JPS6366193A - Novel anthracycline antibiotic - Google Patents

Abstract

NEW MATERIAL:An antibistic of formula. USE:A carcinostatic agent. PREPARATION:The objective antibiotic substance can be produced by using a known strain or a strain separated from soil, belonging to Actinomyces genus and capable of producing daunomycin, carminomycin and their analogues as a variant source, subjecting the strain to conventional mutagenic treatment e.g. with ultraviolet radiation or N-methyl-N'-nitro-N-nitrosoguanidine and culturing the separated variant [e.g. Streptomyces sp. D788, 4L-660 strain (FERM P-7459)].

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel anthracycline antibiotic having anticancer activity.

(Prior Art) Anticancer anthracycline antibiotics include daunomycin (see US Pat. No. 3,616,242) and adriamycin (US Pat. No. 590,028), these compounds have a broad anticancer spectrum against experimental tumors and are widely used clinically as cancer chemotherapeutic agents. However, although daunomycin and adriamycin exhibit strong anticancer effects, they also have strong side effects such as serious electrocardiographic effects, and are by no means satisfactory as anticancer agents. Therefore, a large number of anthracycline antibiotics have been proposed using various methods such as fermentation methods, semisynthetic methods, and microbial conversion methods.
For example, Japanese Patent Publication No. 51-34915 (Aclacinomycin A and B), Japanese Patent Application Publication No. 57-56494 (4
-demethoxy-11-deoxydaunomycin, etc.), JP-A-15299-1983 (Rhodomycin group antibiotics), etc.].

(Problem to be solved by the invention) As mentioned above, various analogous compounds have been proposed for anthracycline antibiotics as antitumor agents, and some of them are already widely used clinically. Some are being tested. However, it is difficult to find a drug that can satisfy both toxicity and anticancer effects. Furthermore, the results of in vitro tests and animal tests on antitumor agents do not always directly correlate with their anticancer effects in humans, so multifaceted research is required. Therefore, it is desired to propose a new class of anthracycline antibiotics, which have been evaluated as antitumor agents, and which are more effective as clinical drugs.

(Means for Solving the Problems) As a result of repeated research to propose new compounds that can serve as more useful anthracycline antibiotics or synthetic intermediates thereof, the present inventors have produced daunomycin and its analogues. A soil-isolated bacterial strain or a known bacterial strain that has the ability to
-2) A bacterium obtained by ordinary mutation treatment using 0-N-nitrosoguanidine (NTG) exhibits a certain anticancer effect and has a functional group that can lead to various derivatives, The present invention was completed by discovering that the present invention can be used as a synthetic intermediate to produce anthracycline antibiotics.

Accordingly, the present invention provides a novel anthracycline antibiotic tS represented by the following formula.

The compound of the present invention is a novel anthracycline antibiotic characterized by consisting of 4-0-methylacrabinone and daunosamine, which has not been described in the prior literature. Below, the formula (I
) is referred to as D788-16.

(Action/Effect) D788-16 has a strong growth-inhibiting effect on cultured mouse leukemia cells L1210, and is itself useful as a cancer drug.

Note that this effect can be easily confirmed by the following test.

For example, add L1210 cells to RPM1164 medium (Rosewell Bar Research PJr) containing 20% calf serum.
The substance of the present invention was added to this at a concentration of 0.001 to 0.25 μi/ml, and 37
The cells were cultured in a carbon dioxide gas incubator at ℃ for 48 hours, and the 50% growth inhibition concentration relative to the control group was determined. The substance of the present invention was added to M150 acetic acid (pH 3,0) at 11°C g/d.
After dissolving in Dulbecco PBS(-)
(manufactured by Hinaga Pharmaceutical Co., Ltd.) and added.

Furthermore, the L1210 cultured cells described above were suspended in RPM11640 medium containing 10 ml of calf serum at a concentration of 8 x 105 cells/g, and cultured for 1.5 hours in a carbon dioxide incubator at 37°C. The solution of this substance prepared above was added at various concentrations, and after 15 minutes, 14C-uridine (0.05μ
C1/d) 4 pieces of 14C-thymidine (0.05μC
1/go) was added and cultured at 37°C for 60 minutes. Cold 10% lJ chloroacetic acid (TCA) was added to the reaction solution to stop the reaction, and at the same time, acid-insoluble substances were precipitated and collected by centrifugation.

After washing twice with cold 5*TCA 2 m, it was dissolved in formic acid, radioactivity was measured, and the 50% uptake inhibition concentration was determined from the radioactivity uptake rate relative to the control group without additives. The results are shown in Table 1.

