desired, there may be added to the medium mineral
salts such as sodium or calcium carbonate, sodium or potassium
phosphate, sodium or potassium chloride, sodium or potassium
iodide, magnesium salts, copper salts, cobalt salt and the like. If
necessary, especially when the culture medium foams seriously, a
defoaming agent, such as liquid paraffin, fatty oil, plant oil,
mineral oil or silicone may be added.
[0197] As the conditions for the production of the FR-900520 and
FR-900523 substances in massive amounts, submerged aerobic cultural
conditions are preferred therefor. For the production in small
amounts, a shaking or surface culture in a flask or bottle is
employed. Furthermore, when the growth is carried out in large
tanks, it is preferable to use the vegetative form of the organism
for inoculation in the production tanks in order to avoid growth
lag in the process of production of the FR-900520 and FR-900523
substances. Accordingly, it is desirable first to produce a
vegetative inoculum of the organism by inoculating a relatively
small quantity of culture medium with spores or mycelia of the
organism and culturing said inoculated medium, and then to transfer
the cultured vegetative inoculum aseptically to large tanks. The
medium, in which the vegetative inoculum is produced, is
substantially the same as or different from the medium utilized for
the production of the FR-900520 and FR-900523 substances.
[0198] Agitation and aeration of the culture mixture may be
accomplished in a variety of ways. Agitation may be provided by a
propeller or similar mechanical agitation equipment, by revolving
or shaking the fermentor, by various pumping equipment or by the
passage of sterile air through the medium. Aeration may be effected
by passing sterile air through the fermentation mixture.
[0199] The fermentation is usually conducted at a temperature
between about 20.degree. C. and 40.degree. C., preferably
25-35.degree. C., for a period of about 50 hours to 150 hours,
which may be varied according to fermentation conditions and
scales.
[0200] Thus produced FR-900520 and/or FR-900523 substance(s) can be
recovered from the culture medium by conventional means which are
commonly used for the recovery of other known biologically active
substances. The FR-900520 and FR-900523 substances produced are
mainly found in the cultured mycelium, and accordingly the
FR-900520 and FR-900523 substances can be isolated and purified
from the mycelium, which are obtained by filtering or centrifuging
the cultured broth, by a conventional method such as concentration
under reduced pressure, lyophilization, extraction with a
conventional solvent, pH adjustment, treatment with a conventional
resin (e.g. anion or cation exchange resin, non-ionic adsorption
resin, etc.), treatment with a conventional adsorbent (e.g.
activated charcoal, silicic acid, silica gel, cellulose, alumina,
etc.), crystallization, recrystallization, and the like.
[0201] Particularly, the FR-900520 substance and the FR-900523
substance can be separated by dissolving the materials containing
both products produced by fermentation in an appropriate solvent
such as ethyl acetate, n-hexane, and the like, and then by
subjecting said solution to chromatography, for example, on silica
gel in a column with an appropriate organic solvent such as ethyl
acetate and n-hexane, or a mixture thereof. And each of the
FR-900520 substance and the FR-900523 substance thus separated can
be further purified by a conventional method, for example,
recrystallization, re-chromatography, high performance liquid
chromatography, and the like.
Physical and Chemical Properties of FR-900520 and FR-900523
Substances
[0202] FR-900520 Substance
[0203] (1) Form and Color:
[0204] colorless plates
[0205] (2) Elemental Analysis:
[0206] C: 64.81%, H: 8.82%, N: 1.55%
[0207] (3) Color Reaction:
[0208] Positive: cerium sulfate reaction, sulfuric acid reaction,
Ehrlich reaction, Dragendorff reaction and iodine vapor
reaction
[0209] Negative: ferric chloride reaction, ninhydrin reaction and
Molish reaction
[0210] (4) Solubility:
[0211] Soluble: methanol, ethanol, acetone, ethyl acetate,
chloroform, diethyl ether and benzene
[0212] Sparingly Soluble: n-hexane, petroleum ether
[0213] Insoluble: water
[0214] (5) Melting Point:
[0215] 163-165.degree. C.
[0216] (6) Specific Rotation:
[0217] [.alpha.].sub.D.sup.23: -84.1.degree. (c=1.0,
CHCl.sub.3)
[0218] (7) Ultraviolet Absorption Spectrum:
[0219] end absorption
[0220] 8) Infrared Absorption Spectrum: 6 CHCl 3 v max : 3680 ,
3575 , 3520 , 2940 , 2875 , 2825 , 1745 , 1725 , 1700 , 1647 , 1610
( sh ) , 1452 , 1380 , 1350 , 1330 , 1285 , 1170 , 1135 , 1090 ,
1030 , 1005 , 990 , 980 ( sh ) , 960 ( sh ) , 913 , 908 ( sh ) cm -
1
[0221] (9) .sup.13C Nuclear Magnetic Resonance Spectrum: 7 ( ppm ,
C D Cl 3 ) : 213.04 ( s ) , { 196.21 ( s ) 193.23 ( s ) , { 169.07
( s ) 168.85 ( s ) , { 164.92 ( s ) 165.97 ( s ) , { 138.67 ( s )
139.53 ( s ) , { 132.46 ( s ) 131.98 ( s ) , { 130.20 ( d ) 130.08
( d ) , { 123.42 ( d ) 123.59 ( d ) , { 97.28 ( s ) 98.75 ( s ) ,
84.37 ( d ) , { 77.80 ( d ) 78.24 ( d ) , { 75.53 ( d ) 76.98 ( d )
, 73.92 ( d ) , 73.69 ( d ) , { 73.11 ( d ) 72.72 ( d ) , { 70.11 (
d ) 69.21 ( d ) , 57.02 ( q ) , { 56.60 ( q ) 57.43 ( q ) , { 56.23
( q ) 55.98 ( q ) , { 56.72 ( d ) 52.91 ( d ) , { 55.10 ( d ) 54.90
( d ) , { 48.90 ( t ) 48.57 ( t ) , { 40.19 ( d ) 40.63 ( d ) , {
27.67 ( t ) 26.32 ( t ) , { 26.51 ( d ) 26.44 ( d ) , 24.60 ( t ) ,
{ 21.19 ( t ) 20.86 ( t ) , { 20.47 ( q ) 19.75 ( q ) , { 16.21 ( q
) 15.97 ( q ) , { 15.83 ( q ) 15.94 ( q ) , { 14.04 ( q ) 14.16 ( q
) , 11.68 ( q ) , { 9.64 ( q ) 9.93 ( q ) ,
[0222] the chart of which being shown in FIG. 7,
[0223] (10) .sup.1H Nuclear Magnetic Resonance Spectrum:
[0224] the chart of which being shown in FIG. 8,
[0225] (11) Thin Layer Chromatography:
11 Developing Stationary Phase Solvent Rf Values silica gel plate
chloroform:methanol 0.38 (20:1, v/v) ethyl acetate 0.51
[0226] (12) Property of the Substance:
[0227] neutral substance
[0228] With regard to the FR-900520 substance, it is to be noted
that in case of measurements of .sup.13C and .sup.1H nuclear
magnetic resonance spectra, this substance shows pairs of the
signals in various chemical shifts, however, in case of
measurements of the thin layer chromatography and the high
performance liquid chromatography, the FR-900520 substance showed a
single spot in the thin layer chromatography and a single peak in
the high performance liquid chromatography, respectively.
[0229] From the above physical and chemical properties and the
success of the determination of the chemical structure of the
FR-900506 substance, the FR-900520 substance could be determined to
have the following chemical structure. 12
[0230]
17-Ethyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-met-
hylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricycl-
o-[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
[0231] FR-900523 Substance
[0232] (1) Form and Color:
[0233] colorless needles
[0234] (2) Elemental Analysis:
[0235] C: 64.57%, H: 8.84%, N: 1.81%
[0236] (3) Color Reaction:
[0237] Positive: cerium sulfate reaction, sulfuric acid reaction,
Ehrlich reaction, Dragendorff reaction and iodine vapor
reaction
[0238] Negative: ferric chloride reaction and ninhydrin
reaction
[0239] (4) Solubility:
[0240] Soluble: methanol, ethanol, acetone, ethyl acetate,
chloroform, diethyl ether and benzene
[0241] Sparingly Soluble: n-hexane and petroleum ether
[0242] Insoluble: water
[0243] (5) Melting Point:
[0244] 152-154.degree. C.
[0245] (6) Specific Rotation:
[0246] [.alpha.].sub.D.sup.23: -73.0.degree. (C=0.65,
CHCl.sub.3)
[0247] (7) Ultraviolet Absorption Spectrum:
[0248] end absorption
[0249] (8) Infrared Absorption Spectrum: 8 CHCl 3 v max : 3670 ,
3580 , 3510 , 2930 , 2875 , 2825 , 1745 , 1722 , 1700 , 1647 , 1450
, 1380 , 1350 , 1330 , 1307 , 1285 , 1170 , 1135 , 1090 , 1050 ,
1030 , 1000 , 990 , 978 , 960 , 930 , 915 , 888 , 870 , 850 cm -
1
[0250] (9) .sup.13C Nuclear Magnetic Resonance Spectrum: 9 ( ppm ,
C D Cl 3 ) : { 213.82 ( s ) 213.32 ( s ) , { 196.31 ( s ) 193.34 (
s ) , { 168.96 ( s ) 168.85 ( s ) , { 164.84 ( s ) 165.98 ( s ) , {
137.80 ( s ) 138.41 ( s ) , { 132.89 ( s ) 131.96 ( s ) , { 129.62
( d ) 130.03 ( d ) , { 124.51 ( d ) 124.84 ( d ) , { 97.13 ( s )
98.67 ( s ) , 84.38 ( d ) , { 76.69 ( d ) 78.06 ( d ) , { 75.45 ( d
) 76.91 ( d ) , { 73.89 ( d ) 73.70 ( d ) , 73.70 ( d ) , { 73.09 (
d ) 72.84 ( d ) , { 70.40 ( d ) 69.24 ( d ) , { 56.75 ( d ) , 52.89
( d ) , { 56.93 ( q ) 57.43 ( q ) , { 56.61 ( q ) 56.56 ( q ) , {
56.24 ( q ) 55.94 ( q ) , { 48.58 ( t ) 48.32 ( t ) , { 47.14 ( d )
47.38 ( d ) , { 40.23 ( d ) 40.65 ( d ) , { 27.85 ( t ) 26.32 ( t )
, { 26.48 ( d ) 26.64 ( d ) , 24.68 ( t ) , { 21.33 ( t ) 20.83 ( t
) , { 20.63 ( q ) 19.77 ( q ) , { 16.24 ( q ) 16.34 ( q ) , { 15.70
( q ) 15.96 ( q ) , { 15.51 ( q ) 15.96 ( q ) , { 14.31 ( q ) 14.18
( q ) , { 9.64 ( q ) 10.04 ( q ) ,
[0251] the chart of which being shown in FIG. 9,
[0252] (10) .sup.1H Nuclear Magnetic Resonance Spectrum:
[0253] the chart of which being shown in FIG. 10,
[0254] (11) Thin Layer Chromatography:
12 Developing Stationary Phase Solvent Rf Values silica gel plate
chloroform:methanol 0.38 (20:1, v/v) ethyl acetate 0.51
[0255] (12) Property of the Substance:
[0256] neutral substance
[0257] With regard to the FR-900523 substance, it is to be noted
that in case of measurements of .sup.13C and .sup.1H nuclear
magnetic resonance spectra, this substance shows pairs of the
signals in various chemical shifts, however, in case of
measurements of the thin layer chromatography and the high
performance liquid chromatography, the FR-900523 substance showed a
single spot in the thin layer chromatography and a single peak in
the high performance liquid chromatography, respectively.
