JPS59193899A - Anthracyclinoneglucuronide - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to anthracyclinone glucuronides and their salts and esters, methods of biosynthesis of these compounds, and compositions containing these compounds.

Anthracyclinone glucuronide (hereinafter [FCE
22724J) was found to be effective in inhibiting the growth of malignant tumors such as P388 leukemia in experimental animals. The structural formula of FCE 22724 has been determined and can be expressed as follows.

The present invention provides antitumor anthracyclinone glucuronides of the general type 2A (wherein R represents a hydrogen atom, a lower alkyl group, or an alkali metal atom).

The present invention also relates to the use of Streptomyces bucetheus var. vinaceus (St.
reptomyaes peucetiusvar,
vinaceus) (DSM 2597). The microorganism Streptomyces bucetius var. vinaceus is a mutant strain of Streptomyces bucetius var. orex (ATCC 31428). The present invention provides for the collection of FCE 22724 from fermentation broth;
Includes methods of concentration and purification from crude solutions. Furthermore,
The present invention includes within its scope the form of a crude concentrate obtained from a biosynthetic process, a pure form such as a free acid or an alkali metal salt isolated from a crude concentrate or FCE 2272
Novel anthracyclinone glucuronide FOB22724 in the form of a pharmaceutical composition in which 4 and its salts or esters are mixed with a pharmaceutically acceptable diluent or carrier.
8.

Microorganisms The microorganisms used in the method of the present invention are those described by Cassinelli et al.
ntib, Vol. 35, No. 2, 176-183 Liao (1982)
A Streptomyces bucetius var. aureus box strain (A
TOO31428,DSM1367,F,R,:[,4
622) was obtained as a reversion mutation after mutagenesis treatment with ultraviolet rays. The bacterial strain thus obtained was given the code no.
Sammlung fiir Mik
It has been contributed to Roorganismen as DSM2597. This new microorganism is typically pink to red to grape-like in color in all young cultures on solid media, and becomes deep purple as it matures, producing a single thread of pigment, so that cultured bacteria and other mutants from Streptomyces bucetius. This color change is due to i1ζ,
sm commonly used for the classification of Streptomyces fungi
Abundant soluble pigments (formed on solid media)
It can be observed against. Such media include Bennett agar,
Tuapek agar, asparagine-glucose agar, glycine-glycerol kiten, potato glucose renten, asparagine-glycerol Gaidai [Wank 2? :yS
,A, :The ActinolIlycet;es
Volume I @ < 1961), Wi], liam Wi
lkins & Oo, Baltimore: ] and 1414 machine salt - starch powder [Pritenomu T, G,, Unterman P0, Holy C+, Lindenfuelser L
, A., Heseltin O. A., and Benetaket R.
G.: Selection and taxonomic study of media for maintenance of Streptomyces. Antibiotics Ann, 195
6/1957947-956].

Therefore, the inventor of the present invention regarded the first ice body M8511'CK as a variety of Streptomyces byuscetius and gave the name Streptomyces byusetius variant vinaceus. Microorganism Streptomyces
Bucetius nebinaceus is itself within the scope of the present invention.

- Fermentative production is carried out by conventional, well-known methods and is carried out at temperatures ranging from 25°C to 37°C (preferably 28°C) for periods varying from 6 to 7 days (preferably 50°C). microorganisms in a pre-sterilized soil under aerobic conditions at a pH ranging from 6.5 to 8.0 at the initial stage and at a pH ranging from 6.5 to 8.0. It consists of culturing the whole thing. The cultivated land contains carbon and nitrogen sources as well as mineral deposits. Simple carbon sources include starch, dextrin, crucose, g1) serine, man = )-/L/
, 7/L/)-sugar, onion infusion, distiller's soluples, soybean oil and soybean meal. Appropriate nitrogen '# is also a @ki charcoal synonym that also -M nitrogen,
Includes dry butterfly yeast, meat peptone or casein. With good results, also ammonium In
ff: 1q is gained by performing flight FI4. Minerals necessary for production (turquoise) can be enriched depending on the medium used. Calcium carbonate and
The addition of sodium acid or potassium phosphate has proven useful. In media containing glucose or ammonium salts, the addition of very clear levels of mineral yellow salts such as potassium, sodium or calcium and imitations of iron, magnesium and manganese salts. is the membership fee. L'! l1: Takashi is
Experiments can be carried out in Erlenmeyer flasks or individual vessels or in one-beam vessels.

