JP2006160723A - Compound having antitumor activity and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel antitumor substance and its manufacturing method. <P>SOLUTION: An antibacterial agent and an antitumor agent contains a compound represented by formula (I) (wherein X<SB>1</SB>and X<SB>2</SB>are each O or S; R<SB>1</SB>is hydrogen, methyl or phenyl; R<SB>2</SB>and R<SB>3</SB>are each hydrogen, methyl, isopropyl, isobutyl or sec-butyl; and R<SB>4</SB>and R<SB>5</SB>are each hydrogen or methyl) or its salt. The method for manufacturing the compound uses a microorganism. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel compound produced by a microorganism, a production method thereof, and an antibacterial agent and an antitumor agent containing the compound.

  Patent Document 1 and Non-Patent Document 1 disclose a compound having the following structural formula isolated from Streptomyces nobilis as an anticancer substance.

  Patent Document 2 discloses Thermoactinomyces sp. As an anticancer substance. Discloses a compound having the following structural formula separated from:

［式中、Ｒ_１は水素、ハロゲン、シアノ、ヒドロキシル、ニトロ、アジド、置換又は無置換のアルキル、置換又は無置換のアルキリデン、置換又は無置換のアルケニル、置換又は無置換のアルキニル、置換又は無置換のアルコキシ、置換又は無置換のアリール、置換又は無置換のヘテロ環基及び置換又は無置換のアシルからなる群から各々独立に選択され；Ｒ_３は水素、ハロゲン、シアノ、ヒドロキシル、ニトロ、アジド、置換又は無置換のアルキル、置換又は無置換のアルケニル、置換又は無置換のアルキニル、置換又は無置換のアルコキシ、置換又は無置換のアリール、置換又は無置換のヘテロ環基及び置換又は無置換のアシルからなる群から各々独立に選択され；Ｒ_４はＮＲ_２、Ｏ及びＳから各々独立に選択され；Ｒ_２は水素、置換又は無置換のアルキル、置換又は無置換のアリール、置換又は無置換のアルコキシ及び置換又は無置換のアシルからなる群から各々独立に選択される。なおこの式中のＲ_１〜Ｒ_４は本発明の化合物における同名の基とは無関係である。］
についても開示しているが、分離・精製あるいは製造されたものではなく、その存在や活性も明らかではないことから単なる類比推理による拡張構造である。また、特許文献２には化学合成法による上記拡張構造を有する化合物の製造法が述べられているが、実施例が記載されているわけでもなく、論文等の引用にのみ依った推論である。現実にはチアゾール、オキサゾールを有する化学物質の合成化学的手法による製造は困難で、たとえば、Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｎｕｌａｔｕｓから単離された最も強力なテロメアーゼ阻害剤として知られるＴｅｌｏｍｅｓｔａｔｉｎ： Patent Document 2 is a structural formula obtained by generalizing the above structural formula:

Wherein R ₁ is hydrogen, halogen, cyano, hydroxyl, nitro, azide, substituted or unsubstituted alkyl, substituted or unsubstituted alkylidene, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted Each independently selected from the group consisting of substituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group and substituted or unsubstituted acyl; R ₃ is hydrogen, halogen, cyano, hydroxyl, nitro, azide Substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group and substituted or unsubstituted They are each independently selected from the group consisting of acyl; R ₄ are each independently selected from NR _{2, O} and S; R ₂ is hydrogen, location Or unsubstituted alkyl, substituted or unsubstituted aryl, are each independently selected from the group consisting of substituted or unsubstituted alkoxy and substituted or unsubstituted acyl. R _{1 to} R _{4 in} this formula are independent of the group of the same name in the compound of the present invention. ]
However, it is not an isolated, purified, or manufactured product, and its existence and activity are not clear. In addition, Patent Document 2 describes a method for producing a compound having the above extended structure by a chemical synthesis method. However, examples are not described, and the inference is based only on the citation of a paper or the like. In reality, it is difficult to produce chemical substances having thiazole and oxazole by synthetic chemical methods, for example, Telomestatin known as the most potent telomerase inhibitor isolated from Streptomyces anuratus:

は２００１に発表され（非特許文献２）合成研究が進められているが、いまだに達成されていない（非特許文献３）。

Has been published in 2001 (Non-patent Document 2), and synthetic studies are in progress, but it has not been achieved yet (Non-Patent Document 3).

  Humans have used many antibiotics and antibacterial agents such as penicillin and streptomycin to treat infections. However, due to frequent use of antibiotics in the medical field, there is a problem that the used bacteria acquire drug resistance and the antibiotics used do not work. In addition, with respect to tumors such as cancer, if the use of antibiotics is continued, resistance to the antibiotics used occurs.

  At present, attempts are being made to search for useful antibiotics from microbial metabolites, as represented by the aforementioned penicillin and streptomycin (see Patent Documents 3, 4 and 5). In addition, fungi and marine organisms are attracting attention as search resources for physiologically active substances (Non-Patent Documents 4 and 5).

  Discovery of new antibiotics that are effective against pathogens that have acquired resistance to existing antibiotics as described above, and that have an effect on tumors for which existing anticancer drugs do not provide sufficient therapeutic effects Is always desired. In particular, discovery of new antibiotics having antitumor activity is desired.

