CA1128882A - Antibiotic neplanocins - Google Patents
Antibiotic neplanocinsInfo
- Publication number
- CA1128882A CA1128882A CA328,113A CA328113A CA1128882A CA 1128882 A CA1128882 A CA 1128882A CA 328113 A CA328113 A CA 328113A CA 1128882 A CA1128882 A CA 1128882A
- Authority
- CA
- Canada
- Prior art keywords
- neplanocin
- formula
- group
- neplanocins
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Compounds Of Unknown Constitution (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention provides antibiotic neplanocins of the following formula wherein X is an NH2, -OH, -SH, or -SCH3 group or a C1 atom, R is a hydrogen atom or a protective group for the -OH group, and Z is the group; in which Y is -O- or a valence bond, and R is a hydrogen atom or a protective group for the -OH group, or the group;
in which Y is -O- or a valence bond. They are prepared by a process which comprises culturing a neplanocins producing microorganism belonging to the genus Ampullariella in a nutrient medium consisting of assimilable carbon and nitrogen sources and inorganic substances, and isolating neplanocin-A, -C, -D, -B
and -F from the cultured mass.
The present invention provides antibiotic neplanocins of the following formula wherein X is an NH2, -OH, -SH, or -SCH3 group or a C1 atom, R is a hydrogen atom or a protective group for the -OH group, and Z is the group; in which Y is -O- or a valence bond, and R is a hydrogen atom or a protective group for the -OH group, or the group;
in which Y is -O- or a valence bond. They are prepared by a process which comprises culturing a neplanocins producing microorganism belonging to the genus Ampullariella in a nutrient medium consisting of assimilable carbon and nitrogen sources and inorganic substances, and isolating neplanocin-A, -C, -D, -B
and -F from the cultured mass.
Description
This invention relates to novel antibiotic neplanocins having inhibitory action for plant pathogenic microorganisms and antitumor activity and the production thereof. More particularly, this invention relates to novel antibiotic neplanocins and a production process thereof which comprises culturing a neplanocins producing actinomycetes Ampullariella sp. A 11079 FERM-P No. 4494 in a nutrient medium and isolating the novel antibiotic neplanocins therefrom. Also this invention includes neplanocin D derivatives derived from neplanocin D and the chemical synthesis thereof.
The novel antibiotic neplanocins in this invention consist of neplanocin -A, -C, -D and its derivatives, -B and -F.
~ eplanocin A is a weakly basic substance and has the following physicochemical properties.
(1) Elemental analysis:
Found: C; 49,96% H; 5.00% N; 26.43%
Calculated: C; 50.19~ H; 4.97% N; 26.60%
The novel antibiotic neplanocins in this invention consist of neplanocin -A, -C, -D and its derivatives, -B and -F.
~ eplanocin A is a weakly basic substance and has the following physicochemical properties.
(1) Elemental analysis:
Found: C; 49,96% H; 5.00% N; 26.43%
Calculated: C; 50.19~ H; 4.97% N; 26.60%
(2) Molecular weight (calculated from mass spectrum analysis):263
(3) Molecular formula: CllH13N5O3
(4) Melting point: (determined by thermal analysis) : 216C.
(5) Specific rotation: [~]DO= -157 (c = 0.45%, H2O)
(6) Ultraviolet absorption spectrum: (14~ /ml) in H2O : shown in Fig. 1.
~ max = 263 ~, ElCm = 602.1 at pH 3:~ max = 261 m~, ElCm = 566.4 at pH 10:~ max = 263 m~, El~m = 595.0
~ max = 263 ~, ElCm = 602.1 at pH 3:~ max = 261 m~, ElCm = 566.4 at pH 10:~ max = 263 m~, El~m = 595.0
(7) Infrared absorption spectrum (KBr): shown in Fig. 2.
Absorption bands at 3360, 3200, 2920, 1640, 1600, 1570, 1480, 1415, 1370, 1330, 1300, 1250, 1205, 1160, 1115, 1080, 1050, 1005, 910, 850, 790, 730 cm 1.
Absorption bands at 3360, 3200, 2920, 1640, 1600, 1570, 1480, 1415, 1370, 1330, 1300, 1250, 1205, 1160, 1115, 1080, 1050, 1005, 910, 850, 790, 730 cm 1.
(8) Nuclear Magn~tic Resonance Spectrum: shown in Fig. 3 (internal standard DSS, in Deuterium dimethylsulfoxide, 100 MHz).
~3 ï~8~82
~3 ï~8~82
(9) Solubility:
Soluble: water, dimethylsulfoxide, dimethylformamide, acetic acid and aqueous acetone.
Insoluble: ethyl acetate, chloroform, benzene, hexane.
Soluble: water, dimethylsulfoxide, dimethylformamide, acetic acid and aqueous acetone.
Insoluble: ethyl acetate, chloroform, benzene, hexane.
(10) Color reactions:
Positive: decolorization of potassium permanganate, periodate oxidation and anisaldehyde.
Negative: ferric chloride, ninhydrin, anilinephthalate, Molisch and Fehling.
Positive: decolorization of potassium permanganate, periodate oxidation and anisaldehyde.
Negative: ferric chloride, ninhydrin, anilinephthalate, Molisch and Fehling.
(11) Nature: weakly basic.
(12) Color: white needle crystal.
(13) Rf value (silica gel, Tokyo Kasei Co., silica gel f):
n-butanol:acetic acid:water (6:1:1); ~f = 0.36.
n-butanol:conc. aq. ammonia: water (10:05:2); Rf = 0.27 n-propanol:conc. aq. ammonia:water (10:1:1); Rf = 0.41 acetone:water tlO:l); Rf = 0.34 ethyl acetate: methanol:water tlO:2:1); Rf = 0.21 chloroform:methanol:acetic acid tlO:2:1); Rf = 0.17 t14) Chemical structure:
~\
OH OH
The biological properties or ncplanocin A are as follows:
(l) Growth inhibitory activity on plant pathogenic fungi:
lOO ~/ml solution of neplanocin A on an agar plate of Helminthosporium oryzae M0306 shows the inhibitory zone of 18.9 mm in diameter.
' ~2-(2) Antitumor activity:
Murine leukemia L-1210 tumor cells (106 cells) were inoculated intraperitoneally in mice and one day after trans-plantation, neplanocin A (each 0.16 mg/kg, 0.32 mg/kg, 0.63 mg/kg, 1.25 mg/kg, 2.5 mg/kg and 5.0 mg/kg, respectively) was administered intraperitoneally once a day for 5 days. 10 mice for the control group and 7 mice in each one group for the treated group were used. The result is shown in Fig. 4. On the 15 days observation, mean survival days for the control group were 7.4 days. Prolongations of life span as compared with the control group were observed for drug administered groups; i.e. 158~ for 0.16 mg/kg, 170~ for the 0.32 mg/kg group, no mortalities were observed for the 2.5 mg/kg and 5 mg/kg groups.
Like L-1210 remarkable life prolongation effects were observed against Ehrlich ascites tumor, Sarcoma-180, P-388 leukemia bearing mice and rat Yoshida sarcoma when neplanocin A was administered.
(3) Acute toxicity:
LD50 : 13.7 mg/kg (i.p., mice) No mortalities were observed wh~n neplanocin A was administered intraperitoneally for 14 days, 5 mg/kg/day.
Directions and dosage~
For therapy of acute leukemia, the drug will be intra-venously administered in a dosage form of 5 - 20 mg/day dissolved in physiological saline, once a day, ~or 7 days.
Neplanocin A can also be effectively used ~or therapy of chronic leukemia, gastroentero carcinoma, pulmonary carcinoma, uterine cancer, mammary cancer and malignant lymphatic leukemia.
Neplanocin A can be used in a rorm of a physiologically acceptable salt of a mineral acid or organic acid such as the hydrochloride, acetate, tartrate, citrate or succinate.
38~
Neplanocin C is a weakly basic substance and has the following physicochemical properties:
(1) Elementary analysis:
Found: C; 47.59% H; 4.64% N; 25.10~
Calculated: C; 47.31% H; 4.66% N; 25.09%
(2) Molecular weight (calculated from mass spectrum analysis): 279 [tetraacetyl neplanocin C: 447]
(3) Molecular ~oLmula: CllH13N504 (4) Melting point: (determined by thermal analysis): 226C
(decomp.) (5) Specific rotation: [~]Dl = -43.6 (C = 0.67%, H2O) (6) Ultraviolet absorption spectrum: (16 ~/ml) in H2O :A max = 263 my, ElCm = 538.9 (shown in Fig. 5) at pH 3:~ max = 259 m~, El% = 510.8 at pH 10:~ max = 263 m~, ElCm = 534.4 (7) Infrared absorption spectrum 5 KBr): shown in Fig. 6.
Absorption bands at 3450 - 3100, 2920, 2740, 2680, 1680, 1630, 1600, 1560, 1500, 1460, 1420, 1380, 1360, 1330, 1300, 1240, 1200, 1170, 1100, 1050, 1020, 960, 920, 900, 880, 830, 780, 720, 680 cm 1.
(8) Nuclear Magnetic Resonance spectrum: shown in Fig. 7.
(internal standard DSS, in Deuterium dimethylsulfoxide, lOOMHz).
(9) Solubility:
Soluble: water, dimethylsulfoxide, dimethylformamide, Insoluble: ethyl acetate, chloro~orm, benzene, hexane.
(10) Color reaction:
Positive: decolorization of potassium permanganate, periodate and Tollens.
Negative: ferric chloride, ninhydrin, anisaldehyde and Molisch.
tll) Nature: weakly basic.
(12) Color: white (colorless plate crystal) -3a-(13) Rf value (silica gel spot film, Tokyo Kasei Co., silical gel f): n-butanol : acetic acid : water (6 : 1 : 1) Rf = 0.33 n-butanol : conc. aq. ammonia : water (10 : 0.5 : 2) Rf = 0.19 n-propanol : conc. aq. ammonia : water (10 : 1 : 1);
Rf = 0.30 acetone : water (10 : 1); Rf = 0.43 ethyl acetate : methanol : water (10 : 2 : 1);
Rf = 0.27 chloroform : methanol : acetic acid (10 : 2 : 1);
Rf = 0.20
n-butanol:acetic acid:water (6:1:1); ~f = 0.36.
n-butanol:conc. aq. ammonia: water (10:05:2); Rf = 0.27 n-propanol:conc. aq. ammonia:water (10:1:1); Rf = 0.41 acetone:water tlO:l); Rf = 0.34 ethyl acetate: methanol:water tlO:2:1); Rf = 0.21 chloroform:methanol:acetic acid tlO:2:1); Rf = 0.17 t14) Chemical structure:
~\
OH OH
The biological properties or ncplanocin A are as follows:
(l) Growth inhibitory activity on plant pathogenic fungi:
lOO ~/ml solution of neplanocin A on an agar plate of Helminthosporium oryzae M0306 shows the inhibitory zone of 18.9 mm in diameter.
' ~2-(2) Antitumor activity:
Murine leukemia L-1210 tumor cells (106 cells) were inoculated intraperitoneally in mice and one day after trans-plantation, neplanocin A (each 0.16 mg/kg, 0.32 mg/kg, 0.63 mg/kg, 1.25 mg/kg, 2.5 mg/kg and 5.0 mg/kg, respectively) was administered intraperitoneally once a day for 5 days. 10 mice for the control group and 7 mice in each one group for the treated group were used. The result is shown in Fig. 4. On the 15 days observation, mean survival days for the control group were 7.4 days. Prolongations of life span as compared with the control group were observed for drug administered groups; i.e. 158~ for 0.16 mg/kg, 170~ for the 0.32 mg/kg group, no mortalities were observed for the 2.5 mg/kg and 5 mg/kg groups.
Like L-1210 remarkable life prolongation effects were observed against Ehrlich ascites tumor, Sarcoma-180, P-388 leukemia bearing mice and rat Yoshida sarcoma when neplanocin A was administered.
(3) Acute toxicity:
LD50 : 13.7 mg/kg (i.p., mice) No mortalities were observed wh~n neplanocin A was administered intraperitoneally for 14 days, 5 mg/kg/day.
Directions and dosage~
For therapy of acute leukemia, the drug will be intra-venously administered in a dosage form of 5 - 20 mg/day dissolved in physiological saline, once a day, ~or 7 days.
Neplanocin A can also be effectively used ~or therapy of chronic leukemia, gastroentero carcinoma, pulmonary carcinoma, uterine cancer, mammary cancer and malignant lymphatic leukemia.
Neplanocin A can be used in a rorm of a physiologically acceptable salt of a mineral acid or organic acid such as the hydrochloride, acetate, tartrate, citrate or succinate.