Fractionation method: Samples of tF nebulos and crude admixture were used as a bath release agent for chloroform: methanol: rn acid: water (8Q with Tani-juni).
:20:3) was subjected to thin-thickness chromatography (T) on a plate previously coated with silica gel.
LO), II'CJ! 1i2272
4 is R of 0.22 as a constituent component that gives Dameng a reddish color.
I found out what I was looking for right away.

Determined total of F122724 present in fermentation broth 1
i111 can be accomplished by the following method. Two times the volume of acetonitrile was added to the sample of broth and the mixture was sonicated for 1 minute at silkworm temperature and then filtered. p
Remove the sample of liquid from the above-mentioned eraser and remove it with silica gel TL.
When subjected to O analysis, the corresponding red colored band was scraped off and the FOE227 at 495 nm was determined by chloroform:ethanol elution.
24 spectrophotometers can also be implemented.

Shun, who completed the isolation operation crystal 1 yesterday, FOE 22724 will be born in the family as a raw material. 1 thread has a pH of 7 with the help of diatomaceous earth
．． At 5, the VC recommends molecular k from Ken Misaki Gan to pass through the sea.

The aJ thread cake is extracted with a water-mixture bath such as acetonitrile, dioxin, methanol, and lower alcohols. Likes. Methanol is conveniently used for power. -The system extract is concentrated under reduced pressure, and the aqueous concentrate is converted into alkali gold! FOE22724 of A sound form 1 fortune
to say.

yc1+:22724iJ: methyl in butyl ketone,
It can be harvested from the filtered broth by extraction at pH 4 with water-incompatible M4 dihydrogens such as methyl propyl ketone and n-butanol. Methyl isobutyl ketone is preferably used. ■From the liquid extract, F'CF22724 is aqueous sodium salt or charcoal salt) pH as IJum salt
7 and can be re-extracted into water. From boiled broth and aqueous concentrate, FOFi22724 was prepared as an/marlite XAD 2, XAD 4 and ER 180
It can be absorbed onto a loosely cross-linked divinylbenzene polymer 4υ゛j such as (manufactured by Rohm Ana Baas). Preferably, KR18[] is 1
Historically used. The absorbed Fag 22724 is dissolved in an aqueous lower alcohol, preferably 10% (B N Te) n-
You can easily report the news using the property tool.

Purification Procedure The crude aqueous concentrate, adjusted to pH 7 after addition of sodium chloride (5%), is passed through a column of KR180 resin. After washing with water, elution is carried out with 10% (by volume) n-propertool in water and the eluent is concentrated in fractions. Crude FCF22724 as the sodium salt is obtained from selected fractions monitored on silica gel TLO K after vacuum concentration and lyophilization. Pure Fro 227
To obtain 24, a water bath solution of the crude 11 cum salt was brought to pH 4 (A) and extracted with methyl in butyl ketone. The organic extract was concentrated in vacuo to Otani, precipitated with n-hexane and extracted with methyl isobutyl ketone. Crystallization from the ketone gives pure FC!E 22724 as free acid in the form of fine red crystalline needles.Pure F (J 2272
4f1) in an alcohol bath, the corresponding "viscera" was treated with alcoholic sodium hydroxide, then reduced and precipitated with diethyl ether to give the corresponding pure solution.
Acid-catalyzed esterification of FOE22724 in methanol yields the corresponding methyl ester (1111).

Chemical and Physical Properties FOE22724 as a free acid is soluble in alkaline aqueous media, lower alcohols and polar organic solvents, slightly soluble in acetone, ethyl acetate, diethyl ether, methylene dichloride and chloroform and Slightly soluble in water, benzene and n-hexane.

F'' (J22724 as the sodium salt f]11 is soluble in water, lower alcohols and orange cedar bath agents and is slightly soluble or incurable in other organic bath agents.FOE22724 Methyl Ester 011) is soluble in lower alcohols, acetone, ethyl acetate, methylene dichloride and chloroform, slightly soluble in diethyl ether, but insoluble in water and n-hexane.

FCF, 22724, has the following chemical and physical characteristics:

Melt 168-169°C (decomposition) U, V, and S absorption tin: λmax in methanol 235.252.288.360.474.
493, (525sh) nm λmaz in 0.01N methanolic sodium hydroxide
253, 252.290, (350sh), 540
and λmax232*252.288.352% 4 in 580 mM/15 phosphorus salt buffer (pH 7)
74.488mm (E 1ka 562, 526.175
, 55% 195, 187) CTn (1), R, spectrum (KBr): Shows peaks at the following wave numbers.

3420.2920.1730.1620.1b85
% 14.50.1405.1330. 1245.
1210.1175. 1120.1060%940
1910.