Japanese Patent Laid-Open No. 11-180997 International Publication WO2005 / 000880 Pamphlet JP 2001-257454 A JP 2000-189182 A Japanese Patent Laid-Open No. 05-247018 Kin-ya Sohda, Koji Nagai, Takao Yamari, Ken-ichi Suzuki and Akihiro Tanaka, The Journal of Antibiotics, 58, 27-31, K. Shin-ya, K .; Wierzba, K.M. Matsuo, T .; Ohtani, Y. et al. Yamada, K .; Furihata, Y .; Hayaka and H.H. Seto, J .; Am. Chem. Soc. , 123, 1262-1263, 2001 Shigeki, Yonezawa Sericulture, Chemistry and Industry, 57, 933-936, 2004 Hiroshi Yoda, Chemistry and Biology, Vol. 40, no. 11, p. 757-764, 2002 John W. Blunt, Brent R. et al. Copp, Murray H.C. G. Munro, Peter T. Northcotte and Michel R. Princep; Natural Products Report, 20, p. 1-48, 2003

  Accordingly, an object of the present invention is to provide a novel antibiotic having antitumor activity.

  As a result of intensive studies to solve the above problems, the present inventors have found that a compound obtained from a culture solution of a microorganism belonging to the genus Thermoactinomyces is novel and has antibacterial activity and antitumor activity. The present inventors have found that the present invention is a useful compound and have completed the present invention.

［式中、
Ｘ_１、Ｘ_２は、互いに独立に、Ｏ又はＳであり、
Ｒ_１は水素、メチル又はフェニルであり、
Ｒ_２、Ｒ_３は、互いに独立に、水素、メチル、イソプロピル、イソブチル又はｓｅｃ−ブチルであり、
Ｒ_４、Ｒ_５は、互いに独立に、水素又はメチルである］
で表される化合物又はその塩。 That is, the present invention includes the following inventions.
(1) Formula (I):

[Where:
X ₁ and X ₂ are independently of each other O or S;
R ₁ is hydrogen, methyl or phenyl;
R ₂ and R ₃ are independently of each other hydrogen, methyl, isopropyl, isobutyl or sec-butyl,
R ₄ and R ₅ are each independently hydrogen or methyl]
Or a salt thereof.

(2) The compound or salt thereof according to (1), wherein in formula (I), X ₂ is O, and R ₄ and R ₅ are both hydrogen.
(3) In formula (I), X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, R ₄ is hydrogen, R The compound or a salt thereof according to (1), wherein ₅ is methyl.

(4) A microorganism belonging to the genus Thermoactinomyces having the ability to produce the compound according to (1) is cultured in a medium, the compound is produced and accumulated in the culture, and the compound is collected. (1) The manufacturing method of the compound of description.

(5) In the compound described in (1), X ₁ is S, X ₂ is O, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is isopropyl, R ₄ and The production method of (4), wherein R ₅ is a compound in which both are hydrogen.

(6) In the compound described in (1), X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, and R ₄ is hydrogen. The production method according to (4), wherein R ₅ is methyl.

(7) In Formula (I), X ₁ is S, X ₂ is O, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is isopropyl, R ₄ and R ₅ A microorganism belonging to the genus Thermoactinomyces having the ability to produce the compound according to (1), wherein both are hydrogen.

(8) In Formula (I), X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, R ₄ is hydrogen, R _A microorganism belonging to the genus Thermoactinomyces having the ability to produce the compound according to (1), wherein ₅ is methyl.
(9) A medicament comprising the compound according to any one of (1) to (3) or a pharmaceutically acceptable salt thereof as an active ingredient.
(10) The medicament according to (9), which is an antitumor agent.

  The compound represented by the formula (I) can form a salt (particularly an acid addition salt) according to a conventional method.

Acid addition salts of compounds of formula (I) include salts with organic or inorganic acids, such as hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, acid phosphate , Acetate, oxalate, maleate, fumarate, succinate, lactate, tartrate, benzoate, citrate, gluconate, saccharide, cyclohexylsulfamate, methane Examples thereof include sulfonates, p-toluenesulfonates and naphthalenesulfonates.
The compound represented by the formula (I) or a salt thereof can also be provided as a solvate.

  The present invention provides a novel antitumor compound, a microorganism having the ability to produce the compound, and a method for producing the compound using the microorganism. The compounds of the present invention are useful as pharmaceuticals and pharmaceutical raw materials.

The present invention is described in detail below.
The compound represented by the above formula (I) has antitumor activity. Accordingly, the compounds of the present invention have use as pharmaceuticals, particularly as antitumor agents. Among the compounds represented by the above formula (I),
Formula (I.1) where X ₂ is O and R ₄ and R ₅ are both hydrogen:

［式中、Ｘ_１、Ｒ_１、Ｒ_２及びＲ_３は式（Ｉ）について定義した通りである］
で表される化合物、及び、
Ｘ_１及びＸ_２が共にＳであり、Ｒ_１がフェニルであり、Ｒ_２がｓｅｃ−ブチルであり、Ｒ_３がメチルであり、Ｒ_４が水素であり、Ｒ_５がメチルである式（Ｉ．２）：

[Wherein X ₁ , R ₁ , R ₂ and R ₃ are as defined for formula (I)]
And a compound represented by:
Formula (I) wherein X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, R ₄ is hydrogen, and R ₅ is methyl. .2):

で表される化合物が好ましい。

The compound represented by these is preferable.

により表される化合物を、最初に海洋性細菌（サーモアクチノマイセス・スピーシーズＹＭ３−２５１株）から単離・精製した。本発明者らはまた、式（Ｉ．１）に包含される他の化合物を調製した。本発明者らはまた、式（Ｉ．２）で表される化合物を海洋性細菌（サーモアクチノマイセス・スピーシーズＹＭ１１−５４２株）から単離・精製した。本発明の化合物の性質及びその製造方法について以下に詳述する。 Among the compounds represented by the above formula (I.1), the present inventors have the following formula (I.1.1):

Was first isolated and purified from a marine bacterium (Thermoactinomyces sp. YM3-251 strain). We have also prepared other compounds encompassed by formula (I.1). The present inventors also isolated and purified the compound represented by the formula (I.2) from a marine bacterium (Thermoactinomyces sp. YM11-542 strain). The properties of the compound of the present invention and the production method thereof will be described in detail below.

