JPH08119992A - New antitumor substance - Google Patents

Abstract

PURPOSE: To obtain a new antitumor substance useful as an antitumor agent, having low adverse actions and high effects, comprising a new peptide having inhibitory activity against multiplication of tumor cells, by culturing a bacterium belonging to the genus Streptomyces, capable of producing a specific antitumor substance. CONSTITUTION: This new antitumor substance comprises a cyclic peptide of the formula, has inhibitory activity against multiplication of tumor cells, has low adverse actions, is more effective and readily usable and is useful as an antitumor agent. The new antitumor substance is obtained by subjecting a bacterium [e.g. Streptomyces nobilis (ATCC19,252)], belonging to the genus Streptomyces, capable of producing the new antitumor substance, to species culture, inoculating the whole amount to a medium, subjecting to shaking culture at 30 deg.C for 8 days, centrifuging the cultured solution, adding ammonium sulfate to the prepared supernatant liquid, stirring the liquid for 1 hour, ultracentrifuging to separate the precipitate, dissolving it in water, extracting with ethyl acetate and purifying the extract by chromatography.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antitumor substance comprising a novel peptide and an antitumor agent containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art Many tumor substances have already been reported, and those derived from microorganisms have been reported such as mitomycin C, bleomycin, peplomycin, neocartinostacin, daunomycin, adriamycin, acranomycin and actinomycin D. .

[0003]

SUMMARY OF THE INVENTION In the use of antitumor substances, there are demands for more effective and easy-to-use antitumor substances having inevitable side effects and few side effects.

The object of the present invention is to provide a more effective novel tumorigenic substance.

[0005]

According to the present invention,

Embedded image An antitumor substance consisting of the novel peptide represented by (hereinafter referred to as the peptide of the present invention) (hereinafter referred to as the antitumor substance of the present invention) and an antitumor agent containing the peptide of the present invention as an active ingredient are provided.

The antitumor substance of the present invention is a strain producing the antitumor substance of the present invention belonging to the genus Streptomyces, for example, Streptomyces nobili.
s, hereinafter abbreviated as “S. nobilis”), and the obtained culture solution, a dried product of the same solution, or an extract extracted from the cultured cells with an organic solvent is subjected to various column chromatography. It can be obtained by recrystallizing a column chromatography fraction containing the desired product.

The actinomycete S. Nobilis is available from public preservation institutions, for example the preservation strain of the Institute of Physical and Chemical Research (JCM4274) (this is ATCC 19252 in the US and C in the Netherlands).
Bacteria such as preserved as BS198.65) can be used.

Actinomycete S. Cultivation of Nobilis is performed using a medium containing appropriate nutrients. In the case of liquid culture, an aqueous solution containing one or several kinds of sugars such as glucose, proteins such as peptone and malt extract, vitamins, nucleic acids, amino acids, and complex carbohydrates is preferable as a component of the liquid culture. Used. A typical medium is starch
Ammonium liquid medium (soluble starch, K ₂ HP
(Including O ₄ and NH ₄ Cl). P of liquid medium
H is preferably 2 to 9, and the culture temperature is preferably 15 to 42 ° C. The preferable culture time for liquid culture is 1 to 14 days. Thus obtained S. The peptide of the present invention is extracted from the culture solution of Nobilis or its dried product or the cells themselves using a solvent.

S. The culture solution of Nobilis or its dried product or the extract of the cells themselves may be treated with ammonium sulfate, and the resulting precipitate may be subjected to solvent extraction. In ammonium sulphate treatment, ammonium sulphate to be added to the culture broth or its dried product or the extract of the cells themselves may be in a solid state or in a solution state. Ammonium sulfate is preferably added in a saturated concentration of 30 to 9
It is in the range of 0% by weight. The treatment time is not particularly limited, but is preferably in the range of 30 minutes to 5 hours. When centrifuging to obtain a precipitate, the centrifugal force is 3,000 G
~ 20,000 G, centrifugation time is 2
It is preferably -60 minutes.