1HNMR at 200 MH2! 'Cuttle (C
DOL5+IMEJO<61c quotient): 0.99(t
, J=7.6Hz, 5H,0JOH2), 1.40.1
．． 66 (2 m, 2H, CH3CH2), 1. ．．
9-2.3 (nr, 2H, H-8), 5.56 (
s + 5 H, COOC' H3), 3.5-3.8
(m, 3H, H22', H-3'+H-4S, 13.5
0 and 16.60 δ (two 8, 2H, 0H-6
,0H-11).

130 NMR spectrum at 50 MHz vC (
'8-i, ``?'1:1 cDct57aD3oa)
: 6.08 (0-14), 3238 (0-16),
34.42 (0-8), 51.52 ((:!-10)
, 51.89 (OOOOOH3), 61.64 (C-4'
), 71.22 (C-7), 71.56 (0-9), 7
2~a9(c-2'), 75.14(03', 5x, 1
01.50 (c-1'), 122.03 (0-1), 1
24.44 (C-3), 153.09 (0-12a),
134.92 (C-6a), 135.81 (C-2),
138.16 (0-10a), 155.47 (0-11
), 156.24 (C!-6), 158.36 (C-
4), 170.61 (051-40CH3), 171.
52 (C1o-0000H3), 186.07 (0-5
)b and 187.13δ(c-12).

Structure determination FOR22724 is shown and the structure corresponding to 4-0-(β-D-glucuronyl)-ε-rhodomidinone (I+ is determined as follows. Gives ε-rhodomidinone (IVI) and D-glucuronic acid tVl as purple to red aglycones, which were confirmed by direct comparison with authentic samples.
Incubation of the 22724 sodium salt enzymatically confirms the presence of the D-glucuronic acid (Vl) moiety bound to ε-rhodomidinone OVI. Glycosidic bond (7) in FcEz2724 Position b Yohi β-configuration is U, V , , V engineering S, ■, R,'H and 130-NMR spectra.
Ber, ir, Vol. 92, No. 1904 (1959)]
, sinerpine A and B (U.S. Pat. No. 3,864,48
No. 0), musetamycin and flucellomycin from a bohemic acid complex (U.S. Pat. No. 4.059.75)
Although it is a known composition of several anthracycline complexes produced by Streptomyces stripes such as No. 6 Shu) 'FCE22724
tll is the first biosynthetic anthracycline-like anti+Md compound containing in its molecule a β-D-glucuronyl moiety linked to the phenolic hydroxyl group at the 4-position of ε-rhodomidinone.

The following examples are presented to illustrate the invention.

Example 1 Strettomyces bucetius variant vinaceus strain M83F (JLv Royogang Zenzen medium SA [glucose 6%,
Brewing factory dry yeast 1.2%, Na06O 11%, KH
2PO40.5%, 0a0030.1%, MgSO4
0,005%, FeSO4” 7H200,0005%
, ZnSO4・7H200,0005%, 0uSO4
・5H200,0005%, bride 2%, tap water 100m
1VC shield, pH/), 115 with Haotocrape instep
Planting was carried out by heating for 20 minutes at 28° C. on agar slants at 28° C. for 14 days. All fc mycelium fragments scraped from the agar culture medium were collected in 6 cups of sterile distilled water and homogenized using a vortex homogenizer. The suspension thus obtained was used as a liquid growth medium [Todori yeast from the brewery 0.6%, Bezotone 0.5%, Ca(NO3)2
・4H200.05%, tap water 100%, sterilize by heating at 1200 for 20 minutes in an autoclave, and sterilize the pH 6.8 to 7.0) 500# + 7 containing 60 gold M ! Transfer to an Erlenmeyer flask. The entire inoculated flask was operated at 25 rpm and rotated in a circle with a diameter of 7 CWL with a rotational shake of 4 m.
1> Shake for 2 days at a temperature of 28°C. The above-mentioned culture growth -1,5- was used as a production medium [glucose 6%, brewery dry yeast 2.5%, soybean meal 1%, Naat 2
%, KH2PO40,1%, ZnSO47H200,
[] [] 1 qbs 0u804・5H200,O
D 1%, amount to make 100ml of tap water, pH 6,7, sterilized! [Carried out by heating at 115° C. for 20 minutes in an autoclave] Place in a 300-meter Erlenmeyer flask with a 50-meter diameter. The flasks are cultured for 7 days at 28° C. under the same conditions as described for the seed stage. FOE22
The highest concentration of 724 was reached during the 6-pewter and 7-pewter fermentations resulting in a production of %50 mcg7'mt.