38~
Neplanocin C is a weakly basic substance and has the following physicochemical properties:
(1) Elementary analysis:
Found: C; 47.59% H; 4.64% N; 25.10~
Calculated: C; 47.31% H; 4.66% N; 25.09%
(2) Molecular weight (calculated from mass spectrum analysis): 279 [tetraacetyl neplanocin C: 447]
(3) Molecular ~oLmula: CllH13N504 (4) Melting point: (determined by thermal analysis): 226C
(decomp.) (5) Specific rotation: [~]Dl = -43.6 (C = 0.67%, H2O) (6) Ultraviolet absorption spectrum: (16 ~/ml) in H2O :A max = 263 my, ElCm = 538.9 (shown in Fig. 5) at pH 3:~ max = 259 m~, El% = 510.8 at pH 10:~ max = 263 m~, ElCm = 534.4 (7) Infrared absorption spectrum 5 KBr): shown in Fig. 6.
Absorption bands at 3450 - 3100, 2920, 2740, 2680, 1680, 1630, 1600, 1560, 1500, 1460, 1420, 1380, 1360, 1330, 1300, 1240, 1200, 1170, 1100, 1050, 1020, 960, 920, 900, 880, 830, 780, 720, 680 cm 1.
(8) Nuclear Magnetic Resonance spectrum: shown in Fig. 7.
(internal standard DSS, in Deuterium dimethylsulfoxide, lOOMHz).
(9) Solubility:
Soluble: water, dimethylsulfoxide, dimethylformamide, Insoluble: ethyl acetate, chloro~orm, benzene, hexane.
(10) Color reaction:
Positive: decolorization of potassium permanganate, periodate and Tollens.
Negative: ferric chloride, ninhydrin, anisaldehyde and Molisch.
tll) Nature: weakly basic.
(12) Color: white (colorless plate crystal) -3a-(13) Rf value (silica gel spot film, Tokyo Kasei Co., silical gel f): n-butanol : acetic acid : water (6 : 1 : 1) Rf = 0.33 n-butanol : conc. aq. ammonia : water (10 : 0.5 : 2) Rf = 0.19 n-propanol : conc. aq. ammonia : water (10 : 1 : 1);
Rf = 0.30 acetone : water (10 : 1); Rf = 0.43 ethyl acetate : methanol : water (10 : 2 : 1);
Rf = 0.27 chloroform : methanol : acetic acid (10 : 2 : 1);
Rf = 0.20
(14) Chemical structure:
N~
N N
~o7, OH OH
The biological properties of neplanocin C are as follows:
(1) Growth inhibitory activity on plant pathogenic fungi:
100 y/ml solution, paper disc diameter 7 mm;
Helminthosporium or~zae M 0306: 31.4 mm (inhibition zone) Cladosporium herbarum M 4278: 14.0 mm ( .
Alternaria kikuch;~ana M 4588: 12.G mm ( " "
(2) Acute toxicity:
LDso: 55 mg/kg ti.p., mice) Neplanocin C can be used in a form of a physiologically acceptable salt of a mineral acid or organic acid such as the hydxochloride, acetate, tartrate, citrate or succinate.
~d~
(3) Antitumor activity:
Neplanocin C completely inhibits the growth of murine leukemic cell culture of L-5178Y at a concentration of 0.8y /ml in vitro.
Neplanocin C has a life prolongation effect of 131~ in T/C (treated/control) against murine leukemia L-1210 when administered intraperitoneally at a dose of 10 mg/kg.
Neplanocin D has the following physico-chemical properties.
~1) Elementary analysis: C; 49.70%, H; 4.64~, N; 21.20~
(2) Molecular weight: (calculated from mass spectrum analysis):264 (3) Molecular formula: CllH12N404 (4) Melting point: 236-237C.
(5) Specific rotation: [~]23 = -145 (C- 0.6%, H20) (6) Ultraviolet absorption spectrum:
in H20: shown in Fig. 8,~ max = 251 m~, ElCm = 479 in acidic water: ~ max = 251 m~, ElCm = 453 in alkaline water: ~ max = 256 m~, El%m = 508 (7) Infrared absorption spectrum (KBr): shown in Fig. 9.
Absorption bands at 3420-3120, 2940-2880, 1680, 1580, 1545, 1510, 1440, 1390, 1305, 1210, 1110, 1075, 1040, 1000, 990, 975, 900, 845, 780 cm 1.
(8) N.M.R. spectrum: shown in Fig. 10.
(internal standard DSS, in Deuterium dimethylsulfoxide, lOOMHz) (9) Solubility:
Soluble: water, dimethylsulfoxide, dimethylformamide, acetic acid and pyridine.
Insoluble: ethyl acetate, chloroform, benzene and hexane.
(10) Color reaction:
Positive: decolorization of potassium permanganate and periodate oxidation.
Negative: ninhydrin, Fehling and ferric chloride.(11) Color: white needle crystal tl2) Rf value (silica gel, Tokyo Kasei Co., silica gel f):
n-butanol : acetic acid " water t6 : 1 : 1); Rf = 0.27 n-butanol : conc. aq. ammonia : water (10 : 0.5 : 2);
Rf = 0.07 n-propanol: conc. aq. ammonia : water (10 : 1 : 1) Rf: = 0.18 - acetone: water (10 : 1); Rf = 0.33 (13) Chemical structure:
OH
i HO-H2C 4\~
\~/' OH OH
Acute toxicity o~ neplanocin D is as follows.
No death was observed when 100 mg/kg of neplanocin D
was intraperitoneally administered in mice.
Neplanocin B has the following physico-chemical properties.
(1) Elementary analysis: C; 47~44%, H; 4.63%, N; 25.07%
(2) Molecular weight: (calculat~d from mass spectrum analysis): 279 (3) Molecular formula: CllH13N5O4 (4) Melting point: 269 - 272C (decomposed) (5) Specific rotation: [~]24 = -3.5(C=1.0%, dimethylsulfoxide) (6) Ultraviolet absorption spectrum:
in H2O : shown in Fig~ max = 262 m~, ElCm = 530 4 in acidic water: A max = 259 m~, El~ = 506.7 (one drop of 0.1 N HCl) cm 8~3~
in alkaline water: ~ x 264 m~ El% = 522 2 (7) Infrared absorption spectrum (XBr): shown in Fig. 12.
Absorption bands at 3380, 3280, 3100, 2920, 2880, 2740, 1700, 1610, 1570, 1520, 1480, 1420, 1380, 1350, 1300, 1250, 1220, 1170, 1160, 1100, 1080, 1030, 1020, 980, 970, 870, 840, 790, 730, 710 cm 1, (8) N.M.R. spectrum: shown in Fig. 13.
(internal standard DSS, in Deuterium dimethylsulfoxide, 100 MHz) (9) Solubility:
Soluble: water, dimethylsulfoxide.
Insoluble: ethyl acetate, chloroform, benzene and ethyl ether.
(10) Color reaction:
Slightly positive: decolorization of potassium permanganate.
Negative: ninhydrin, Molisch and anisaldehyde.
(11) Nature: weakly basic.
(12) Color: white (white plate crystal) (13) Rf value (silica gel f, Tokyo Kasei Co.):
n-butanol : acetic acid : water ~6 : 1 : 1); Rf = 0.42 n-butanol : conc. aq. ammonia : water (10 : O.5 : 2) Rf = 0.27 n-propanol : conc. aq. ammonia : water (10 : 1 : 1);
Rf = 0.41 acetone : water (10 : 1); Rf = 0.48 (141 Chemical structure:
OH
HOH2~
OH
,, j , ~28882 Neplanocin F has the following physico-chemical properties.
(1) Elementary analysis: C; 48.74~; H; 4.83%, N; 25.71% (contains water of crystallization) (2) Molecular weight (calculated from mass spectrum analysis): 263 (3) Molecular ormula: CllH13N5O3.21 H2O
(4) Melting point: 223C (decomposed) (5) Specific rotation: [~]D ~ -6.6 (C=0.8%, H2O) (6) Ultraviolet absorption spectrum:
in H20: shown in Fig. 14, ~max = 263 ~ cm = 548-7 in acidic water (one drop of 0.1 N HCL): ~ max = 260 m~,Elcm = 527.1 . in alkaline water (one ~rop of 0.1 N NaO~ ma~ = 263 m~rElc~ = _31.8 (7) Infrared absorption spectrum (KBr) : shown in Fig. 15.
Absorption bands at 3320, 3210, 2920, 1650, 1610, 1580, 1480, 1420, 1380, 1340, 1310, 1270, 1220, 1180, 1110, 1~70, 1020, 980, 900, 840, 800, 730 cm 1.
(a) N.M.R. spectrum: shown in Fig. 16.
(internal standard DSS, in Deuterium dimethylsulfoxide, 100 l~qHZ) (9) Solubility:
Soluble: water, dimethylsulfoxide and acetic acid.
Insoluble: ethyl acetate, chloroform, benzene and ethyl ether.
(10) Color reaction:
Positive decolorization of potassium permanganate.
Negative: ferric chloride, ninhydrin and Fehling.
(11) Nature: weakly basic.
(12) Color: white (white needle crystal).
(13) Rf value (silica gel f, Tokyo Kasei Co.):
n-butanol: acetic acid : water (6 : 1 : 1); Rf = 0.51 n-butanol: conc. aq. ammonia : water (10 : 0.5 : 2);
Rf = 0.43 n-propanol: conc. aq. ammonia : water (10 : 1 : l);Rf = 0.59 acetone : water (10 : 1); Rf = 0.48 (14) Chemical structure:
~H2 N~X ~
OH
rOH~C
H
Biological properties of neplanocin-B and -F are as follows.
(1) Acute toxicity:
No mortalities were observed when 100 mg/kg of neplanocin-B or -F were administered intraperitoneally in mice.
(2) Cell growth inhibitory activity:
Growth of mouse lymphoma cell L 5178Y culture was inhibited at concentrations of neplanocin B of 0.8 y/ml and of neplanocin F, of 20 y/ml, respectively.
(3) Antitumor activity: -Neplanocin B has a life prolongation effect of 14~% in T/C (treated/control) against murine leukemia L-1210 when administered intraperitoneally at a dose of 50 mg/ky.
(4) Another activity:
Neplanocin B has antidepressant activity.
A neplanocin producing microorganism was isolated from a soil sample collected in an onion field of Niigata-ken, Japan, and belongs to the genus Ampullariella. The strain is referred to as Ampullariella sp. A 11079 and has been deposited for permanent collection at the Institute for Microbial Industry and Technology, Japan as FERM-P No. 4494.
Ampullariella sp. A 11079 FERM-P No. 4494 has the following taxonomic characteristics.
[I] Morphological characteristics :
9 _ Observations on inorganic salts-starch agar medium at 30~C for 10 - 15 days culture were as follows.
Strain A 11079 produces a curved and branching substrate mycelium, 0.5 - 0.8 ~ in diameter and a slightly unmatured aerial mycelium.
Sporangiophore grown on substrate mycelium has sporangia and shapes of sporangium are cylindrical or bottle shaped and measuring from 5 - 15 x 10 - 25 ~ . Many sporangiospores are arranged in parallel chains within the sporangium. Sporangio-spores are motile by a tuft of polar flagella in water and are rod shaped measuring from 0.5 - 1.0 x 1.0 - 2.0~ .
[II] Composition of diaminopimelic acid:
Diaminopimelic acid detected by whole cell analysis is meso- and hydroxy-type.
[III] Cultural characteristics on various media:
Observations of cultural characteristics on various media at 30C for 20 days culture are shown in Table 1. No aerial mycelium was observed except slight formation of imm~ture growth aerial mycelia on inorganic salts-starch agar medium and oatmeal agar medium.
Indication of color is based on the indication in "Color ~armony Manual", 4th Ed., 1958, published by Container Corporation of America. [IV] Physiological properties:
Physiological properties are illustrated as follows:
1) Utility of carbon sources:
`' (+: positive, -: negative) Carbon source utilization Carbon source utilization . ~ j .
L-arabinose + salicin +
D-xylose + D-galactose +
D-glucose - + glycerol +
D-fructose + L-sorbose D-mannose + trehalose +
~-mannitol + -melibiose -inositol _ D-ribose -L-rhamnose + I maltose +
sucrose + meleZitose -B-lactose _ D-cellobiose +
raffinose - D-sorbitol ~
cellulose - dulcitol -starch +
..... _ , .
:' -V Q, n a~ n o a~ acl i e ~ e _ _ 8 ~
6 ~ r7 ~ c a ~ ~
. U V O c~: o ~ a I 0 ~1 U .a ~ ~ ~ r o ~ ~U ~ U ~ U
h h ~ ~ c ~ '~ V
~U ~ 3 a3 a) ~ O Q~ O O ~ Oc u~ ~ C t; C
~; O , . ___ .