The solvent used for extraction is preferably an organic solvent, and typical examples thereof include esters such as ethyl acetate, alcohols such as methanol, ethanol and propanol, ethers such as ethyl ether and dioxane, acetone and methyl ethyl ketone. In addition to the above ketones, dichloromethane, chloroform, etc. can be mentioned,
Solvents that can be used are not limited to these. It is also possible to use a mixture of the above solvents. Particularly suitable solvents are ethyl acetate, dichloromethane, acetone and the like. The extraction time varies depending on the type of solvent and extraction temperature,
It is preferably in the range of 3 to 120 minutes. The liquid may be left standing or stirred during the extraction. Preferably, the extraction operation is repeated a plurality of times for the same sample. The extraction temperature is not particularly limited.

Next, column chromatography for the solvent extract will be described.

As a packing material for column chromatography, ODS (octadecylsilanes such as octadecyldimethylchlorosilane) is preferable. The filling amount is not particularly limited, but it is preferable to fill 10 to 500 times the weight ratio with respect to the solvent extract to be charged. When charging the column with the solvent extract, it is preferable to first adsorb it on the ODS. At this time, the amount of ODS is not particularly limited, but it is preferable that the extract is adsorbed on the extract by using ODS in an amount of 0.5 to 20 times the weight of the solvent extract to be adsorbed, and then the extract-adsorbed ODS is added in a small amount. Suspend in the same solvent and charge the column. The elution solvent is not particularly limited, but a solvent having a polarity between an aqueous solution containing 80% of a mixed solvent of methanol: acetonitrile = 1: 1 and methanol is preferably used.

Next, purification by recrystallization of the peptide of the present invention will be described.

The solvent used for recrystallization is not particularly limited as long as it can dissolve the peptide of the present invention, but is preferably methanol, ethanol or the like. As a method of recrystallization, the column elution fraction containing the substance may be dissolved in a small amount of a solvent under heating, and the resulting solution may be gradually cooled to recrystallize the substance. The column elution fraction may be dissolved in a solvent having a high solubility for the substance, and a liquid having a low solubility for the substance (for example, water) may be gradually added thereto to recrystallize the substance.

The peptide of the present invention is an antitumor substance which is effective for allergic and non-allergic tumors, as can be seen from the results of the pharmacological test described later.

In order to formulate the antitumor substance of the present invention, it is usually used in the form of a pharmaceutical composition together with a pharmaceutical carrier.
As a carrier, a filler, a disintegrating agent, a bulking agent, a binder, a coloring agent, a flavoring agent, a pH adjusting agent, a solubilizing agent, a suspending agent, which are usually used for preparing a drug depending on the dosage form, Examples include buffers, stabilizers, preservatives, fillers, surfactants, lubricants, and excipients. By selecting an appropriate solvent,
The obtained antitumor substance of the present invention can be used as it is as a liquid preparation for external use.

As the dosage unit form of the antitumor agent prepared by using the antitumor substance of the present invention, in addition to the above external liquid preparation, tablets, pills, liquid drinks, powders, suspensions, emulsions,
Granules, extracts, fine granules, syrups, dips, decoctions,
Examples include eye drops, troches, poultices, liniments, lotions, eye ointments, plasters, capsules, suppositories, injections (solutions, suspensions, etc.), patches, ointments and the like.

The amount of the antitumor substance of the present invention to be contained in the antitumor agent is not particularly limited and may be appropriately selected within a wide range, but is preferably 0.1 to 50% by weight in the antitumor agent. .

The antitumor agent obtained from the antitumor substance of the present invention is administered by a method suitable for various forms in its use. For example, in the case of the above external liquid preparation, this is directly applied to a required site such as skin or mucous membrane, and tablets,
Orally administered in the case of pills, drinking solutions, suspensions, emulsions, granules and capsules, intravenous in the case of injections,
It is administered intramuscularly, intradermally, subcutaneously or intraperitoneally, rectally in the case of suppositories, and applied or applied in the case of patches and ointments.

The dose of the antitumor agent obtained from the antitumor substance of the present invention is appropriately selected depending on the purpose of use, symptom, etc., but is usually 0.001 per day as the antitumor substance of the present invention.
The range is about 50 mg / kg. Further, it goes without saying that the above-mentioned pharmaceutical composition may be administered 1 to 4 times / day in divided doses.