Example 2 The entire fermentation operation was as described in Example 1 except that the following production media were used: Soybean meal 1%, Na042%, KH2PO40.1%,
CaO030.2%, MgSO40.01%, FeSO
4.7B20" 0.001%, ZnSO4.7H
200, OD I'10.0uSO4-5H20o,
o o i%, amount to make 1ooy of tap water, pH 6,7
, sterilization was performed by heating at 115C for 20 minutes in an autoclave.

The highest concentration of FOB22724 is reached during the fermentation of the 6th and 7th whites, resulting in a production of 100 mcg/mt.

Example 6 Culture boxes of one strain M 83 FOB are obtained as described in Example 1. Collect 3 or 1 side 4 pieces of yarn in sterilized distilled water and carry out as described in Example 1. Transfer 5[]0rnt of the prepared 4 stopper culture medium to a 2L round bottom flask with a pan flask. This flask'fc 120r
Incubate for 48 hours at a temperature of 28° C. on a rotary shaker operated in a circle with a diameter of 7α and 7α.

As described in Example 1, whole yuzu was sterilized by steam pressure at 120°C for 60 minutes.
[4 times] in a stainless steel fermenter.

The culture at the miko stage is cultured at 28° C. under stirring conditions at 230 rpm and 1 minute of medium 1 and an air flow of 0.5 t. After 3 minutes, this service'k, 12
500 Afg of the production medium described in Example 2, sterilized by steam pressure for 30 minutes at 0° C., is placed in a 800 t stainless steel fermenter. WiiJ was carried out at 28° C., stirred at 25 rpm and aerated through 90.7 tons of medium 1 for 1 minute. pcg22
724's highest concentration il'' i6 white of the eyes and 7 white of the eyes!
The blue one was slow and the production of 70 mcg/mt was 12
t et al.

Example 4 Total broth from the fermentation obtained according to Example 1 (
3t) Th, p using 6% diatomaceous earth as offsetting additive
Passed offshore at H7. Wet Okinawa Cake Total Methanol (3t
) to extract. After clearing, two more extractions (with 6 t and 2 t of diluted methanol respectively) are performed to ensure complete collection of the red pigment. The combined extract was concentrated under reduced pressure for 30 minutes.
Reduce to 0 ml. The Y#-perbroth obtained as described above was adjusted to pH 4 with sulfuric acid and extracted with methyl isobutyl ketone (3T). After two more extractions, 4
The machine extractions were combined, concentrated and re-extracted with 6% permanent sodium carbonate.

9. Water combined with aqueous residue of mouth thread extract. Sodium chloride (5%) is added to the layer and the whole '1Er180 (200
〃J) Adsorb Hatsumode on a column (6, 2 x 27) filled with light. After discarding the initial wash with water, the general P4. 10 volumes in water 1% n-Real Ml with proper tool! do. The selected fractions monitored by TLC were depressurized and dried in a Japanese-made FOE2272.
4 is obtained as the sodium salt (12).

Example 5 Sodium salt obtained according to actual gun example 4 and F manufactured by Teno Phase
(:iE 22724 (1?) No water bath liquid (200
〃I/) was adjusted to pH 4 with sulfuric acid and extracted with methylimbutin 7L ketone. Extract fIf: Concentrated under reduced pressure and almost pure FcFli22724 (0,3i
i') Precipitate with all n-hexane. FCE2272 is crystallized from methyl isobutyl ketone as Yujingen.
4 pure red needles (0,259).

1 touch point 168℃ (decomposition).

Fruit is 4 + / 1 6 FOE22724 play ski which was investigated by Example 5
m(o, 12r) meta/-/14ih (20Td
) i, Phase decoupling of methanolic water sensitized sodium (0
, 2 mmol). 4 condensation and precipitation with diethyl ether, na) Pure FC as IJium salt
! Bj 22724 [:0.11F, 7 points 190~
191°C (min') M)].

Example 7 FOE 22724 (100 Biao) in anhydrous methanol (100 Biao)
o, i r ) total 1N methanolic hydrochloric acid (11
nl). After 2 hours at room temperature, the reaction mixture diluted with η'ethyl torate (100 scallions) was concentrated under reduced pressure to 50
m1K. After washing with aqueous sodium bicarbonate and water, it was dry-exploded over anhydrous sodium bicarbonate and concentrated to a 7c shoulder-to-shoulder phase. Addition of n-hexane gives the triturated methyl ester (0,09) of FCE22724 as a red microcrystalline powder. Melting point 150-152℃
. U, V, and
25,252, 288, 474, aX 178% 96). Its molecular formula based on elemental analysis 02
9H30015 is Tweel Dobutsution installation analysis (
m/e61B) and structure confirmed by (fllJ
This was confirmed by NMR spectroscopy.