1. Properties of the compounds of the invention
1. Properties of the compound represented by the formula (I.1.1) The structural formula and physicochemical properties of the compound represented by the formula (I.1.1) are as follows:
(1) Color of substance: colorless (2) Molecular weight: 708
(3) Molecular _{formula: C 35 H 32 N 8 O} 7 S
(4) Mass spectrometry: high resolution FABMS (fast neutral particle impact ionization mass spectrometry), actual measurement value: 709.2104 (M + H) ⁺ , calculated value: 709.2193 (C ₃₅ H ₃₃ N ₈ O ₇ S)
(5) UV absorption spectrum (in methanol) λmax (log ε) 260 nm (4.73)
(6) [α] _D 110 ° (c = 0.038, DMSO)
(7) ¹ H NMR (measured in deuterated dimethyl sulfoxide, 750 MHz)
δppm: 0.89 (d, 3H, J = 6.8Hz), 0.93 (t, 3H, J = 7.5Hz), 0.95 (d, 3H, J = 6.8Hz), 1.00 (d, 3H, J = 6.8Hz), 1.13 (m, 1H), 1.64 (m, 1H), 2.06 (m, 2H), 4.65 (dd, 1H, J = 6.6, 3.9Hz), 4.78 (t, 1H, J = 8.6Hz), 5.79 (s , 1H), 5.92 (s, 1H), 7.50 (t, 1H, J = 7.5Hz), 7.55 (t, 2H, J = 7.5Hz), 8.31 (t, 2H, J = 7.5Hz), 8.49 (d , 1H, J = 6.9Hz), 8.50 (s, 1H), 8.77 (d, 1H, J = 9.0Hz), 8.95 (s, 1H), 9.06 (s, 1H), 9.09 (s, 1H), 9.95 (s, 1H)
(8) ¹³ C NMR (measured in deuterated dimethyl sulfoxide, 125 MHz)
δppm: 12.01 (q), 14.35 (q), 18.55 (q), 19.13 (q), 26.07 (t), 32.05 (d), 38.66 (d), 56.92 (d), 58.14 (d), 109.31 (t ), 121.37 (d), 126.41 (s), 127.15 (d), 128.48 (d), 128.54 (s), 129.53 (s), 129.57 (s), 129.90 (d), 130.59 (s), 135.50 (s) ), 139.32 (d), 140.22 (d), 140.27 (d), 141.31 (s), 150.35 (s), 152.00 (s), 154.63 (s), 157.14 (s), 157.35 (s), 158.78 (s) ), 159.90 (s), 170.08 (s), 170.71 (s)
(9) Solubility: Soluble in chloroform and dimethyl sulfoxide (DMSO). Insoluble in methanol.
(10) When human lung cancer-derived cell line A549 cells were cultured for 48 hours at a concentration of 40 nM of the compound of formula (I.1.1) of the present invention, cell proliferation of A549 cells was inhibited by 50%, and 48 at a concentration of 200 nM. When cultured for a period of time, A549 cells were completely killed.

1.2. Properties of other compounds represented by formula (I.1) Other compounds represented by formula (I.1) may also have antitumor activity in the same manner as the compounds of formula (I.1.1). confirmed.

1.3. Properties of the compound represented by the formula (I.2) The structural formula and physicochemical properties of the compound represented by the above formula (I) are as follows:
(1) Color of the substance: colorless (2) Molecular weight: 710
(3) Molecular _{formula: C 34 H 30 N 8 O} 6 S 2
(4) Mass spectrometry: FABMS (fast neutral particle collision ionization mass spectrometry), measured value 711 (M + H) ⁺ (C ₃₄ H ₃₁ N ₈ O ₆ S ₂ ), 733 (M + Na) ⁺ (C ₃₄ H ₃₀ N ₈ O ₆ S ₂ Na)
(5) UV absorption spectrum (in methanol) λmax (log ε) 267 nm (4.35), 291 nm (4.52)
(6) Infrared absorption spectrum (KBr) 3393, 2921, 2851, 1654, 1507, 1459 cm ⁻¹
(7) [α] _D ²² = + 38 ° (c = 0.5, CHCl ₃ )
(8) ¹ H NMR (measured in deuterated dimethyl sulfoxide, 750 MHz)
δppm 0.85 (3H, d, J = 6.8 Hz), 0.86 (3H, t, J = 7.5 Hz), 1.09 (1H, m), 1.46 (1H, m), 1.50 (3H, d, J = 6.8 Hz) , 1.82 (3H, d, J = 7.5 Hz), 2.06 (1H, m), 4.52 (1H, dd, J = 8.3, 9.0 Hz), 4.75 (1H, dq, J = 6.8, 7.5 Hz), 6.62 ( 1H, q, J = 7.5 Hz), 7.51 (1H, dd, J = 6.8, 6.8 Hz), 7.55 (2H, dd, J = 6.8, 7.5 Hz), 8.34 (2H, d, J = 7.5 Hz), 8.59 (1H, s), 8.69 (1H, s), 8.75 (1H, br d, J = 7.5 Hz), 8.88 (1H, s), 9.00 (1H, br), 9.07 (1H, s), 9.49 ( 1H, br s)
(9) ¹³ C NMR (measured in deuterated dimethyl sulfoxide, 125 MHz)
δppm 11.09 (q), 13.38 (q), 15.09 (q), 19.80 (q), 25.24 (t), 36.92 (d), 48.13 (d), 58.45 (d), 121.06 (d), 123.05 (d) , 123.70 (s), 126.64 (s), 127.78 (d), 127.78 (d), 128.30 (d), 128.30 (d), 128.78 (d), 129.56 (s), 129.84 (d), 130.52 (s) , 135.50 (s), 139.43 (d), 140.12 (d), 141.93 (s), 147.79 (s), 151.00 (s), 154.02 (s), 155.11 (s), 157.26 (s), 159.65 (s) , 159.95 (s), 161.08 (s), 169.45 (s), 170.30 (s)
(10) Solubility: Soluble in chloroform and dimethyl sulfoxide (DMSO). Slightly soluble in methanol.
(11) When human lung cancer-derived cell line A549 cells were cultured for 48 hours at a concentration of 12 nM of the compound of formula (I) of the present invention, cell growth of A549 cells was inhibited by 50% and cultured at a concentration of 100 nM for 48 hours. A549 cells were completely killed. Further, when Jurket cells, which are human leukemic T cell lines, were cultured for 48 hours at a concentration of 5.6 nM of the compound of the formula (I) of the present invention, the cell proliferation of the Jurket cells was inhibited by 50% and 48 at a concentration of 50 nM. When cultured for a period of time, A549 cells were completely killed.