~, O O o ~
v ~ V I 0 3 ~'8 3 8 ~ 8 o o 3 -I~ ~ E 8 ~
. ~1 ~_ ___ h tJ er~ ~ hu ,~
~ ~ c~a v 6 V ~ v ~ ~ ~ 0-~
:) .~ h ~ 4n~ V ~O V
~a v n. u~
~:~ ~5~ 0 ~ va E c 6 E~ 0 al Iol ~: c ~ X ~o ~ ~ ~o c _l ~ v o ~ o E v~ ~ x o x u .a u ~ . O
~ ~ ~ 3 --) 3~ ~
E~ ~ O ~ ~ ~ Z
- ~
. ~ o "' .a " r~
~ ~ C-- c c E~ O ~C~ GJ 3 al .~
.,1 R l~l ~ ~ ~ V o ~ -- o \ o x ~o ~ o ~ ~ c ~o ~ ~ ~ oc u ~ v u~
-.
E rq ~ A~ ~,) a) _ .~ u~
A C ,~
1.1 V Q~ V V I U 'J X
Q ~ V ~ ~, 3 u) ~ h I '1 h ¦ a r~) â) A V i:: .a V .
O C ~
O O ~ S ~ ~ ~
O C E~ ~ ~ E
.~ U ~ .
,~
E
cn rCa 2) 0~ 3 3 Q' ~ a~ Q
C ~ ~
~ ~0 0 ~ CO ~ _ m ~ ~
.
a a ~0 oO ~ _ .~ .
.. ~ ~
Q~ ~ ~ O U-f~ E o V o ~
c o o ~ ~ ~ ~ '~'1 ~0 ~0 ~ ~ ~ ~ ~ ~0 O o ~ U o E u~
! ~ ~ J~ V
l ~ o ~
.. . ~ E-l Q) N
z 111 ~ ~ 4 ~ O
~- ~
~ cr ~a ~ ~ v ~ ~ ~ u~
v '~I ~ E~ N h ~ ~ ~ -~ U) Ul ~ V ~, ~ ~ .~, .,. m _~ :~: c ~, 0 Q, X C t~ a O E ~ E C~ C C ut ~ 1 c) QJ ~ ~ o v 'a~ x ,,, ~
a ~ ~ ~ 3 E~ ~3-- m~ ~
~ - -- 1 3 - .
.~
2) Growth temperature: 10 - 45C.
3) Action on skim milk: peptonization and coagulation positives.
4) Melanin production: Tyrosine agar medium; negative.
Peptone yeast iron agar medium,positive.
5) Starch hydrolysis: positive.
6) Cellulose hydxolysis: negative.
7) Casein hydrolysis: positive.
8) Gelatin liquefaction: positive.
9~ Tyrosine decomposition: negative.
10) Xanthine decomposition: negative.
11) Hypoxanthine decomposition: negative.
12) H2S formation: negative.
13) Nitrate reduction: negative.
According to the above taxonomical data wherein the strain A 11079 has sporangia bearing sporangiophore grown on branching substrate mycelium, cylindrical or bottle shaped sporangium, sporangiospores arranged in parallel chains within the sporan-gium, rod shaped sporangiospore with tufty polar flagellum and meso diaminopimelic acid, this strain belongs to the genus Ampullariella by consulting the "key to the genera of family"
Actinoplanaceae in Bergey's Manual of Determinative Bacteriology, gth Ed., 1974, pO 707-708. Therefore this strain is referred to as Ampullariella sp. A 11079.
It is an object of the present invention to provide the novel antibiotics neplanocin-A, -C, -D and derivatives thereof, -B and -F.
Another object of the present invention is to provide a neplanocin group of antibiotics which are active against plant pathogenic fungi, tumor cells, or the like.
The present invention will now be described in more detail with reference to the accompanying drawings, in which:-Fig. 1 is the ultraviolet absorption spectrum of neplanocin A;
Fig. 2 is the infrared absorption spectrum of neplanocin A;
Fig. 3 is the N. M. R. spectrum of neplanocin A;
Fig. 4 shows the effect of neplanocin A on L-1210 leukemia bearing mice;
Fig. 5 is the ultraviolet absorption spectrum of neplanocin C; Fig. 6 is the infrared absorption spectrum of neplanocin C;
Fig. 7 is the N. M. R. spectrum of neplanocin C;
Fig. 8 is the ultraviolet absorption spectrum of neplanocin D;
Fig. 9 is the infrared absorption spectrum of neplanocin D;
Fig. 10 is the N. M. R. spectrum of neplanocin D;
Fig. 11 is the ultraviolet absorption spectrum of neplanocin B;
Fig. 12 is the infrared absorption spectrum of neplanocin B;
Fig. 13 is the N. M. R. spectrum of neplanocin B;
Fig. 14 is the ultraviolet absorption spectrum of neplanocin F;
Fig. 15 is the infrared absorption spectrum of neplanocin F;
and Fig. 16 is the N. M. R. spectrum of neplanocin F.
N~
N N
~o7, OH OH
The biological properties of neplanocin C are as follows:
(1) Growth inhibitory activity on plant pathogenic fungi:
100 y/ml solution, paper disc diameter 7 mm;
Helminthosporium or~zae M 0306: 31.4 mm (inhibition zone) Cladosporium herbarum M 4278: 14.0 mm ( .
Alternaria kikuch;~ana M 4588: 12.G mm ( " "
(2) Acute toxicity:
LDso: 55 mg/kg ti.p., mice) Neplanocin C can be used in a form of a physiologically acceptable salt of a mineral acid or organic acid such as the hydxochloride, acetate, tartrate, citrate or succinate.
~d~
(3) Antitumor activity:
Neplanocin C completely inhibits the growth of murine leukemic cell culture of L-5178Y at a concentration of 0.8y /ml in vitro.
Neplanocin C has a life prolongation effect of 131~ in T/C (treated/control) against murine leukemia L-1210 when administered intraperitoneally at a dose of 10 mg/kg.
Neplanocin D has the following physico-chemical properties.
~1) Elementary analysis: C; 49.70%, H; 4.64~, N; 21.20~
(2) Molecular weight: (calculated from mass spectrum analysis):264 (3) Molecular formula: CllH12N404 (4) Melting point: 236-237C.
(5) Specific rotation: [~]23 = -145 (C- 0.6%, H20) (6) Ultraviolet absorption spectrum:
in H20: shown in Fig. 8,~ max = 251 m~, ElCm = 479 in acidic water: ~ max = 251 m~, ElCm = 453 in alkaline water: ~ max = 256 m~, El%m = 508 (7) Infrared absorption spectrum (KBr): shown in Fig. 9.
Absorption bands at 3420-3120, 2940-2880, 1680, 1580, 1545, 1510, 1440, 1390, 1305, 1210, 1110, 1075, 1040, 1000, 990, 975, 900, 845, 780 cm 1.
(8) N.M.R. spectrum: shown in Fig. 10.
(internal standard DSS, in Deuterium dimethylsulfoxide, lOOMHz) (9) Solubility:
Soluble: water, dimethylsulfoxide, dimethylformamide, acetic acid and pyridine.
Insoluble: ethyl acetate, chloroform, benzene and hexane.
(10) Color reaction:
Positive: decolorization of potassium permanganate and periodate oxidation.
Negative: ninhydrin, Fehling and ferric chloride.(11) Color: white needle crystal tl2) Rf value (silica gel, Tokyo Kasei Co., silica gel f):
n-butanol : acetic acid " water t6 : 1 : 1); Rf = 0.27 n-butanol : conc. aq. ammonia : water (10 : 0.5 : 2);
Rf = 0.07 n-propanol: conc. aq. ammonia : water (10 : 1 : 1) Rf: = 0.18 - acetone: water (10 : 1); Rf = 0.33 (13) Chemical structure:
OH
i HO-H2C 4\~
\~/' OH OH
Acute toxicity o~ neplanocin D is as follows.
No death was observed when 100 mg/kg of neplanocin D
was intraperitoneally administered in mice.
Neplanocin B has the following physico-chemical properties.
(1) Elementary analysis: C; 47~44%, H; 4.63%, N; 25.07%
(2) Molecular weight: (calculat~d from mass spectrum analysis): 279 (3) Molecular formula: CllH13N5O4 (4) Melting point: 269 - 272C (decomposed) (5) Specific rotation: [~]24 = -3.5(C=1.0%, dimethylsulfoxide) (6) Ultraviolet absorption spectrum:
in H2O : shown in Fig~ max = 262 m~, ElCm = 530 4 in acidic water: A max = 259 m~, El~ = 506.7 (one drop of 0.1 N HCl) cm 8~3~
in alkaline water: ~ x 264 m~ El% = 522 2 (7) Infrared absorption spectrum (XBr): shown in Fig. 12.
Absorption bands at 3380, 3280, 3100, 2920, 2880, 2740, 1700, 1610, 1570, 1520, 1480, 1420, 1380, 1350, 1300, 1250, 1220, 1170, 1160, 1100, 1080, 1030, 1020, 980, 970, 870, 840, 790, 730, 710 cm 1, (8) N.M.R. spectrum: shown in Fig. 13.
(internal standard DSS, in Deuterium dimethylsulfoxide, 100 MHz) (9) Solubility:
Soluble: water, dimethylsulfoxide.
Insoluble: ethyl acetate, chloroform, benzene and ethyl ether.
(10) Color reaction:
Slightly positive: decolorization of potassium permanganate.
Negative: ninhydrin, Molisch and anisaldehyde.
(11) Nature: weakly basic.
(12) Color: white (white plate crystal) (13) Rf value (silica gel f, Tokyo Kasei Co.):
n-butanol : acetic acid : water ~6 : 1 : 1); Rf = 0.42 n-butanol : conc. aq. ammonia : water (10 : O.5 : 2) Rf = 0.27 n-propanol : conc. aq. ammonia : water (10 : 1 : 1);
Rf = 0.41 acetone : water (10 : 1); Rf = 0.48 (141 Chemical structure:
OH
HOH2~
OH
,, j , ~28882 Neplanocin F has the following physico-chemical properties.
(1) Elementary analysis: C; 48.74~; H; 4.83%, N; 25.71% (contains water of crystallization) (2) Molecular weight (calculated from mass spectrum analysis): 263 (3) Molecular ormula: CllH13N5O3.21 H2O
(4) Melting point: 223C (decomposed) (5) Specific rotation: [~]D ~ -6.6 (C=0.8%, H2O) (6) Ultraviolet absorption spectrum:
in H20: shown in Fig. 14, ~max = 263 ~ cm = 548-7 in acidic water (one drop of 0.1 N HCL): ~ max = 260 m~,Elcm = 527.1 . in alkaline water (one ~rop of 0.1 N NaO~ ma~ = 263 m~rElc~ = _31.8 (7) Infrared absorption spectrum (KBr) : shown in Fig. 15.
Absorption bands at 3320, 3210, 2920, 1650, 1610, 1580, 1480, 1420, 1380, 1340, 1310, 1270, 1220, 1180, 1110, 1~70, 1020, 980, 900, 840, 800, 730 cm 1.
(a) N.M.R. spectrum: shown in Fig. 16.
(internal standard DSS, in Deuterium dimethylsulfoxide, 100 l~qHZ) (9) Solubility:
Soluble: water, dimethylsulfoxide and acetic acid.
Insoluble: ethyl acetate, chloroform, benzene and ethyl ether.
(10) Color reaction:
Positive decolorization of potassium permanganate.
Negative: ferric chloride, ninhydrin and Fehling.
(11) Nature: weakly basic.
(12) Color: white (white needle crystal).
(13) Rf value (silica gel f, Tokyo Kasei Co.):
n-butanol: acetic acid : water (6 : 1 : 1); Rf = 0.51 n-butanol: conc. aq. ammonia : water (10 : 0.5 : 2);
Rf = 0.43 n-propanol: conc. aq. ammonia : water (10 : 1 : l);Rf = 0.59 acetone : water (10 : 1); Rf = 0.48 (14) Chemical structure:
~H2 N~X ~
OH
rOH~C
H
Biological properties of neplanocin-B and -F are as follows.
(1) Acute toxicity:
No mortalities were observed when 100 mg/kg of neplanocin-B or -F were administered intraperitoneally in mice.
(2) Cell growth inhibitory activity:
Growth of mouse lymphoma cell L 5178Y culture was inhibited at concentrations of neplanocin B of 0.8 y/ml and of neplanocin F, of 20 y/ml, respectively.
(3) Antitumor activity: -Neplanocin B has a life prolongation effect of 14~% in T/C (treated/control) against murine leukemia L-1210 when administered intraperitoneally at a dose of 50 mg/ky.
(4) Another activity:
Neplanocin B has antidepressant activity.