[0021]

EXAMPLES Example 1 Actinomyces S. cerevisiae obtained from RIKEN. Nobilis (JCM
4274) shake culture (pre-preculture) for 40 hours in 100 ml of starch / ammonium medium with 0.2% yeast extract.
Then, subsequently, 3 liters of the same medium was inoculated with 60 ml of the pre-preculture liquid, and shake culture (seed culture) was carried out for 25 hours. Furthermore, 285 liters of starch / ammonium medium (containing 1 g of soluble starch, 0.05 g of dipotassium hydrogen phosphate and 0.05 g of ammonium chloride in 100 ml of distilled water) were inoculated with the whole seed culture, and inoculated at about 30 ° C. for 8 hours. Culture was carried out with shaking for one day. This culture solution was centrifuged, 662 g of ammonium sulfate per liter of the supernatant was added to 250 liters of the obtained supernatant, and the mixture was stirred for 1 hour. Then 20,000G (flow rate 36
Continuous centrifugation was performed at 0-500 liters / hour) to obtain about 12 kg of precipitate.

To 1.2 kg of the precipitate obtained above, 3.6 liters of water was added and stirred at 80 ° C. for 30 minutes.
Next, an equal amount of ethyl acetate was added, and after shaking for 10 minutes, the whole was centrifuged at 5,000 G for 10 minutes. After separating the ethyl acetate phase, this operation was repeated several times for the aqueous phase to obtain a solvent extract.

Next, the solvent extract obtained as described above is
ODS (octadecyldimethylchlorosilane) carrier 25
Adsorbed to g. Then, the extract adsorption carrier was charged on the carrier in a column having a diameter of 3.2 cm packed with 275 g of ODS carrier to prepare an ODS column. This O
Purification was performed under the following conditions using a DS column. As elution solvent a) methanol: acetonitrile: water = 7:
1.5 liter of 7: 6, 1.2 liter of b) methanol: acetonitrile: water = 8: 8: 4, c) 150 ml of methanol: acetonitrile: water = 9: 9: 2,
d) Methanol: acetonitrile: water = 19: 19: 2
1 liter, and e) 1 liter of methanol were flown in this order at a flow rate of 10.5 ml / min. Fractionation was performed every time the solvent composition was changed, and particularly the elution fraction of methanol: acetonitrile: water = 19: 19: 2 was fractionated in small portions (every 2 minutes) using an appropriate fraction collector. For each elution fraction, ODS-80TM, inner diameter 4.6 mm x
HPL using a 25.0 cm long Tosoh column
C (Hitachi, pump L-6000L-6200, detector L-3000, column open 655A-52), detection wavelength 210 nm, column temperature 40 ° C., flow rate 1
Water: acetonitrile as eluent under conditions of ml / min:
Methanol = 6: 7: 7 (0 minutes) to 0: 1: 1 (30
Min) was used for analysis. Of the above fractions, only those containing a peak with a retention time of 18 to 20 minutes were collected, made into the same fraction (100 mg), and repeatedly recrystallized using methanol-water to obtain 45 mg of needle crystals.

The structure of this material was determined to be of formula [I] from various instrumental analysis data.

Example 2 Culture was carried out in the same manner as in Example 1, and the obtained bacterial cells 215 were obtained.
2.15 liter of dichloromethane was added to g (wet weight), followed by ultrasonic treatment for 30 minutes, and then 8.6 liter of dichloromethane was further added, and the whole was stirred at room temperature for 1 hour. After the bacterial cells were filtered off, the obtained filtrate was concentrated to dryness. The dried solid matter of this extract was dissolved in a small amount of hexane, and the hexane phase was extracted with methanol three times. The hexane-insoluble matter and the methanol extract were redissolved in methanol.