[0258] From the above physical and chemical properties and the
success of the determination of the chemical structure of the
FR-900506 substance, the FR-900523 substance could be determined to
have the following chemical structure. 13
[0259]
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-
-23,25-dimethoxy-13,19,17,21,27-pentamethyl-11,28-dioxa-4-azatricyclo
[22.3.1.0.sup.4,9]-octacos-18-ene-2,3,10,16-tetraone
[0260] [II] Synthetic Processes:
[0261] (1) Process 1: (Introduction of Hydroxy-Protective
Group)
[0262] The compound (Ib) or a salt thereof can be prepared by
introducing a hydroxy-protective group into the compound (Ia) or a
salt thereof.
[0263] Suitable introducing agent of the hydroxy-protective group
used in this reaction may be a conventional one such as
di(lower)alkyl sulfoxide, for example, lower alkyl methyl sulfoxide
(e.g. dimethyl sulfoxide, ethyl methyl sulfoxide, propyl methyl
sulfoxide, isopropyl methyl sulfoxide, butyl methyl sulfoxide,
isobutyl methyl sulfoxide, hexyl methyl sulfoxide, etc.),
trisubstituted silyl compound such as tri(lower)alkylsilyl halide
(e.g. trimethylsilyl chloride, triethylsilyl bromide, tributylsilyl
chloride, tert-butyl-dimethylsilyl chloride, etc.), lower
alkyl-diarylsilyl halide (e.g. methyl-diphenylsilyl chloride,
ethyl-diphenylsilyl bromide, propyl-ditolylsilyl chloride,
tert-butyl-diphenylsilyl chloride, etc.), and acylating agent which
is capable of introducing the acyl group as mentioned before such
as carboxylic acid, sulfonic acid, carbamic acid and their reactive
derivative, for example, an acid halide, an acid anhydride, an
activated amide, an activated ester, isocyanate, and the like.
Preferable example of such reactive derivative may include acid
chloride, acid bromide, a mixed acid anhydride with an acid such as
substituted phosphoric acid (e.g. dialkylphosphoric acid,
phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric
acid, halogenated phosphoric acid, etc.), dialkylphosphorous acid,
sulfurous acid, thiosulfuric acid, sulfuric acid, alkyl carbonate
(e.g. methyl carbonate, ethyl carbonate, propyl carbonate, etc.),
aliphatic carboxylic acid (e.g. pivalic acid, pentanoic acid,
isopentanoic acid, 2-ethylbutyric acid, trichloroacetic
trifluoroacetic acid, etc.), aromatic carboxylic acid (e.g. benzoic
acid, etc.), a symmetrical acid anhydride, an activated acid amide
with a heterocyclic compound containing imino function such as
imidazole, 4-substituted imidazole, dimethylpyrazole, triazole and
tetrazole, an activated ester (e.g. p-nitrophenyl ester,
2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl
ester, mesylphenyl ester, phenylazophenyl ester, phenyl thioester,
p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl
thioester, pyridyl ester, piperidinyl ester, 8-quinolyl thioester,
or an ester with a N-hydroxy compound such as
N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone,
N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole,
1-hydroxy-6-chlorobenzotriazole, etc.), isocyanate, and the
like.
[0264] In this reaction, in case that the di(lower)alkyl sulfoxide
is used as an introducing agent of the hydroxy-protective group,
the reaction is usually conducted in the presence of lower alkanoic
anhydride such as acetic anhydride.
[0265] Further, in case that the trisubstituted silyl compound is
used as an introducing agent of the hydroxy-protective group, the
reaction is preferable conducted in the presence of a conventional
condensing agent such as imidazole, and the like.
[0266] Still further, in case that the acylating agent is used as
an introducing agent of the hydroxy-protective group, the reaction
is preferably conducted in the presence of an organic or inorganic
base such as alkali metal (e.g. lithium, sodium, potassium, etc.),
alkaline earth metal (e.g. calcium, etc.), alkali metal hydride
(e.g. sodium hydride, etc.), alkaline earth metal hydride (e.g.
calcium hydride, etc.), alkali metal hydroxide (e.g. sodium
hydroxide, potassium hydroxide, etc.), alkali metal carbonate (e.g.
sodium carbonate, potassium carbonate, etc.), alkali metal hydrogen
carbonate (e.g. sodium hydrogen carbonate, potassium hydrogen
carbonate, etc.), alkali metal alkoxide (e.g. sodium methoxide,
sodium ethoxide, potassium tert-butoxide, etc.), alkali metal
alkanoic acid (e.g. sodium acetate, etc.), trialkylamine (e.g.
triethylamine, etc.), pyridine compound (e.g. pyridine, lutidine,
picoline, 4-N,N-dimethylaminopyridine, etc.), quinoline, and the
like.
[0267] In case that the acylating agent is used in a free form or
its salt in this reaction, the reaction is preferably conducted in
the presence of a conventional condensing agent such as a
carbodiimide compound [e.g. N,N'-dicyclohexylcarbodiimide,
N-cyclohexyl-N'-(4-diethylaminocyclohexyl)- carbodiimide,
N,N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide,
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, etc.], a ketenimine
compound (e.g. N,N'-carbonylbis (2-methylimidazole),
pentamethyleneketene-N-cyclohexylimine,
diphenylketene-N-cyclohexylimine, etc.); an olefinic or acetylenic
ether compounds (e.g. ethoxyacetylene, .beta.-cyclovinylethyl
ether), a sulfonic acid ester of N-hydroxybenzotriazole derivative
[e.g. 1-(4-chlorobenzenesulfonyloxy) 6-chloro-1H-benzotriazole,
etc.], and the like.
[0268] The reaction is usually conducted in a conventional solvent
which does not adversely influence the reaction such as water,
acetone, dichloromethane, alcohol (e.g. methanol, ethanol, etc.),
tetrahydrofuran, pyridine, benzene, N,N-dimethylformamide, etc., or
a mixture thereof, and further in case that the base or the
introducing agent of the hydroxy-protective group is in liquid, it
can also be used as a solvent.
[0269] The reaction temperature is not critical and the reaction is
usually conducted under from cooling to heating.
[0270] This process includes, within a scope thereof, a case that
during the reaction, the hydroxy group for R.sup.2 of the compound
(Ia) may occasionally be transformed into the corresponding
protected hydroxy group in the object compound (Ib).
[0271] Further, this process also includes, within a scope thereof,
a case that when the di(lower)alkyl sulfoxide is used as an
introducing agent of the hydroxy-protective group in the presence
of lower alkanoic anhydride, the compound (Ia) having a partial
structure of the formula: 14
[0272] wherein R.sup.2 is hydroxy, may occasionally be oxidized
during the reaction to give the compound (Ib) having a partial
structure of the formula: 15
[0273] wherein R.sup.2 is hydroxy.
[0274] (2) Process 2: (Introduction of Hydroxy-Protective
Group)
[0275] The compound (Id) or a salt thereof can be prepared by
introducing a hydroxy-protective group into the compound (Ic) or a
salt thereof.
[0276] The reaction can be conducted by substantially the same
method as that of Process 1, and therefore the reaction conditions
(e.g. base, condensing agent, solvent, reaction temperature, etc.)
are referred to those of Process 1.
[0277] This process includes, within a scope thereof, a case that
during the reaction, the hydroxy group for R.sup.1 of the compound
(Ic) may frequently be transformed into the corresponding protected
hydroxy group in the object compound (Id).
[0278] (3) Process 3: (Formation of Double Bond)
[0279] The compound (If) or a salt thereof can be prepared by
reacting the compound (Ie) or a salt thereof with a base.
[0280] Suitable base to be used in this reaction may include one as
exemplified in Process 1.
[0281] This reaction can also be conducted by reacting the compound
(Ie), where R.sup.2 is hydroxy, with an acylating agent in the
presence of a base.
[0282] The reaction is usually conducted in a conventional solvent
which does not adversely influence the reaction such as water,
acetone, dichloromethane, alcohol (e.g. methanol, ethanol,
propanol, etc.), tetrahydrofuran, pyridine, N,N-dimethylformamide,
etc., or a mixture thereof, and further in case that the base is in
liquid, it can also be used as a solvent.
[0283] The reaction temperature is not critical and the reaction is
usually conducted under from cooling to heating.
[0284] (4) Process 4: (Oxidation of Hydroxyethylene Group)
[0285] The compound (Ih) or a salt thereof can be prepared by
oxidizing the compound (Ig) or a salt thereof.
[0286] The oxidizing agent to be used in this reaction may include
di(lower)alkyl sulfoxide such as those given in Process 1.