I NMR spectrum/L at 200 h'Mz,
(ODOt5): 1.12 (t, J=7.2Hz, 3H
, (EJOH2), 1.12.1.52 (2 m.

2H, CH2CH3), 2-02A (m r 2H,
H-8), 3.69(s, 3H+C1O-C00CH3
), 3.82 (s, 3JC5, -coocH3), 3.
8-4.11 (m.

Wide 8. IH, H-7), 7.2-7.6 (m, 3H
, H-1, H-2, H-3), 13.21.13.47
δ (two 8,2H,0H-6,0H-11).

Example Dioxane (2 Biao) and 1% water solution (1Hm1.
) in ycg 22724 (0,1? ) i<N
'&tio.

Heat at 2:1 tj + ℃. Shen in a different color red! Reaction to crotch orgasm 'jJ? Dilute the s compound with water (20 m) and add to '1. Extract with acid enal. Washed with 6% sodium bicarbonate and water and 11% sodium sulfate in water.
The extract dried over 7 μm was made into cotton under reduced pressure.
I will do it. In addition to an excess of rl-hexane, a red crystalline compound (0,045 r,
Obtain the i touch point 2tO'C)'. On the other hand, the acidic components present in the bath fraction of the hydrolyzate of i1] were ascertained by the Silica Gel TLO as a comparison with the authentic sample.

Example pH 70M/15M late salt 1bi hardness (5, Oral
) in FCE22724 (0,05v) (to DR4'
β-D -f Lucronidar 7 Cal1J type)'fl
Enzyme-Sigma 50 (9) water bath solution (50, d) and cultured at 67°C. ε-Rhodomidinone [αV) from Inki enal extract, 0.02
2v) is released early.

On the other hand, the presence of D-glucurone (Vl) in the water phase is reconfirmed.

Biological Activity FLDE22724 exhibits cell harvesting activity on Hera cell cloning efficiency in test tubes.

The sos'fr of the growth rhinoceros i11: +s+ is stopped (I D 50 ) and the volcanic intensity (ke 40 Ag/mt).

FCE22724 also inhibits in vivo antifungal activity in mice by inhibiting P588 whitening disease, as shown in Table 1.

4.4 135 0/106
．． 6 160 0/1015
．． 0 165 0/l1j2
2.5 175 1/10 (
8) Peritoneal tissue treatment (bl Median survival time: % above untreated control (cl
The evaluation was based on the necropsy findings of dead mice.

Societa Bel Azio Ichiji

Claims (1)

[Scope of Claims] 1) An anthrazyrinone glucuronide of the general formula A (wherein R represents a hydrogen atom, a lower alkyl group, or an alkali-sensitive atom). 2) Assimilable M-system and nitrate and mineral l trade gold standard/
Anthrax of one-plate formula A (wherein R represents a sodium atom) consisting of culturing the microorganism Streptomyces bucetius (DSM 2597) under aerobic conditions in an aqueous culture medium containing Pi. Production method of cyclinone glucuronide. 6) Culture temperature of 25-67℃ and initial temperature of 6.5-Z
3. A method according to claim 2, which is carried out for 6 to 7 days at a pH of 6.5 to 8.0 at the end of the culture. 4) Furthermore, the hyphae were removed from the culture medium, the hyphae were extracted with a water-miscible organic @ agent, and the extract was concentrated under reduced pressure to obtain the crude anthracycline of General 2A (in the formula, R represents a sodium atom). The method described in Section 2 or Section 6 of the four evils of seeking non-glucuronide. 5) Apply a thick bathing liquid of the crude ornament to the side fat of the hydrophobic cross-linked divinylbenzene car, remove the bath with aqueous lower alcohol, and adjust the pH of the bathing liquid to 4.0. (In the formula, R is aqueous.) The andhracyclinone glucuronide is liberated, the eluent is extracted with methyl isobutyl ketone, and the anthracyclinone glucuronide is extracted by the addition of n-hexane. Precipitating the cronate in pure form and reconverting it to the pure sodium salt by treatment with a significant amount of methanolic sodium hydroxide, followed by precipitation with diethyl ether. A method for purifying crude anthracyclinone glucuronide of General 2A (in the formula, R represents a sodium atom) obtained by any of the methods described in Items 2 to 4. capacity it% n-
6. The method of claim 5, wherein pro-zeta-nol is used. 7) A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 in admixture with a pharmaceutically acceptable diluent or carrier. 8) The microorganism Streptomyces bucedeus var. Vinacecus (DSM 2597).