Next, the solvent extract obtained as described above is
It was adsorbed on 28 g of ODS carrier. Then, ODS carrier 2
About 30 g of the above extract adsorption carrier was charged on the above carrier in a column having a diameter of 3.2 cm filled with 75 g to prepare an ODS column. Purification was carried out using the ODS column under the following conditions. As elution solvent a) 1.5 liters of methanol: acetonitrile: water = 7: 7: 6, b)
Methanol: acetonitrile: water = 8: 8: 4 to 1.2
Liter, c) methanol: acetonitrile: water = 9:
200 ml of 9: 2, 1 liter of d) methanol: acetonitrile: water = 19: 19: 2, and 500 ml of e) methanol were flown in this order at a flow rate of 10.5 ml / min. Fractionation is performed each time the solvent composition is changed, and especially the elution fraction of methanol: acetonitrile: water = 19: 19: 2 is gradually added (2
Fractionated). For each elution fraction, ODS-8
HPLC using a column made by Tosoh Co., Ltd., 0TM, inner diameter 4.6 mm × length 25.0 cm (Hitachi, pump L-6)
000L-6200, detector L-3000, column open 655A-52), detection wavelength 210 nm,
Under conditions of a column temperature of 40 ° C. and a flow rate of 1 ml / min, water: acetonitrile: methanol = 6: 7: 7 (0
Analysis was performed using (min) to 0: 1: 1 (30 min). Of the above fractions, only those containing a peak with a retention time of 18 to 20 minutes were collected and made into the same fraction (840 m
g), repeated recrystallization using methanol-water,
330 mg of the present peptide in the form of needle crystals was obtained.

The structure of this substance was determined to be the formula [I] from various instrumental analysis data in the same manner as in Example 1.

Structural analysis data The instrumental analysis data of the substances obtained in Examples 1 and 2 are shown below.

[0029]

[Table 1] 1. MS-ESI-MS: m / z = 913.6 (M + H-H
₂ O) ⁺ , 931.6 (M + H) ⁺ , 953.6 (M +
Na) ⁺

[Table 2] HRFAB-MS Found: m / z = 913.5079 (M + H-H
₂ O) ⁺ , m / z = 913, 953, 931 (913 is main, 931 is very small) Calcd for: C ₄₅ H ₆₉ N ₈ O ₁₂ m / z = 913.0553

[Table 3] 2. IR IR: 3,400 cm ^-1 : -OH, -NH, 2,900 cm ^-1 : alkyl group, 1,750 cm ^-1 : -C (= O) -O-, 1,650 cm ^-1 : -C (= O) -NH-

3. Amino acid analysis D-serine, L-alanine and D- as hydrolysates
N-methyl-phenylalanine was found.

4. Compositional formula It was found from the mass spectrometric analysis and NMR data described later that the compositional formula of the novel peptide was C ₄₅ H ₇₀ N ₈ O ₁₃ .

5. NMR a) partial structure A result of HMBC spectrum measured, alanine (Ala) of the carbonyl carbon (173.6ppm) and N-CH ₃ - CH ₃ phenylalanine (Phe) (3.04ppm)
The partial structure of A was estimated by observing a long-range correlation between and.

[0033] b) partial structure B, ¹ H- ¹ H COSY for C, 2 with similar spin system from HOHAHA
Two components were estimated.

[0034]

[Table 4] i) NH (4.38ppm) CH ₂ (46.1, 2.89, 3.15ppm) CH ₂ (21.3, 1.45, 1.63ppm) CH ₂ (24.2, 1.93, 2.25ppm) CH (51.7, 4.90ppm) ii) _{NH (3.93ppm) CH 2 (47.9} , 2.56, 3.30ppm) CH 2 (21.5, 1.55, 1.66ppm) CH 2 (24.4, 1.67, 2.58ppm) CH (52.5, 5.18ppm)

Since the ¹³ C shifts of both are in good agreement, they are considered to have the same structure (residue). Furthermore, CH ₂
Since the protons are not equivalent, it can be assumed to be a cyclic structure.

This shift is due to monamycin
This component was presumed to be Pip (the left part of the partial structure B and the partial structure C) because it matches the one of Piperazic acid (Pip) contained in.

[0037]

[Table 5] α β γ δ Pip ¹⁾ 49.8 24.1 21.0 47.0 (CDCl ₃ solution) Actual value 52-53 24-25 21 46-48 (CDCl ₃ solution)

6.40 ppm of serine (Ser)
The partial structure B was inferred from the fact that a long range was observed between the NH 3 and the Pip carbonyl of 168.9 ppm.

1) CHHassall, WAThomas, MCMosch
idis, J. Chem. Soc., Perkin Trans.I, 1977, 2371 (1977)

C) Regarding the partial structure D, from the ¹ H- ¹ H spin bond, (CH ₃ ) ₂ —CH—CH—
Partial structure of CH-NH- is introduced and long range ¹
This partial structure was confirmed by the H- ¹³ C bond, and 1
The structure of β-OH-leucine (Leu) was found from the spin bond between 70.6 ppm carbonyl and 4.88 ppm CH, and 5.46 ppm CH to β-OH-Le.
It was presumed that u was ester-bonded at its β-position.