[0287] This reaction is usually conducted in the presence of lower
alkanoic anhydride such as acetic anhydride in a conventional
solvent which does not adversely influence the reaction such as
acetone, dichloromethane, ethyl acetate, tetrahydrofuran, pyridine,
N,N-dimethylformamide, etc., or a mixture thereof, and further in
case that the lower alkanoic anhydride is in liquid, it can also be
used as a solvent.
[0288] The reaction temperature is not critical and the reaction is
usually conducted under from cooling to heating.
[0289] This process includes, within a scope thereof, a case that
during the reaction the hydroxy group for R.sup.1 of the starting
compound (Ig) may occasionally be transformed into 1-(lower
alkylthio) (lower)alkyloxy group in the object compound (Ih).
[0290] (5) Process 5: (Reduction of Allyl Group)
[0291] The compound (Ij) or a salt thereof can be obtained by
reducing the compound (Ii) or a salt thereof.
[0292] Reduction in this process can be conducted by a conventional
method which is capable of reducing an allyl group to a propyl
group, such as catalytic reduction, or the like.
[0293] Suitable catalysts used in catalytic reduction are
conventional ones such as platinum catalysts (e.g. platinum plate,
spongy platinum, platinum black, colloidal platinum, platinum
oxide, platinum wire, etc.), palladium catalysts (e.g. spongy
palladium, palladium black, palladium oxide, palladium on carbon,
colloidal palladium, palladium on barium sulfate, palladium on
barium carbonate, etc.), nickel catalysts (e.g. reduced nickel,
nickel oxide, Raney nickel, etc.), cobalt catalysts (e.g. reduced
cobalt, Raney cobalt, etc.), iron catalysts (e.g. reduced iron,
Raney iron, etc.), copper catalysts (e.g. reduced copper, Raney
copper, Ullman copper, etc.), and the like.
[0294] The reduction is usually conducted in a conventional solvent
which does not adversely influence the reaction such as water,
methanol, ethanol, propanol, pyridine, ethyl acetate,
N,N-dimethylformamide, dichloromethane, or a mixture thereof.
[0295] The reaction temperature of this reduction is not critical
and the reaction is usually conducted under from cooling to
warming.
[0296] (6) Process 6: (Removal of the Carboxy-Protective Group)
[0297] The compound (Il) or a salt thereof can be prepared by
removing the carboxy-protective group from the compound (Ik) or a
salt thereof.
[0298] The removal reaction in this process can be conducted in a
conventional manner which is capable of transforming a
tri(lower)alkylsilyl(lower)alkoxycarbonyl group to a carboxy group,
that is, in the presence of tetra(lower)alkylammonium fluoride
(e.g. tetrabutylammonium fluoride, etc.), potassium fluoride,
hydrogen fluoride, and the like.
[0299] This reaction is usually conducted in a conventional solvent
which does not adversely influence the reaction such as
tetrahydrofuran, and the like.
[0300] The reaction temperature is not critical and the reaction is
usually conducted under from cooling to warming.
[0301] The object tricyclo compounds (I) obtained according to the
synthetic processes 1 to 6 as explained above can be isolated and
purified in a conventional manner, for example, extraction,
precipitation, fractional crystallization, recrystallization,
chromatography, and the like.
[0302] Suitable salts of the compounds (I) and (Ia) to (Il) may
include pharamaceutically acceptable salts such as basic salts, for
example, alkali metal salt (e.g. sodium salt, potassium salt,
etc.), alkaline earth metal salt (e.g. calcium salt, magnesium
salt, etc.), ammonium salt, amine salt (e.g. triethylamine salt,
N-benzyl-N-methylamine salt, etc.) and other conventional organic
salts.
[0303] It is to be noted that in the aforementioned reactions in
the synthetic processes 1 to 6 or the post-treatment of the
reaction mixture therein, the conformer and/or stereo isomer(s) due
to asymmetric carbon atom(s) or double bond(s) of the starting and
object compounds may occasionally be transformed into the other
conformer and/or stereoisomer(s), and such cases are also included
within the scope of the present invention.
[0304] The tricyclo compounds (I) of the present invention possess
pharmacological activities such as immunosuppressive activity,
antimicrobial activity, and the like, and therefore are useful for
the treatment and prevention of the resistance by transplantation
of organs or tissues such as heart, kidney, liver, medulla ossium,
skin, etc., graft-versus-host diseases by medulla ossium
transplantation, autoimmune diseases such as rheumatoid arthritis,
systemic lupus erythematosus, Hashimoto's thyroiditis, multiple
sclerosis, myasthenia gravis, type I diabetes, uveitis, etc.,
infectious diseases caused by pathogenic microorganisms, and the
like.
[0305] As examples for showing such pharmacological activities,
some pharmacological test data of the tricyclo compounds are
illustrated in the following.
Test 1
Suppression of Tricyclo Compounds (I) in In Vitro Mixed Lymphocyte
Reaction (MLR)
[0306] The MLR test was performed in microtiter plates, with each
well containing 5.times.10.sup.5 C57BL/6 responder cells
(H-2.sup.b), 5.times.10.sup.5 mitomycin C treated (25 .mu.g/ml
mitomycin C at 37.degree. C. for 30 minutes and washed three times
with RPMI 1640 medium) BALB/C stimulator cells (H-2.sup.d) in 0.2
ml RPMI 1640 medium supplemented with 10% fetal calf serum, 2mM
sodium hydrogen carbonate, penicillin (50 unit/ml) and streptomycin
(50 .mu.g/ml). The cells were incubated at 37.degree. C. in
humidified atmosphere of 5% carbon dioxide and 95% of air for 68
hours and pulsed with .sup.3H-thymidine (0.5 .mu.Ci) 4 hours before
the cells were collected. The object compound of this invention was
dissolved in ethanol and further diluted in RPMI 1640 medium and
added to the cultures to give final concentrations of 0.1 .mu.g/ml
or less.
[0307] The results are shown in Tables 7 to 10. The tricyclo
compounds of the present invention suppressed mouse MLR.
13TABLE 7 Effect of the FR-900506 Substance on MLR FR-900506
concentration Radioactivities Suppression IC.sub.50 (ng/ml) (mean
C.P.M. .+-. S.E.) (%) (ng/ml) 2.5 54 .+-. 4 99.5 1.25 168 .+-. 23
98.3 0.625 614 .+-. 57 93.8 0.313 3880 .+-. 222 60.9 0.26 0.156
5490 + 431 44.7 0.078 7189 .+-. 365 27.6 0 9935 .+-. 428 0
[0308]
14TABLE 8 Effect of FR-900520 Substance on MLR FR-900520
concentration Radioactivities Suppression IC.sub.50 (ng/ml) (mean
C.P.M. .+-. S.E.) (%) (ng/ml) 100 175 .+-. 16 99.2 10 515 .+-. 55
97.8 1 2744 .+-. 527 88.1 0.38 0.500 9434 .+-. 1546 59.2 0.25 14987
.+-. 1786 35.1 0 23106 .+-. 1652 0
[0309]
15TABLE 9 Effect of FR-900523 Substance on MLR FR-900523
concentration Radioactivities Suppression IC.sub.50 (ng/ml) (mean
C.P.M. .+-. S.E.) (%) (ng/ml) 100 25 .+-. 12 99.9 10 156 .+-. 37
99.3 1 5600 .+-. 399 75.8 0.5 0.500 11624 .+-. 395 49.7 0.250 17721
.+-. 1083 23.3 0 23106 .+-. 1052 0
[0310]
16TABLE 10 Effect of the FR-900525 Substance on MLR FR-900525
concentration Radioactivities Suppression IC.sub.50 (ng/ml) (mean
C.P.M. .+-. S.E.) (%) (ng/ml) 100 469 .+-. 56 97.0 10 10372 .+-. 32
97.6 5 828 .+-. 369 94.7 1.55 2.5 3564 .+-. 512 77.4 1.2 10103 .+-.
421 35.8 0 15741 .+-. 411 0
Test 2
Antimicrobial Activities of Tricyclo Compounds (I)
[0311] Antimicrobial activities of the tricyclo compounds (I)
against various fungi were determined by a serial agar dilution
method in a Sabouraud agar. Minimum inhibitory concentrations (MIC)
were expressed in terms of .mu.g/ml after incubation at 30.degree.
C. for 24 hours.
[0312] Tricyclo compounds of the present invention showed
antimicrobial activities against fungi, for example, Aspergillus
fumigatus IFO 5840 and Fusarium oxysporum IFO 5942 as described in
the following Tables 11 and 12.
17TABLE 11 MIC values (.mu.g/ml) of Tricyclo Compounds (I) against
Aspergillus fumigatus IFO 5840 Substances MIC (.mu.g/ml) FR-900506
0.025 FR-900520 0.1 FR-900523 0.3 FR-900525 0.5
[0313]
18TABLE 12 MIC values (.mu.g/ml) of Tricyclo Compounds (I) of
against Fusarium oxysporum Substances MIC (.mu.g/ml) FR-900506 0.05
FR-900525 1
Test 3
Effect of Tricyclo Compounds (I) on Skin Allograft Survival in
Rats
[0314] Vental allografts from donor (Fischer) rats were grafted
onto the lateral thoracic area of recipient (WKA) rats. The
dressings were removed on day 5. The grafts were inspected daily
until rejection which was defined as more than 90% necrosis of the
graft epitherium.
[0315] The FR-900506 substance was dissolved in olive oil and
administered intramuscularly for 14 consecutive days, beginning at
the day of transplantation.
[0316] As shown in Table 13, all skin allografts were rejected
within 8 days in rats treated with olive oil intramuscularly for 14
consecutive days, but daily treatment with the FR-900506 substance
clearly prolonged skin allograft survival.
19TABLE 13 Effect of FR-900506 Substance on Skin Allograft Survival
Number of Skin Allograft Dose (mg/kg) Animals Survival Day Control
-- 11 7, 7, 7, 7, 7, 7, 8, 8, 8, 8, 8 (olive oil) FR-900506 1 8 19,
19, 19, 20, 21, 21, 22, 22 Substance 3.2 6 22, 23, 23, 26, 27, 35
10 5 56, 61, 82, 85, 89
Test 4
Effect of Tricylo Compounds (I) on Type II
Collagen-Induced-Arthritis in Rats
[0317] Collagen was dissolved in cold 0.01 M acetic acid at a
concentration of 2 mg/ml. The solution was emulsified in an equal
volume of incomplete Freund's adjuvant. A total volume of 0.5 ml of
the cold emulsion was injected intradermally at several sites on
the back and one or two sites into the tail of female Lewis rats.