2. Production of Compound of the Present Invention The compound represented by formula (I) can be produced by culturing a microorganism in a medium, producing and accumulating the compound in the culture, and collecting the compound from the culture. it can. The microorganism that can be used in the present invention is not particularly limited as long as it belongs to the genus Thermoactinomyces and has the ability to produce the compound represented by the above formula (I). The compound represented by the formula (I) can also be produced by chemical synthesis.

2.1. Production of Compound of Formula (I.1.1) The compound of formula (I.1.1) of the present invention is obtained by culturing a microorganism in a medium, producing and accumulating the compound in a culture, and It can be produced by collecting the compound.
(1) Microorganism The microorganism that can be used in the production of the compound of formula (I.1.1) belongs to the genus Thermoactinomyces and is represented by the above formula (I.1.1). The microorganism is not particularly limited as long as it is a microorganism capable of producing a compound.

  Here, “microorganism having the ability to produce the compound represented by the above formula (I.1.1)” typically means that in a conical flask with a 1 L baffle containing 500 mL of marine broth medium. And inoculated at 30 ° C. for 5 days with rotary shaking (100 rpm) to form an inoculum, inoculated with 5 mL each of 80 1L baffled flasks (40 L in total) containing 500 mL of marine broth medium at 30 ° C. The culture solution 40L obtained by culturing for 5 days by rotary shaking (100 rpm) was centrifuged (6000 × g, 20 minutes) to separate the cells and supernatant, and the cells were washed with chloroform / methanol (9: 1). The bacterial cell extract obtained by extraction is purified by silica gel column chromatography to collect a fraction having antitumor activity, and the fraction is purified by high performance liquid chromatography. When the above is repeated, the compound represented by the above formula (I.1.1) is produced in an amount of 3 mg or more, more preferably 4 mg or more, more preferably 5 mg or more, most preferably 6 mg or more per 120 L of the culture solution. Means a microorganism that can.

  Examples of such microorganisms include Thermoactinomyces sp. YM3-251 and mutant strains derived from the strain or similar strains of the strain represented by the above formula (I.1.1). And a compound having the ability to produce a compound. Thermoactinomyces sp. YM3-251 was consigned on May 19, 2004 to the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) Deposited as number NITE P-2.

  The “mutant strain” here is obtained by mutagenesis treatment using any appropriate mutagen, and the term “mutagen” in the broad sense includes not only a drug having a mutagenic effect, for example. It should be understood to include treatments having a mutagenic effect, such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, but any other effect A typical mutagen can also be used.

  As used herein, “similar strain” refers to a 16S r DNA gene represented by 95% or more homologous base sequence of the 16S rDNA gene of Thermoactinomyces sp. YM3-251 (shown in SEQ ID NO: 1). Can be mentioned. The homology of the 16S rDNA gene may be 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 100% homology.

Thermoactinomyces sp. YM3-251 strain is a newly isolated strain from nature, and its bacteriological properties are as follows.
a. Morphological and cultural properties 1) Cell shape and size: forming hyphae.
2) Presence / absence of spore: Yes.
3) Marine agar plate culture: Grows well, colonies are round, trapezoidal, mycelium, milky white.
4) Marine broth culture: Grows well.
5) Litmus milk: No change.
b. Physiological properties 1) Reduction of nitrate: Not reduced.
2) Generation of indole: not generated.
3) Generation of hydrogen sulfide: Not generated.
4) Starch hydrolysis: No degradation.
5) Casein degradation: Decomposes.
6) Degradation of esculin: Does not degrade.
7) Degradation of DNA: Does not degrade.
8) Degradation of chitin: Does not decompose.
9) Tyrosine degradation: Does not degrade.
10) Use of citric acid: Simmons Medium: Not used.
11) Production of pigment: None.
12) Urease activity: Negative 13) Oxidase activity: Positive 14) Catalase activity: Positive 15) Alkaline phosphatase activity: Positive 16) Esterase (C4) activity: Positive 17) Esterase lipase (C8) activity: Positive 18) Esterase (C4) Activity: Negative 19) Leucine Allylamidase Activity: Negative 20) Valine Allylamidase Activity: Negative 21) Cystine Allylamidase Activity: Negative 22) Trypsin Activity: Positive 23) Chymotrypsin Activity: Negative 24) Acid Phosphatase Activity: Positive 25) Naphthol- AS-BI-phosphohydrolase activity: positive 26) α-galactosidase activity: negative 27) β-galactosidase activity: negative 28) β-glucuronidase activity: negative 29) α-glucosidase activity: negative 30) beta-glucosidase activity: negative 31) alpha-mannosidase activity: negative 32) alpha-fucosidase activity: negative 33) Growth range (pH): pH 6-9
34) Salt tolerance: Yes (Growth salt concentration range: 0-7%)
35) Requirement for Mg ²⁺ : Yes 36) Utilization of carbon source (using BiOLOG GP2 microplate)
L-arabinose-
D-xylose-
D-glucose −
D-Mannose-
D-fructose-
D-galactose-
Maltose −
Sucrose −
Lactose −
Trehalose +
D-sorbitol-
D-mannitol +
Inositol −
Glycerol −
Glycogen +
Inulin −
Melobiose +
Raffinose +
Acetic acid +
Pyruvate +
37) GC content: 47 (mol%)
38) Isoprenoid quinone: MK-9 (73%), MK-8 (21.7%), MK-10 (2.9%), MK-7 (2.5%)