A neplanocin producing microorganism was isolated from a soil sample collected in an onion field of Niigata-ken, Japan, and belongs to the genus Ampullariella. The strain is referred to as Ampullariella sp. A 11079 and has been deposited for permanent collection at the Institute for Microbial Industry and Technology, Japan as FERM-P No. 4494.
Ampullariella sp. A 11079 FERM-P No. 4494 has the following taxonomic characteristics.
[I] Morphological characteristics :
9 _ Observations on inorganic salts-starch agar medium at 30~C for 10 - 15 days culture were as follows.
Strain A 11079 produces a curved and branching substrate mycelium, 0.5 - 0.8 ~ in diameter and a slightly unmatured aerial mycelium.
Sporangiophore grown on substrate mycelium has sporangia and shapes of sporangium are cylindrical or bottle shaped and measuring from 5 - 15 x 10 - 25 ~ . Many sporangiospores are arranged in parallel chains within the sporangium. Sporangio-spores are motile by a tuft of polar flagella in water and are rod shaped measuring from 0.5 - 1.0 x 1.0 - 2.0~ .
[II] Composition of diaminopimelic acid:
Diaminopimelic acid detected by whole cell analysis is meso- and hydroxy-type.
[III] Cultural characteristics on various media:
Observations of cultural characteristics on various media at 30C for 20 days culture are shown in Table 1. No aerial mycelium was observed except slight formation of imm~ture growth aerial mycelia on inorganic salts-starch agar medium and oatmeal agar medium.
Indication of color is based on the indication in "Color ~armony Manual", 4th Ed., 1958, published by Container Corporation of America. [IV] Physiological properties:
Physiological properties are illustrated as follows:
1) Utility of carbon sources:
`' (+: positive, -: negative) Carbon source utilization Carbon source utilization . ~ j .
L-arabinose + salicin +
D-xylose + D-galactose +
D-glucose - + glycerol +
D-fructose + L-sorbose D-mannose + trehalose +
~-mannitol + -melibiose -inositol _ D-ribose -L-rhamnose + I maltose +
sucrose + meleZitose -B-lactose _ D-cellobiose +
raffinose - D-sorbitol ~
cellulose - dulcitol -starch +
..... _ , .
:' -V Q, n a~ n o a~ acl i e ~ e _ _ 8 ~
6 ~ r7 ~ c a ~ ~
. U V O c~: o ~ a I 0 ~1 U .a ~ ~ ~ r o ~ ~U ~ U ~ U
h h ~ ~ c ~ '~ V
~U ~ 3 a3 a) ~ O Q~ O O ~ Oc u~ ~ C t; C
~; O , . ___ .
~, O O o ~
v ~ V I 0 3 ~'8 3 8 ~ 8 o o 3 -I~ ~ E 8 ~
. ~1 ~_ ___ h tJ er~ ~ hu ,~
~ ~ c~a v 6 V ~ v ~ ~ ~ 0-~
:) .~ h ~ 4n~ V ~O V
~a v n. u~
~:~ ~5~ 0 ~ va E c 6 E~ 0 al Iol ~: c ~ X ~o ~ ~ ~o c _l ~ v o ~ o E v~ ~ x o x u .a u ~ . O
~ ~ ~ 3 --) 3~ ~
E~ ~ O ~ ~ ~ Z
- ~
. ~ o "' .a " r~
~ ~ C-- c c E~ O ~C~ GJ 3 al .~
.,1 R l~l ~ ~ ~ V o ~ -- o \ o x ~o ~ o ~ ~ c ~o ~ ~ ~ oc u ~ v u~
-.
E rq ~ A~ ~,) a) _ .~ u~
A C ,~
1.1 V Q~ V V I U 'J X
Q ~ V ~ ~, 3 u) ~ h I '1 h ¦ a r~) â) A V i:: .a V .
O C ~
O O ~ S ~ ~ ~
O C E~ ~ ~ E
.~ U ~ .
,~
E
cn rCa 2) 0~ 3 3 Q' ~ a~ Q
C ~ ~
~ ~0 0 ~ CO ~ _ m ~ ~
.
a a ~0 oO ~ _ .~ .
.. ~ ~
Q~ ~ ~ O U-f~ E o V o ~
c o o ~ ~ ~ ~ '~'1 ~0 ~0 ~ ~ ~ ~ ~ ~0 O o ~ U o E u~
! ~ ~ J~ V
l ~ o ~
.. . ~ E-l Q) N
z 111 ~ ~ 4 ~ O
~- ~
~ cr ~a ~ ~ v ~ ~ ~ u~
v '~I ~ E~ N h ~ ~ ~ -~ U) Ul ~ V ~, ~ ~ .~, .,. m _~ :~: c ~, 0 Q, X C t~ a O E ~ E C~ C C ut ~ 1 c) QJ ~ ~ o v 'a~ x ,,, ~
a ~ ~ ~ 3 E~ ~3-- m~ ~
~ - -- 1 3 - .
.~
2) Growth temperature: 10 - 45C.
3) Action on skim milk: peptonization and coagulation positives.
4) Melanin production: Tyrosine agar medium; negative.
Peptone yeast iron agar medium,positive.
5) Starch hydrolysis: positive.
6) Cellulose hydxolysis: negative.
7) Casein hydrolysis: positive.
8) Gelatin liquefaction: positive.
9~ Tyrosine decomposition: negative.
10) Xanthine decomposition: negative.
11) Hypoxanthine decomposition: negative.
12) H2S formation: negative.
13) Nitrate reduction: negative.
According to the above taxonomical data wherein the strain A 11079 has sporangia bearing sporangiophore grown on branching substrate mycelium, cylindrical or bottle shaped sporangium, sporangiospores arranged in parallel chains within the sporan-gium, rod shaped sporangiospore with tufty polar flagellum and meso diaminopimelic acid, this strain belongs to the genus Ampullariella by consulting the "key to the genera of family"
Actinoplanaceae in Bergey's Manual of Determinative Bacteriology, gth Ed., 1974, pO 707-708. Therefore this strain is referred to as Ampullariella sp. A 11079.
It is an object of the present invention to provide the novel antibiotics neplanocin-A, -C, -D and derivatives thereof, -B and -F.
Another object of the present invention is to provide a neplanocin group of antibiotics which are active against plant pathogenic fungi, tumor cells, or the like.
The present invention will now be described in more detail with reference to the accompanying drawings, in which:-Fig. 1 is the ultraviolet absorption spectrum of neplanocin A;
Fig. 2 is the infrared absorption spectrum of neplanocin A;
Fig. 3 is the N. M. R. spectrum of neplanocin A;
Fig. 4 shows the effect of neplanocin A on L-1210 leukemia bearing mice;
Fig. 5 is the ultraviolet absorption spectrum of neplanocin C; Fig. 6 is the infrared absorption spectrum of neplanocin C;
Fig. 7 is the N. M. R. spectrum of neplanocin C;
Fig. 8 is the ultraviolet absorption spectrum of neplanocin D;
Fig. 9 is the infrared absorption spectrum of neplanocin D;
Fig. 10 is the N. M. R. spectrum of neplanocin D;
Fig. 11 is the ultraviolet absorption spectrum of neplanocin B;
Fig. 12 is the infrared absorption spectrum of neplanocin B;
Fig. 13 is the N. M. R. spectrum of neplanocin B;
Fig. 14 is the ultraviolet absorption spectrum of neplanocin F;
Fig. 15 is the infrared absorption spectrum of neplanocin F;
and Fig. 16 is the N. M. R. spectrum of neplanocin F.
-15--~L~ .?~32 According to the present invention, the neplanocins are produced, for example, by inoculating the strain Ampullariella sp. A 11079 FERM-P No. 4494 in a suitable nutrient medium.
The cultivation of the microorganism can be carried out in a number of different ways, such as in a synthetic medium or natural medium, liquid or solid culture. In an industrial production, a liquid medium is preferable. For the composition of the medium, there may be used assimilable carbon sources, nitrogen sources, an inorganic salt and others for antibiotic `~ 10 neplanocins producing microorganisms. Examples of carbon sources are glucose, sucrose, glycerin, soluble starch, molas-: ses and the like. Assimilable nitrogen sources such as peptone, corn steep liquor, soy bean powder, meat extract, rice bran, casein hydrolysate, nitrates, ammonium salts and the like are used. An inorganic salt such as sodium chloride, phosphate ` (calcium, magnesium, ferrous or manganese) can also be used.
An anti~foamer such as silicone oil or soybean oil can be added.
; For liquid culture submerged aeration culture is preferably applied. In this case the cultivating temperature will be 20 selected for optimum temperature for microorganisms, and pre-ferably is 25 - 30C. The cultivation time is dependent on the conditions and generally may range from 2 - 4 days. The pH
of the medium during cultivation is preferably controlled at neutral or slightly acidic conditions.
The thus cultured medium contains antibiotic neplanocins.
Isolation of the antibiotic neplanocins can be performed by conventional isolation or separation processes for micro-organism metabolites. Since neplanocins are weakly basic substances they can be isolated by adsorbing suitable adsorbents 30 followed by eluting with a suitable solvent. Examples of adsorbents are active carbon, cation exchange resins, active .. ~, ~, alumina or silica gel. The eluting solvent can be selected by the adsorbent used, for example, these are water miscible organic solvents such as aqueous methanol, aqueous acetone or aqueous dioxane or acidic, alkaline or salt solutions.
Further, the neplanocins can be isolated and purified on the basis of the weakly basic nature of the substances. For example the antibiotics are adsorbed on a cation exchange resin such as "Amberlite IRC-50" (trade mark of Rohm and Haas Co., U.S.A.) or "Dowex 50" (trade mark of Dow Chemical Company, U.S.A.), and are eluted by a suitable acidic, alkaline or salt solution.
A combination of an adsorbent and ion exchange resin can preferably be applied for isolation, elution and purification of the antibiotics. For example, the culture filtrate is charged on the cation exchange resin "Amberlite IR-120" (trade mark) to adsorb the antibiotics, eluted with an alkaline solu-tion such as 3.7 N aqueous ammonia to obtain the active fraction, and after the pH thereof is adjusted to a neutral or weakly alkaline pH, the antibiotics are adsorbed on active carbon fol-lowed by eluting with 70% methanol. The eluent is adsorbed on the anion exchange resin "Amberlite IRA-410" (trade mark) and again eluted with water to collect the active fractions.
The combined fractions are concentrated to obtain the crude material and finally purified by silica gel adsorption chromato-graphy. Further purification is performed by recrystallization.
Purity as a single substance can be checked by s ~wing a single melting point or a single spot on paper chromatography, thin layer chromatography and paper electrophoresis at 263 m~ ultra-violet light.
Neplanocin A can also be synthesized from- neplanocin D
through neplanocin D derivatives as follows.
The three hydroxy groups in the cyclopentene ring of -~2~813~:
neplanocin D are protected by reacting with benzoylchloride.
The hydroxy group in the adenine structure of neplanocin D
is changed to a mercapto group by reacting, for example, with phosphorus pentasulfide, and the protective group for the hydroxy group is thereafter removed to obtain neplanocin D derivative (III). Methylation thereof by methyl iodide gives neplanocin D derivative (IV). The neplanocin D derivative (IV) is treated with methanolic ammonia to produce neplanocin A.
Furthermore, the three hydroxy groups in the cyclopentene ring of neplanocin D are acetylated for protection by reacting with acetic anhydride, and thereafter the said compound is changed to neplanocin D derivative [3] by reacting with thionyl chloride. The neplanocin D derivative [3] can be changed to neplanocin A through neplanocin D derivative [4] by treating with methanolic ammonia.
These reactions can be illustrated as follows:
~1) o~ 0 ~\il ~enzoylchlo~ide ~
~a~zC~ ~2C~
OE~ C1Bz' Neplano~in D ~I) 8~32 6~ 8~1 P2 s 8 ~ 9 ~ ~X N
B~ ONzC ~ N~2C
:: . 0~ 0~
;~ ~II) Neplanocin D derivatiYe ~III) .
' ' ~C~ ~
~ N
CH I ~ Neplanocin A
3 . ~ethanollc ammonia ~OH2~ ~
.O~I H
Neplanocin D derivative ~IV) ,~
~ ~8~3~32 t2) OE C~
Neplanocin D ti anhydride 2 A C 1~ 1~ z C '~ OI~j OAc OAc OAc OA~
[2] Neplanocin D derivative [3]
--.~ Neplanocin A
The physico-chemical properties of the neplanocin D d~riv- :
atives are as follows.
Neplanocin D derivative .
~III) (IV) . ... _ . , _ , I Molecular weight (calculated from mass 280 294 spectrum analysis) . _ . . . _ .
