JP4511413B2 - Pharmaceutical composition - Google Patents

Description

  The present invention relates to a novel compound, and particularly to a novel compound extracted and isolated from an Asteraceae plant or an Acrobaceae plant. In addition, the present invention relates to a pharmaceutical use of a known substance extracted and isolated from a asteraceae plant or a red plant of the genus Asteraceae.

  An Asteraceae plant or a plant of the genus Chrysantaceae, in particular, Nitobegiku, is known to have pharmacological effects such as suppression of skin diseases (aquatic diseases), hepatitis, jaundice, and cystitis. For example, according to Japanese Patent No. 2609780 (Patent Document 1) and Japanese Patent Application No. 8-127538 (Patent Document 2), a therapeutic agent for diabetes using decoction of Nitobegiku or an extract thereof is proposed. .

において、Ｒ_１およびＲ_２が水酸基を示す化合物（以下、「ＴＤ−７」という）、Ｒ_１ が水素原子を示しＲ_２が水酸基を示す化合物（以下、「ＴＤ−９」という）、Ｒ_１が水素原子を示しＲ_２がメトキシ基を示す化合物（以下、「ＴＤ−１０」という）が開示されている。 Also, the same compounds as those isolated from Nitobegi extract are disclosed in the literature. For example, Raghwendra Pal, et al. Indian Journal of Chemistry, Section B: Organic Chemical Including Medicinal Chemistry, 14B, 259-262 (1976) (Non-Patent Document 1), Raghwendra Pal, et al. Indian Journal of Chemistry, Section B : Organic Chemical Including Medicinal Chemistry, 15B, 208-211 (1977) (Non-Patent Document 2), Schuster A, et al. Phytochemistry, 31, 3139-3141 (1992) (Non-Patent Document 3) In order, compounds represented by the following general formula (I):

A compound wherein R ₁ and R ₂ represent a hydroxyl group (hereinafter referred to as “TD-7”), R ₁ Discloses a compound in which R ₂ represents a hydrogen atom and R ₂ represents a hydroxyl group (hereinafter referred to as “TD-9”), and a compound in which R ₁ represents a hydrogen atom and R ₂ represents a methoxy group (hereinafter referred to as “TD-10”). Has been.

において、Ｒ_３およびＲ_４がメトキシ基を示す化合物（以下、「ＴＤ−１」という）、Ｒ_３がメトキシ基を示しＲ_４が水酸基を示す化合物（以下、「ＴＤ−４」という）が開示されている。 Paulo Sergio Pereira, et al. Phytochemistry, 45, 1445-1448 (1997) (Non-Patent Document 4) and C. Zdero et al. Phytochemistry, 26, 1999-2006 (1987) (Non-Patent Document 5) A compound represented by the general formula (II):

In which R ₃ and R ₄ represent a methoxy group (hereinafter referred to as “TD-1”), a compound wherein R ₃ represents a methoxy group and R ₄ represents a hydroxyl group (hereinafter referred to as “TD-4”). Has been.

で表される化合物（以下、「ＴＤ−２」という）が開示されている。
Baruah Nabin C., et al. Journal of Organic Chemistry, 44, 1831 - 1835 (1979)（非特許文献７）には、下記の一般式（Ｖ）：

で表される化合物（以下、「ＴＤ−８」という）が開示されている。
Antonia G. Gonzalez, et al, Journal of Natural Products, 57, 400 - 402 (1994)（非特許文献８）では、（４Ｓ，５Ｒ）−４−ｈｙｄｒｏｘｙ−４−［（１Ｅ，３Ｒ）−３−ｈｙｄｒｏｘｙ−１−ｂｕｔｅｎｙｌ］−３，３，５−ｔｒｉｍｅｔｈｙｌ−ｃｙｃｌｏｈｅｘａｎｏｎｅ）が開示されている。 Baruah Nabin C., et al. Journal of Organic Chemistry, 44, 1831-1835 (1979) (Non-Patent Document 6) includes the following general formula (IV):

(Hereinafter, referred to as “TD-2”).
Baruah Nabin C., et al. Journal of Organic Chemistry, 44, 1831-1835 (1979) (Non-Patent Document 7) includes the following general formula (V):

(Hereinafter referred to as “TD-8”).
In Antonia G. Gonzalez, et al, Journal of Natural Products, 57, 400-402 (1994) (Non-Patent Document 8), (4S, 5R) -4-hydroxy-4-[(1E, 3R) -3- hydroxy-1-buteneyl] -3,3,5-trimethyl-cyclohexone).

  On the other hand, in Raghwendra Pal., Et al. Journal of Phamaceutical Science, 65, 918-920 (1976) (Non-patent Document 9), it was reported that tagitinin A to F were isolated from the extract of Nitobegiku, In addition, among these compounds, it has been reported that only Tagitinin F gave a life-prolonging effect to mice transplanted intraperitoneally with P388 mouse leukemia. Here, tagitinin A corresponds to TD-7, tagitinin C corresponds to TD-2, tagitinin D corresponds to TD-9, and tagitinin E corresponds to TD-8, while tagitinin F corresponds to TD- It does not correspond to 1-10.