(2) Microbial culture In the present invention, a normal microorganism culture method is used for the culture of the microorganism. As the medium, any of a synthetic medium or a natural medium can be used as long as it contains an assimilated carbon source, nitrogen source, inorganic substance, and necessary growth / production promoting substances as appropriate. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses and the like can be used alone or in combination. Furthermore, hydrocarbons, alcohols, organic acids, amino acids (such as tryptophan) and the like can be used as necessary. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soy flour, cottonseed meal, casamino acid, etc. should be used alone or in combination. Can do. In addition, if necessary, inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate can be added. it can. Furthermore, trace components that promote the growth of the microorganisms to be used and the production of the compounds of the present invention can be added appropriately, and those skilled in the art can select appropriate components as such components.

  As a culture method, liquid culture is suitable, but is not limited thereto. The culture temperature is suitably 25 to 37 ° C., the pH of the medium during the culture is desirably maintained at 7 to 9, and it is desirable to carry out the rotation or reciprocal shaking culture at a shaking speed of 30 to 120 rpm. When culturing is usually carried out for 5 to 14 days in liquid culture, the target compound is produced and accumulated in the culture solution and in the cells. Usually, the culture is stopped when the production amount in the culture reaches the maximum.

(3) Isolation / Purification of Compound Isolation / purification of the compound of the present invention from a culture can be performed according to a method commonly used for isolating / purifying a microbial metabolite from the culture. Here, “culture” means any of culture supernatant, cultured cells, or disrupted cells. For example, the culture is divided into culture broth and cells by filtration or centrifugation, and the cells are extracted with a chloroform / methanol (9: 1) mixed solvent or the like. The culture filtrate can be extracted with ethyl acetate, chloroform or the like. Subsequently, the bacterial cell extract, the culture broth, or the extract of the culture broth is concentrated alone or in combination of two or more thereof, and is concentrated by column chromatography, preparative thin layer chromatography, high performance liquid chromatography, or the like. Purification can be carried out to obtain the compound of the present invention. By examining whether or not the obtained compound exhibits the properties described in “1. Properties of the compound represented by the formula (I.1.1)” by an ordinary chemical method such as NMR analysis. The compound of the present invention can be confirmed.

  On the other hand, in order to confirm that the obtained compound has antitumor activity, for example, the obtained compound is added at an appropriate concentration to a microplate seeded with cancer cells and cultured. After culturing, the number of viable cancer cells is measured by, for example, an MTT assay, and compared with the number of cells in the control compound-free group, it is observed whether or not the proliferation of the cancer cells is suppressed.

2.2. Production of other compounds represented by formula (I.1) Specific examples of the compound represented by formula (I.1) include compounds having combinations of substituents shown in Table 1. It is not limited to.

  The compound represented by the formula (I.1) can be prepared, for example, by the procedure shown in the following synthetic route diagram.

1 (N-α-t-butoxycarbonylserine methyl ester) and 2,2-dimethoxypropane are heated to reflux in benzene in the presence of paratoluenesulfonic acid monohydrate to give 2. After removing the methyl group by alkali treatment, 3 is obtained by a condensation reaction with serine methyl ester. 3 is reacted with methanesulfonic acid in the presence of triethylamine to form methanesulfonate, and then diazabicycloundecene is reacted to obtain 4. Bromination of 4 with N-bromosuccinimide in methanol to form an oxazole ring with calcium carbonate and demethanol to give 5. After removing the methyl group of 5, a condensation reaction with phenylserine benzyl ester is performed to obtain 6 (R ₁ = phenyl). 6 is converted to 7 in the same manner as 3 to 5 are synthesized. The Boc (t-butoxycarbonyl) group of 7 and acetonide are removed and brominated to give 8.

  In addition, 9 is obtained by repeating the oxazole formation reaction used in the synthesis of 2 to 5 using 5 as a raw material. 9 is sulfonamidated to give 10.

Next, the condensation reaction of 8 and 10 is performed to obtain 11. The benzyl group of 11 is removed, and condensation reaction with valine benzyl ester is performed to obtain 12 (R ₂ = isopropyl). The benzyl group of 12 is removed, and a condensation reaction with valine benzyl ester is performed again to obtain 13 (R ₃ = isopropyl). Removal of 13 Boc, acetonide, and benzyl groups yields 14. 14 was obtained by intramolecular cyclization reaction, followed by the method used for the synthesis from 3 to 4 with the formula I.I. 1.2 compounds are obtained.

In addition, by changing the amino acid used when synthesizing 6 from phenylserine to threonine, a derivative in which R ₁ is a methyl group is changed, and by changing the amino acid used when synthesizing 12 or 13 from valine to alanine, R ₂ , Derivatives in which R ₃ is methyl can be synthesized, and various derivatives can be obtained by appropriately combining these amino acids.

2.3. Production of Compound of Formula (I.2) The compound of formula (I.2) of the present invention is obtained by culturing a microorganism in a medium, producing and accumulating the compound in the culture, and collecting the compound from the culture. Can be manufactured.
(1) Microorganism As a microorganism that can be used in the production of the compound of formula (I.2), a compound belonging to the genus Thermoactinomyces and represented by the above formula (I.2) is produced. There is no particular limitation as long as it is a microorganism having ability.