Molecular formula 11 12 4 3 12 14 4 3 . _~ . . ._ Melting point 260 - 262DC.(decomp.) 177 - 178~C.
__ .. ~ ...
.Specific rotation I - 13)D = -141.7 i (c=071H2o) . _ . _ . . .
j : 222 ma Ultraviolet absorption ' 226 m~ ~ 288 m~
spectrum (in H20) 324 m~ 1 295 m~ i The following examples illustrate the present invention but are not construed as limiting.
,,)j ,, j Example 1.
100 ml. of aqueous medium (pH 6.5) containing glucose 2%, starch 2%, yeast extract 1%, casein hydrolysate 1% and calcium carbonate 0.2% were introduced in a 500 ml flask and sterilized at 120C for 15 minutes. Into two flasks of this medium, one loopful of slant of Ampullariella sp. A 11079 FERM-P No. 4494 was inoculated and shake cultured at 30C. After 4 days, the cultured medium was transferred to 20 1. of the same sterilized medium hereinabove in a 30 1. jar fermenter and cultured at 30C
with agitation at 300 r.p.m. and aeration of 20 l./min. for 48 hours.
The thus cultured medium was transferred into 200 1. of aqueous medium (pH 6.5) containing glucose 4~, soy bean powder 1%, meat extract 0.4%, peptone 0.4%, yeast extract 0.1%, NaCl 0.25% and calcium carbonate 0.1% and cultured at 30C, with agitation at 180 r.p.m., and aeration 130 l./min., for 40 hours.
The obtained cultured broth (about 200 1.) was filtered and the myce~a was washed with water. The filtrate and washed water were combined to obtain about 140 1. of clear filtrate (potency 57 mcg/ml as neplanocin A).
Example 2.
The filtrate obtained in Example 1 was passed through a column of 20 1. of cation exchange resin 'IAmberlite IR-120' (H~ type) (trade mark) to adsorb the material and washed with 100 1. of water. Elution was carried out with 3.7 N
aqueous ammonia and the primary eluate (30 1.) was discarded.
90 1. of the following eluate were collected, adjusted to pH
8 by adding 6N HCl, then passed through 4 1. of active carbon in a column, washed with water and thereafter eluted with 90 1.
of 70~ methanol solution. The thus obtained eluate was concen-trated under reduced pressure to give 1.5 1 of a concentrate.
''`- I
The concentrate was charged on a column of 10 1. of "Amberlite IRA-410" (OH type) (trade mark), and eluted with water and adjusted to pH 7 by adding 4N NCl. The eluate was concentrated in vacuo, the material was precipitated under cooling conditions filtered to obtain 41.8 g of crude neplanocin A (purity about 12%).
The crude neplanocin A (41.8 g) was dissolved in a small amount of water and charged on 2 1. of silica gel in a column (8.3 X 40 cm) packed with a solvent mixture of n-butanol: 28%
aqueous ammonia : water (10 : 0.2 : 1), and thereaft~r eluted with the same solvent mixture.
Each 150 ml of the eluate was fractionated and active fractions were found in fractions Nos. 24 - 52. The said active fractions were collected, concentrated in vacuo, an~ was allowed to stand under cooling.
The precipitate was collected by filtration to obtain 2.6 g of crude crystalline neplanocin A. (yield 32~).
The crude neplanocin A was dissolved in about 60 ml of hot water and allowed to stand at room temperature. The pre-cipitated white needle crystals was collected by filtration to obtain 2.01 g of crystals of neplanocin A. (yield 25.2~).
Example 3.
The filtrate (140 1., potency 300 mcg/ml as neplanocin C) obtained by the same procedure in Example 1 was passed through a column of 20 1. of cation exchange resin "Amberlite I -120"
(H type) (trade mark) to adsorb the material and washed with about 1001- of water. Elution was carried out with 3.7N
aqueous ammonia and the primary eluate (20 1.) was discarded.
100 1. of th~ following eluate were collected, adjusted to p~ 8 by adding conc. HCl, then passed through 4 1. of active carbon in a column, washed with water and thereafter eluted with 70 1.
~X~8~
of 70% methanol solution. The thus obta~ined eluate was con-centrated under reduced pressure to give 1.5 1. of a concen-trate. This concentrate was freeze dried to obtain 83.6 g of a crude powder. (purity 29.5%).
83 g of the crude powder was dissolved in water saturated n-butanol solution and charged on 2 1- of silica gel in a column (8.3 X 40 cm) packed with n-butanol, and thereafter eluted with a solvent mixture of n-butanol : conc. aq. ammonia:
water (10 : 0.2 : 1). Each 150 ml of the eluate was fraction-ated and active fractions w ge found in fraction Nos. 53 - 108.
The said active fractions were collected, concentrated and was allowed to stand at low temperature.
The precipitate was collected by filtration to obtain 18.2 g of neplanocin C as white crystals. (purity 96%, yield 41.6%).
This was dissolved in hot water and allowed to stand at room temperature to precipitate the neplanocin C as colorless plate crystals which was filtered to obtain pure neplanocin C.
Example 4.
To the filtrate obtained by the same procedure in Example 1 was added 3.6 kg of active carbon powder. After stirring for 40 minutes, active carbon was collected by filtration and washed thoroughly with water. Elution was carried out with 70%
methanol solution. 40 1. of eluate were dried up in vacuo to obtain 80 g of crude powder containing neplanocin D.
The 80 g of crude powder was charg~d on 6 1. of silica gel in a column packed with n-propanol : conc. aq. ammonia : water (10 : 1 : 1), and thereafter eluted with the sa~e solvent mixture. Each 600 g of the eluate was fractionated and neplan-ocin D was found in fractions Nos. 20 - 37. The said active fractions were collected, concentrated in vacuo up to 30 ml, and was allowed to stand under cooling. The precipitate was collected b~ filtration to obtain 460 mg of crude crystalline -~3-~28~382 neplanocin D.
The 460 mg of crude crystals were dissolved in 3 ml of hot water and put on a column of "Sephadex G-15" (1850 ml, packed with water), then eluted with water. Each lO0 ml. of the eluate was fractionated, the eluate fractions Nos. 15 - 16 t200 ml) were collected and concentrated under reduced pressure up to 20 ml which was allowed to stand at room temperature.
314 mg of the precipitated neplanocin D as white needle crystals were obtained.
Example 5.
(1) 376 mg of neplanocin D dissolved in 10 ml of anhydrous pyridine were stirred thoroughly at 55C. 0.9 ml of benzoyl chloride was added dropwisely thereto and the reaction mixture was stixred for 2 hours at 60 - 65C.
Thereafter the solution was kept at 40 - 45C for 2 hours and further allowed to stand overnight at room temperature.
10 ml of water was added with stirring therein and after 25 minutes the solution was evaporated to dryness under reduced pressure in an e~aporator. The dried material was dissolved in 30 ml of chloroform, washed three times with 10 ml of 1 N
HCl and twice with 1 N sodium bicarbonate solution, and there-after washed twice with 20 ml of water. The chloroform layer was dehydrated by anhydrous magnesium sulfate and concentrated to obtain 747 mg of a pale creamy compound (I). (yield 91%).
Physico-chemical properties of the compound tI) are as follows.
[ ~] = 199 (c=0.6, methanol) D
Molecular formula: C32H24N4O7 * Trademark "Sephadex" is a trademark for a dry insoluble powder composed of macroscopic beads of cross-linked dextran containing functional ionic groups.
38;~
Molecular weight: 576 (mass spectrum) Melting point: 116 - 117C.
Ultraviolet absorption spectrum: ~max = 232 m~. E = 704 1 cm RF value (chloroform : methanol = 10 : 1); RF = 0.53 (2) 735 mg of the compound (I) obtained hereinabove (1) and 1.0736 g of phosphorus pentasulfide diss ~ved in 19 ml of pyridine were stirred thoroughly and 0.19 ml of water was drop-wisely added thereto. After refluxing for 4 hours with stirring, the reaction mixture was cooled and concentrated in vacuo to obtain a brown oily material. This oily material was poured in boiling water with stirring and further stirred for 30 minutes to obtain a yellowish substance. This w æ filtered to collect the precipitate, washed with a mixture of ethanol : ether (1 : 1), further washed with ether and dried to obtain 561 mg of the compound (II). (yield 74.3%).
The physico-chemical properties of the compound (II) are as follows.
[a ]D = -286.9 (c-0.6, CHC13~
Molecular formula: C32~24N4O6S
Molecular weight: 592 Melting point: 235 - 239C (foaming decomp.) W spectrum (in methanol): 232 m ~(E = 858.5) 1 cm 323 m ~(El~ = 327.5) 1 cm Rf value (chloroform : methanol = 10 : 1) ; Rf = 0.82 (3) 525 mg of the compound (II) obtained hereina~ove (2) were dissolved in 31 ml of anhydrous methanol. 0.62 ml of a freshly-prepared 2 N solution of sodium methylate in methanol was added therein and refluxed with stirring for 2 hours to remove the benzoyl group.
After the methanol was distilled off about 50 ml of water were .
.~
, 11281~82 added and adjusted to pH 7.5 by adding acetic acid. The solution was extracted twice with 20 ml of ethyl acetate and the aqueous layer was concentrated to obtain neplanocin D
derivative (III) as needle crystals. Recrystallization was made from hot water to obtain 110 mg of neplanocin D derivative (III). (yield 44.3%).
The physico-chemical properties of neplanocin D deri~ative (III) are as follows.
Specific rotation: impossible to measure due to varying at measurement.
Molecular formula: CllH12N4O3S
Molecular weight: 280 (mass spectrum) Melting point: 260 - 262 C (decomp.) W spectrum in H2O: 226 m~ , (El% = 388.6) 324 m~ (El~ = 968 o) 1 cm Rf value (propanol : conc. aq. ammonia : water = 10 : 1 : l); Rf = 0.21 Example 6.
To 37 mg of neplanocin D derivative (III) obtained in Example 5 dissolved in 0.3 ml of 0.4 N NaOH were added 0.009 ml of methyl iodide and the reaction mixture was sh~ken at room te~ature for 10 m~utes. Thereafter 0.05 ml of 0.4 N NaOH was ~ thereto and there was f~er added 0.009 nl of methyl iodide, then the reaction m~h~e was again s ~ en and a~owed to stand at ~ m temperature. Tb this solution was ~d 10 ml of water, and the solution was concentrated in vacu~
up to 2 ml, and ch~ed on a oolumn of "Seph~ G-15" (1.4 x 49 cm). Elution was carried out with water and each 3 g of eluate was fractianated. Fractions Nbs. 17-19 were oDllected, ~ oentrated and cooled to bbtain 35 mg of crystalline nep ~ ocin D derivative (r".(yield 90%).
Ihe physi ~ chemical properties of neplanocin D derivative (IV) are as follows.
* ~ demark 88~3~
[ ~ ] = -141.7 (c=0.7, H2O) Molecular formula: C12H14N4O3S
Molecular weight: 294 (mass spectrum) Melting point: 177 - 178C
UV spectrum in water : 222 m~ (E % = 415.8) 1 cm 288 m~ ( ~% = 676.3) 1 cm 295 my (El% = 670.7) 1 cm Rf value (propanol : conc. aq. ammonia : water = 10 :1 :1) ; Rf= 0.42 Example 7.
Synthesis of neplanocin A from neplanocin D derivative (IV) 50 mg of neplanocin D derivative (IV) obtained by the same process as described in Example 6 dissolved in 1.5 ml of metha-nolic ammonia (0C saturation) wereplaoed in a sealed tube and heated at 146 - 148C for 7 hours. After removing the solvent under reduced pressure, the residue was dissolved in 3 ml of hot water, then cooled to obtain 33 mg of neplanocin A as needle crystals. (yield 74%).
Example 8.
(1) To 1.14 g of neplanocin D dissolved in 12 ml of anhydrous pyridine was added 1 ml of acetic anhydride and the reaction mixture was allowed to stand at room temperature for over-night.
30 ml of n-hexane was added thereto and cooled in ice-water to obtain 1 g of the compound [2] as crystals.
(2) 1 g of the compound [2] was added to 25 ml of chloroform solution containing 0.8 ml of thionyl chloride and 0.4 ml of dimethylformamide and refluxed at room temperature for 3 hours.
Chloroform was distilled off under reduced pressure. The residue was poured into 100 ml of ice-water under stirring.
Extraction was made with 100 ml of chloroform, and the extract was washed with NaHCO3 and water, and dried with sodium sulfate.
Neplanocin D derivative [3] was obtained after drying in vacuo.
Example 9.
Preparation of neplanocin A from neplanocin D derivative [3];
To neplanocin D derivative [3] obtained in Example 8 placedin a sealed tube made of steel was added 35 ml of saturated methanolic ammonia and reacted at 100C for 5 hours. After cooling, the reaction mixture was dried up in vacuo to obtain a crystalline powder which was purified by silica gel column chromatography to obtain 286 mg of neplanocin A.