50% inhibitory concentration (μg 1) 0788-16 1.2 2.60 1.7
0 The antibiotic D788-16 of the present invention is produced by using a soil-isolated bacterial strain or a known bacterial strain that has the ability to produce daunomycin or carminomycin and related compounds belonging to the genus Actinomyces as a mutagen, for example, by ultraviolet rays or N- Methyl-N'-nitro N-nitrosoguanidine (
This can be carried out by culturing a mutant strain capable of producing the compound D788-16 of the present invention, which is easily isolated by a conventional mutation treatment using NTG), in a medium containing an appropriate nutrient source. Specifically, among these mutant strains, the new anthracycline antibiotic D
Streptomyces sp. D788.4L-660 strain (Feikoken Bacterial Serial No. 7459), which is the producing strain of 788-5 (Patent Application No. 7459), was treated with NTG, and the resulting mutant strain was used to produce YDK-18 strain. I can list them.

This strain was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology on September 3, 1986, as part of the Microbiological Research Institute No. 8952 (FEBM P).
-19,! ;l). less than,
The mycological properties of YDK-18 strain are shown.

Medium form Straight aerial hyphae extend from branched basal hyphae, and whorled branches are not observed. A mature spore chain is a chain of 10 or more spores, and the size of the spores is 0.6 to 0.8
The surface of the spore is smooth, measuring 0.9 to 2.5 microns. Ascospores, flagellated spores, etc. are not observed.

(11) Growth status in various media Standards indicated within 0 for color descriptions are H and D.

Trasner & E, Ja Backum
Author, system of color whe
sla for streptomyees taxo
nomy (J, Appl.

Mieroblol, vol. 11, pp. 335-338, 1
963) was used, and ``Color Standard'' published by the Japan Color Research Institute was used as a supplement.

(iii) Physiological properties (1) Growth temperature range (using yeast and malt agar medium,
20°C, 28°C, 33°C, 37°C at pH 6.0.

Experiment at various temperatures of 42°C): Growth was observed from 20°C to 37°C.

(2) Liquefaction of gelatin (using glucose, bezotone, and gelatin medium and culturing at 20°C): Positive (3)
Hydrolysis of starch (starch, inorganic salt agar medium): Positive (4) Coagulation and (penotonization) of skim milk: Slight coagulation and penotonization are positive (5) Production of melanin-like pigments (trinoton, yeast extract, iron agar) Medium): Positive 4ψ Availability of various carbon sources (on Fridham and Madrive agar media); L-arabinose + 9+ grows well D-xylose + Slightly grows D-glucose
+ D-7 lactose + inositol + L-rhamnose + raffinose ± D-mannitol The strain used for the production of D788-16 according to the present invention is cultured using a strain known per se that is commonly used as a nutritional source for actinomycetes. can be carried out in a medium composition of For example, as a carbon source, glucose, glycerin, sucrose, starch, maltose, animal or vegetable oil, etc. can be used, and as a nitrogen source, soybean flour, meat extract, yeast extract, pegtone, corn steno liquor, jff seed meal 1 fish meal, etc. Organic nitrogen sources such as ammonium sulfate, ammonium chloride, sodium nitrate, ammonium phosphate and the like can be used.

In addition, salt, potassium chloride, phosphate, and other M
g+, Ca", Zn, Fe, Ca,
In addition to adding divalent metal salts such as Mu+ or Nl+, amino acids, and vitamins, various commercially available antifoaming agents such as silicone may be appropriately added to suppress foaming during fermentation.

Fermentation conditions such as temperature, aeration, aeration and fermentation time are selected such that the strain used accumulates the maximum amount of the compound. For example, the temperature is 20 to 40°C, preferably 28°C, -
is 5-9, preferably 6-7, and one fermentation time is 3-9.
It is advantageous to carry out the fermentation for 10 days, preferably 6 days.

To isolate and collect the D788-16 substance from the culture solution,
Can the culture be centrifuged after fermentation?

By filtration in the presence of a suitable filter aid such as diatomaceous earth, the bacterial cells and supernatant liquid are separated into 1ltF liquid.

The supernatant is extracted with M organic solvents such as chloroform, toluene, ethyl acetate, n-butanol, etc. in P) 17-9.

The bacterial cells are extracted using a 0M organic solvent such as acetone, methanol, ethanol, or n-butanol, if necessary. By treating this with chromatography using an adsorption phase such as a synthetic adsorption resin or silica gel, or by using treatment using an anion exchange resin or a cation exchange resin alone or in appropriate combinations, D78
8-16 substances can be purified and collected.

The present invention will be explained in more detail below using examples.

Example 1 Streptosythesis D788, TDK-1
YS (0.3% yeast extract, 1% soluble starch, 1.51 agar, pH
7,2) For slant culture, take a platinum loop and inoculate it into a sterilized 500 d Erlenmeyer flask by dispensing 100 d of the seed culture medium described below, and culture at 28°C in a rotary shaker (220 rpm).
A seed mother was prepared by culturing with shaking for 2 days.

Seed medium Soluble starch 0.5% glucose 0.5% soybean flour (Nissan Meat, Ajinomoto Co., Ltd. ff) 1.01 servings
Salt 0.1 Potassium diphosphate 0.1 Magnesium sulfate (contains 7H20) 0.1 Tap water pH 7.4 (before sterilization) Next, 15 pieces of production medium with the following composition were added and sterilized.
0/volume jar - 1 fermenter with the above seed culture solution 7
501d (equivalent to 5%) was added and inoculated.