  Here, the “microorganism having the ability to produce the compound represented by the above formula (I.2)” typically refers to a 1 L baffled Erlenmeyer flask containing 500 mL of marine broth medium. Inoculate 5 ml each into 50 flasks with 1 L baffle (total 25 L) containing 500 mL of marine broth medium at 30 ° C. for 7 days. Then, 25 L of a culture solution obtained by culturing by rotary shaking (100 rpm) is centrifuged (6000 × g, 20 minutes) to separate the cells and supernatant, and the cells are extracted with chloroform / methanol (1: 1). The cell extract obtained above was purified by silica gel column chromatography to collect a fraction having antitumor activity, and the fraction was purified by high performance liquid chromatography. When repeated, the compound represented by the above formula (I.2) is produced in an amount of 3 mg or more, more preferably 4 mg or more, more preferably 5 mg or more, most preferably 6 mg or more per 25 L of the culture solution. Means a microorganism capable of

  Examples of such microorganisms include, for example, Thermoactinomyces sp. YM11-542, and mutants derived from the strain or similar strains of the strain and represented by the above formula (I.2) Can be mentioned that have the ability to produce Thermoactinomyces sp. YM11-542 was commissioned on January 25, 2005 to the Patent Microorganisms Depositary Center for Product Evaluation Technology (2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture). Deposited under the number NITE P-70.

  For the “mutant strain”, see the description in “2.1. Preparation of compound of formula (I.1.1)” above.

  “Similar strain” refers to a strain having a 16S rDNA gene represented by a base sequence of at least 95% homology with the base sequence of the 16S rDNA gene of Thermoactinomyces sp. YM11-542 (shown in SEQ ID NO: 2). Can be mentioned. The homology of the 16S rDNA gene may be 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 100% homology.

Thermoactinomyces sp. YM11-542 is a newly isolated strain from nature, and its bacteriological properties are as follows.
a. Morphological and cultural properties 1) Spore presence: Yes.
2) Marine agar plate culture: Grows well, colonies are round, trapezoidal, mycelium, milky white.
3) Marine broth culture: Grows well.
4) Litmus milk: No change.

b. Physiological properties 1) Reduction of nitrate: Not reduced.
2) Generation of indole: not generated.
3) Generation of hydrogen sulfide: Not generated.
4) Starch hydrolysis: No degradation.
5) Casein degradation: Decomposes.
6) Degradation of esculin: Does not degrade.
7) Degradation of DNA: Does not degrade.
8) Degradation of chitin: Does not decompose.
9) Tyrosine degradation: Does not degrade.
10) Use of citric acid: Simmons Medium: Not used.
11) Production of pigment: None.
12) Urease activity: Negative 13) Oxidase activity: Positive 14) Catalase activity: Positive 15) Alkaline phosphatase activity: Positive 16) Esterase (C4) activity: Positive 17) Esterase lipase (C8) activity: Positive 18) Lipase (C4) Activity: Negative 19) Leucine Allylamidase Activity: Negative 20) Valine Allylamidase Activity: Negative 21) Cystine Allylamidase Activity: Negative 22) Trypsin Activity: Negative 23) Chymotrypsin Activity: Negative 24) Acid Phosphatase Activity: Positive 25) Naphthol- AS-BI-phosphohydrolase activity: positive 26) α-galactosidase activity: negative 27) β-galactosidase activity: negative 28) β-glucuronidase activity: negative 29) α-glucosidase activity: negative 3 ) Beta-glucosidase activity: negative 31) alpha-mannosidase activity: negative 32) alpha-fucosidase activity: negative 33) Growth range (pH): pH 6-9
34) Salt tolerance: Existence (growth salt concentration range: 0-7%)
35) Requirement of Mg ²⁺ : Yes 36) Generation of acid from carbon source (API50CH used)
L-arabinose-
D-xylose-
D-glucose −
D-Mannose-
D-fructose-
D-galactose-
Maltose −
Sucrose-
Lactose −
Trehalose −
D-sorbitol-
D-mannitol-
Inositol −
Starch −
5-ketogluconic acid +
37) GC content: 45 (mol%)
38) Isoprenoid quinone: MK-9, MK-8
(2) Culture of microorganisms Refer to the explanation of (2) in “2.1. Production of compound of formula (I.1.1)” above.
(3) Isolation / Purification of Compound See the description of (3) in “2.1. Preparation of Compound of Formula (I.1.1)” above. However, a chloroform / methanol (1: 1) mixed solvent is preferable as an extraction solvent for the cells.

3. Use of the Compound of the Present Invention (1) Antitumor Agent The compound represented by the formula (I) of the present invention has an excellent antitumor activity and is useful as an antitumor agent. Can be used as etc.

  The antitumor agent of the present invention comprises a compound represented by the above formula (I) as an active ingredient. The antitumor agent of the present invention comprises a culture containing the compound represented by the above (I), which is prepared according to the method described in the above section “2. Production of the compound of the present invention”. Also good. Furthermore, the antitumor agent of the present invention may further contain other antitumor agents and / or additives. Any other antitumor agent and additive can be used as long as they do not reduce the antitumor activity of the compound of the present invention. Examples thereof include adriamycin, cisplatin, taxol, herceptin and the like. It is done.

  When the antitumor agent of the present invention is used for the purpose of preventing or treating tumors, the subject of prevention or treatment is not particularly limited. For example, prevention or treatment can be used as a specific purpose for at least one kind of tumor such as cancer, sarcoma, or benign tumor. These illnesses can be used, whether they are singly or co-occurring, or are combined with other illnesses other than those described above. These cancer types are not particularly limited, for example, brain tumor, nasopharyngeal cancer, tongue cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, rectal cancer, colon cancer, uterine cancer, ovarian cancer, testicular cancer, osteosarcoma And at least one selected from the group consisting of leukemia. In addition, not only a single cancer but also multiple cancers are included.