The FR-900506 substance was dissolved in olive oil and administered
orally. Control rats immunized with same amount of type II collagen
received oral administrations of olive oil alone. Incidences of the
arthritis were observed.
[0318] The test results are shown in Table 14. The inflammatory
polyarthritis was induced in all rats treated with olive oil for 14
days starting on the same day as the type II collagen
immunization.
[0319] Daily treatment with the FR-900506 substance for 14 days
gave complete suppression of arthritis induction during an
observation period of 3 weeks.
20TABLE 14 Effect of FR-900506 Substance on Type II
Collagen-induced-Arthritis in Rats Dose Incidence of (mg/kg per
day) Arthritis Control -- 5/5 (olive oil) FR-900506 3.2 0/5
Substance
Test 5
Effect of Tricylo Compounds (I) on Experimental Allergic
Encephalomyelytis (EAE) in SJL/J Mice
[0320] Spinal cord homogenate was prepared from SJL/J mice. The
spinal cords were removed by insufflation, mixed with an
approximately equal volume of water and homogenized at 4.degree. C.
An equal volume of this cold homogenate (10 mg/ml) was emulsified
with complete Freund's adjuvant (CFA) containing 0.6 mg/ml of
Mycobacterium tuberculosis H37RA.
[0321] EAE was induced by two injections of 0.2 ml of spinal
cord-CFA emulsion into SJL/J mice on day 0 and day 13. All mice
used in these tests were evaluated and scored daily for clinical
signs of EAE.
[0322] The severity of EAE was scored according to the following
criteria: grade 1--decreased tail tone: grade 2--a clumsy gait:
grade 3--weakness of one or more limb: grade 4--paraplegia or
hemiplegia.
[0323] The FR-900506 substance was dissolved in olive oil and
administered orally for 19 days starting on day 0 (the day of first
immunization). As shown in Table 15, the FR-900506 substance
clearly prevented the development of clinical signs of EAE.
21TABLE 15 Effect of FR-900506 Substance on Experimental Allergic
Encephalomyelytis in SJL/J Mice Number of Animals Dose (mg/kg) with
Disease at Day 24 Control -- 10/10 (olive oil) FR-900506 32 0/5
Substance
Test 6
Effect of Tricyclo Compounds (I) on Local Graft-versus-Host
Reaction (GvHR) in Mice
[0324] The viable spleen cells (1.times.10.sup.7 cells) from
C57BL/6 donors were injected subcutaneously into the right hind
foot pad of BDF.sub.1 mice to induce local GvHR. The mice were
killed 7 days later and both right (injected paw) and left
(uninjected paw) popliteal lymph nodes (PLN) were weighed. The GvHR
was expressed as the weight difference between right and left
PLN.
[0325] The FR-900506 substance was dissolved in olive oil and
administered orally for five days starting on the same day as
sensitization.
[0326] ED.sub.50 Value of the FR-900506 substance for prevention of
the local graft-versus-host reaction was 19 mg/ka.
Test 7
Acute Toxicicities of Tricyclo Compounds (I)
[0327] Test on acute toxicities of the FR-900506, FR-900520,
FR-900523 and FR-900525 substances in ddY mice by intraperitoneal
injection were conducted, and the dead at dose of 100 mg/kg could
not be observed in each case.
Test 8
Effect of Tricyclo Compounds (I) on Antibody Formation in Mice
(Assay for the Haemagglutination Test)
[0328] Male BDF.sub.1 mice 6 to 8 weeks of age were used for this
test. The mice were immunized on day 0 with 1.times.10.sup.8 sheep
erythrocytes (SRBC) intraperitoneally. Serum aggulutinin titers
were determined for each individual animal with sedimentation of
erythrocytes by serial two fold dilutions. The titers are expressed
as -log.sub.2.
[0329] The FR-900506 substance was administered orally for 5 days
from one day before immunization to day 3.
[0330] The results of each experiments were evaluated by means of
Student's t-test as shown in Table 16.
22TABLE 16 Effect of FR-900506 Substance on antibody formation in
mice Dose Number Haemagglutination ED.sub.50 (mg/kg) of Animals
Mean log.sub.2 titer .+-. SE (mg/kg) Control -- 5 8.2 .+-. 0.2 --
FR-900506 10 5 7.6 .+-. 0.4 32 5 6.0 .+-. 0.45 16.9 100 5 4.4 .+-.
0.75
Test 9
Effect of Tricyclo Compounds (I) on In Vivo Plaque Forming Cell
(PFC) Response in Mice
[0331] Male C3H/He mice 6 to 7 weeks of age were used for this
test. The mice were immunized on day 0 with 0.2 ml of
1.times.10.sup.8 washed sheep erythrocytes intravenously. Spleens
were removed on day 4 and spleen cells were incubated in the
presence of SRBC as described by Cunningham and Szenberg (1968).
Tests were evaluated by enumeration of direct generated plaque
forming cells in the presence of complement. Suspensions of spleen
cells were counted with a Microcellcounter CC-130 (Sysmex, Japan)
and PFC results were calculated as PFC/10.sup.6 recovered
cells.
[0332] The FR-900506 substance was administered orally for 4 days
starting from the immunization.
[0333] The results of each experiment were evaluated by means of
Student's t-test as shown in Table 17.
23TABLE 17 Effect of FR-900506 Substance on in vivo PFC Response in
Mice Dose Number PFC/10.sup.6 ED.sub.50 (mg/kg) of Animals cells
(mg/kg) Control -- 5 5054 .+-. 408 -- FR-900506 3.2 5 3618 .+-. 476
10 5 1382 .+-. 243 5.9 32 5 657 .+-. 41 100 5 278 .+-. 41
Test 10
Effect of Tricyclo Compounds (I) on Contact-Delayed
Hypersensitivity in Mice
[0334] Female ICR mice were used for this test. A tenth of a 7%
(w/v) solution of picryl chloride in ethanol prepared at the time
of the experiment was applied to the ventral surface of previously
shaved animals. Such a sensitization was performed twice at
intervals of 7 days. Seven days later, the thickness of the ears
were measured with an ordinary engineer's micrometer and a 1% (w/v)
solution of picryl chloride in olive oil was painted to both
surfaces of each ear. The inflammation was evaluated 24 hours later
by measuring the both of ears. The results were expressed as the
average increase in thickness of the ears measured in units of 10
.sup.-3 cm.
[0335] The FR-900506 substance was injected for 14 days orally,
starting from the first sensitization.
[0336] Statistical significance was evaluated by Student's t-test
as shown in Table 18.
24TABLE 18 Effect of FR-900506 Substance on contact-delayed
hypersensitivity in mice Dose Number Increase of ears at 24 hours
(mg/kg) of Animals after challenge (.times.10.sup.-3 cm) Control --
5 15.5 .+-. 0.8 FR-900506 32 5 6.4 .+-. 1.6 100 5 2.2 .+-. 1.5
Test 11
Effect of Tricyclo Compounds (I) on Delayed Type Hypersensitivity
(DTH) Response to Methylated Bovine Serum Albumin (MBSA)
[0337] Female BDF1 mice were used for this test. The mice were
sensitized with a subcutaneous injection of 0.1 ml emulsion
consisting of equal volume of MBSA (2 mg/ml) and Freund's
incomplete adjuvant (FIA). Seven days later, a 0.05 ml challenge
dose of 0.4 mg/ml MIBSA in saline was injected into the plantar
region of the right hind foot and 0.05 ml saline into the left hind
foot to act as a control. Twenty four hours after challenge, both
hind feet were measured with a dial gauge and the mean challenge in
footpad thickness was measured.
[0338] The FR-900506 substance was injected for 8 days orally,
starting from the sensitization.
[0339] Statistical significance was evaluated by Student's t-test
as shown in Table 19.
25TABLE 19 Effect of FR-900506 Substance on DTH to MBSA in mice
Footpad Dose Number Swelling ED.sub.50 (mg/kg) of Animals
(.times.10.sup.-3 cm) (mg/kg) Control -- 5 57.4 .+-. 3.4 --
FR-900506 10 5 31.0 .+-. 4.4 32 5 20.8 .+-. 4.4 18.4 100 5 7.8 .+-.
1.6
Test 12
Inhibition of Interleukin-2 (IL-2) Production by Tricyclo Compounds
(I)
[0340] The mixed lymphocyte reaction (MLR) was performed in
microtiter plates, with each well containing 5.times.10.sup.5
C57BL/6 responder cells (H-2.sup.b), 5.times.10.sup.5 mitomycin C
treated (25 .mu.g/ml mitomycin C at 37.degree. C. for 30 minutes
and washed three times with RPMI 1640 medium) BALB/C stimulator
cells (H-2.sup.b) in 0.2 ml RPMI 1640 medium supplemented with 10%
fetal calf serum, 2 mM sodium hydrogen carbonate, penicillin 20 (50
unites/ml) and streptomycin (50 .mu.g/ml). The cells were incubated
at 37.degree. C. in humidified atmosphere of 5% carbon dioxide and
95% of air for 48 hours and the supernatant was collected. The
FR-900506 substance was dissolved in ethanol and further diluted in
RPMI 1640 medium and added to the cultures to give final
concentrations of 0.1 .mu.g/ml or less.
[0341] The IL-2 activity was measured according to the method
described by S. Gillis et. al. [Journal of Inmunology, Vol. 120,
page 2027, (1978)]. The IL-2 dependent CTLL-2 cell line was used to
quantitate IL-2 activity. Cells (5.times.10.sup.3/well) were
cultured at 37.degree. C. for 24 hours with various dilutions of
IL-2 containing supernatant from MLR. The uptake of
.sup.3H-thymidine was measured by pulsing cultures with 0.5 .mu.Ci
of .sup.3H-thymidine for 6 hours. The unit value was calculated by
dilution analysis of test sample and was compared with a laboratory
standard preparation in which 100 units are equivalent to the
amount of IL-2 necessary to achieve 50% proliferation of CTLL-2
cells.
[0342] As shown in Table 20, the FR-900506 substance of the present
invention inhibited the production of IL-2 from mouse MLR.