The physico-chemical properties are identical with those of neplanocin A obtained in Example 7.
Example 10.
The filtrate (140 1., potency about 50 mcg/ml as neplan-ocin B) obtained by the same procedure as in Example 1 was passed through a column of 20 1. of cation exchange resin "Amberlite IR-120" (H+ type) to adsorb the material and washed with about 100 1. of water. Elution was carried out with 3.7 N
aqueous ammonia and the primary eluate (30 1.) was discarded.
~0 1. of the following eluate were collected, adjusted to pH 8 by adding 6 N HCl, then charged on a column of 4 1. of active carbon, washed with water, and thereafter eluted with 90 1. of 70% aqueous methanol. The thus obtained eluate was concentrated to gi~e 500 ml of a concentrate which was allowed to stand at low temperature to precipitate the material. The precipitate was collected and dried to obtain crude neplanocin B (5.1 g, purity about 60~ r yield 44%).
Example 11.
Neplanocin B obtained in Example 10 (5.1 g) was charged on a column of silica~gel (8.3 X 40 cm, 2 1.) packed with a mixture of acetone : water (10 : 0.5) and eluted with the same ~, solvent mixture. Each 80 g of the eluate was fractionated and the fractions No. 16 - 24 were combined. The said active fractions were allowed to stand at low temperature to precip-itate neplanocin B as white plate crystals. 2.57 g of crystal-line neplanocin B were obtained. (yield 32.8%).Example 12 Cultivation was carried out as illustrated in Example 1 to obtain about 200 l.of a cultured mass which was filtered and the cells were washed with water. The filtrate and washing water were combined to yield 140 1. (potency; about 1.5 mcg/
ml).
The solution was passed through a column of 20 l. of cation exchange resin "Amberlite IR-120" (H type~ to adsorb the material and washed with about 100 1. of water. Elution was carried out with 3.7 N aqueous ammonia and the primary eluate (30 1.) was discarded. 90 1. of the following eluate were collected, adjusted to pH 8 by adding 6 N HCl, then charged on a column of 4 l. of active carbon, washed with water and thereafter eluted with 90 l. of 70% aqueous methanol. The thus obtained eluate was concentrated to give 500 ml of a concentrate which was freeze dried to obtain 31.2 g of crude neplanocin F. (purity about 0.5%).
Example 13.
The crude powder of neplanocin F obtained in Example 12 (31.2 g) was charged on a column of silica gel (800 ml) packed with a mixture of n-butanol : 28% aqueous ammonia : water (10 : 0.2 : 1) and eluted with the same solvent-mixture. Each 150 ml of the eluate was fractionated and the fractions No. 5 - 10 were combined, and then concentrated in vacuo.
The concentrate was charged on a silica gel column (240 ml) previously packed ~ith a mixed solution of chloroform: methanol:
38~
acetic acid (10 : 2 : 0.2) and eluted with the same solvent mixture, fractionated each 20 g of the fraction. Fractions No. 67 - 120 were combined, concentrated and dried to obtain crude crystals of neplanocin F. (108 mg, yield 51%).
Example 14.
The crude crystals of neplanocin F (108 mg) obtained in Example 13 were charged on a column of "Sephadex G-15" (186 ml) packed with water and eluted with water. Each 10 g of the fraction was fractionated and the fractions No. 22 - 27 were collected, combined and concentrated to 5 ml which was allowed to stand in a cold room to precipitate neplanocin F as white needle crystals. The precipitate was filtered and dried to obtain crystalline neplanocin F (87 mg). (yield 41~).
The cultivation of the microorganism can be carried out in a number of different ways, such as in a synthetic medium or natural medium, liquid or solid culture. In an industrial production, a liquid medium is preferable. For the composition of the medium, there may be used assimilable carbon sources, nitrogen sources, an inorganic salt and others for antibiotic `~ 10 neplanocins producing microorganisms. Examples of carbon sources are glucose, sucrose, glycerin, soluble starch, molas-: ses and the like. Assimilable nitrogen sources such as peptone, corn steep liquor, soy bean powder, meat extract, rice bran, casein hydrolysate, nitrates, ammonium salts and the like are used. An inorganic salt such as sodium chloride, phosphate ` (calcium, magnesium, ferrous or manganese) can also be used.
An anti~foamer such as silicone oil or soybean oil can be added.
; For liquid culture submerged aeration culture is preferably applied. In this case the cultivating temperature will be 20 selected for optimum temperature for microorganisms, and pre-ferably is 25 - 30C. The cultivation time is dependent on the conditions and generally may range from 2 - 4 days. The pH
of the medium during cultivation is preferably controlled at neutral or slightly acidic conditions.
The thus cultured medium contains antibiotic neplanocins.
Isolation of the antibiotic neplanocins can be performed by conventional isolation or separation processes for micro-organism metabolites. Since neplanocins are weakly basic substances they can be isolated by adsorbing suitable adsorbents 30 followed by eluting with a suitable solvent. Examples of adsorbents are active carbon, cation exchange resins, active .. ~, ~, alumina or silica gel. The eluting solvent can be selected by the adsorbent used, for example, these are water miscible organic solvents such as aqueous methanol, aqueous acetone or aqueous dioxane or acidic, alkaline or salt solutions.
Further, the neplanocins can be isolated and purified on the basis of the weakly basic nature of the substances. For example the antibiotics are adsorbed on a cation exchange resin such as "Amberlite IRC-50" (trade mark of Rohm and Haas Co., U.S.A.) or "Dowex 50" (trade mark of Dow Chemical Company, U.S.A.), and are eluted by a suitable acidic, alkaline or salt solution.
A combination of an adsorbent and ion exchange resin can preferably be applied for isolation, elution and purification of the antibiotics. For example, the culture filtrate is charged on the cation exchange resin "Amberlite IR-120" (trade mark) to adsorb the antibiotics, eluted with an alkaline solu-tion such as 3.7 N aqueous ammonia to obtain the active fraction, and after the pH thereof is adjusted to a neutral or weakly alkaline pH, the antibiotics are adsorbed on active carbon fol-lowed by eluting with 70% methanol. The eluent is adsorbed on the anion exchange resin "Amberlite IRA-410" (trade mark) and again eluted with water to collect the active fractions.
The combined fractions are concentrated to obtain the crude material and finally purified by silica gel adsorption chromato-graphy. Further purification is performed by recrystallization.
Purity as a single substance can be checked by s ~wing a single melting point or a single spot on paper chromatography, thin layer chromatography and paper electrophoresis at 263 m~ ultra-violet light.
Neplanocin A can also be synthesized from- neplanocin D
through neplanocin D derivatives as follows.
The three hydroxy groups in the cyclopentene ring of -~2~813~:
neplanocin D are protected by reacting with benzoylchloride.
The hydroxy group in the adenine structure of neplanocin D
is changed to a mercapto group by reacting, for example, with phosphorus pentasulfide, and the protective group for the hydroxy group is thereafter removed to obtain neplanocin D derivative (III). Methylation thereof by methyl iodide gives neplanocin D derivative (IV). The neplanocin D derivative (IV) is treated with methanolic ammonia to produce neplanocin A.
Furthermore, the three hydroxy groups in the cyclopentene ring of neplanocin D are acetylated for protection by reacting with acetic anhydride, and thereafter the said compound is changed to neplanocin D derivative [3] by reacting with thionyl chloride. The neplanocin D derivative [3] can be changed to neplanocin A through neplanocin D derivative [4] by treating with methanolic ammonia.
These reactions can be illustrated as follows:
~1) o~ 0 ~\il ~enzoylchlo~ide ~
~a~zC~ ~2C~
OE~ C1Bz' Neplano~in D ~I) 8~32 6~ 8~1 P2 s 8 ~ 9 ~ ~X N
B~ ONzC ~ N~2C
:: . 0~ 0~
;~ ~II) Neplanocin D derivatiYe ~III) .
' ' ~C~ ~
~ N
CH I ~ Neplanocin A
3 . ~ethanollc ammonia ~OH2~ ~
.O~I H
Neplanocin D derivative ~IV) ,~
~ ~8~3~32 t2) OE C~
Neplanocin D ti anhydride 2 A C 1~ 1~ z C '~ OI~j OAc OAc OAc OA~
[2] Neplanocin D derivative [3]
--.~ Neplanocin A
The physico-chemical properties of the neplanocin D d~riv- :
atives are as follows.
Neplanocin D derivative .
~III) (IV) . ... _ . , _ , I Molecular weight (calculated from mass 280 294 spectrum analysis) . _ . . . _ .
Molecular formula 11 12 4 3 12 14 4 3 . _~ . . ._ Melting point 260 - 262DC.(decomp.) 177 - 178~C.
__ .. ~ ...
.Specific rotation I - 13)D = -141.7 i (c=071H2o) . _ . _ . . .
j : 222 ma Ultraviolet absorption ' 226 m~ ~ 288 m~
spectrum (in H20) 324 m~ 1 295 m~ i The following examples illustrate the present invention but are not construed as limiting.
,,)j ,, j Example 1.
100 ml. of aqueous medium (pH 6.5) containing glucose 2%, starch 2%, yeast extract 1%, casein hydrolysate 1% and calcium carbonate 0.2% were introduced in a 500 ml flask and sterilized at 120C for 15 minutes. Into two flasks of this medium, one loopful of slant of Ampullariella sp. A 11079 FERM-P No. 4494 was inoculated and shake cultured at 30C. After 4 days, the cultured medium was transferred to 20 1. of the same sterilized medium hereinabove in a 30 1. jar fermenter and cultured at 30C
with agitation at 300 r.p.m. and aeration of 20 l./min. for 48 hours.
The thus cultured medium was transferred into 200 1. of aqueous medium (pH 6.5) containing glucose 4~, soy bean powder 1%, meat extract 0.4%, peptone 0.4%, yeast extract 0.1%, NaCl 0.25% and calcium carbonate 0.1% and cultured at 30C, with agitation at 180 r.p.m., and aeration 130 l./min., for 40 hours.
The obtained cultured broth (about 200 1.) was filtered and the myce~a was washed with water. The filtrate and washed water were combined to obtain about 140 1. of clear filtrate (potency 57 mcg/ml as neplanocin A).
Example 2.
The filtrate obtained in Example 1 was passed through a column of 20 1. of cation exchange resin 'IAmberlite IR-120' (H~ type) (trade mark) to adsorb the material and washed with 100 1. of water. Elution was carried out with 3.7 N
aqueous ammonia and the primary eluate (30 1.) was discarded.
90 1. of the following eluate were collected, adjusted to pH
8 by adding 6N HCl, then passed through 4 1. of active carbon in a column, washed with water and thereafter eluted with 90 1.
of 70~ methanol solution. The thus obtained eluate was concen-trated under reduced pressure to give 1.5 1 of a concentrate.
''`- I
The concentrate was charged on a column of 10 1. of "Amberlite IRA-410" (OH type) (trade mark), and eluted with water and adjusted to pH 7 by adding 4N NCl. The eluate was concentrated in vacuo, the material was precipitated under cooling conditions filtered to obtain 41.8 g of crude neplanocin A (purity about 12%).
The crude neplanocin A (41.8 g) was dissolved in a small amount of water and charged on 2 1. of silica gel in a column (8.3 X 40 cm) packed with a solvent mixture of n-butanol: 28%
aqueous ammonia : water (10 : 0.2 : 1), and thereaft~r eluted with the same solvent mixture.
Each 150 ml of the eluate was fractionated and active fractions were found in fractions Nos. 24 - 52. The said active fractions were collected, concentrated in vacuo, an~ was allowed to stand under cooling.
The precipitate was collected by filtration to obtain 2.6 g of crude crystalline neplanocin A. (yield 32~).
The crude neplanocin A was dissolved in about 60 ml of hot water and allowed to stand at room temperature. The pre-cipitated white needle crystals was collected by filtration to obtain 2.01 g of crystals of neplanocin A. (yield 25.2~).
Example 3.
The filtrate (140 1., potency 300 mcg/ml as neplanocin C) obtained by the same procedure in Example 1 was passed through a column of 20 1. of cation exchange resin "Amberlite I -120"
(H type) (trade mark) to adsorb the material and washed with about 1001- of water. Elution was carried out with 3.7N
aqueous ammonia and the primary eluate (20 1.) was discarded.
100 1. of th~ following eluate were collected, adjusted to p~ 8 by adding conc. HCl, then passed through 4 1. of active carbon in a column, washed with water and thereafter eluted with 70 1.
~X~8~
of 70% methanol solution. The thus obta~ined eluate was con-centrated under reduced pressure to give 1.5 1. of a concen-trate. This concentrate was freeze dried to obtain 83.6 g of a crude powder. (purity 29.5%).