Production medium Soluble starch 5 Thymaltose 3 Super dry yeast
3q6 soybean flour (Nissanmeat, mentioned above) 2 Excellent yeast extract
0.2 serving salt
0.11 Magnesium sulfate (contains 7H20
) 0.1 Calcium carbonate
0.2 hours mineral solution”
0.05 Sorry Tap water PH7.4 (before sterilization) Aeration rate 151/min, stirring 350 rpm, 28℃, 6
Incubate for days. The jar fermenter culture solution is collected and separated into bacterial cells and supernatant by centrifugation. Acetone 81 is added to the bacterial cells, stirred well, and then filtered to obtain acetone-extracted P solution. After concentrating this under reduced pressure to approximately 2 mm, the pH was adjusted to 8.0 with 4N caustic soda and extracted with chloroform 41 (total amount). On the other hand, regarding the culture supernatant, after adjusting the voice to 8.0 with 4N caustic soda,
27(1) Extract twice with chloroform. The chloroform extract from the bacterial cells and the chloroform extract from the supernatant are mixed and concentrated to a small volume. Excess n-hexane was added to this to precipitate the extract, which was dried and D788-1
A coarse powder (3, 4, V) containing 6 substances was collected.

Example 2 The crude powder 3.411 obtained in Example 1 was dissolved in chloroform 500
mt, and add this to 300ml of 0.1M acetic acid buffer.
Extracted three times. The aqueous extract fractions were combined and washed twice with 300 g of toluene. Next, 4N caustic soda was added to the aqueous layer to adjust the pH to 300 ml of chloroform. Extracted twice. The mixture was concentrated and dried to obtain 983 Ing of a partially purified yellow powder.

Example 3 200 Da of Part N powder obtained in Example 2 was subjected to preparative high performance liquid chromatography as described below.

That is, dissolve the entire amount in the following liquid chromatography developing solvent 9 rrt, 1
．． Chromatography was performed in 6 times of 5 d each.

High performance liquid chromatography conditions: Waters M-600OA Detector: Waters Model 440 (λ285
(using nm) Flow rate: 5 d/min Fraction d Fractions 3 to 14 were collected and 6 batches were mixed. After adding 4N caustic soda solution to adjust the pH to 0, the mixture was extracted with chloroform in a total amount of 5QQd. Washed with water, concentrated and dried, approximately 90
D78B-13 powder 134+119 of purity was obtained.

Example 4 134 µm of the powder obtained in Example 3 was then further subjected to MM using preparative silica gel thin layer chromatography. Preparative Silica Del Thin Layer Grate PF254 (Merck: 20cr
nX20α) 20 sheets, bottom end 2 closed position.

A sample solution dissolved in chloroform-methanol (15:1) was applied to the entire sample, air-dried, and developed with a chloroform-water-acetic acid-ammonia water (120:50:5:1:1) system. The D786-16 fraction was scraped off and eluted with a chloroform-methanol (4:1) mixture. Concentrate to dryness and add 3Qa/0.1M acetate buffer (μm3.
0) was added and dissolved, and after removing insoluble matter by centrifugation, the mixture was washed twice with 101ml of toluene. After adjusting the voice to 8.0 with 4N caustic soda, it was extracted with chloroform. The five chlorophor extract layers were washed with water, dehydrated by adding mirabilite, and concentrated to a small volume. Approximately 10 times the volume of n-hexane was added to precipitate, collected by centrifugation, and dried under vacuum.
D788-16 with a purity of 8.8% was obtained from 86%.

The physicochemical properties of the D788-16 compound obtained in the above examples are as follows.

A) Properties: yellow powder B) Melting point: 138-141°C (decomposed) C) (α), +106' (C0,02, in chloroform) D) UV-visible absorption spectrum: λ901CH30Hnm (El m,.

wax 1crs204 (3
99), 229 (700), 258 (405).

λ0.01NHCz-904CH,OHnm (,1%
).

max 1c
rII205 (605), 229 (797), 258 (
421).

λO-01N NaOH-90%CHs OHnm C
E1%) :max
1 cm207 (750), 230 (49
5), 251 (fi20).

323(84), 511(94) E) Infrared absorption spectrum (KBr) (vc+n)
:3430.2950.1740.16B0,1640
゜1595.1450,1390,1300,1255
゜1130.1020.990,850,760F)
FD-MS spectrum: m7 z 5s s (M+1t > (C29H
! , 301ON as m, w555) #390 (M-daunosamine) G) Proton nuclear magnetic resonance spectrum (400MHz)
: Proton δ ppm H-17,97,d (J=8.0Hz) H-27
,76,t CJ=8.0Hz)H-37,38,
d (J=8.0Hz) OCH3-44,08,5 H-75,29,bs Ha-82,33,d (J=15.0Hz) Hb
-82,53,dd (J=15.0 & 4.0H
z) H-104,10, m CHs-163,69, a H-117,63,5

Claims (1)

[Claims] ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A novel anthracycline antibiotic.