で表される化合物（以下、「ＴＤ−１１」という）が開示されている。
Isao Agata, et al. Yakugaku zasshi, 101, 1067-1071 (1981)（非特許文献１１）には、下記の一般式（ＶＩＩ）：

で表される化合物（以下、「ＴＤ−１２」という）が開示されている。
S. Shibata, et al. Yakugaku Zasshi, 80, 620-624 (1960)（非特許文献１２）には、下記の一般式（ＶＩＩＩ）：

で表される化合物（以下、「ＴＤ−１３」という）が開示されている。
Christa Zdero, et al. Phytochemistry, 26, 1999-2006 (1987)（非特許文献１３）には、下記の一般式（ＩＸ）：

で表される化合物（以下、「ＴＤ−１４」という）が開示されている。 In addition, Raghwendra Pal, et al. Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry, 15B, 208-211 (1977) (Non-Patent Document 10) includes the following general formula (VI):

(Hereinafter referred to as “TD-11”).
Isao Agata, et al. Yakugaku zasshi, 101, 1067-1071 (1981) (Non-Patent Document 11) includes the following general formula (VII):

(Hereinafter referred to as “TD-12”).
S. Shibata, et al. Yakugaku Zasshi, 80, 620-624 (1960) (Non-Patent Document 12) includes the following general formula (VIII):

(Hereinafter referred to as “TD-13”).
Christa Zdero, et al. Phytochemistry, 26, 1999-2006 (1987) (Non-patent Document 13) includes the following general formula (IX):

(Hereinafter referred to as “TD-14”).

However, there are unknown compounds in the Asteraceae plants or the Aceraceae cactiaceae plants, and identification of these compounds is anxious. In addition, it is urgently required to clarify new uses (pharmacological effects and the like) of known and unknown compounds extracted and isolated from these plants.
Japanese Patent No. 2609780 Japanese Patent Application No. 8-127538 Raghwendra Pal, et al. Indian Journal of Chemistry, Section B: Organic Chemical Including Medicinal Chemistry, 14B, 259-262 (1976). Raghwendra Pal, et al. Indian Journal of Chemistry, Section B: Organic Chemical Including Medicinal Chemistry, 15B, 208-211 (1977). Schuster A, et al. Phytochemistry, 31, 3139-3141 (1992). Paulo Sergio Pereira, et al. Phytochemistry, 45, 1445-1448 (1997). C. Zdero et al. Phytochemistry, 26, 1999-2006 (1987). Raghwendra Pal, et al. Indian Journal of Chemistry, Section B: Organic Chemical Including Medicinal Chemistry, 15B, 208-211 (1977). Baruah Nabin C, et al. Journal of Organic Chemistry, 44, 1831-1835 (1979). Antonia G. Gonzalez, et al, Journal of Natural Products, 57, 400-402 (1994) Raghwendra Pal, et al. Journal of Phamaceutical Science, 65, 918-920 (1976). Raghwendra Pal, et al. Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry, 15B, 208-211 (1977) Isao Agata, et al. Yakugaku zasshi, 101, 1067-1071 (1981) S. Shibata, et al. Yakugaku Zasshi, 80, 620-624 (1960) Christa Zdero, et al. Phytochemistry, 26, 1999-2006 (1987)

Summary of the Invention

［上記式中、Ｒ_１は水酸基を示し、Ｒ_２はメトキシ基を示す］
で表される化合物（以下、「ＴＤ−３」という）。
下記の一般式（ＩＩ）：

［上記式中、Ｒ_３は水酸基を示し、Ｒ_４はメトキシ基を示す］
で表される化合物（以下、「ＴＤ−６」という）。
下記の一般式（III）：

［上記式中、３−ヒドロキシ−１−ブテニル基の３−ヒドロキシ基が３Ｓ立体配座とされてなるものである］
で表される、化合物（以下、「ＴＤ−５」という）。 The present inventors have now found that a novel compound having anticancer activity and a known compound have anticancer activity against specific cancer cells. The present invention is based on this finding. Therefore, the present invention provides a pharmaceutical use of a novel compound and a known compound having anticancer activity against a specific cancer.
1st aspect of this invention According to the 1st aspect of this invention, a novel compound can be provided and the compound is represented by the following general formula.
The following general formula (I):

[Wherein R ₁ represents a hydroxyl group and R ₂ represents a methoxy group]
(Hereinafter referred to as “TD-3”).
The following general formula (II):

[In the above formula, R ₃ represents a hydroxyl group and R ₄ represents a methoxy group]
(Hereinafter referred to as “TD-6”).
The following general formula (III):

[In the above formula, the 3-hydroxy-1-butenyl group of the 3-hydroxy group is the 3S conformation]
A compound represented by the following (hereinafter referred to as “TD-5”).

で表される化合物（ＴＤ−２）、および
下記の一般式（Ｖ）：

で表される化合物（ＴＤ−８）。 Second aspect of the present invention According to the second aspect of the present invention, there is provided an anti-acute myeloid leukemia agent comprising one or more of the known compounds represented by the following general formula: Can do.
In the above general formula (I), _{a compound wherein R 1} and _{R 2} represents a hydroxyl group (TD-7), compounds _{in which R 2} represents a hydroxyl group _{R 1} is a hydrogen atom (TD-9), _{R 1} is a hydrogen atom And R ₂ represents a methoxy group (TD-10),
Compound (TD-1) in which R ₃ and R ₄ represent a methoxy group in the above general formula (II), Compound (TD-4) in which R ₃ represents a methoxy group and R ₄ represents a hydroxyl group,
The following general formula (IV):

A compound represented by formula (TD-2), and the following general formula (V):

A compound represented by formula (TD-8).