26TABLE 20 Effect of the FR-900506 Substance on IL-2 Production
FR-900506 concentration IL-2 (nM) (units/ml) Inhibition % 10 0 100
1 0 100 0.3 0.2 99.5 0.1 25.0 34.4 0 38.1 --
[0343] The FR-900506 substance also inhibited lymphokine production
such as interleukin 3 and interferones, especially
.gamma.-interferone, in the supernatant of MLR.
[0344] The pharmaceutical composition of this invention can be used
in the form of a pharmaceutical preparation, for example, in solid,
semisolid or liquid form, which contains the tricyclo compounds (I)
of the present invention, as an active ingredient, in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral or parenteral applications. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, and any
other form suitable for use. The carriers which can be used are
water, glucose, lactose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form,
and in addition auxiliary, stabilizing, thickening and coloring
agents and perfumes may be used. The active object compound is
included in the pharmaceutical composition in an amount sufficient
to produce the desired effect upon the process or condition of
diseases.
[0345] For applying this composition to human, it is preferable to
apply it by parenteral or enteral administration. While the dosage
of therapeutically effective amount of the tricyclo compounds (I)
varies from and also depends upon the age and condition of each
individual patient to be treated, a daily dose of about 0.01-1000
mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the
active ingredient is generally given or treating diseases, and an
average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100
mg, 250 mg and 500 mg is generally administered.
[0346] The following examples are given for the purpose of
illustrating the present invention.
EXAMPLE 1
Isolation of Streptomyces tsukubaensis No. 9993
[0347] Streptomyces tsukubaensis No. 9993 was isolated by using
dilution plate techniques as shown in the following.
[0348] About one gram soil which was collected at Toyosato-cho,
Tsukuba Gun, Ibaraki Prefecture, Japan, was added to a sterile test
tube and the volume made up to 5 ml with sterile water. The mixture
was then blended for 10 second by a tube buzzer and kept on 10
minutes. The supernatant was sequentially diluted by 100 fold with
sterile water. The diluted solution (0.1 ml) was spread on Czapek
agar supplemented with thiamine hydrochloride (saccharose 30 g,
sodium nitrate 3 g, dipotassium phosphate 1 g, magnesium sulfate
0.5 g, potassium chloride 0.5 g, ferrous sulfate 0.01 g, thiamine
hydrochloride 0.1 g, agar 20 g, tap water 1000 ml; pH 7.2) in a
Petri dish. The growing colonies developed on the plates after 21
days incubation at 30.degree. C. were transferred to slants
[yeast-malt extract agar (ISP-medium 2)], and cultured for 10 days
at 30.degree. C. Among of the colonies isolated, the Streptomyces
tsukubaensis No. 9993 could be found.
Fermentation
[0349] A culture medium (160 ml) containing glycerin (1%), soluble
starch (1%), glucose (0.5%), cottonseed meal (0.5%), dried yeast
(0.5%), corn steep liquor (0.5%) and calcium carbonate (0.2%)
(adjusted to pH 6.5) was poured into each of twenty 500
ml-Erlenmeyer flasks and sterilized at 120.degree. C. for 30
minutes. A loopful of slant culture of Streptomyces tsukubaensis
No. 9993, FERM BP-927 was inoculated to each of the media and
cultured at 30.degree. C. for 4 days on a rotary shaker. The
resultant culture was inoculated to a medium containing soluble
starch (4.5%), corn steep liquor (1%), dried yeast (1%), calcium
carbonate (0.1%) and Adekanol (defoaming agent, Trade Mark, maker;
Asahi Denka Co.) (0.1%) (150 liters) in a 200-liter jar-fermentor,
which had been sterilized at 120.degree. C. for 20 minutes in
advance, and cultured at 30.degree. C. for 4 days under aeration of
150 liters/minutes and agitation of 250 rpm.
Isolation and Purification
[0350] The cultured broth thus obtained was filtered with an aid of
diatomaseous earth (5 kg). The mycelial cake was extracted with
methanol (50 liters), yielding 50 liters of the extract. The
methanol extract from mycelium and the filtrate were combined and
passed through a column of a non-ionic adsorption resin "Diaion
HP-20" (Trade Mark, maker Mitsubishi Chemical Industries Ltd.) (10
liters). After washing with water (30 liters) and aqueous methanol
(30 liters), elution was carried out with methanol.
[0351] The eluate was evaporated under reduced pressure to give
residual water (2 liters). This residue was extracted with ethyl
acetate (2 liters). The ethyl acetate extract was concentrated
under reduced pressure to give an oily residue. The oily residue
was mixed with twice weight of acidic silica gel (special silica
gel grade 12, maker Fuji Devison Co.), and this mixture was
slurried in ethylacetate. After evaporating the solvent, the
resultant dry powder was subjected to column chromatography of the
same acid silica gel (800 ml) which was packed with n-hexane. The
column was developed with n-hexane (3 liters), a mixture of
n-hexane and ethyl acetate (9:1 v/v, 3 liters and 4:1 v/v, 3
liters) and ethyl acetate (3 liters). The fractions containing the
object compound were collected and concentrated under reduced
pressure to give an oily residue. The oily residue was dissolved in
a mixture of n-hexane and ethyl acetate (1:1 v/v, 30 ml) and
subjected to column chromatography of silica gel (maker Merck Co.,
Ltd. 230-400 mesh) (500 ml) packed with the same solvents
system.
[0352] Elution was carried out with a mixture of n-hexane and ethyl
acetate (1:1 v/v, 2 liters and 1:2 v/v, 1.5 liters). Fractions
containing the first object compound were collected and
concentrated under reduced pressure to give a yellowish oil. The
oily residue was mixed twice weight of acidic silica gel and this
mixture was slurried in ethyl acetate. After evaporating the
solvent, the resultant dry powder was chromatographed on acidic
silica gel packed and developed with n-hexane. Fractions containing
the object compound were collected and concentrated under reduced
pressure to give crude FR-900506 substance (1054 mg) in the form of
white powder.
[0353] 100 mg Of this crude product was subjected to high
performance liquid chromatography. Elution was carried out using a
column (8.phi..times.500 mm) with Lichrosorb SI 60 (Trade Mark,
made by Merck & Co.) as a carrier. This chromatography was
monitored by UV detector at 230 nm and mobile phase was a mixture
of methylene chloride and dioxane (85:15 v/v) under flow rate of 5
ml/minute., and the active fractions were collected and evaporated.
This high performance chromatography was repeated again, and 14 mg
of the purified FR-900506 substance was obtained as white
powder.
[0354] Further, elution was continuously carried out with ethyl
acetate (1.5 liters), and fractions containing the second object
compound were collected and concentrated under reduced pressure to
give crude FR-900525 substance (30 mg) in the form of yellowish
oil.
EXAMPLE 2
Fermentation
[0355] A preculture medium (100 ml) containing glycerin (1%), corn
starch (1%), glucose (0.5%), cottenseed meal (1%), corn steep
liquor (0.5%), dried yeast (0.5%) and calcium carbonate (0.2%) at
pH 6.5 was poured into a 500 ml-Erlenmeyer flask and sterilized at
120.degree. C. for 30 minutes. A loopful of slant culture of
Streptomyces tsukubaensis No. 9993 was inoculated to the medium and
cultured at 30.degree. C. for four days. The resultant culture was
transferred to the same preculture medium (20 liters) in 30 liters
jar-fermentor which had been sterilized at 120.degree. C. for 30
minutes in advance. After the culture was incubated at 30.degree.
C. for 2 days, 16 liters of the preculture was inoculated to a
fermentation medium (1600 liters) containing soluble starch (4.5%),
corn steep liquor (1%), dried yeast (1%), calcium carbonate (0.1%)
and Adekanol (defoaming agent, Trade Mark, maker Asahi Denka Co.)
(0.1%) at pH 6.8 in 2 ton tank which had been sterilized at
120.degree. C. for30 minutes in advance and cultured at 30.degree.
C. for 4 days.
Isolation and Purification
[0356] The cultured broth thus obtained was filtered with an aid of
diatomaseous earth (25 kg). The mycelial cake was extracted with
acetone (500 liters), yielding 500 liters of the extract. The
acetone extract from mycelium and the filtrate (1350 liters) were
combined and passed tnrough a column of a non-ionic adsorption
resin "Diaion HP-20" (Trade Mark, maker Mitsubishi Chemical
Industries Ltd.) (100 liters). After washing with water (300
liters) and 50% aqueous acetone (300 liters), elution was carried
out with 75% aqueous acetone. The eluate was evaporated under
reduced pressure to give residual water (300 liters). This residue
was extracted with ethyl acetate (20 liters) three times. The ethyl
acetate extract was concentrated under reduced pressure to give an
oily residue. The oily residue was mixed with twice weight of
acidic silica gel (special silica gel grade 12, maker Fuji Devison
Co.), and this mixture was slurried in ethyl acetate. After
evaporating the solvent, the resultant dry powder was subjected to
column chromatography of the same acidic silica gel (8 liters)
which was packed with n-hexane. The column was developed with
n-hexane (30 liters), a mixture of n-hexane and ethyl acetate (4:1
v/v, 30 liters) and ethyl acetate (30 liters). The fractions
containing the object compound were collected and concentrated
under reduced pressure to give an oily residue. The oily residue
was mixed with twice weight of acidic silica gel and this mixture
was slurried in ethyl acetate. After evaporating the solvent, the
resultant dry powder was rechromatographed on acidic silica gel
(3.5 liters) packed with n-hexane. The column was developed with
n-hexane (10 liters), a mixture of n-hexane and ethyl acetate (4:1
v/v, 10 liters) and ethyl acetate (10 liters). Fractions containing
the object compound were collected and concentrated under reduced
pressure to give a yellowish oil. The oily residue was dissolved in
a mixture of n-hexane and ethyl acetate (1:1 v/v, 300 ml) and
subjected to column chromatography of silica gel (maker Merck Co.,
Ltd. 230-400 mesh) (2 liters) packed with the same solvents system.
Elution was curried out with a mixture of n-hexane and ethyl
acetate (1:1 v/v, 10 liters and 1:2 v/v 6 liters) and ethyl acetate
(6 liters).