83 g of the crude powder was dissolved in water saturated n-butanol solution and charged on 2 1- of silica gel in a column (8.3 X 40 cm) packed with n-butanol, and thereafter eluted with a solvent mixture of n-butanol : conc. aq. ammonia:
water (10 : 0.2 : 1). Each 150 ml of the eluate was fraction-ated and active fractions w ge found in fraction Nos. 53 - 108.
The said active fractions were collected, concentrated and was allowed to stand at low temperature.
The precipitate was collected by filtration to obtain 18.2 g of neplanocin C as white crystals. (purity 96%, yield 41.6%).
This was dissolved in hot water and allowed to stand at room temperature to precipitate the neplanocin C as colorless plate crystals which was filtered to obtain pure neplanocin C.
Example 4.
To the filtrate obtained by the same procedure in Example 1 was added 3.6 kg of active carbon powder. After stirring for 40 minutes, active carbon was collected by filtration and washed thoroughly with water. Elution was carried out with 70%
methanol solution. 40 1. of eluate were dried up in vacuo to obtain 80 g of crude powder containing neplanocin D.
The 80 g of crude powder was charg~d on 6 1. of silica gel in a column packed with n-propanol : conc. aq. ammonia : water (10 : 1 : 1), and thereafter eluted with the sa~e solvent mixture. Each 600 g of the eluate was fractionated and neplan-ocin D was found in fractions Nos. 20 - 37. The said active fractions were collected, concentrated in vacuo up to 30 ml, and was allowed to stand under cooling. The precipitate was collected b~ filtration to obtain 460 mg of crude crystalline -~3-~28~382 neplanocin D.
The 460 mg of crude crystals were dissolved in 3 ml of hot water and put on a column of "Sephadex G-15" (1850 ml, packed with water), then eluted with water. Each lO0 ml. of the eluate was fractionated, the eluate fractions Nos. 15 - 16 t200 ml) were collected and concentrated under reduced pressure up to 20 ml which was allowed to stand at room temperature.
314 mg of the precipitated neplanocin D as white needle crystals were obtained.
Example 5.
(1) 376 mg of neplanocin D dissolved in 10 ml of anhydrous pyridine were stirred thoroughly at 55C. 0.9 ml of benzoyl chloride was added dropwisely thereto and the reaction mixture was stixred for 2 hours at 60 - 65C.
Thereafter the solution was kept at 40 - 45C for 2 hours and further allowed to stand overnight at room temperature.
10 ml of water was added with stirring therein and after 25 minutes the solution was evaporated to dryness under reduced pressure in an e~aporator. The dried material was dissolved in 30 ml of chloroform, washed three times with 10 ml of 1 N
HCl and twice with 1 N sodium bicarbonate solution, and there-after washed twice with 20 ml of water. The chloroform layer was dehydrated by anhydrous magnesium sulfate and concentrated to obtain 747 mg of a pale creamy compound (I). (yield 91%).
Physico-chemical properties of the compound tI) are as follows.
[ ~] = 199 (c=0.6, methanol) D
Molecular formula: C32H24N4O7 * Trademark "Sephadex" is a trademark for a dry insoluble powder composed of macroscopic beads of cross-linked dextran containing functional ionic groups.
38;~
Molecular weight: 576 (mass spectrum) Melting point: 116 - 117C.
Ultraviolet absorption spectrum: ~max = 232 m~. E = 704 1 cm RF value (chloroform : methanol = 10 : 1); RF = 0.53 (2) 735 mg of the compound (I) obtained hereinabove (1) and 1.0736 g of phosphorus pentasulfide diss ~ved in 19 ml of pyridine were stirred thoroughly and 0.19 ml of water was drop-wisely added thereto. After refluxing for 4 hours with stirring, the reaction mixture was cooled and concentrated in vacuo to obtain a brown oily material. This oily material was poured in boiling water with stirring and further stirred for 30 minutes to obtain a yellowish substance. This w æ filtered to collect the precipitate, washed with a mixture of ethanol : ether (1 : 1), further washed with ether and dried to obtain 561 mg of the compound (II). (yield 74.3%).
The physico-chemical properties of the compound (II) are as follows.
[a ]D = -286.9 (c-0.6, CHC13~
Molecular formula: C32~24N4O6S
Molecular weight: 592 Melting point: 235 - 239C (foaming decomp.) W spectrum (in methanol): 232 m ~(E = 858.5) 1 cm 323 m ~(El~ = 327.5) 1 cm Rf value (chloroform : methanol = 10 : 1) ; Rf = 0.82 (3) 525 mg of the compound (II) obtained hereina~ove (2) were dissolved in 31 ml of anhydrous methanol. 0.62 ml of a freshly-prepared 2 N solution of sodium methylate in methanol was added therein and refluxed with stirring for 2 hours to remove the benzoyl group.
After the methanol was distilled off about 50 ml of water were .
.~
, 11281~82 added and adjusted to pH 7.5 by adding acetic acid. The solution was extracted twice with 20 ml of ethyl acetate and the aqueous layer was concentrated to obtain neplanocin D
derivative (III) as needle crystals. Recrystallization was made from hot water to obtain 110 mg of neplanocin D derivative (III). (yield 44.3%).
The physico-chemical properties of neplanocin D deri~ative (III) are as follows.
Specific rotation: impossible to measure due to varying at measurement.
Molecular formula: CllH12N4O3S
Molecular weight: 280 (mass spectrum) Melting point: 260 - 262 C (decomp.) W spectrum in H2O: 226 m~ , (El% = 388.6) 324 m~ (El~ = 968 o) 1 cm Rf value (propanol : conc. aq. ammonia : water = 10 : 1 : l); Rf = 0.21 Example 6.
To 37 mg of neplanocin D derivative (III) obtained in Example 5 dissolved in 0.3 ml of 0.4 N NaOH were added 0.009 ml of methyl iodide and the reaction mixture was sh~ken at room te~ature for 10 m~utes. Thereafter 0.05 ml of 0.4 N NaOH was ~ thereto and there was f~er added 0.009 nl of methyl iodide, then the reaction m~h~e was again s ~ en and a~owed to stand at ~ m temperature. Tb this solution was ~d 10 ml of water, and the solution was concentrated in vacu~
up to 2 ml, and ch~ed on a oolumn of "Seph~ G-15" (1.4 x 49 cm). Elution was carried out with water and each 3 g of eluate was fractianated. Fractions Nbs. 17-19 were oDllected, ~ oentrated and cooled to bbtain 35 mg of crystalline nep ~ ocin D derivative (r".(yield 90%).
Ihe physi ~ chemical properties of neplanocin D derivative (IV) are as follows.
* ~ demark 88~3~
[ ~ ] = -141.7 (c=0.7, H2O) Molecular formula: C12H14N4O3S
Molecular weight: 294 (mass spectrum) Melting point: 177 - 178C
UV spectrum in water : 222 m~ (E % = 415.8) 1 cm 288 m~ ( ~% = 676.3) 1 cm 295 my (El% = 670.7) 1 cm Rf value (propanol : conc. aq. ammonia : water = 10 :1 :1) ; Rf= 0.42 Example 7.
Synthesis of neplanocin A from neplanocin D derivative (IV) 50 mg of neplanocin D derivative (IV) obtained by the same process as described in Example 6 dissolved in 1.5 ml of metha-nolic ammonia (0C saturation) wereplaoed in a sealed tube and heated at 146 - 148C for 7 hours. After removing the solvent under reduced pressure, the residue was dissolved in 3 ml of hot water, then cooled to obtain 33 mg of neplanocin A as needle crystals. (yield 74%).
Example 8.
(1) To 1.14 g of neplanocin D dissolved in 12 ml of anhydrous pyridine was added 1 ml of acetic anhydride and the reaction mixture was allowed to stand at room temperature for over-night.
30 ml of n-hexane was added thereto and cooled in ice-water to obtain 1 g of the compound [2] as crystals.
(2) 1 g of the compound [2] was added to 25 ml of chloroform solution containing 0.8 ml of thionyl chloride and 0.4 ml of dimethylformamide and refluxed at room temperature for 3 hours.
Chloroform was distilled off under reduced pressure. The residue was poured into 100 ml of ice-water under stirring.
Extraction was made with 100 ml of chloroform, and the extract was washed with NaHCO3 and water, and dried with sodium sulfate.
Neplanocin D derivative [3] was obtained after drying in vacuo.
Example 9.
Preparation of neplanocin A from neplanocin D derivative [3];
To neplanocin D derivative [3] obtained in Example 8 placedin a sealed tube made of steel was added 35 ml of saturated methanolic ammonia and reacted at 100C for 5 hours. After cooling, the reaction mixture was dried up in vacuo to obtain a crystalline powder which was purified by silica gel column chromatography to obtain 286 mg of neplanocin A.
The physico-chemical properties are identical with those of neplanocin A obtained in Example 7.
Example 10.
The filtrate (140 1., potency about 50 mcg/ml as neplan-ocin B) obtained by the same procedure as in Example 1 was passed through a column of 20 1. of cation exchange resin "Amberlite IR-120" (H+ type) to adsorb the material and washed with about 100 1. of water. Elution was carried out with 3.7 N
aqueous ammonia and the primary eluate (30 1.) was discarded.
~0 1. of the following eluate were collected, adjusted to pH 8 by adding 6 N HCl, then charged on a column of 4 1. of active carbon, washed with water, and thereafter eluted with 90 1. of 70% aqueous methanol. The thus obtained eluate was concentrated to gi~e 500 ml of a concentrate which was allowed to stand at low temperature to precipitate the material. The precipitate was collected and dried to obtain crude neplanocin B (5.1 g, purity about 60~ r yield 44%).
Example 11.
Neplanocin B obtained in Example 10 (5.1 g) was charged on a column of silica~gel (8.3 X 40 cm, 2 1.) packed with a mixture of acetone : water (10 : 0.5) and eluted with the same ~, solvent mixture. Each 80 g of the eluate was fractionated and the fractions No. 16 - 24 were combined. The said active fractions were allowed to stand at low temperature to precip-itate neplanocin B as white plate crystals. 2.57 g of crystal-line neplanocin B were obtained. (yield 32.8%).Example 12 Cultivation was carried out as illustrated in Example 1 to obtain about 200 l.of a cultured mass which was filtered and the cells were washed with water. The filtrate and washing water were combined to yield 140 1. (potency; about 1.5 mcg/
ml).
The solution was passed through a column of 20 l. of cation exchange resin "Amberlite IR-120" (H type~ to adsorb the material and washed with about 100 1. of water. Elution was carried out with 3.7 N aqueous ammonia and the primary eluate (30 1.) was discarded. 90 1. of the following eluate were collected, adjusted to pH 8 by adding 6 N HCl, then charged on a column of 4 l. of active carbon, washed with water and thereafter eluted with 90 l. of 70% aqueous methanol. The thus obtained eluate was concentrated to give 500 ml of a concentrate which was freeze dried to obtain 31.2 g of crude neplanocin F. (purity about 0.5%).
Example 13.
The crude powder of neplanocin F obtained in Example 12 (31.2 g) was charged on a column of silica gel (800 ml) packed with a mixture of n-butanol : 28% aqueous ammonia : water (10 : 0.2 : 1) and eluted with the same solvent-mixture. Each 150 ml of the eluate was fractionated and the fractions No. 5 - 10 were combined, and then concentrated in vacuo.
The concentrate was charged on a silica gel column (240 ml) previously packed ~ith a mixed solution of chloroform: methanol:
38~
acetic acid (10 : 2 : 0.2) and eluted with the same solvent mixture, fractionated each 20 g of the fraction. Fractions No. 67 - 120 were combined, concentrated and dried to obtain crude crystals of neplanocin F. (108 mg, yield 51%).
Example 14.
The crude crystals of neplanocin F (108 mg) obtained in Example 13 were charged on a column of "Sephadex G-15" (186 ml) packed with water and eluted with water. Each 10 g of the fraction was fractionated and the fractions No. 22 - 27 were collected, combined and concentrated to 5 ml which was allowed to stand in a cold room to precipitate neplanocin F as white needle crystals. The precipitate was filtered and dried to obtain crystalline neplanocin F (87 mg). (yield 41~).