  The subject to which the antitumor agent of the present invention is administered is not limited, but mammals such as humans, domestic animals (cattle, horses, etc.), pet animals (dogs, cats, etc.), laboratory animals (mouse, rats). Hamster, etc.).

(2) Administration The antitumor agent of the present invention can be administered to mammals as a pharmaceutical preparation containing a pharmaceutically acceptable carrier or additive together. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum , Gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, etc. Other examples include artificial cell structures such as liposomes. The additive to be used is selected appropriately or in combination from the above according to the dosage form of the pharmaceutical composition.
The antitumor agent of the present invention can be administered by either oral route or parenteral route.

  When the antitumor agent of the present invention is administered orally, the antitumor agent of the present invention is applied to solid preparations such as tablets, granules, powders and pills, or liquid preparations such as liquids and syrups. Can be formulated as In particular, granules and powders can be made into unit dosage forms as capsules, and in the case of liquid preparations, they may be dried products that are redissolved when used.

  Of these dosage forms, oral solid preparations usually contain additives such as binders, excipients, lubricants, disintegrants, wetting agents and the like that are commonly used in pharmaceutical compositions. Oral liquid preparations usually contain additives such as stabilizers, buffers, taste-masking agents, preservatives, fragrances, coloring agents and the like that are generally used in the formulation.

  Moreover, when administering parenterally, it can be set as dosage forms, such as an injection or a suppository. For example, injections are prepared by dissolving or suspending polyalkoxyflavonoids in solutions, suspensions, emulsions, etc., and are usually provided in the form of unit dose ampoules or multi-dose containers. The injection may be a powder that is redissolved in a suitable carrier, such as sterile pyrogen-free water, when used. Examples of the injection technique include intravenous drip injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intradermal injection. These parenteral dosage forms usually contain additives such as emulsifiers and suspending agents which are generally used pharmaceutically in their compositions.

  The amount of the compound of the present invention in the antitumor agent is 1 to 5% by weight, preferably 2 to 3% by weight, based on the total weight, although it varies depending on the use, dosage form and administration route. In addition, the effective amount of the compound of the present invention varies depending on the age of the administration subject, the administration route, and the frequency of administration, and can be varied over a wide range. For example, it is 0.1 to 1000 mg per kg body weight per day, and can be administered several times a day to several weeks.

  Hereinafter, the present invention will be described specifically by way of examples. However, the technical scope of the present invention is not limited by the examples.

Production of Compound of Formula (I.1.1) Thermoactinomyces sp. YM3-251 strain was used as a bacterium producing the compound of formula (I.1.1). The strain was cultured in a 1-L baffled Erlenmeyer flask containing 500 mL of marine broth medium (Difco) at 30 ° C. for 5 days by rotating and shaking (100 rpm), and used as an inoculum for mass culture to be performed next. 5 mL of this inoculum culture was inoculated into 80 1L baffled flasks (40 L in total) containing 500 mL of marine broth medium, and cultured at 30 ° C. for 5 days by rotary shaking (100 rpm). During the culture, the pH of the medium was not particularly controlled.

  The culture solution 40L thus obtained was centrifuged (6000 × g, 20 minutes), and separated into cells and supernatant. The supernatant was extracted twice with an equal volume of ethyl acetate. The cells were extracted with chloroform / methanol (9: 1). Since most of the antitumor activity was observed in the bacterial cell extract, the resulting bacterial cell extract was then purified by silica gel column chromatography. Using Wakogel C200 (Wako Pure Chemical Industries) as a carrier, the mobile layer was first eluted with chloroform, then eluted with chloroform containing 2% (v / v) methanol, and then containing 3% (v / v) methanol. Elution was performed with chloroform and methanol containing 6% (v / v) in 1% increments. Antitumor activity was observed in the fraction eluted with chloroform containing 3% (v / v) methanol and chloroform containing 4% (v / v) methanol.

  Next, the fraction in which the antitumor activity was recognized was subjected to high performance liquid chromatography (column: TSKgel ODS-80Ts (diameter 4.6 mm, length 150 mm), mobile phase A: water, mobile phase B: acetonitrile, gradient: 0 min. Purification for 2 minutes 60% (v / v) acetonitrile, 2 minutes to 18 minutes 60% (v / v) acetonitrile to 70% (v / v) acetonitrile, linear gradient, flow rate 1 mL / min). The compound of the formula (I.1.1) of the present invention was eluted with a retention time of 13 to 14 minutes under the above conditions.

  Such mass culture and purification from the culture were repeated, and 6 mg of the compound of formula (I.1.1) of the present invention was obtained from a total of 120 L of culture. Finally, crystallization from a dichloromethane / methanol mixed solvent was performed to obtain crystals of the compound of formula (I.1.1).

Production of compound of formula (I.2) Thermoactinomyces sp. YM11-542 was used as a bacterium producing the compound of formula (I.2). The strain was cultured in a 1-L baffled Erlenmeyer flask containing 500 mL of marine broth medium (Difco) at 30 ° C. for 5 days by rotating and shaking (100 rpm), and used as an inoculum for mass culture to be performed next. 5 mL of this inoculum culture was inoculated into 50 mL of a 1 L baffled flask (total 25 L) containing 500 mL of 0.1% sucrose-added marine broth, and cultured at 30 ° C. for 7 days with rotary shaking (100 rpm). During the culture, the pH of the medium was not particularly controlled.