[0357] Fractions containing the first object compound were
collected and concentrated under reduced pressure to give FR-900506
substance in the form of white powder (34 g). This white powder was
dissolved in acetonitrile and concentrated under reduced pressure.
This concentrate was kept at 5.degree. C. overnight and prisms
(22.7 g) were obtained. Recrystallization from the same solvent
gave purified FR-900506 substance (13.6 g) as colorless prisms.
[0358] Further, fractions containing the second object compound
were collected and concentrated under reduced pressure to give
crude FR-900525 substance (314 mg) in the form of yellowish
powder.
EXAMPLE 3
Fermentation
[0359] A culture medium (160 ml) containing glycerin (1%), corn
starch (1%), glucose (0.5%), cottonseed meal (1%), dried yeast
(0.5%), corn steep liquor (0.5%) and calcium carbonate (0.2%)
(adjusted to pH 6.5) was poured into each of ten 500 ml-Erlenmeyer
flasks and sterilized at 120.degree. C. for 30 minutes. A loopful
of slant culture of Streptomyces tsukubaensis No. 9993 was
inoculated to each of the medium and cultured at 30.degree. C. for
4 days on a rotary shaker. The resultant culture was inoculated to
a medium containing soluble starch (5%), peanut powder (0.5%),
dried yeast (0.5%), gluten meal (0.5%), calcium carbonate (0.1%)
and Adekanol (deforming agent, Trade Mark, maker Asasi Denka Co.)
(0.1%) (150 liters) in a 200-liter jar-fermentor, which had been
sterilized at 120.degree. C. for 20 minutes in advance, and
cultured at 30.degree. C. for 4 days under aeration of 150
liters/minutes and agitation of 250 rpm.
Isolation and Purification
[0360] The cultured broth thus obtained was filtered with an aid of
diatomaseous earth (5 kg). The mycelial cake was extracted with
acetone (50 liters), yielding 50 liters of the extract. The acetone
extract from mycelium and the filtrate (135 liters) were combined
and passed through a column of a non-ionic adsorption resin "Diaion
HP-20" (Trade Mark, maker Mitsubishi Chemical Industries Ltd.) (10
liters). After washing with water (30 liters) and 50% aqueous
acetone (30 liters), elution was carried out with 75% aqueous
acetone. The eluate (30 liters) was evaporated under reduced
pressure to give residual water (2 liters). This residue was
extracted with ethyl acetate (2 liters) three times. The ethyl
acetate extract was concentrated under reduced pressure to give an
oily residue. The oily residue was mixed with twice weight of
acidic silica gel (special silica gel grade 12, maker Fuji Devison
Co.), and this mixture was slurried in ethyl acetate. After
evaporating the solvent, the resultant dry powder was subjected to
column chromatography of the same acidic silica gel (800 ml) which
was packed with n-hexane. The column was developed with n-hexane (3
liters), a mixture of n-hexane and ethyl acetate (4:1 v/v, 3
liters) and ethyl acetate (3 liters). The fractions containing the
object compound were collected and concentrated under reduced
pressure to give an oily residue. The oily residue was dissolved in
a mixture of n-hexane and ethyl acetate (1:1 v/v, 30 ml) and
subjected to column chromatography of silica gel (maker Merck Co.,
Ltd. 230-400 mesh) (500 ml) packed with the same solvents system.
Elution was carried out with a mixture of n-hexane and ethyl
acetate (1:1 v/v, 2 liters and 1:2 v/v, 1.5 liters) and ethyl
acetate (1.5 liters).
[0361] Fractions containing the first object compound were
collected and concentrated under reduced pressure to give crude
FR-900506 substance (3 g) in the form of yellowish powder.
[0362] Further, fractions containing the second object compound
were collected and concentrated under reduced pressure to give an
oily residue. This oily residue was rechromatographed with silica
gel to give a yellowish oil. The oily residue was mixed with twice
weight of acidic silica gel and this mixture was slurried in ethyl
acetate. After evaporating the solvent, the resultant dry powder
was chromatographed on acidic silica gel (100 ml) packed and
developed with n-hexane. Fractions containing the object compound
were collected and concentrated under reduced pressure to give
FR-900525 substance in the form of pale yellowish powder (380 mg).
This powder was dissolved in a mixture of n-hexane and ethyl
acetate (1:2. v/v, 5 ml) and subjected to acidic silica gel
(special silica gel grade 922, maker Fuji Devison Co.) (100 ml)
packed and washed with the same solvent system. Elution was carried
out with ethyl acetate. The active fractions were collected and
evaporated under reduced pressure to give the purified FR-900525
substance (230 mg) in the form of white powder.
EXAMPLE 4
Isolation of Streptomyces hygroscopicus subsp. yakushimaensis No.
7238
[0363] Streptomyces hygroscopicus subsp. yakushimaensis No. 7238
was isolated by using dilution plate techniques as shown in the
following.
[0364] About one gram soil which was collected at Yakushima,
Kagoshima Prefecture, Japan, was added to a sterile test tube and
the volume made up to 5 ml with sterile water. The mixture was then
blended for 10 seconds by a tube buzzer and kept on 10 minutes. The
supernatant was sequentially diluted by 100 fold with sterile
water. The diluted solution (0.1 ml) was spread on Czapek agar
supplemented with thiamine hydrochloride (saccharose 30 g, sodium
nitrate 3 g, dipotassium phosphate 1 g, magnesium sulfate 0.5 g,
potassium chloride 0.5 g, ferrous sulfate 0.01 g, thiamine
hydrochloride 0.1 g, agar 20 g, tap water 1000 ml; pH 7.2) in a
Petri dish. The growing colonies developed on the plates after 21
days incubation at 30.degree. C. were transferred to slants
[yeast-malt extract agar (ISP-medium 2)], and cultured for 10 days
at 30.degree. C. Among of the colonies isolated, the Streptomyces
hygroscopicus subsp. yakushimaensis No. 7238 could be found.
Fermentation
[0365] A culture medium (160 ml) containing glycerin (1%), soluble
starch (1%), glucose (0.5%), cottonseed meal (0.5%), dried yeast
(0.5%), corn steep liquor (0.5%) and calcium carbonate (0.2%)
(adjusted to pH 6.5) was poured into each of twenty 500
ml-Erlenmeyer flasks and sterilized at 120.degree. C. for 30
minutes. A loopful of slant culture of Streptomyces hygroscopicus
subsp. yakushimaensis No. 7238, FERM BP-928 was inoculated to each
of the media and cultured at 30.degree. C. for 4 days on a rotary
shaker. The resultant culture was inoculated to a medium containing
glucose (4.5%), corn steep liquor (1%), dried yeast (1%), gluten
meal (1%), wheat germ (0.5%), calcium carbonate (0.1%) and Adekanol
(defoaming agent, Trade Mark, maker Asahi Denka Co.) (0.1%) (150
liters) in a 200-liter jar-fermentor, which had been sterilized at
120.degree. C. for 20 minutes in advance, and cultured at
30.degree. C. for 4 days under aeration of 150 liters/minutes and
agitation of 250 rpm.
Isolation and Purification
[0366] The cultured broth thus obtained was filtered with an aid of
diatomaseous earth (5 kg). The mycelial cake was extracted with
acetone (50 liters), yielding 50 liters of the extract. The acetone
extract from mycelium and the filtrate (135 liters) were combined
and passed through a column of a non-ionic adsorption resin "Diaion
HP-20" (Trade Mark, maker Mitsubishi Chemical Industries Ltd.) (10
liters). After washing with water (30 liters) and aqueous acetone
(30 liters), elution was carried out with acetone. The eluate was
evaporated under reduced pressure to give residual water (2
liters). This residue was extracted with ethyl acetate (4 liters).
The ethyl acetate extract was concentrated under reduced pressure
to give an oily residue. The oily residue was mixed with twice
weight of acidic silica gel (special silica gel grade 12, maker
Fuji Devison Co.), and this mixture was slurried in ethyl acetate.
After evaporating the solvent, the resultant dry powder was
subjected to column chromatography of the same acid silica gel (800
ml) which was packed with n-hexane. The column was developed with
n-hexane (3 liters), a mixture of n-hexane and ethyl acetate (4:1
v/v, 3 liters) and ethyl acetate (3 liters). The fractions
containing the FR-900520 and FR-900523 substances were collected
and concentrated under reduced pressure to give an oily residue.
The oily residue was dissolved in a mixture of n-hexane and ethyl
acetate (1:1 v/v, 50 ml) and subjected to column chromatography of
silica gel (maker Merck Co., Ltd. 70-230 mesh) (1000 ml) packed
with the same solvents system. Elution was carried out with a
mixture of n-hexane and ethyl acetate (1:1 v/v, 3 liters and 1:2
v/v, 3 liters) and ethyl acetate (3 liters). Fractions containing
the object compounds were collected and concentrated under reduced
pressure to give a yellowish powder (4.5 g). This powder was
dissolved in methanol (20 ml) and mixed with water (10 ml). The
mixture was chromatographed on a reverse phase silica gel "YMC"
(60-200 mesh) (500 ml) (Trade mark, maker Yamamura Chemical
Institute) packed and developed with a mixture of methanol and
water (4:1 v/v).
[0367] Fractions containing the FR-900520 substance were collected
and concentrated under reduced pressure to give crude product of
the FR-900520 substance (1.8 g) in the form of pale yellowish
powder. This powder was dissolved in a small amount of diethyl
ether. After standing overnight, the precipitated crystals were
collected by filtration, washed with diethyl ether and then dried
under reduced pressure. Recrystallization from diethyl ether gave
600 mg of the purified FR-900520 substance in the form of colorless
plates.
[0368] The chromatography of the reverse phase silica gel was
carried on with the same solvents system, and the subsequent
fractions containing the FR-900523 substance were collected and
then concentrated under reduced pressure to give crude product of
the FR-900523 substance (0.51 g) in the form of pale yellowish
powder. This crude product was dissolved in acetonitrile (3 ml) and
subjected to a reverse phase silica gel "YMC" (70 ml) packed and
developed with a mixture of acetonitrile, tetrahydrofuran and 50 mM
phosphate buffer solution (pH 2.0) (3:2:5, v/v). Fractions
containing the object compound were collected and were extracted
with ethyl acetate. This extract was concentrated under reduced
pressure to give a yellowish white powder (190 mg). The yellowish
white powder was chromatographed again on a reverse phase silica
gel "YMC" to give white powder (80 mg). This white powder was
dissolved in a small amount of diethyl ether and allowed to stand
overnight at room temperature to give 56mg of crystals.