Third aspect of the present invention According to the third aspect of the present invention, an anti-ovary comprising the compound (TD-1) in which R ₃ and R ₄ each represent a methoxy group in the general formula (II) as an active ingredient Cancer agents or anti-prostate cancer agents can be provided. Moreover, in this invention, the anti-ovarian cancer agent or anti-prostate cancer agent used for the treatment of ovarian cancer or a prostate cancer is provided.

Fourth Aspect of the Invention According to the fourth aspect of the invention, a method for isolating TD-1 to TD-14 is proposed.

First aspect of the present invention The novel compound provided in the first aspect of the present invention is a compound of the above general formula (I) wherein R ₁ In which R ₂ represents a hydroxyl group and R ₂ represents a methoxy group (TD-3), wherein R ₃ represents a hydroxyl group and R ₄ represents a methoxy group (TD-6), It is a compound (TD-5) represented by general formula (III).
TD-5 is an isomeric compound having a different conformation from the compound described in Non-Patent Document 8, specifically, the hydroxy group of the 3-hydroxy-1-butenyl group is in the 3S conformation. It is a novel compound.

Uses of the new compounds The new compounds TD-3, TD-5 and TD-6 according to the present invention have improved immunity, activation of metabolic functions, killing and bactericidal action against pathogenic bacteria, suppression of excessive inflammation or allergy, life Use as an active ingredient conferring suppression, cure, etc. of habitual diseases (such as diabetes) and immune diseases is considered. Among these novel compounds, “TD-3” and “TD-6” have anticancer properties, and preferably have anticancer properties in leukemia (more preferably acute myeloid leukemia). Therefore, the novel compounds “TD-3” and “TD-6” are considered to be active ingredients of anticancer agents, and these anticancer agents can be used for cancer treatment.

In order to use the novel compounds “TD-3” and “TD-6” as anticancer agents for cancer therapy, these novel compounds are formulated in combination with known pharmaceutical carriers in accordance with various administration forms. It is possible to For example, parenteral administration by subcutaneous injection, intravenous injection, intramuscular injection, suppository, or systemic administration by tablet, capsule, powder, granule, etc., ointment, lotion, suppository, aerosol The form of topical administration such as can be exemplified.
The dose can be appropriately determined according to the age, weight and symptoms of animals and humans.

Second aspect of the present invention According to the second aspect of the present invention, an anti-acute myeloid leukemia agent comprising one or more known compounds can be provided. As described above, “TD-1”, “TD-2”, “TD-4”, “TD-7”, “TD-8”, “TD-9” and “TD-10” are known compounds. It is. The results of this experiment have revealed that these known compounds have excellent suppressive and therapeutic effects on acute myeloid leukemia in animals or humans (preferably). Therefore, these TD compounds can be used as anti-acute myeloid leukemia agents containing these as active ingredients. Drugs and administration forms as pharmaceuticals may be the same as described in the first aspect of the present invention.

Third Aspect of the Invention According to the third aspect of the present invention, an anti-ovarian cancer agent or an anti-prostate cancer agent containing the known compound “TD-1” as an active ingredient can be provided. Additional experimental results have revealed that TD-1 has excellent suppressive and therapeutic effects on ovarian cancer or prostate cancer in animals or humans (preferred). Therefore, TD-1 can be used as an anti-ovarian cancer or anti-prostate cancer agent containing this as an active ingredient. Drugs and administration forms as pharmaceuticals may be the same as described in the first aspect of the present invention.

4th aspect of this invention 1) Manufacturing method (extraction method) of the composition which comprises TD-1-10
According to the present invention, a method for obtaining a composition comprising TD-1 to 10 can be proposed, which comprises preparing a raw material comprising TD-1 to 10,
The raw material is extracted with a solvent under heating as necessary,
The extract is supplied to ion-exchange chromatography, and a first lower alcohol, a second lower alcohol, and, if necessary, a lower ester are solvent-extracted in this order,
Obtaining a composition comprising TD-1 to 10 in the second lower alcohol fraction.

The raw material may be any material as long as it contains a significant amount of TD-1 to 10 (and TD-11 to 14), and preferably a asteraceae plant or a red rosaceae plant. .
Preferable specific examples of the Asteraceae plant include nitobegiku [(Tissonia diversifolia (Hemsul) A, Gray: “Tithonia diversifolia (Hemsl) A, Gray”, (as a family, Tissonia Fraticosa Canby and Rose: “Tithonia]). fruticosaCanby & Rose "), (Tissonia scaberimaa Benth:" Tithonia scaberima Benth "), (Tissonia longgera diata (Bartle), etc. Sunflower [scientific name is Tissonia rotundifolia (mil) break: "Tithonia rotundifoli (Mill) Blake]) and the like.
Preferable specific examples of the plant belonging to the genus Rabbitaceae are: Kidney chicken [(Ludwigia octobarvis raben: “Ludwigia octovalvis Raven”);