  The culture solution 25L thus obtained was centrifuged (6,000 × g, 20 minutes) and separated into cells and supernatant. The supernatant was extracted twice with an equal volume of ethyl acetate. The cells were repeatedly extracted with chloroform / methanol (1: 1). Since most of the antitumor activity was observed in the bacterial cell extraction, the resulting bacterial cell extract was then purified by silica gel column chromatography. Using kieselguhr (Merck) as a carrier, the mobile layer is first eluted with chloroform, then with 2% methanol-containing chloroform, then with 3% methanol-containing chloroform and 1% increments up to 6% -containing methanol. Eluted. Antitumor activity was observed in the fraction eluted with chloroform containing 4% methanol and chloroform containing 5% methanol.

Next, the fraction in which the antitumor activity was recognized was subjected to high performance liquid chromatography (column: TSKgel ODS-80Ts (diameter 4.6 mm, length 150 mm), mobile phase A: water, mobile phase B: acetonitrile, gradient: 0 min. To 2 minutes 45% acetonitrile, 2 minutes to 40 minutes, linear gradient from 45% acetonitrile to 50% acetonitrile, flow rate 1 mL / min). The compound of the formula (I.2) of the present invention was eluted with a retention time of 27 to 29 minutes under the above conditions.
Finally, 6 mg of the compound of the formula (I.2) of the present invention was obtained from a total of 25 L of the culture.

Evaluation of Antitumor Activity of Compounds of Formula (I.1.1) The antitumor activity of the compounds of the present invention was evaluated as follows. Human lung cancer-derived cells A549 cells were seeded in a 96-well microplate at a concentration of 4 × 10 ³ cells / 200 μL / well and pre-cultured in a CO ₂ incubator (5% carbon dioxide, 37 ° C.) for 14 hours. To this was added a compound of formula (I.1.1) that produced a dilution series and cultured for 48 hours. After 48 hours, the number of viable cells was measured using Alamar Blue (purchased from Wako Pure Chemical Industries). As a result, the compound of formula (I.1.1) inhibits cell proliferation of A549 cells by about 50% at a concentration of 43 nM (ie IC ₅₀ value is about 43 nM) and completely kills A549 cells at a concentration of 200 nM. I let you.

The IC ₅₀ value of the existing antitumor agent measured under the same conditions is, for example, that camptothecin is 18 nM, doxorubicin is 283 nM, and paclitaxel is 12 nM. Therefore, the compound of the present invention is an excellent antitumor agent. It can be seen that it is.

Evaluation of antitumor activity of the compound of formula (I.2) The antitumor activity of the compound of the present invention was evaluated as follows. Human lung cancer-derived cells A549 cells were seeded in a 96-well microplate at a concentration of 4 × 10 ³ cells / 200 μL / well and pre-cultured in a CO ₂ incubator (5% carbon dioxide, 37 ° C.) for 14 hours. To this was added the compound of formula (I.2) that produced a dilution series and cultured for 48 hours. After 48 hours, the number of viable cells was measured using Alamar Blue (purchased from Wako Pure Chemical Industries). As a result, the compound of formula (I.2) inhibited cell proliferation of A549 cells by 50% at a concentration of 12 nM, and completely killed A549 cells at a concentration of 100 nM. In addition, Jurket cells, which are human leukemic T cell lines, were seeded in a 96-well microplate at a concentration of 4 × 10 ³ cells / 200 μL / well, and in a CO ₂ incubator (5% carbon dioxide, 37 ° C.) for 14 hours. Pre-cultured. To this was added the compound of formula (I.2) that produced a dilution series and cultured for 48 hours. After 48 hours, the number of viable cells was measured using Alamar Blue (purchased from Wako Pure Chemical Industries). As a result, the compound of formula (I.2) inhibited Jurket cell proliferation by 50% at a concentration of 5.6 nM and completely killed A549 cells at a concentration of 50 nM.

  The present invention provides a novel antitumor compound represented by formula (I) and a method for producing the compound using a microorganism. The compounds of the present invention are useful as pharmaceuticals and pharmaceutical raw materials.

Claims (10)

Formula (I):

[Where:
X ₁ and X ₂ are independently of each other O or S;
R ₁ is hydrogen, methyl or phenyl;
R ₂ and R ₃ are independently of each other hydrogen, methyl, isopropyl, isobutyl or sec-butyl,
R ₄ and R ₅ are each independently hydrogen or methyl]
Or a salt thereof. The compound or a salt thereof according to claim 1, wherein X ₂ in formula (I) is O, and R ₄ and R ₅ are both hydrogen. In Formula (I), X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, R ₄ is hydrogen, and R ₅ is methyl. The compound according to claim 1 or a salt thereof.   A microorganism belonging to the genus Thermoactinomyces having the ability to produce the compound according to claim 1 is cultured in a medium, the compound is produced and accumulated in the culture, and the compound is collected. A process for producing the compound according to 1. A compound of claim 1 wherein there is _{X 1} S is _{X 2} is O, _{R 1} is phenyl, _{R 2} is sec- butyl, _{R 3} is isopropyl, _{R 4} and _{R 5} The process according to claim 4, wherein the compounds are both hydrogen. A compound according to claim ₁ wherein X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, R ₄ is hydrogen, R ₅ The process according to claim 4, wherein is a compound in which is methyl. In formula (I), X ₁ is S, X ₂ is O, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is isopropyl, and R ₄ and R ₅ are both hydrogen. A microorganism belonging to the genus Thermoactinomyces having the ability to produce the compound according to claim 1. In Formula (I), X ₁ and X ₂ are both S, R ₁ is phenyl, R ₂ is sec-butyl, R ₃ is methyl, R ₄ is hydrogen, and R ₅ is methyl. A microorganism belonging to the genus Thermoactinomyces having the ability to produce the compound according to claim 1.   The pharmaceutical which contains the compound in any one of Claims 1-3, or its pharmaceutically acceptable salt as an active ingredient.   The medicament according to claim 9, which is an antitumor agent.