Recrystallization from diethyl ether gave 34 mg of the FR-900523
substance in the form of colorless needles.
EXAMPLE 5
[0369] To a solution of the FR-900506 substance (10.4 mg) in
dichloromethane (0.2 ml) were added pyridine (0.1 ml) and acetic
anhydride (0.05 ml) at room temperature, and the mixture was
stirred for 5 hours. The solvent was removed from the reaction
mixture under reduced pressure. The residue was subjected to silica
gel thin layer chromatography (developing solvent: diethyl ether
and dichloromethane, 1:2 v/v) to give
12-[2-(4-acetoxy-3-methoxycyclohexyl)-1-methylvinyl]-17--
allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-
-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(6.0 mg).
[0370] IR.upsilon.(CHCl.sub.3): 3520, 1728, 1705 (sh), 1640, 1095
cm.sup.-1
EXAMPLE 6
[0371] To a solution of the FR-900506 substance (52.5 mg) in
dichloromethane (1 ml) were added pyridine (0.5 ml) and acetic
anhydride (0.3 ml) at room temperature, and the mixture was stirred
at room temperature for 9 hours. The solvent was removed from the
reaction mixture under reduced pressure. The residue was subjected
to silica gel thin layer chromatography (developing solvent:
diethyl ether and hexane, 3:1 v/v) to give
14-acetoxy-12-[2-(4-acetoxy-3-methoxycyclohexyl)-1-methy-
lvinyl]-17-allyl-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-d-
ioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(48.0 mg) and
12-[2-(4-acetoxy-3-methoxycyclohexyl)-1-methylvinyl]-17-all-
yl-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatric-
yclo-[22.3.1.0.sup.4,9]octacosa-14,18-diene-2,3,10,16-tetraone (5.4
mg), respectively.
Former Compound
[0372] IR.upsilon.(CHCl.sub.3): 1730, 1720 (sh), 1640 cm.sup.-1
Latter Compound
[0373] IR.upsilon.(CHCl.sub.3): 1730, 1690, 1640, 1627
cm.sup.-1
EXAMPLE 7
[0374] To a solution of the FR-900506 substance (9.7 mg) in
dichloromethane (0.2 ml) and pyridine (0.1 ml) was added benzoyl
chloride (50 .mu.l) at room temperature, and the mixture was
stirred at room temperature for 2 hours. The solvent was removed
from the reaction mixture under reduced pressure to give a crude
oil. This oil was purified on silica gel thin layer chromatography
(developing solvent: diethyl ether and hexane, 2:1 v/v) to afford
17-allyl-12-[2-(4-benzoyloxy-3-metho-
xycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-te-
tramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10-
,16-tetraone (8.0 mg).
[0375] IR.upsilon.(CHCl.sub.3): 3500, 1735(sh), 1710, 1640, 1600
cm.sup.-1
EXAMPLE 8
[0376] To a solution of the FR-900506 substance (30.5 mg) in
pyridine (1 ml) was added p-nitrobenzoyl chloride (ca. 100 mg), and
the mixture was stirred at room temperature for 2 hours. The
reaction mixture was diluted with ethyl acetate, and washed with a
saturated aqueous sodium hydrogen carbonate, water, 1N-hydrochloric
acid, water, a saturated aqueous sodium hydrogen carbonate, water
and an aqueous sodium chloride, successively, and then dried. The
resulting solution was concentrated under reduced pressure, and the
residue was purified on silica gel column chromatography to give
17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,2-
7-tetramethyl-12-[2-[4-(p-nitrobenzoyloxy)-3-methoxycyclohexyl]-1-methylvi-
nyl]-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-t-
etraone (37.7 mg).
[0377] IR.upsilon.(CHCl.sub.3): 1720, 1640, 1610, 1530-1520
cm.sup.-1
EXAMPLE 9
[0378]
17-Allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12--
[2-[4-(3,5-dinitrobenzoyloxy)-3-methoxycyclohexyl]-1-methylvinyl]-11,28-di-
oxa-4-azatricyclo-[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(36.0 mg) was obtained by reacting the FR-900506 substance (30.6
mg) with 3,5-dinitrobenzoyl chloride (33 mg) in pyridine (0.5 ml)
in accordance with a similar manner to that of Example 8.
[0379] IR.upsilon.(CHCl.sub.3): 1730, 1640, 1610, 1530-1520
cm.sup.-1
EXAMPLE 10
[0380]
17-Allyl-1,14-dihydroxy-23,25-dimethoxy-12-[2-[4-(2-l-menthyloxyace-
toxy)-3-methoxycyclohexyl]-1-methylvinyl]-13,19,21,27-tetramethyl-11,28-di-
oxa-4-azatricyclo-[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(50.9 mg) was obtained by reacting the FR-900506 substance (48 mg)
with 2-l-methyloxyacetyl chloride (0.08 ml) in pyridine (0.5 ml) in
accordance with a similar manner to that of Example 8.
[0381] IR.upsilon.(neat): 3520, 1760, 1740(sh), 1720(sh), 1652
cm.sup.-1
EXAMPLE 11
[0382] To a solution of
(-)-2-trifluoromethyl-2-methoxy-2-phenylacetic acid (51 mg) in
ethyl acetate (10 ml) was added at room temperature
N,N'-dicyclohexylcarbodiimide (47 mg). After stirring for 1.5 hours
at room temperature, then the FR-900506 substance (25.0 mg) and
4-(N,N-dimethylamino)-pyridine (11 mg) were added, followed by
stirring at room temperature for 3.5 hours. The resulting solution
was concentrated to provide a residue, which was taken up in
diethyl ether and then washed successively with hydrochloric acid,
an aqueous sodium hydrogen carbonate and an aqueous sodium
chloride. The organic layer was dried over sodium sulfate and
concentrated to provide a residue, which was chromatographed on
silica gel (developing solvent: dichloromethane and diethyl ether,
10:1 v/v) to give 17-allyl-12-[2-[4-[(-)-2-trifluorome-
thyl-2-methoxy-2-phenylacetoxy]-3-methoxycyclohexyl]-1-methylvinyl]-1,14-d-
ihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-
[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (6.5 mg) and
17-allyl-14-[(-)-2-trifluoromethyl-2-methoxy-2-phenylacetoxy]-12-[2-[4-[(-
-)-2-trifluoromethyl-2-methoxy-2-phenylacetoxy]-3-methoxycyclohexyl]-1-met-
hylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4--
azatricyclo-[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(20.2 mg).
Former Compound
[0383] IR.upsilon.(neat): 3510, 1750, 1730(sh), 1710, 1652, 1500
cm.sup.-1
Latter Compound
[0384] IR.upsilon.(neat): 1750, 1720, 1652, 1500 cm.sup.-1
EXAMPLE 12
[0385] To a stirred solution of the FR-900506 substance (248 mg) in
pyridine (7 ml) were added succinic anhydride (145 mg) and
4-(N,N-dimethylamino) pyridine (7 mg), and the resulting mixture
was stirred at room temperature for 18 hours. The reaction mixture
was concentrated under reduced pressure and the residue was
subjected to chromatography on silica gel (20 g) with ethyl acetate
to give
17-allyl-12-[2-[4-(3-carboxypropionyloxy)-3-methoxycyclohexyl]-1-methylvi-
nyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4--
azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (90
mg).
[0386] IR.upsilon.(CHCl.sub.3): 3500, 3100-2300, 1720, 1705(sh),
1635 cm.sup.-1
EXAMPLE 13
[0387] To a solution of the FR-900506 substance (100.7 mg) in
pyridine (3 ml) was added p-iodobenzenesulfonyl chloride (500 mg),
and the mixture was stirred at room temperature for 36 hours. The
solution was diluted with ethyl acetate and washed with a saturated
aqueous sodium hydrogen carbonate, water and an aqueous sodium
chloride. The organic layer was dried over sodium sulate filtered
and concentrated under reduced pressure. The residue was
chromatographed on silica gel (developing solvent: diethyl ether
and hexane, 3:1 v/v) to give
17-allyl-1,14-dihydroxy-12-[2-[4-(p-iodobenzenesulfonyloxy)-3-methoxycycl-
ohexy]-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa--
4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(61 mg) and
17-allyl-1-hydroxy-12-[2-[4-(p-iodobenzenesulfonyloxy)-3-methoxycyclo-
hexyl]-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa--
4-azatricyclo[22.3.1.0.sup.4,9]octacosa-14,18-diene-2,3,10,16-tetraone
(12 mg), respectively.
Former Compound
[0388] IR.upsilon.(CHCl.sub.3): 3470, 1730, 1717, 1692, 1635, 1568
cm.sup.-1
Latter Compound
[0389] 10 1 H NMR ppm ( C D Cl 3 ) : 6.15 ( d , J = 15 Hz ) 6.25 (
d , J = 15 Hz ) } ( 1 H ) , 6.70 ( dd , J = 15 Hz , 10 Hz ) 6.80 (
dd , J = 15 Hz , 10 Hz ) } ( 1 H ) , 7.60 ( 2 H , m ) , 7.90 ( 2 H
, m ) ,
EXAMPLE 14
[0390]
17-Allyl-12-[2-(4-d-camphorsulfonyloxy-3-methoxycyclohexyl)-1-methy-
lvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-
-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(34 mg) was obtained by reacting the FR-900506 substance (27 mg)
with d-camphorsulfonyl chloride (97 mg) in pyridine (0.6 ml) in
accordance with a similar manner to that of Example 13.
[0391] IR.upsilon.(neat): 3500, 1747, 1720(sh), 1710(sh), 1655
cm.sup.-1
EXAMPLE 15
[0392] To a stirred solution of the FR-900506 substance (89.7 mg)
in dichloromethane (3 ml) were added imidazole (118 mg) and
tert-butyl-diphenylsilyl chloride (52.2 mg). After the mixture was
stirred at room temperature for 2 hours, the reaction mixture was
diluted with a saturated aqueous ammonium chloride and extracted
three times with diethyl ether. The extract was washed with water
and an aqueous sodium chloride, dried over sodium sulfate, and then
concentrated under reduced pressure. The residue was purified on
silica gel column chromatography (developing solvent: ethyl acetate
and hexane, 1:3 v/v) to give
17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxycyclohexyl)-1-meth-
ylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-diox-
a-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone
(107 mg).