As a specific example of the solvent used when extracting the raw material comprising the extraction methods TD-1 to 10, a lower alcohol having 1 to 5 carbon atoms, preferably ethanol, is used.
Further, the first lower alcohol and the second lower alcohol may be the same or different, but preferably different ones. Examples of the first lower alcohol and the second lower alcohol include those having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. In the present invention, a combination in which the first lower alcohol is methanol and the second lower alcohol is ethanol is preferably mentioned. According to a preferred embodiment of the present invention, in order to rapidly perform the ion exchange chromatography extraction, it is preferable to use the lower ester as the extraction solvent after the second lower alcohol is placed in the column. In the solvent extraction, heating may be performed, and the heating temperature is −20 ° C. or higher and 100 ° C. or lower, preferably the upper limit is 80 ° C. or lower, and the lower limit is 1 ° C. or higher.
The ion exchange chromatography used includes a column packed with an ion exchange material (preferably a porous ion exchange resin: for example, “Diaion HP-20” manufactured by Mitsubishi Chemical Corporation).
After solvent extraction, the solvent is preferably evaporated and concentrated to obtain a composition comprising a compound of TD-1 to 10 as an extract.

1-1) Isolation of TD-1-10 According to the present invention, a method of isolating a compound of TD-1-10 can be proposed, and the method comprises TD-1-10. Prepare the composition,
By repeating the chromatographic separation of the composition multiple times,
A compound of TD-1 to 10 is obtained.

  According to the preferable aspect of this invention, it is preferable to utilize what was obtained with the manufacturing method of the composition which comprises TD-1-10 demonstrated previously for the composition which comprises TD-1-10. As the separation solvent, water, acetone, chloroform, a lower alcohol having 5 or less carbon atoms, a lower ester having 5 or less carbon atoms, and a mixed solvent thereof can be used. The chromatography used may be normal phase or reverse phase, and a porous material (preferably silica gel) may be used as a filler. In addition, (high performance) liquid chromatography can be used. In the present invention, it is preferable to isolate the TD compound by a combination of these chromatography.

The following method is mentioned as a specific example of the method of isolating the compound of TD-1-10 by this invention. Accordingly, the column filler, chromatography, extraction solvent and the like may be the same as described in the previous method for isolating TD compounds.
1-2) Fractionation of a composition comprising TD-1-10 According to the present invention, a first composition comprising TD-1-8 from a composition comprising TD-1-10, TD-9 and And a method comprising separating a composition comprising TD-1-10 by normal phase column chromatography followed by reverse phase column chromatography. The first composition containing TD-1 to 8 and the second composition containing TD-9 and 10 are separated.

According to the present invention, there is provided a method for isolating TD-1 to 10 from a first composition comprising TD-1 to 8 and a second composition comprising TD-9 and 10, respectively. Specifically, it is as follows.
1-3) Repeat the normal phase or reverse phase (silica gel) column chromatography of the first composition containing TD-1, 2, 4 and 5 isolated TD-1 to 8 and further (high-speed) liquid By performing chromatography, TD-1, TD-2, TD-4, and TD-5 can be isolated.
According to a more preferred embodiment of the present invention, the first composition comprising TD-1 to 8 is subjected to reverse phase silica gel column chromatography, reverse phase silica gel column chromatography, high performance liquid chromatography equipped with a reverse phase silica gel column, By sequentially separating and purifying, TD-1, TD-2, TD-4, and TD-5 can be isolated.

1-4) Isolation of TD-3 and 7 Repeating normal phase or reverse phase silica gel column chromatography of the first composition containing TD-1 to TD-8 to isolate TD-3 and 7 respectively. Can do.
According to a more preferred embodiment of the present invention, the first composition containing TD-1 to 8 is sequentially separated and purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and reverse phase silica gel column chromatography. Can isolate TD-3 and TD-7, respectively.

1-5) Isolation of TD-6 and 8 Repeating normal phase or reverse phase silica gel column chromatography of the first composition containing TD-1 to 8 to isolate TD-6 and 8 respectively. Can do.
According to a more preferred embodiment of the present invention, the first composition containing TD-1 to 8 is sequentially separated and purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and reverse phase silica gel column chromatography. Can isolate TD-6 and TD-8, respectively.

1-6) Isolation of TD-9 and 10 The second composition comprising TD-9 and 10 is repeatedly subjected to normal phase or reverse phase silica gel column chromatography to isolate TD-9 and 10 respectively. can do. According to a more preferred embodiment of the present invention, the second composition comprising TD-9 and 10 is sequentially purified by normal phase silica gel column chromatography, normal phase silica gel column chromatography, and reverse phase silica gel column chromatography. Can isolate TD-9 and TD-10, respectively.

2) Production method (extraction) of a composition comprising TD-11-14
The method for producing the composition comprising TD-11 to 14 may be the same as that described in the section 1 ) Production method (extraction method) for the composition comprising TD-1 to 10 . Therefore, the raw material and the extraction method described in the section 1) may be the same in the method for producing a composition containing TD-11-14. Moreover, TD-11-14 may be obtained as a composition mixed with TD-1-10 by this manufacturing method.