¹ H- ¹ H spin coupling, long range ¹
H- ¹³ C bond and NOE (Nuclear Overhauser Effect)
From the measurement result of 1., the partial structure of the side chain portion (left) of the partial structure D was found.

From the long range ¹ H- ¹³ C bond, β-
OH-Leu 4.88 ppm CH and 8.34 p
The partial structure D was estimated by the long-range correlation observed between pm NH and 177 ppm carbonyl in the side chain.

D) Chemical Structure of Novel Peptide From the long-range ¹ H- ¹³ C bond, Ser.
5.46 p of 2 ppm carbonyl and β-OH-Leu
Since the long-range correlation was observed with pm CH, the partial structure B and the partial structure D were Ser and β-OH-Le.
It was found that there was an ester bond between u. Also,
5.46 ppm CH of β-OH-Leu and 4.38 ppm NH of Pip (substructure C), β-OH-Leu
8.34 ppm of NH and Pip (substructure C) of 2.
CH ₂ of 89ppm, NOE to the CH of 4.90ppm
Was observed, the partial structure C was β-OH-Leu.
It was found to be bonded to the carbonyl group of 170.6 ppm of The remaining partial structure A was bound by one method, and the chemical structure of the novel peptide was determined.

Further, 6.40 ppm of Ser and NH and N
-CH ₃ -Phe contains N during 3.04 ppm CH _3.
The observation of OE suggests that the above structure is correct.

E) The NMR measurement results are as follows.

Tables 6 and 7 show ¹³ C shifts and carbon types, and ¹ H bound directly, and Tables 8 to 10 show ¹ H shift, ¹ H bound to spin bound, ¹³ C bound to ¹³ H, and ¹³ bound to long range bound. C is shown, Tables 11 and 12 show NOE measurement results, and Tables 13 to 16 show attributions of ¹³ C and ¹ H.

[0047]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Chemical 4]

Embedded image

[Chemical 6]

[Chemical 7]

Embedded image Pharmacological Test Test Example 1 Effect on Tumor Cell Proliferation The effect of the peptide of the present invention on tumor growth was examined by the following method.

First, the peptide of the present invention was dissolved in methanol to prepare solutions having various concentrations to prepare test solutions.

Mouse leukemia cells P38 were used as test bacteria.
8D1, mouse leukemia cells L-1210, and mouse melanoma cells B-16 were used and cultured in RPMI medium containing 10% FCS at 5 × 10 ⁴ cells / ml. A cell suspension was added to a 96-well plate at 0.20 ml / well, 0.01 ml of a test solution was further added, and the cells were cultured in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 24 hours. It was Cell proliferation was confirmed by trypan blue exclusion test. Cell proliferation of B-16 was confirmed by the neutral red staining method.

As a control for this test, drug-free methanol was used in place of the test solution impregnation solution,
Other points were the same as the above operation.

Each of the tests was carried out with n = 3, and the minimum inhibitory effective dose for the cell growth of tumor cells was determined. The test results are shown in Table 17.

As is clear from this table, in the group to which the test solution containing the peptide of the present invention was administered, cell proliferation was obviously suppressed as compared with the control group.

[0053]

Table 17 Inhibitory effect of tumor cells on cell growth Test cells Minimum effective amount Mouse leukemia cell P388D1 0.005 μg / ml Mouse leukemia cell L-1210 0.005 μg / ml Mouse melanoma cell B-16 0.010 μg / ml

[0054]

INDUSTRIAL APPLICABILITY According to the present invention, a novel antitumor substance which is effective in inhibiting tumor cell growth can be provided.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1: 465) (72) Inventor Takashi Inagaki 2-1 Hyakusan, Shimamoto-cho, Mishima-gun, Osaka 2-1 Sekisui Chemical Industry Co., Ltd.

Claims (2)

[Claims] Claims: An antitumor substance consisting of the peptide shown in. 2. [Chemical formula 2] An antitumor agent comprising the peptide represented by as an active ingredient.