[0393] IR.upsilon.(neat): 3520, 1742, 1705, 1650 cm.sup.-1
EXAMPLE 16
[0394]
17-Allyl-12-[2-(4-tert-butyl-dimethylsilyloxy-3-methoxycyclohexyl)--
1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,2-
8-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2-3,10,16-tetraone
(85 mg) was obtained by reacting the FR-900506 substance (80 mg)
with tert-butyl-dimethylsilyl chloride (17 mg) in the presence of
imidazole (15 mg) in N,N-dimethylformamide (1 ml) in accordance
with a similar manner to that of Example 15.
[0395] IR.upsilon.(CHCl.sub.3): 1735, 1720(sh), 1700, 1640
cm.sup.-1
EXAMPLE 17
[0396] To a solution of the FR-900506 substance (100 mg) in
dimethyl sulfoxide (1.5 ml) was added acetic anhydride (1.5 ml),
and the mixture was stirred at room temperature for 14 hours. The
reaction mixture was diluted with ethyl acetate and washed with a
saturated aqueous sodium hydrogen carbonate, water and an aqueous
sodium chloride. The organic layer was dried over sodium sulfate,
filtered and then concentrated under reduced pressure. The residue
was subjected to thin layer chromatography on silica gel
(developing solvent: diethyl ether) to give
17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-(4--
methylthiomethoxy-3-methoxycyclohexyl)-1-methylvinyl]-11,28-dioxa-4-azatri-
cyclo[22.3.1.0.sup.4,9]octacose-14,18-diene-2,3,10,16-tetraone (51
mg),
17-allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-2-
3,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.-
sup.4,9]octacose-14,18-diene-2,3,10-16-tetraone (18 mg) and
17-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-(4--
methylthiomethoxy-3-methoxycyclohexyl)-1-methylvinyl]-11,28-dioxa-4-azatri-
cyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (10 mg),
respectively.
First Compound
[0397] IR.upsilon.(CHCl.sub.3): 3470, 1730, 1635, 1630(sh),
1580(sh) cm.sup.-1
Second Compound
[0398] IR.upsilon.(CHCl.sub.3): 1728, 1640, 1090 cm.sup.-1
Third Compound
[0399] IR.upsilon.(CHCl.sub.3): 3480, 1735, 1710, 1640
cm.sup.-1
EXAMPLE 18
[0400] To a solution of
17-allyl-12-[2-(4-tert-butyl-dimethylsilyloxy-3-me-
thoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-
-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3-
,10,16-tetraone (38.9 mg) in pyridine (1.5 ml) was added acetic
anhydride (0.5 ml), and the mixture was stirred at room temperature
for 6 hours. The solvent was removed from the reaction nixture
under reduced pressure to give a crude oil, which was purified on
silica gel thin layer chromatography (developing solvent: diethyl
ether and hexane, 1:1 v/v) to afford
14-acetoxy-17-allyl-12-[2-(4-tert-butyl-dimethylsilyloxy-3-methoxy-
cyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetrameth-
yl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tet-
raone (26.5 mg).
[0401] IR.upsilon.(CHCl.sub.3): 1728, 1715(sh), 1635 cm.sup.-1
EXAMPLE 19
[0402]
14-Acetoxy-17-allyl-12-[2-(4-tert-butyl-diphenylsilyloxy-3-methoxyc-
yclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,27-tetramethy-
l-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetr-
aone (10 mg) was obtained by reacting
17-allyl-12-[2-(4-tert-butyl-dipheny-
lsilyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1,14-dihydroxy-23,25-dimetho-
xy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octa-
cos-18-ene-2,3,10,16-tetraone (10.6 mg) with acetic anhydride (0.1
mg) in pyridine (0.2 ml) in accordance with a similar manner to
that of Example 18.
[0403] IR.upsilon.(CHCl.sub.3): 3500, 1730, 1720(sh), 1660(sh),
1640, 1620(sh), 1100 cm.sup.-1
EXAMPLE 20
[0404] To a solution of
14-acetoxy-17-allyl-12-[2-(4-tert-butyl-diphenylsi-
lyloxy-3-methoxycyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19-
,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-e-
ne-2,3,10,16-tetraone (43.8 mg) in tetrahydrofuran (1.5 ml) was
added potassium carbonate (ca 100 mg) at room temperature and the
mixture was stirred at the same temperature for 3 hours. The
reaction mixture was diluted with diethyl ether and the resulting
solution was washed with a saturated aqueous ammonium chloride,
water and an aqueous sodium chloride successively, and dried over
sodium sulfate. The resulting solution was concentrated under
reduced pressure and the residue was purified on silica gel thin
layer chromatography (developing solvent: diethyl ether and hexane,
3:2 v/v) to give 17-allyl-12-[2-(4-tert-butyl-diphenylsilylox-
y-3-methoxycyclohexyl)-1-methylvinyl]-1-hydroxy-23,25-dimethoxy-13,19,21,2-
7-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacosa-14,18-di-
ene-2,3,10,16-tetraone (30 mg).
[0405] IR.upsilon.(CHCl.sub.3): 1733, 1720(sh), 1685, 1640(sh),
1620 cm.sup.-1
EXAMPLE 21
[0406] A solution of the FR-900506 substance (50 mg) in ethyl
acetate (2 ml) was subjected to catalytic reduction using 10%
palladium on carbon (10 mg) under atmospheric pressure at room
temperature for 20 minutes. The reaction mixture was filtered and
the filtrate was evaporated to dryness, which was purified on thin
layer chromatography. Development with a mixture of chloroform and
acetone (5:1 v/v) gave
1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-
-dimethoxy-13,19,21,27-tetramethyl-17-propyl-11,28-dioxa-4-azatricyclo[22.-
3.1.0.sup.4,9]octacos-18-ene-2,3,10,16-tetraone (50.0 mg).
[0407] IR.upsilon.(CHCl.sub.3): 3480, 1735(sh), 1717, 1700,
1650(sh), 1625 cm.sup.-1
EXAMPLE 22
[0408] White powder of crude FR-900506 substance (1 g) obtained by
a similar fermentation process to Example 1 was dissolved in
acetonitrile (5 ml) and subjected to high performance liquid
chromatography (HPLC) using Shimazu LC4A (Trade Mark, made by
Shimazu Seisaku-sho). Steel column (25 mm inside diameter, 250 mm
length) packed with YMC-S343 (ODS) (Trade Mark, made by Shimakyu
Co., Ltd.) was used at a flow rate of 12 ml/min. Mobile phase was
an aqueous mixture of 28% acetonitrile 10% n-butanol, 0.075%
phosphoric acid, 3.75 mM sodium dodecyl sulfate (SDS) and detection
was carried out using Hitachi UV-recorder at 210 nm. One hundred
.mu.l of the sample was injected each time and the HPLC was
repeated 50 times so that all the sample could be subjected to the
column. Each eluate with a retention time of 85 min. to 90 min. was
collected and extracted with an equal volume of ethyl acetate (3.6
liters). The ethyl acetate layer was separated and washed with an
aqueous sodium hydrogen carbonate (1%, 2 liters) and concentrated
in vacuo to a small amount. SDS crystallized on concentration was
removed by filtration. Crude powder obtained was dissolved in
acetonitrile at a concentration of 100 mg/ml and applied again to
HPLC. Mobile phase was an aqueous mixture of 12.5% acetonitrile,
9.75% n-butanol, 0.075% phosphoric acid, 3.75 mM DS. The column was
eluted at a flow rate of 10 ml/min. The eluates with a retention
time of 131 min. to 143 min. were collected and extracted with
equal volume of ethyl acetate. The solvent layer was separated and
washed with 1% aqueous sodium hydrogen carbonate and concentrated
in vacuo to a small volume. SDS crystallized on concentration was
removed by filtration.
[0409] Crude powder thus obtained was dissolved in a small amount
of ethyl acetate and subjected to column chromatography using
silica gel (10 ml) (Kiesel gel, 230-400 mesh, maker: Merck Co.,
Ltd.). The column was washed with a mixture of n-hexane and ethyl
acetate (30 ml) (1:1 v/v) and a mixture of n-hexane and ethyl
acetate (60 ml) (1:2 v/v). Elution was carried out using ethyl
acetate and fractionated (each fraction: 3 ml). Fractions 18 to 24
were collected and concentrated in vacuo to dryness to give
FR-900520 substance (24 mg).
EXAMPLE 23
[0410] A solution of the FE-900506 substance (310 mg),
2-trimethylsilylethyl 4-isocyanatobutyrate (350 mg) and
triethylamine (6 drops) in anhydrous benzene (4 ml) was heated at
50.degree. C. with stirring for 2 hours. The reaction mixture was
allowed to stand at room temperature overnight. The mixture was
concentrated to dryness under reduced processure to leave a
residue, which was chromatographed on silica gel in chloroform.
Elution was carried out with chloroform to give
17-allyl-12-[2-[3-methoxy-4-{3-(2-trimethylsilylethoxycarbonyl)propylcarb-
amoyloxy}cyclohexyl]-1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,2-
1,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9]octacos-18-ene-
-2,3,10,16-tetraone (99 mg). This product was treated with
tetra(n-butyl)ammonium fluoride (0.12 m mole) in tetrahydrofuran
(2.5 ml) at room temperature for 20 minutes. The reaction mixture
was concentrated to dryness under reduced pressure to leave a
residue, which was purified with preparative thin layer
chromatography on silica gel. Elution with a mixture of chloroform
and methanol (5:1) gave 17-allyl-12-[2-{4-(3-carbox-
ypropylcarbamoyloxy)-3-methoxycyclohexyl}-1-methylvinyl]-1,14-dihydroxy-23-
,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.-
sup.4,9]octacos-18-ene-2,3,10,16-tetraone (18.1 mg).
[0411] IR.upsilon.(CHCl.sub.3): 3550-3100, 2870, 2750-2400, 1730,
1690, 1630 cm.sup.-1
* * * * *