2-1) Isolation of TD-11-14 According to the present invention, a method of isolating the compounds of TD-11-14 can be proposed, which method comprises TD-11-14. Prepare the composition,
By repeating the chromatographic separation of the composition multiple times,
A compound of TD-11 to 14 is obtained.
In the present invention, isolation of TD-11 to 14 may be the same as that described for isolation of TD-1 to 10. Moreover, by this isolation, TD-11 to 14 may be obtained as a composition mixed with TD-1 to 10.

2-2) Fractionation of composition containing TD-11-14 According to the present invention, a third composition containing TD-11 and TD-14 from a composition containing TD-11-14, and TD- A method of separating each of the fourth compositions comprising 12 and TD-13 can be provided. The method comprises separating a composition comprising TD-11-14 by normal phase column chromatography followed by reverse phase column chromatography, a third composition comprising TD-11 and 14, TD-12 and 13 to separate into a fourth composition.

According to the preferable aspect of this invention, the method of obtaining a 1st composition-a 4th composition using the composition containing TD-1 to TD-14 is proposed. This method can be performed in the same manner as 1-2) fractionation of a composition containing TD-1 to 10 and 2-2) fractionation of a composition containing TD-11 to 14 . Specifically, the composition comprising TD-1 to TD-14 is separated by normal phase column chromatography and then reverse phase column chromatography, whereby the first composition comprising TD-1 to 8 is obtained. Separating a second composition comprising TD-9 and TD, a third composition comprising TD-11 and TD-14, and a fourth composition comprising TD-12 and TD-13, respectively. Can do. According to a preferred embodiment of the present invention, TD-1 to TD-14 can be isolated by using the first composition to the fourth composition, respectively, using the method described above or described later.

2-3) Isolation of TD-11 and 14 The third composition comprising TD-11 and 14 is repeatedly subjected to normal phase or reverse phase silica gel column chromatography to isolate TD-11 and 14 respectively. can do. According to a more preferred embodiment of the present invention, the third composition comprising TD-11 and 14 is sequentially separated and purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and reverse phase silica gel column chromatography. By doing so, TD-11 and TD-14 can be isolated, respectively.

2-4) Isolation of TD-12 and 13 The fourth composition comprising TD-12 and 13 is repeatedly subjected to normal phase or reverse phase silica gel column chromatography to isolate TD-12 and 13 respectively. can do. According to a more preferred embodiment of the present invention, the fourth composition comprising TD-12 and 13 is sequentially purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and reverse phase silica gel column chromatography. Can isolate TD-12 and 13 respectively.

Example
Extraction and fractionation of compounds of TD-1-14 and fractionation of Nitobegiku (Tissonia diversifolia (Hemsul) A, Gray) 3.0 kg of dry powder by heating with 45 liters of 80% ethanol at 65 ° C for 2 hours Then, after filtering and removing impurities, the solvent was removed to obtain a concentrated extract.
This concentrated extract (320 g) was packed into ion exchange column chromatography (ion exchange agent: Diaion HP-20). Next, a first alcohol solvent (methanol: water ratio of 50% or more) is flowed, then a second alcohol solvent (80% ethanol) 45 liters is flown, and finally, ethyl acetate which is a lower ester is flowed. The mixture was heated and extracted at 65 ° C. for 2 hours to obtain a second alcohol fraction. The second alcohol fraction was filtered to remove impurities, and then the second alcohol solvent was evaporated to obtain a concentrated extract.
Next, this extract (320 g) was subjected to normal phase silica gel column chromatography (solvent having a 9: 1 ratio of chloroform to methanol), and then to reverse phase silica gel column chromatography (filler: ODS silica gel, methanol to water ratio). 1: 1 solvent), 2-1 fraction (containing TD-1 to 8), 2-2 fraction (containing TD-11 and 14), 2-3 fraction (TD-9 and 10) and 2-4 fractions (containing TD-12 and 13), respectively. In normal-phase silica gel column chromatography, due to the difference in adsorption power to silica gel, which is a stationary phase, reverse-phase ODS silica gel column chromatography uses a silica gel surface substituted with octadecyl group as a packing material, thereby fixing The four fractions were separated using the partitioning action between the phase (ODS silica gel) and the mobile phase (solvent).
Each of 2-1 fraction to 2-4 fraction was subjected to the following treatment to isolate each TD compound.

2-1 fraction / TD-1, 2, 4 and 5 A portion of the 2-1 fraction was subjected to normal phase silica gel column chromatography (solvent with 9: 1, 49: 1 ratio of chloroform to acetone). ), Reverse phase silica gel column chromatography (solvent with a ratio of acetonitrile to water of 2: 3), reverse phase silica gel column chromatography (solvent with a ratio of methanol to water of 11: 9), equipped with a reverse phase silica gel column TD-1 (28.0 mg), TD-2 (14.0 mg) by successive purification by high performance liquid chromatography (HPLC) (solvent with methanol to water ratio of 7: 3, 3: 2). ), TD-4 (35.3 mg), TD-5 (13.0 mg).

2-1 Fraction / Isolation of TD-3 and 7 A portion of the 2-1 fraction was subjected to normal phase silica gel column chromatography (solvent having a chloroform / acetone ratio of 49: 1) and reverse phase silica gel column chromatography. (TD-3 (56.2 mg) by sequentially purifying by a solvent having a ratio of methanol to water of 11: 9) and reverse phase silica gel column chromatography (a solvent having a ratio of acetonitrile to water of 1: 2). And TD-7 (50.0 mg) was isolated.

2-1 Fraction / Isolation of TD-6 and 8 A portion of the 2-1 fraction was subjected to normal phase silica gel column chromatography (solvent having a chloroform to acetone ratio of 49: 1 and 19: 1), reverse phase silica gel TD-6 (14) was purified by column chromatography (solvent with a methanol / water ratio of 3: 2) and reverse phase silica gel column chromatography (solvent with a acetonitrile / water ratio of 1: 2). 0.0 mg) and TD-8 (8.7 mg) were isolated.

2-3 fraction / isolation of TD-9 and 10 2-3 fractions were subjected to normal phase silica gel column chromatography (solvent with a ratio of chloroform to acetone of 19: 1, 4: 1, 2: 1), Phase silica gel column chromatography (solvent with hexane: acetone ratio 9: 1, 7: 3), reverse phase silica gel column chromatography (acetonitrile: water ratio 9: 1, 4: 1, 3: 1, TD-9 (8.4 mg) and TD-10 (7.8 mg) were isolated by sequential purification with 2: 1, 1: 1 solvent).

2-2 Fraction / Isolation of TD-11 and 14 The 2-2 fraction was subjected to normal phase silica gel column chromatography (solvent having a chloroform to acetone ratio of 49: 1) and reverse phase silica gel column chromatography (methanol and TD-11 (67) by sequentially purifying with a solvent having a water ratio of 1: 1) and reverse phase silica gel column chromatography (solvents having a ratio of acetonitrile to water of 3: 1, 5: 8, 1: 2). .9 mg) and TD-14 (8.7 mg) were isolated.

2-4 Fraction / TD-12 and Isolation of TD-12 and 13 Fractions 2-4 were subjected to normal phase silica gel column chromatography (solvents having a ratio of chloroform to acetone of 4: 1, 3: 2), and reverse phase silica gel column chromatography. By sequentially purifying by chromatography (solvent with a methanol to water ratio of 1: 1) and reverse phase silica gel column chromatography (solvent with a ratio of acetonitrile to water of 1: 2, 1: 1, 2: 1), TD -12 (18.8 mg) and TD-13 (60.8 mg) were isolated.

Determination of structure of TD-1 to 14 The structure of TD-1 to TD-14 obtained above was determined.
(1) Determination of structure of new compound The structures of TD-3, TD-5, and TD-6, which are new compounds, were determined based on the following spectral data.

_{TD-3 (C 20 H 30} O 7)
(1S, 3R, 4S, 6R, 7S, 8R, 10R) -1-hydroxy-3-methoxy-3, 10-epoxy-8-isobutylyloxygermacra-11 (13) -en-6,12-olide
1) Properties: White powder [α] _D ²⁶ -102 degrees (methanol)
2) HR-ESI-MS m / z: 383.2058 (Calcd for C ₂₀ H ₃₁ O ₇ , 383.2070)
3) UV (methanol) λ _max (log ε): 216 (log ε = 3.81)
4) IR (film) cm ⁻¹ : 3446, 2972, 2933, 1761, 1736, 1460, 1317, 1261, 1194, 1155, 1072, 1007, 984.
5) ¹ H-NMR (deuterated chloroform) ppm: 6.19 (1H, d, J = 3.4 Hz, H-13a), 5.54 (1H, ddd, J = 11.4, 5.3, 2.6 Hz, H-8), 5.47 ( 1H, d, J = 2.9 Hz, H-13b), 4.46 (1H, ddd, J = 10.6, 6.9, 1.1 Hz, H-6), 4.06 (1H, dd, J = 9.5, 8.6 Hz, H-1 ), 3.92 (1H, m, H-7), 3.10 (3H, s, OMe), 2.49 (1H, dd, J = 14.5, 9.5 Hz, H-2b), 2.37 (1H, m, H-2 ' ), 2.06 (1H, m, H-5b), 1.99 (1H, m, H-4), 1.97 (1H, dd, J = 14.5, 5.4 Hz, H-9a), 1.81 (1H, dd, J = 14.5, 8.6 Hz, H-2a), 1.72 (1H, br d, J = 13.3 Hz, H-5a), 1.63 (1H, dd, J = 14.5, 11.4 Hz, H-9b), 1.37 (3H, s , Me-14), 1.01 and 0.99 (each 3H, d, J = 7.0 Hz, Me-3 'and Me-4'), 0.99 (3H, d, J = 7.0 Hz, Me-15).
6) ¹³ C-NMR (deuterated chloroform) ppm: 79.7 (C-1), 41.7 (C-2), 108.8 (C-3), 46.4 (C-4), 38.2 (C-5), 82.3 (C -6), 48.2 (C-7), 70.2 (C-8), 35.1 (C-9), 81.3 (C-10), 137.7 (C-11), 169.8 (C-12), 121.7 (C- 13), 24.1 (C-14), 18.3 (C-15), 176.7 (C-1 '), 34.5 (C-2'), 19.1 (C-3 'or C-4'), 19.6 (C- 3'and C-4 '), 49.2 (OMe).

_{TD-5 (C 13 H 22} O 3)
(4S, 5R) -4-hydroxy-4-[(1E, 3S) -3-hydroxy-1-butenyl] -3,3,5-trimethyl-cyclohexone)
1) Properties: White powder [α] _D ²⁶ -8 degrees (methanol)
2) HR-ESI-MS m / z: 227.1050 (Calcd for C ₁₃ H ₂₂ O ₃ , 227.1647)
3) IR (film) cm ^-1 : 3415, 2968, 11900, 2877, 1695, 1454, 1412, 1369, 1286, 1140, 1061, 1030, 977, 939.
4) ¹ H-NMR (deuterated chloroform) ppm: 2.77 (1H, d, J = 13.6 Hz, H-2ax), 1.84 (1H, dd, J = 13.6, 2.2 Hz, H-2ax), 2.35 (1H, t-like, J = 12.9 Hz, H-4ax), 2.13 (1H, ddd, J = 13.6, 4.5, 2.2 Hz, H-4eq), 2.19 (1H, ddq, J = 12.9, 6.5, 4.5 Hz, H -5), 5.63 (1H, dd, J = 15.8, 0.8 Hz, H-7), 5.77 (1H, dd, J = 15.8, 5.7 Hz, H-8), 4.36 (1H, ddd, J = 6.4, 5.7, 0.8 Hz, H-9), 1.25 (3H, d, J = 6.4 Hz, Me-10), 0.87 (3H, s, Me-11), 0.90 (3H, s, Me-12), 0.81 ( 3H, d, J = 6.5 Hz, Me-13).
5) ¹³ C-NMR (deuterated chloroform) ppm: 42.5 (C-1), 51.4 (C-2), 211.4 (C-3), 45.1 (C-4), 36.4 (C-5), 77.0 (C -6), 131.8 (C-7), 135.2 (C-8), 68.3 (C-9), 23.9 (C-10), 24.5 (C-11), 24.4 (C-12), 15.9 (C- 13).

_{TD-6 (C 20 H 28} O 7)
(1S, 3R, 6R, 7R, 8R, 10R) -1-hydroxy-3-methoxy-3,10-epoxy-8-isobutylyloxygermacra-4,11 (13) -dien-6,12-olide
1) Property: White powder [α] _D ²⁶ -110 degrees (methanol)
2) HR-ESI-MS m / z: 381.1947 (Calcd for C ₂₀ H ₂₉ O ₇ , 381.1913)
3) UV (methanol) λ _max (log ε): 213 (log ε = 3.88)
4) IR (film) cm ^-1 : 3421, 2972, 1763, 1732, 1587, 1282, 1186, 1111, 1064, 991
5) ¹ H-NMR (deuterated chloroform) ppm: 6.20 (1H, d, J = 2.8 Hz, H-13a), 5.66 (1H, m, H-5), 5.59 (1H, ddd, J = 11.2, 5.0 , 4.6 Hz, H-8), 5.55 (1H, d, J = 2.3 Hz, H-13b), 5.20 (1H, m, H-6), 4.16 (1H, m, H-7), 3.77 (1H , dd, J = 11.1, 3.9 Hz, H-1), 3.11 (3H, s, OMe), 2.91 (1H, br d, J = 11.3 Hz, H-2b), 2.34 (1H, m, H-2 '), 2.31 (1H, m, H-2a), 1.82 (1H, dd, J = 14.1, 5.0 Hz, H-9a), 1.70 (3H, t-like, J = 1.8 Hz, Me-15), 1.66 (1H, dd, J = 14.1, 11.2 Hz, H-9b), 1.44 (3H, s, Me-14), 1.00 (3H, d, J = 7.1 Hz, Me-3 'or Me-4') , 0.97 (3H, d, J = 7.2 Hz, Me-3 'or Me-4').
6) ¹³ C-NMR (deuterated chloroform) ppm: 77.1 (C-1), 45.2 (C-2), 109.7 (C-3), 138.4 C-4), 131.2 (C-5), 75.8 (C- 6), 49.6 (C-7), 71.2 (C-8), 39.7 (C-9), 87.5 (C-10), 136.2 (C-11), 169.5 (C-12), 122.7 (C-13 ), 20.8 (C-14), 21.6 (C-15), 175.9 (C-1 '), 334.0 (C-2'), 18.6 (C-3 'or C-4'), 19.0 (C-3 'or C-4'), 50.1 (OMe).

(2) Structure determination of known compounds A) TD-1, TD-2, TD-4, TD-7, TD-8, TD-9, TD-10 are known compounds, and various spectral data are described above. These structures were determined based on the contents described in Patent Documents 1-13. Specifically, it was as follows.
TD-1
(1R, 3R, 6R, 7R, 8R, 10R) -1,3-dimoxy-3,10-epoxy-8-isobutyryloxygermacra-4,11 (13) -dien-6,12-olide
TD-2
(6S, 7R, 8R, 10R) -10-hydroxy-3-oxo-8-isobutylyloxygermacra-1,4,11 (13) -trien-6,12-olide
TD-4
(1R, 3R, 6R, 7R, 8R, 10R) -3-hydroxy-1-methyoxy-3, 10-epoxy-8-isobutylyloxygermacra-4, 11 (13) -dien-6,12-olide
TD-7
(1S, 3R, 4S, 6R, 7S, 8R, 10R) -1,3-dihydroxy-3,10-epoxy-8-isobutylyloxygermacra-11 (13) -en-6,12-olide
TD-8
(1R, 3S, 6S, 7R, 8R, 10R) -3-hydroxy-1,10-epoxy-8-isobutylgermacra-4, 11 (13) -dien-6,12-olide
TD-9
(3R, 4S, 6R, 7S, 8R, 10S) -3-hydroxy-3,10-epoxy-8-isobutylyloxygermacra-11 (13) -en-6,12-olide
TD-10
(3R, 4S, 6R, 7S, 8R, 10S) -3-methyoxy-3,10-epoxy-8-isobutylyloxygermacra-11 (13) -en-6,12-olide

B) TD-11 to 14 are known compounds, and their structures were determined based on the contents of Non-Patent Documents 10 to 13 described above for various spectral data. Specifically, it was as follows.
TD-11 (generic name: tagitinin F)
(3R, 6R, 7R, 8R, 10R) -3-hydroxy-3,10-epoxy-8-isobutylyloxygermacra-1,4,11 (13) -trien-6,12-olide
TD-12 (generic name: luteolin)
3 ', 4'5, 7-tetrahydroxy-flavone
TD-13 (generic name: nepetin)
3 ', 4', 5, 7-tetrahydroxy-6-methoxy-flavone
TD-14
7a, 10a-dihydroxy-3-oxo-8b-isobutyryloxyguaia-11 (13) -en-12, 6a-olide

Evaluation test
Evaluation test 1: Evaluation test of TD-1 to 10 The anticancer activity of TD-1 to 10 was examined by the value of cytotoxic activity using human acute myeloid leukemia cell line HL-60. Specifically, the test was performed as follows.
The number of HL-60 cells pre-cultured in FBS-containing RPMI1640 medium is suspended in FBS-containing RPMI1640 medium so that the number of HL-60 cells is 4 × 10 ^{4 in} 1 mL, and the suspension is 196 mL in each well of a 96-well microplate. Dispensing and preincubating at 37 ° C. in 5% carbon dioxide gas for 24 hours. Subsequently, EtOH-H ₂ O (1: 1) as a control and 4 mL of each TD compound dissolved in EtOH-H ₂ O (1: 1) as a sample were added to each well. After incubating at 37 ° C. in 5% carbon dioxide gas for 72 hours, 10 mL of MTT reagent was added to each well and further incubated for 4 hours. After removing the medium, the produced MTT formazan was dissolved in 175 mL DMSO and detected at a light absorption wavelength of 550 nm using a microplate reader, and the number of viable cells relative to the control was calculated. IC ₅₀ values were calculated from dose-effect curves.
The IC ₅₀ values (μg / mL) of TD-1 to 10 were as shown in Table 1 below.

Table 1
Compound IC ₅₀ value (μg / mL)
TD-1 (0.60)
TD-2 (0.38)
TD-3 (4.97)
TD-4 (0.05)
TD-6 (2.50)
TD-7 (3.87)
TD-8 (0.92)
TD-9 (2.50)
TD-10 (0.78)

Evaluation test 2: Evaluation test of TD-1 (anticancer test)
To examine the anticancer activity of TD-1, a human cultured cancer cell panel screening test was conducted. Specifically, the human cultured cancer cell panel screening test was conducted in accordance with the method described in “Cancer and Chemotherapy (Takao Yamori), 24, 129-135, 1997”. This was done by measuring the 50% cell growth inhibitory concentration (GI ₅₀ ) for human tumor cells. The results were as shown in Table 2 below.
Table 2
TD-1 GI ₅₀ (μM)
Average of all 39 types of cancer cells 6.8
Ovarian cancer cell line OVCAR-3 3.3
OVCAR-4 5.6
OVCAR-5 2.9
OVCAR-8 3.6
SK-OV-3 4.0
Prostate cancer cell line DU-145 4.2
PC-3 3.3

Evaluation results As shown in Table 2, TD-1 has lower GI ₅₀ values for ovarian cancer cell lines and prostate cancer cell lines than the average value for GI ₅₀ of all 39 types of cancer cells. It is understood that it has an anti-cancer effect on the system.

Claims (6)

The following general formula (I):

A compound in which R ₁ and R ₂ represent a hydroxyl group, a compound in which R ₁ represents a hydrogen atom and R ₂ represents a hydroxyl group, a compound in which R ₁ represents a hydrogen atom and R ₂ represents a methoxy group,
The following general formula (II):

In which R ₃ and R ₄ represent a methoxy group, R ₃ represents a methoxy group and R ₄ represents a hydroxyl group, or the following general formula (V):

An anti-acute myeloid leukemia agent comprising one or more compounds represented by the formula as an active ingredient.   The anti-acute myeloid leukemia agent according to claim 1, which is used for treatment of acute myeloid leukemia in animals or humans. The anti-ovarian cancer agent which uses the compound represented by the following general formula (II) as an active ingredient.

[Wherein R ₃ and R ₄ represent a methoxy group]   The anti-ovarian cancer agent according to claim 3, which is used for treatment of ovarian cancer in animals or humans. An anti-prostatic cancer agent comprising a compound represented by the following general formula (II) as an active ingredient.

[Wherein R ₃ and R ₄ represent a methoxy group]   The anti-prostatic cancer agent according to claim 5, which is used for treatment of prostate cancer in animals or humans.