Claims (19)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the production of antibiotic neplanocins of the formula wherein X is an NH2, -OH, -SH, or -SCH3 group or a Cl atom, R is a hydrogen atom or a protective group for an -OH group, and Z is the group in which Y is -0- or a valence bond, and R is a hydrogen atom or a protective group for an -OH group, or the group; in which Y is -0- or a valence bond , which comprises culturing a neplanocins producing microorganism belonging in the genus Ampullariella in a nutrient medium consisting of assimilable carbon and nitrogen sources and inorganic substances,and isolating neplanocin-A, -C, -D, -B
and -F from the cultured mass; and where a compound of the above formula wherein X is an -SH group is desired, isolating neplanocin D of the formula wherein R' is a protective group, and reacting said compound with a mercaptonizing agent to prepare a compound of the formula (III) wherein R' has the same meaning as hereinabove, and where desired, removing the protective groups R' for the hydroxy groups; and where a compound of the above formula wherein X
is an -SCH3 group is desired, preparing a compound of formula (III) as aforesaid, wherein the protective groups R' for the hydroxy groups are removed, and reacting the compound so prepared with a methylating agent;
and where a compound of the above formula wherein X is a C1 atom is desired, preparing a compound, in which the three hydroxy groups in the cyclopentene ring of neplanocin D are protected, of the formula (B) from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass, reacting said neplanocin D with an acylating agent to prepare a compound, in which the three hydroxy groups in the cyclopentene ring of neplanocin D are protected, of the formula wherein R' is a protective group, by reacting neplanocin D, obtained as aforesaid, with an acylating reagent, and reacting said compound of formula (B) with a chlorinating agent.
and -F from the cultured mass; and where a compound of the above formula wherein X is an -SH group is desired, isolating neplanocin D of the formula wherein R' is a protective group, and reacting said compound with a mercaptonizing agent to prepare a compound of the formula (III) wherein R' has the same meaning as hereinabove, and where desired, removing the protective groups R' for the hydroxy groups; and where a compound of the above formula wherein X
is an -SCH3 group is desired, preparing a compound of formula (III) as aforesaid, wherein the protective groups R' for the hydroxy groups are removed, and reacting the compound so prepared with a methylating agent;
and where a compound of the above formula wherein X is a C1 atom is desired, preparing a compound, in which the three hydroxy groups in the cyclopentene ring of neplanocin D are protected, of the formula (B) from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass, reacting said neplanocin D with an acylating agent to prepare a compound, in which the three hydroxy groups in the cyclopentene ring of neplanocin D are protected, of the formula wherein R' is a protective group, by reacting neplanocin D, obtained as aforesaid, with an acylating reagent, and reacting said compound of formula (B) with a chlorinating agent.
2. A process as defined in claim 1 wherein the neplanocins producing microorganism is Ampullariella sp. A 11079 FERM-P
No. 4494.
No. 4494.
3. A process according to claim 1 which comprises the step of isolating neplanocin A of the formula from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass.
4. A process according to claim 1 which comprises the step of isolating neplanocin C of the formula from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass.
5. A process according to claim 1 which comprises the step of isolating neplanocin D of the formula from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass.
6. A process according to claim 1 which comprises the step of isolating neplanocin B of the formula from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass.
7. A process according to claim 1 which comprises the step of isolating neplanocin F of the formula from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass.
8. A process according to claim 1 which comprises the steps of isolating neplanocin D of the formula from a mixture of neplanocins -A, -C, -D, -B and -F as obtained from the cultured mass; reacting said neplanocin D with an acylating agent, to prepare a compound, in which the three hydroxy groups in the cyclopentene ring of neplanocin D are protected, of the formula (B) wherein R' is a protective group; and reacting said compound of formula (B) with a mercaptonizing agent, thereby to prepare a neplanocin D derivative of the formula (III) wherein R' is a protective group, and then removing said protective groups R'.
9. A process according to claim 8, which further comprises the step of reacting the neplanocin D derivative of formula (III) with a methylating agent, thereby to produce a neplanocin D
derivative of the formula (IV)
derivative of the formula (IV)
10. A process according to claim 1 which comprises the steps of isolating neplanocin D of the formula from a mixture of neplanocins -A, -C, -D, -B, and -F as obtained from the cultured mass, reacting said neplanocin D with an acylating agent, to prepare a compound, in which the three hydroxy groups in the cyclopentene ring of neplanocin D are protected, of the formula (B) wherein R' is a protective group, and reacting said compound of formula (B) with a chlorinating agent, thereby to produce a neplanocin D derivative of the formula wherein R' is a protective group.
11. Antibiotic neplanocins of the following formula, wherein X is an NH2, -OH, -SH, or -SCH3 group or a Cl atom, R is a hydrogen atom or a protective group for an -OH group, and Z is the group; in which Y is -O-or a valence bond, and R is a hydrogen atom or a protective group for an -OH group; or the group:
in which Y is -O- or a valence bond, when pre-pared by the process of claim 1 or by an obvious chemical equivalent thereof.
in which Y is -O- or a valence bond, when pre-pared by the process of claim 1 or by an obvious chemical equivalent thereof.
12. Neplanocin A of the following formula, when prepared by the process of claim 3 or by an obvious chemical equivalent thereof.
13. Neplanocin C of the following formula, when prepared by the process of claim 4 or by an obvious chemical equivalent thereof.
14. Neplanocin D of the following formula, when prepared by the process of claim 5 or by an obvious chemical equivalent thereof.
15. A neplanocin D derivative of the following formula, when prepared by the process of claim 8 or by an obvious chemical equivalent thereof.
16. A neplanocin D derivative of the following formula, When prepared by the process of claim 9 or by an obvious chemical equivalent thereof.
17. A neplanocin D derivative of the following formula, wherein R' is a protective group, when prepared by the process of claim 10 or by an abvious chemical equivalent thereof.
18. Neplanocin B of the following formula, when prepared by the process of claim 6 or by an obvious chemical equivalent thereof.
19. Neplanocin F of the following formula, When prepared by the process of claim 7 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP53-62899 | 1978-05-25 | ||
| JP6289978A JPS54154792A (en) | 1978-05-25 | 1978-05-25 | Novel antibiotic a-11079-b1b and its preparation |
| JP53-98027 | 1978-08-10 | ||
| JP9802778A JPS5524157A (en) | 1978-08-10 | 1978-08-10 | Novel antibiotic substance a-11079-b2 and its preparation |
| JP820579A JPS55100388A (en) | 1979-01-29 | 1979-01-29 | Novel substance neplanocin d and its derivative |
| JP54-8205 | 1979-01-29 | ||
| JP54-21201 | 1979-02-23 | ||
| JP2120179A JPS55113786A (en) | 1979-02-23 | 1979-02-23 | Novel substance, nepranosin b or f and its preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1128882A true CA1128882A (en) | 1982-08-03 |
Family
ID=27454901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA328,113A Expired CA1128882A (en) | 1978-05-25 | 1979-05-23 | Antibiotic neplanocins |
Country Status (13)
| Country | Link |
|---|---|
| CA (1) | CA1128882A (en) |
| CH (1) | CH641804A5 (en) |
| DE (2) | DE2954197C2 (en) |
| DK (1) | DK149313C (en) |
| ES (1) | ES480384A1 (en) |
| FR (1) | FR2426688A1 (en) |
| GB (1) | GB2021582B (en) |
| HU (1) | HU192741B (en) |
| IT (1) | IT1115245B (en) |
| MX (1) | MX5788E (en) |
| NL (1) | NL182231C (en) |
| SE (1) | SE443576B (en) |
| SU (1) | SU906388A3 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3148363A1 (en) * | 1980-12-12 | 1982-09-16 | Toyo Jozo K.K., Shizuoka | NEPLANOCIN A DERIVATIVES |
| US4742064A (en) * | 1985-09-10 | 1988-05-03 | Regents Of The University Of Minnesota | Antiviral carbocyclic analogs of xylofuranosylpurines |
| ZA894534B (en) * | 1988-06-20 | 1990-03-28 | Merrell Dow Pharma | Novel neplanocin derivatives |
| US5059690A (en) * | 1990-03-01 | 1991-10-22 | E. R. Squibb & Sons, Inc. | Purinyl tetrahydrofurans |
| US5145960A (en) * | 1989-04-24 | 1992-09-08 | E. R. Squibb & Sons, Inc. | Pyrimidinyl tetrahydrofurans |
| US5164520A (en) * | 1989-04-24 | 1992-11-17 | E. R. Squibb & Sons, Inc. | Intermediates for purinyl and pyrimidinyl tetrahydrofurans |
| NZ232993A (en) * | 1989-04-24 | 1992-10-28 | Squibb & Sons Inc | Purinyl and pyrimidinyl tetrahydrofurans |
-
1979
- 1979-04-12 HU HU79TO1102A patent/HU192741B/en unknown
- 1979-04-26 DE DE2954197A patent/DE2954197C2/de not_active Expired - Lifetime
- 1979-04-26 NL NLAANVRAGE7903318,A patent/NL182231C/en not_active IP Right Cessation
- 1979-04-26 SE SE7903672A patent/SE443576B/en not_active IP Right Cessation
- 1979-04-26 DE DE2917000A patent/DE2917000C2/en not_active Expired
- 1979-04-30 DK DK177079A patent/DK149313C/en not_active IP Right Cessation
- 1979-05-08 FR FR7911578A patent/FR2426688A1/en active Granted
- 1979-05-09 ES ES480384A patent/ES480384A1/en not_active Expired
- 1979-05-17 SU SU792763801A patent/SU906388A3/en active
- 1979-05-18 GB GB7917481A patent/GB2021582B/en not_active Expired
- 1979-05-23 CA CA328,113A patent/CA1128882A/en not_active Expired
- 1979-05-23 MX MX797996U patent/MX5788E/en unknown
- 1979-05-24 IT IT7922957A patent/IT1115245B/en active
- 1979-05-25 CH CH490879A patent/CH641804A5/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| SE7903672L (en) | 1979-11-26 |
| MX5788E (en) | 1984-07-11 |
| DK177079A (en) | 1979-11-26 |
| GB2021582B (en) | 1982-10-20 |
| GB2021582A (en) | 1979-12-05 |
| CH641804A5 (en) | 1984-03-15 |
| DK149313C (en) | 1986-10-20 |
| DK149313B (en) | 1986-04-28 |
| FR2426688A1 (en) | 1979-12-21 |
| IT7922957A0 (en) | 1979-05-24 |
| IT1115245B (en) | 1986-02-03 |
| DE2917000C2 (en) | 1985-10-03 |
| SU906388A3 (en) | 1982-02-15 |
| DE2954197C2 (en) | 1990-09-27 |
| SE443576B (en) | 1986-03-03 |
| ES480384A1 (en) | 1980-04-01 |
| FR2426688B1 (en) | 1982-03-12 |
| NL7903318A (en) | 1979-11-27 |
| DE2917000A1 (en) | 1979-11-29 |
| NL182231C (en) | 1988-02-01 |
| HU192741B (en) | 1987-07-28 |
| NL182231B (en) | 1987-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0182315B1 (en) | Novel antibiotic nk84-0218 pharmaceutical compositions containing it and process for the production of the same | |
| US4321376A (en) | Neplanocin-B and -F | |
| CA2111283C (en) | Process for preparation of indolopyrrolocarbazole derivatives | |
| CA1128882A (en) | Antibiotic neplanocins | |
| US4423218A (en) | Antibiotic neplanocin A | |
| EP0327009B1 (en) | WS-9326A, WS-9326B and their derivatives | |
| KR830001688B1 (en) | Manufacturing method of novel antibiotic neplanosin | |
| US4897470A (en) | Anthracycline antibiotics DCP-1 and 2 | |
| JPS625990A (en) | Antibiotic substance and production thereof | |
| JPH1045738A (en) | Antibiotic substance epoxyquinomicin c and d, its production and antirheumatic agent | |
| US4605559A (en) | Compound, FR-900447, production and use thereof | |
| EP0205981B1 (en) | A novel anti-tumor and antimicrobial compound, its microbiological preparation and its use as medicament | |
| JPS6210048A (en) | Novel physiologically active substance mh435 | |
| EP0191399B1 (en) | Benz[a]anthraquinone compounds, a microbial process for their preparation and their use as medicaments | |
| JP2971204B2 (en) | New substance WK-2955 and method for producing the same | |
| CA1125746A (en) | Antitumor antiobiotics ma 144-m.sub.1 and ma 144- m.sub.2 | |
| JPH09157266A (en) | New antibiotic epoxynomicin a and b and their production | |
| JPH0272167A (en) | Bu-3862t anti-tumor antibiotic | |
| KR840000127B1 (en) | How to prepare Istamycin | |
| JPS62186787A (en) | Novel microorganism | |
| JPS61115081A (en) | Novel antibiotic ss21020d and production thereof | |
| JPS59125898A (en) | Novel antibiotic, am-6424 and its preparation | |
| JPS63157992A (en) | Antibiotic substance n-hydroxydihydroabikoviromycin | |
| JPS62138196A (en) | Anthracyclin compound and production thereon | |
| JPH06316595A (en) | Cyclic peptide compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |