JPH07258153A - Pentacyclic compound, production and use thereof - Google Patents

Abstract

PURPOSE:To obtain a novel compound, having inhibiting activities against microtubular polymerization, squalene synthases and cell growth and suppressing activities against tumor metastasis and useful for treating or preventing the metastasis of the tumor and preventing and treating, etc., ischemic cardiopathy. CONSTITUTION:This compound is expressed by the formula I (R<1> is carboxyl; R<2> to R<6> each is hydroxyl group) or its salt, e.g. a compound expressed by the formula II. appearance: yellow powder; molecular weight; m/Z 519(M+H); elementary analytical value (%) (expressed in terms of 1.5mol moisture content): found: C, 66.04; H, 6.1; molecular formula: C30H30O8 and color reaction: positive to potassium permanganate, iodine, etc., and negative to the Dragendorff reagent and ninhydrin. The compound is obtained by culturing a microorganism, belonging to the genus Streptomyces and having the ability to produce the compound expressed by the formula I [preferably Streptomyces roseogriseus AL-28015 (FERM P-14224), etc.], preferably at pH6-8 and 24-30 deg.C temperature for 48-144hr, producing and accumulating the compound expressed by the formula I in the culture medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pentacyclic compound having microtubule polymerization inhibitory activity, squalene synthase inhibitory activity, cell growth inhibitory activity and tumor metastasis inhibitory activity, a process for producing the same and use thereof.

[0002]

2. Description of the Related Art Microtubules are intracellular structures existing in all eukaryotes, which are polymerized with a heterodimer consisting of one molecule of alpha-tubulin and one molecule of beta-tubulin as a basic unit. It has a tubular structure consisting of protofilaments. Polymerization of intracellular microtubules is governed by the hydrolytic reaction of guanine nucleotides bound to tubulin at the end of microtubules, and this dynamic instability (microtubule dynamics) is associated with mitosis, flagella and flagella movement. , Organelle transport, secretory function, cytoskeleton formation, biomembrane function, etc. are known to be important for performing various central functions [Annual Review of
Cell Biology (Annual Review of Cell Bio
logy), 7 , 93 (1991); Annual Review of Biophysics and Biomolecular Structure (Annual Review of Biophysics).
and Biomolecular Structure), 21 , 145 (1
992)].

In particular, changes in microtubule dynamics play a fundamental role in biological phenomena in the progression of the mitotic phase (M phase) of eukaryotes. That is, long and stable interphase microtubules become unstable and disappear in M phase, and instead, short and unstable spindle microtubules appear. Spindle formation by reconstruction of this microtubule This is because eukaryotic cells cannot perform mitosis unless the above occurs [Science, 246 , 622 (1989); cell (C
ell), 71 , 547 (1992)]. Drugs that act on the microtubule system and inhibit mitosis have tubulin-binding ability with a few exceptions, but many clinically useful anticancer drugs. Among plant-derived microtubule-acting agents, as microtubule polymerization inhibitors, Colchicine, Podophyllotoxin, Vinblastine
Kind etc. [Cancer Medicin
e), 1 , 782 (1993)] has recently attracted attention as a microtubule polymerization accelerator due to its excellent therapeutic effect. Taxol [Journal of the National Cancer Institute (J
ournal of the National Cancer Institute),
82 , 1247 (1990)]. Furthermore, as a microtubule polymerization inhibitor derived from microorganisms, ansamitocin (An
samitocin) [Nature, 270 , 721]
(1977)], Rhizoxins [Journal ・
Journal of the National Cancer Institu
te), 84 , 494 (1992)], Phomopsin A (Phom
opsin A) [Biochemical Pharmacology (Bioch
emical Pharmacology), 43 , 219 (1992)]. In addition, as a source of marine animals, dolastatins [Biochemical Pharmacology
(Biochemical Pharmacology), 43 , 2637 (19)
92)], Halichondorin [Biochemical Pharmacolog
y), 45 , 421 (1993)] and the like are also reported.

On the other hand, hypercholesterolemia is generally
It is known as the three major risk factors for ischemic heart disease along with hypertension and smoking. Appropriate control of blood cholesterol level is to prevent or treat this ischemic heart disease.
It is extremely important for the prevention or treatment of coronary arteriosclerosis. As a drug for lowering blood cholesterol level, melinamide (French Patent No. 1476596)
Drugs that inhibit acyl coenzyme A cholesterol acyltransferase (ACAT) and suppress cholesterol biosynthesis have been drawing attention. As a cholesterol biosynthesis inhibitor, in particular, Lovastatin (Lovastatin) that inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (US Pat. No. 4,23,193).
8), Simvastatin (US Pat. No. 44)
44784), pravastatin (U.S. Pat. No. 4,346,227) and the like are used as medicines. However, inhibition of HMG-CoA reductase inhibits not only cholesterol biosynthesis but also biosynthesis of other components necessary for the living body such as ubiquinone, dolichol and heme A. Has been done.

Squalene synthase is an enzyme involved in an essential step of the new cholesterol biosynthetic pathway. This enzyme is an enzyme that catalyzes the reductive dimerization of two molecules of farnesyl pyrophosphate to form squalene. On the other hand, as compounds expected to inhibit cholesterol biosynthesis by inhibiting squalene synthase, phosphorus-containing compounds [Journal of Medical Chemistry
urnal of Medical Chemistry), 51 , No. 10,
1869-1871, 1988, JP-A 1-213
288, etc.], quinuclidine derivative (WO9215)
579), and natural products such as zaragodic acid (US Pat. No. 5,096,923).

[0006]

If a novel substance having an inhibitory action on microtubule polymerization can be found, it is possible to treat a cancer type which has acquired a useful antitumor action, tumor metastasis inhibitory action or multidrug resistance with lower toxicity. It is expected that a new anti-cancer agent having an effective anti-tumor action will be developed, and that a new substance having an excellent activity will be developed by using the new substance as an intermediate material.
Further, in the cholesterol biosynthetic pathway, if a novel substance that specifically inhibits the enzyme system of farnesyl pyrophosphate or later, particularly squalene synthase, can be found,
It is possible to eliminate side effects due to deficiencies such as ubiquinone, dolichol, and heme A biosynthesized from farnesyl pyrophosphate on the cholesterol biosynthetic pathway.

[0007]

[Means for Solving the Problems] In view of such circumstances,
The present inventors searched for a microtubule polymerization inhibitor and a squalene synthase inhibitor that are potent and have few side effects in the microbial metabolites, and as a result of diligent search by the method described below, a certain pentacyclic novel compound was cultured. Successfully isolated from the liquid,
It was found that this has a microtubule polymerization inhibitory action, a squalene synthase inhibitory action, a cell growth inhibitory action and a tumor metastasis inhibitory action. As a result of further studies based on these findings, the present invention has been completed. That is, the present invention provides (1) the general formula (I):

[0008]

[Chemical 4]

(Wherein R ¹ is a carboxyl group which may be esterified or amidated, and R ² , R ³ , R ⁴ and R ⁵
And R ⁶ each represent a hydroxyl group which may be etherified or esterified) or a salt thereof, (2) the compound described in (1) above, wherein R ¹ is a carboxyl group, and (3) the formula (IA):

[0010]

[Chemical 5]

A compound represented by the above (1):
(4) Formula (IB):

[0012]

[Chemical 6]

A compound represented by the above (1) represented by:
(5) A microorganism belonging to the genus Streptomyces and capable of producing the compound represented by the formula (IA) and / or the compound represented by the formula (IB) is cultured in a medium, Compound represented by IA) and / or formula (I-
A compound represented by formula (IA) and / or a compound represented by formula (IB), characterized in that the compound represented by B) is produced and accumulated in a medium and collected. (6) The method for producing a salt according to (6), wherein the microorganism is a microorganism belonging to Streptomyces roseoglyceus.
(7) Microbial Streptomyces roseoglyceus AL-28015, (8) Microtubule polymerization inhibitor comprising the compound according to (1) above or a salt thereof, (9) above (1) Squalene synthase inhibitor containing the compound or a salt thereof, and (10) a metastasis inhibitor containing the compound according to (1) or a salt thereof.

In the above general formula (I), the esterified or amidated carboxyl group represented by R ¹ is
Such as those used as synthetic intermediates, those can enjoy pharmacologically, or the first time such as those changes that could enjoy pharmacologically are used in vivo, for example, the general formula: -COOR ⁷ (wherein , R ⁷ is an ester residue, for example, a group represented by an alkyl group, an aryl group, an aralkyl group, a heterocyclic group or the like), a general formula: —CONR ⁸ R ⁹ (wherein R ⁸ and R ⁹ are For example, hydrogen, an alkyl group, a cycloalkyl group, an aryl group, an aralkyl group,
An acyl group or the like, and R ⁸ and R ⁹ may each form a heterocycle with an adjacent nitrogen atom). Examples of the alkyl group represented by R ⁷ include linear or branched alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and hexyl. Preferred is an alkyl group having 1 to 3 carbon atoms. Examples of the aryl group represented by R ⁷ include phenyl, tolyl, xylyl, biphenyl, 1- or 2-naphthyl, 1-, 2- or 9-anthryl, 1-, 2
-, 3-, 4- or 9-phenanthryl, 1, -2
And aryl having 6 to 14 carbon atoms such as-, 4-, 5- or 6-azulenyl. Preferred is an aryl group having 6 to 10 carbon atoms.

[0015] As the aralkyl group represented by R ⁷ is, for example, an aryl group and an aralkyl group consisting of an alkyl group represented by R ^7, preferably aralkyl group 19 C 7 carbon atoms. Specifically, for example, benzyl,
1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, diphenylmethyl, α- or β-naphthyl and the like,
Further o-, m- or p-methylbenzyl, o-, m- or p-ethylbenzyl, o-, m- or p-isopropylbenzyl, o-, m- or p-tert-butylbenzyl,
2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-dimethylbenzyl, 2,3,4-, 3,4,5-or 2,4, 6-trimethylbenzyl, 5-isopropyl-2-methylbenzyl, 2-isopropyl-5-methylbenzyl, 2-methyl-5-tert-butylbenzyl,
2,4-, 2,5- or 3,5-diisopropylbenzyl, 3,5-di-tert-butylbenzyl, 1- (2-methylphenyl) ethyl, 1- (3-methylphenyl) ethyl, 1- (4-methylphenyl) ethyl, 1- (2-isopropylphenyl) ethyl, 1- (3-isopropylphenyl) ethyl, 1- (4-isopropylphenyl) ethyl, 1- (2-tert-butylphenyl) ethyl, 1- (4
-Tert-butylphenyl) ethyl, 1- (2-isopropyl-4-methylphenyl) ethyl, 1- (4-isopropyl-2-methylphenyl) ethyl, 1- (2,4-dimethylphenyl) ethyl, 1- (2,5-dimethylphenyl) ethyl, 1- (3,5-dimethylphenyl) ethyl, 1-
(3,5-di-tert-butylphenyl) ethyl and the like can be mentioned. Preferred is an aralkyl group having 7 to 12 carbon atoms.

The heterocyclic group represented by R ⁷ is 1 to 4
5- or 6-membered heterocyclic groups containing one nitrogen, oxygen and / or sulfur atom and bonded on a carbon atom are mentioned. Specific examples thereof include, for example, pyrrolidinyl, pyrrolyl, pyrazolyl, imidazolyl, furyl, thienyl, oxazolyl, isoxazolyl, isothiazolyl, thiazolyl, piperidinyl, pyridyl, pyridazinyl, pyrazinyl, piperazinyl, pyrimidinyl, indolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,3
-Dioxolanyl, morpholinyl and the like. Further, the heterocyclic group may be condensed with a 5- or 6-membered ring (eg, benzene, pyridine, cyclohexane etc.) to form a bicyclic condensed ring group (eg, 8-quinolyl, 8-purinyl etc.). Good.

The alkyl group, aryl group and aralkyl group represented by R ⁸ or R ⁹ are the above R ⁷
Examples of the group represented by Examples of the cycloalkyl group represented by R ⁸ or R ⁹ include cycloalkyl groups having 3 to 8 carbon atoms. Specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Preferred is a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

As the acyl group represented by R ⁸ or R ⁹ ,
An acyl group derived from an organic carboxylic acid is preferable. Preferred examples include a formyl group, an alkanoyl group, an arylcarbonyl group, an aralkylcarbonyl group, an alkyloxycarbonyl group and a heterocyclic carbonyl group. The acyl group is preferably an alkanoyl group, an arylcarbonyl group and an alkyloxycarbonyl group. An alkanoyl (alkylcarbonyl) group, an alkyl group in an alkyloxycarbonyl group, an aryl group in an arylcarbonyl group,
Examples of the aralkyl group in the aralkylcarbonyl group and the heterocyclic group in the heterocyclic carbonyl group include those exemplified as the groups represented by R ⁷ above. The acyl group is more preferably one having 2 to 7 carbon atoms (eg, acetyl, propionyl, butyryl,
Isobutyryl, valeryl, isovaleryl, pivaloyl,
Benzoyl, formyloxy, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, tert-butoxycarbonyl, isobutoxycarbonyl and the like). R ⁸
Examples of the heterocyclic group formed by R ⁹ and R ⁹ together with the adjacent nitrogen atom include a 5- or 6-membered heterocyclic group containing 1 to 4 nitrogen atoms, oxygen atoms and sulfur atoms. Specific examples thereof include morpholino and piperazino.

Each of the substituents represented by R ⁷ , R ⁸ or R ⁹ is a suitable substituent (eg, a hydroxyl group, a thiol group which may be substituted with a C _1-6 alkyl group, a carboxyl group, carbamoyl). Group, an amidino group, a guanidino group, a heterocyclic group, an amino group which may be mono- or di-substituted with C _1-6 alkyl, etc.). The substituents on the optionally substituted amino group and the optionally substituted thiol group are preferably an alkyl group or an aralkyl group, more preferably an alkyl group. Among them, a C _1-6 alkyl group (eg, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, etc.) or a C _7-12 aralkyl group (eg, benzyl, 1-phenylethyl, 2-phenylethyl, etc.) Is more preferable. Examples of the heterocyclic group include those exemplified as the group represented by —NR ⁸ R ⁹ of the group represented by R ⁷ .

The R ¹ is preferably a carboxyl group. In the above general formula (I), the etherified hydroxyl group represented by R ² , R ³ , R ⁴ , R ⁵ or R ⁶ includes, for example, the general formula: —OR ¹⁰ (wherein R ¹⁰ is, for example, an alkyl group). A group represented by a group, an aryl group, an aralkyl group, a heterocyclic group or the like) is an esterified hydroxyl group, for example, the general formula —OR
Examples thereof include groups represented by ¹¹ (in the formula, R ¹¹ represents an acyl group or the like). Examples of the alkyl group, aryl group, aralkyl group, heterocyclic group represented by R ¹⁰ and the acyl group represented by R ¹¹ include those exemplified as the groups represented by R ⁷ . The above R ² , R ³ , R ⁴ and R ⁶ are preferably hydroxyl groups. R ⁵ above
Is preferably a hydroxyl group or a C _1-3 alkoxy group.

The compound represented by the above general formula (I) or a salt thereof has stereoisomers, and each isomer and a mixture thereof are also included in the present invention. Also the general formula
The compound represented by (I) may form a salt. For example, when the molecule has a basic group such as an amino group, it may form an acid addition salt, and when it has an acidic group such as a carboxyl group, it may form a base salt. Examples of the acid for forming the acid addition salt include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid; organic acids such as acetic acid, benzoic acid, succinic acid, tartaric acid and maleic acid. . Examples of the base for forming a base salt include sodium,
Alkali metals such as potassium; alkaline earth metals such as magnesium and calcium; ammonia, alkylamines (eg, methylamine, dimethylamine, triethylamine, etc.), amines such as pyridine, and the like.

In the general formula (I) of the present invention, R
Wherein ¹ is a carboxyl group, R ² , R ³ , R ⁴ and R ⁶ are each a hydroxyl group, and R ⁵ is a methoxy group.
Compound represented by A) (hereinafter, may be referred to as compound (IA) or TAN-1532A), and R ¹
Is a carboxyl group, and R ² , R ³ , R ⁴ , R ⁵ and R
A compound represented by the above formula (IB) in which ⁶ is a hydroxyl group (hereinafter, compound (IB or TAN-1532).
B)) is preferable.

The method for producing the compound represented by the general formula (I) or a salt thereof will be described below. The compound represented by the general formula (I) of the present invention or a salt thereof, particularly the compound (I
-A), compound (IB) and salts thereof preferably belong to the genus Streptomyces, and compound (IA)
And / or by culturing in a medium a microorganism capable of producing compound (IB), compound (IA) and / or compound (IB) is produced and accumulated in the medium, and by collecting this Manufactured. The compound of the present invention (I-
Examples of the microorganism that can be used for the production of (A) or (IB) include Streptomyce (Streptomyce).
s) Actinomycetes belonging to the genus, preferably microorganisms belonging to Streptomyces roseoglyceus. Examples of the microorganism include strain AL-28015 newly isolated from Indian soil. About the AL-28015 strain, International Journal of Systematic Bacteriology
Journal of Systematic Bacteriology), 16, 3
The mycological properties examined according to the method described in 13-340 (1966) are as follows. Unless noted otherwise, the findings on the medium were cultured at 28 ° C for 14 days,
It is what I observed.

(I) Morphological characteristics Aerial mycelium extends from a well-branched basal mycelium in a simple branching manner, and a spore chain (usually 1
(0 to 50 or more) has a linear or wavy shape. No raw silk is found. Spores are cylinder gauges (φ1.1-1.4 × 0.5)
μm) and its surface is smooth. (ii) Growth state on various media The following are listed below regarding the degree of growth (G), growth and color tone (AM) of the aerial mycelium, color tone (R) on the back surface, presence or absence of soluble pigment and color tone (SP) in various media. To do. Regarding the description of colors, the standard tone symbol shown in () is the Continer Corporation of America.
n of America) The Color Harmony Manual (The Color Harmony Manual) 4th Edition, 195
It's been eight years.

[0025]

[Table 1]

(Iii) Physiological properties (a) Growth temperature range: 14 to 43 ° C. Optimal growth temperature range: 24 to 34 ° C. (b) Nitrate reduction: Negative (c) Gelatin liquefaction: Positive (glucose peptone (Gelatin medium) (d) Starch hydrolysis: Weakly positive (e) Coagulation of skim milk: Negative Peptonization of skim milk: Positive (f) Formation of melanin-like pigment Tyrosine agar medium: Positive peptone, yeast extract, iron agar medium : Positive (g) carbon source assimilation (predham-Gottlieb agar) L-arabinose: ++ D-xylose: ++ D-glucose: ++ D-fructose: ++ Sucrose: ++ inositol: ++ L-rhamnose: ++ Raffinose: ++ D-mannite: ++ Control: − (Note) ++: Relatively good growth +: Growth is recognized ±: Difficult to judge + or − −: No growth

(Iv) Bacterial cell analysis Hasegawa et al.'S method (Journal of General Applied Microbiology)
al Applied Microbiology) 29, 319-322.
(1983)), diaminopimelic acid in the hydrochloric acid hydrolyzate of the cells was LL-form.

From the above results, the aerial hyphae of this strain exhibit a slightly reddish gray color, the spore chain is linear or wavy, the spore surface is smooth, and melanin pigment is produced, and soluble pigment is produced. Judging from various properties such as not doing and diaminopimelic acid being LL-form, it is clear that it belongs to the genus Streptomyces, and Streptomyces sp. AL-2801
5 ( Streptomyces sp. AL-28015). Based on the above properties, ISP (International St
When you search for a reptomyces Project) described strains, as a similar strains in the present strain, Streptomyces Feo Billiton disk (S. phaeoviridis of gray series), Streptomyces Noboritoenshisu (S. noboritoensi
s ), Streptomyces nashbilensis ( S. nas
hvillensis ), Streptomyces purpeofuscus ( S. purpeofuscus ), Streptomyces cinereoruber ( S. cinereoruber ), Streptomyces zanthophaeus ( S. xanthophaeus ), Streptomyces roseogriseus rose ( S. Eight strains such as S. actuosus can be mentioned. These 8 strains and this strain AL-28
As a result of comparison with 015, it was determined that S. roseogriseus IFO 13406 was the most closely related, and the results are shown below.

[0029]

[Table 2]

[0030]

[Table 3]

[0031]

[Table 4]

[0032]

[Table 5]

When AL-28015 and IFO-13406 were compared, a difference was observed in the aerial mycelial settlement in sucrose nitrate agar medium, starch inorganic salt agar medium, peptone-yeast-iron agar medium, etc., and gelatin liquefaction. Differences were also observed in the assimilation properties of D-xylose, L-arabinose and D-fructose.
L-28015 and IFO-13406 were determined to be strains of the same species. Therefore, this strain is S. roseogriseus AL-280.
Identified as 15.

This strain was designated by the Fermentation Research Institute (IFO) on January 31, 1994 under the accession number IFO15646.
In addition, on March 9, 1994, Institute of Biotechnology, Institute of Industrial Science and Technology
(NI) under the deposit number FERM P-14224. The compound of the present invention is not limited to these strains, and includes, by a method known per se, including genetic engineering techniques, a mutant strain having the ability to produce the present compound derived from them, and a microorganism having the ability to produce the medium. It can be produced by culturing in the medium, allowing the compound to form and accumulate in the medium, and collecting the compound.

The medium used for culturing the compound-producing bacterium of the present invention may be liquid or solid as long as it contains a nutrient source that can be utilized by the bacterium, but it is more preferable to use a liquid medium when treating in large quantities. Is. A carbon source, a nitrogen source, an inorganic substance, and a trace nutrient source that can be assimilated by the compound-producing bacterium are appropriately added to the medium. Examples of carbon sources include glucose, lactose,
Sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, oils and fats (eg, soybean oil, lard oil, chicken oil, etc.), n-paraffin and the like are used. As the nitrogen source, for example, meat extract, yeast extract,
Dry yeast, soybean flour, corn steep liquor, peptone, cottonseed flour, molasses, urea, ammonium salts (eg,
Ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) are used.

Further, salts containing sodium, potassium, calcium, magnesium and the like, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid and salts of organic acids such as acetic acid and propionic acid. May be used as appropriate. In addition, amino acids (eg, glutamic acid, aspartic acid, alanine, lysine, methionine, proline, etc.), peptides (eg, dipeptides, tripeptides, etc.), vitamins (eg, B ₁ , B ₂ , nicotinic acid,
B ₁₂ , C, etc.), nucleic acids (eg, purines, pyrimidines, derivatives thereof, etc.) and the like may be contained. Of course, an inorganic or organic acid or alkali, a buffer, etc. may be added for the purpose of adjusting the pH of the medium, or an appropriate amount of fats and oils, a surfactant, etc. may be added for the purpose of defoaming. In liquid culture, the pH of the medium is near neutral, especially pH 6-
8 is preferable. The culturing temperature is preferably about 24 ° C to 30 ° C, and the culturing time is preferably about 48 hours to 144 hours.

The compound (I
-A) and compound (IB) were quantified by using the HeLa (HeLa) cells as an indicator of the cell growth inhibitory action, and the method of Mossman [Journal of Immunological Method (Journal of Immunological Method. Meth
ods), 65 , 55 (1983)] by a modified method. For example, 2 × 10 HeLa cells
Suspend in a medium at a concentration of ⁴ cells / ml, and dispense 0.1 ml each into each well of a 96-well flat bottom plate (Nunc). HeL
Minimum essential medium of Eagle for a-cells (Whittaker Bioproducts, USA)
Fetal bovine serum (Whittaker BioProducts)
Use a medium supplemented with 10%. 37 above plate
After culturing for 1 day in a carbon dioxide gas incubator set at 5 ° C. and 5% CO ₂ , compounds of various concentrations (I-
Phosphate saline in which A) and the compound (IB) are dissolved is added to each well in which the cell suspension is dispensed in an amount of 10 μl. After culturing for 3 days after the addition, the tetrazolium salt MTT (Sigma, USA) was added to P to a concentration of 5 mg / ml.
25 μl of a solution dissolved in BS is added and kept warm in the carbon dioxide gas incubator. 0.01N after 4 hours
0.1 ml of a solution of SDS in 10% hydrochloric acid was added to each well, and the mixture was kept warm in the carbon dioxide incubator overnight, and the absorbance at 620 nm was measured by a titer tech multi-scan absorbance meter (Flow Co.). , USA). Using the absorbance obtained by adding 10 μl of PBS alone as a control, the cell growth inhibition rate (%) of the compound (IA) and the compound (IB) at each concentration is calculated. Usually, the maximum production of compound (IA) and compound (IB) is reached in 4 to 6 days of culture.

The method for collecting the desired compound (IA) or compound (IB) from the culture is described below.
Since these are fat-soluble substances and show acidity, a general means utilizing this property may be adopted. Since each of these components is contained in the bacterial cells and the filtrate, for example, the following purification method is adopted. First, the culture solution is adjusted to pH 1.5 to 10
After adjusting the pH to 2 to 8, preferably, an organic solvent immiscible with water, such as chloroform, ethyl acetate, methyl isobutyl ketone or butanol, is added, and the mixture is stirred and mixed for 10 minutes to 10 hours, preferably 30 minutes to 2 hours, and the filter aid is added. The agent is added and the cells are removed by filtration or centrifugation. The obtained organic solvent layer is washed with water and then concentrated, or the obtained organic layer is an aqueous solution of an appropriate inorganic acid (eg, hydrochloric acid, sulfuric acid, phosphoric acid, etc.) or an appropriate inorganic salt (eg, After washing appropriately with an aqueous solution of sodium chloride, sodium hydrogen carbonate, ammonium chloride, etc., wash with water,
Concentration gives a crude material containing compound (IA) or compound (IB).

The crude material was further purified to give the pure compound (I
Various general chromatographic methods are advantageously used to obtain -A) or compound (IB). As the carrier, silica gel, crystalline cellulose, adsorptive resin such as Diaion HP-20 (manufactured by Mitsubishi Kasei), Amberlite XAD-I or II (manufactured by Rohm and Haas, USA), Sephadex LH-20 ( Pharmacia, Sweden) and the like are used, and these are usually carried out by a column chromatography method. To elute the active substance from the carrier, depending on the kind of the carrier, a suitable organic solvent, for example, dichloroethane, toluene, ethyl acetate, acetone, 2-propanol, methanol alone or in a mixed solvent, or mixed with water is used. Obtained organic solvent (eg, acetone, acetonitrile, methanol, etc.)
A mixed solvent of an aqueous solution (eg, water, dilute alkaline water, dilute acid water, buffer solution, etc.) is used.

High-performance liquid chromatography for preparative separation
Compound (IA) or compound (IB) can also be purified by (HPLC). Octadecylsilane (ODS) -based and silica gel-based carriers are advantageously used as the carrier. For example, in the case of ODS, a mixed solution of methanol or acetonitrile and water or an aqueous solution containing salts is advantageously used. The pure compound (IA) or compound (IB) is yellow when the organic solvent eluate containing the active substance is concentrated or, if it contains an aqueous solution, extracted with a suitable organic solvent immiscible with water and concentrated to dryness. Alternatively, it is obtained as a yellow-orange powder. In addition, since this substance has low solubility in a solvent, it is preferably purified by crystallization. As the crystal solvent, one of methanol, ethyl acetate, acetone, dichloromethane, chloroform, toluene, diethyl ether, hexane or a mixed solvent thereof is advantageously used.

Compound (IA) and compound (IB)
Is an acidic fat-soluble substance, and as a common property, it is positive for potassium permanganate, phosphomolybdic acid, sulfuric acid, Burton, iodine in the color reaction, and negative for Dragendorff, ninhydrin. The behaviors in the following analytical HPLC and thin layer chromatography (TLC) are shown in Table 6.

[0042]

[Table 6]

HPLC conditions: carrier; ODS, YMC
-Pack A-312 mobile phase; 70% acetonitrile / 0.01 M phosphate buffer (pH 3.0) flow rate; 2 ml / min detection method; UV absorption: 214,254 nm TLC conditions: carrier; silica gel 60 F254.
(E. Merck AG.) Developing solvent; dichloromethane: methanol: acetic acid (24: 1:
0.5)

The compound (I-
The physicochemical properties of A) and compound (IB) are shown below. Compounds (IA), (TAN-1532A): (1) Appearance: yellow powder (2) Optical rotation: [α] _D (18 ° C) + 54.6 ° (c 0.1
30, in methanol) (3) Molecular weight: m / z 519 (M + H), (from SI-mass spectrum) (4) Elemental analysis value: (%) (water content: 1.5 mol) Actual value: C, 66. 04; H, 6.10 Calculated value: C, 66.01; H, 6.02 (5) Molecular formula: C ₃₀ H ₃₀ O ₈ (6) Ultraviolet absorption (UV) spectrum: in methanol (Fig. 1) Maximum value: 227 nm (ε48,800), 395 nm (ε38,4)
00) (7) Red external absorption (IR) spectrum: in KBr tablets,
(Fig. 2) Main absorptions (wave number, cm ^-1 ) 3260, 2950, 1710, 1600, 1450, 13
80,1280,1190 (8) ¹³ C nuclear magnetic resonance (NMR) spectrum: 75 MHz,
(Fig. 3) δppm; 189.0 (Q), 173.7 (Q), 165.9 (Q), 1 in deuterated dimethylsulfoxide.
65.0 (Q), 160.2 (Q), 157.9 (Q), 149.8
(Q), 146.7 (Q), 145.5 (Q), 142.8 (Q), 1
39.8 (Q), 123.9 (Q), 121.9 (CH), 119.
1 (Q), 117.0 (CH), 112.4 (Q), 112.4 (Q), 1
07.9 (Q), 106.0 (CH), 102.0 (CH), 76.
3 (Q), 51.9 (CH ₃ ), 37.6 (CH ₃ ), 35.6 (C
H ₂ ), 31.5 (CH ₂ ), 31.1 (CH ₂ ), 28.7 (CH ₂ ),
_{21.9 (CH 2), 19.3 (} CH 2), 13.9 (CH 3).

Compounds (IB), (TAN-1532)
B): (1) Appearance: Yellow powder (2) Optical rotation: [α] _D (18 ° C) + 152 ° (c 0.13)
(3, in methanol) (3) Molecular weight: m / z 505 (M + H), (from SI-mass spectrum) (4) Elemental analysis: (%) (water content: 0.5 mol) Measured value: C, 67. 65; H, 5.74 Calculated value: C, 67.83; H, 5.69 (5) Molecular formula: C ₂₉ H ₂₈ O ₈ (6) Ultraviolet absorption (UV) spectrum: in methanol (Fig. 4) Maximum value: 215 nm (sh, ε21,800), 393 nm (ε3
2,400) (7) Red external absorption (IR) spectrum: in KBr tablets,
(Fig. 5) Main absorptions (wave number, cm ^-1 ) 3430, 2930, 1710, 1600, 1470, 13
70,1280,1180,1050 (8) ¹³ C nuclear magnetic resonance (NMR) spectrum: 75 MHz,
(Fig. 6) δppm; 189.0 (Q), 173.6 (Q), 165.6 (Q), 1 in deuterated dimethylsulfoxide.
64.6 (Q), 160.2 (Q), 157.4 (Q), 155.0
(Q), 148.2 (Q), 146.5 (Q), 145.4 (Q), 1
39.7 (Q), 123.1 (Q), 121.8 (CH), 119.
2 (Q), 116.9 (CH), 112.4 (Q), 110.9
(Q), 106.4 (Q), 106.1 (CH), 101.2 (C
H), 69.6 (Q), 38.9 (CH ₃ ), 35.5 (CH ₂ ), 3
1.5 (CH ₂ ), 31.1 (CH ₂ ), 28.9 (CH ₂ ), 21.9
_{(CH 2), 19.3 (CH} 2), 13.8 (CH 3). From the physicochemical and biological properties described above, it was found that the compound (IA) and the compound (IB) are novel compounds.

Of the compounds of the present invention, R ² , R ³ , R ⁴ ,
The compound in which R ⁵ or R ⁶ is an etherified hydroxyl group is, for example, a compound of the general formula (I) in which the corresponding R ² , R ³ , R ⁴ , R ⁵ or R ⁶ is a hydroxyl group or a salt thereof (eg, ,
Compound (IA), compound (IB) or salts thereof, etc.) may be used as a starting compound for etherification by a method known per se. For example, a method of reacting a raw material compound with a halide and a base, a method of reacting a corresponding lower sulfonic acid ester such as optionally substituted lower alcohols, arylalkanols or benzhydrols, and a base, A method of reacting with a diazo compound, a method of reacting with a sulfate ester, a method of subjecting a raw material compound to an acetal type etherification reaction or a glucosyl reaction, and the like are adopted.

Examples of the halide in the above method include alkyl halide (eg, C _1-6 alkyl halide such as methyl iodide, ethyl iodide, propyl iodide and tert-butyl bromide) and alkenyl halide. (Eg, allyl bromide, butenyl bromide, C _3-6 alkenyl halide such as 2-methylpropenyl bromide, etc.), aralkyl halide (eg, benzyl bromide, diphenylmethyl bromide, triphenylmethyl bromide, etc.) Halogenated C such as p-nitrobenzyl bromide and p-bromobenzyl bromide
_6-18 aryl-C _1-3 alkyl, etc.), halogenated alkoxyalkyl (eg, methoxymethyl chloride, ethoxymethyl chloride, methoxyethoxymethyl chloride, t-butoxymethyl chloride, 2,2,2-trichloroethoxymethyl, etc. Halogenated C _1-6 alkoxy-C _1-3 alkyl etc.),
_{Alkylthioalkyl} halides (eg, methylthiomethyl chloride, ethylthiomethyl chloride, C _1-6 alkyl-C _1-3 alkyl halides such as t-butylthiomethyl chloride), halogenated cyclic ethers (eg, tetrahydropyranyl chloride) ), Silyl halides (e.g., trimethylsilyl chloride, triethylsilyl chloride, triisopropylsilyl chloride, -tert-butyldimethylsilyl chloride, -te chloride)
(rt-butyldiphenylsilyl, etc.) and the like.

Examples of the sulfonic acid ester in the above method include optionally substituted lower (C _1-8 ) alcohols (eg, methanol, ethanol, isopropanol, tert-butanol, cyanomethyl alcohol,
Benzoylmethyl alcohol, etc., optionally substituted arylalkanols (eg, lower (C _1-6 ) alkyl group, lower (C _1-6 ) alkoxy group, or benzyl alcohol optionally substituted by halogen atom) ),
Or optionally substituted benzhydrols (e.g.,
Benzhydrol, p-nitrobenzyl alcohol, p-
Corresponding sulfonic acid esters such as methoxybenzyl alcohol, 2,4,6-trimethylbenzyl alcohol, etc. (eg, benzenesulfonic acid ester, p-toluenesulfonic acid ester, methanesulfonic acid ester, trifluoromethanesulfonic acid ester, etc.), etc. Is mentioned. The amount of these halides and sulfonates to be used is about 0.1 to 5 relative to the starting compound.
It is 0 equivalent, preferably about 1 to 10 equivalents.

Examples of the base in the above method or the above include inorganic bases (eg, alkali metal or alkaline earth metal salts of carbonic acid such as sodium carbonate and potassium carbonate,
Alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide and calcium hydroxide,
Alkali metal or alkaline earth metal hydrides such as sodium hydride and calcium hydride), organic bases
(E.g., sodium methoxide, sodium ethoxide,
Alkali metal alkoxides such as potassium tert-butoxide, lithium diisopropylamide, lithium bistrimethylsilylamide, lithium dicyclohexylamide, lithium amides such as lithium cyclohexylisopropylamide, methyllithium, ethyllithium, n-, sec-, and tert-butyl. Alkyl lithiums such as lithium and phenyl lithium, Grignard reagents such as methyl magnesium bromide, ethyl magnesium bromide, butyl magnesium bromide and phenyl magnesium bromide, pyridine, 2,4,6-trimethylpyridine, picoline, 4-dimethylaminopyridine,
Aromatic amines such as 2,6-lutidine and imidazole, triethylamine, diisopropylethylamine,
Tertiary amines such as dimethylaniline) and the like are used. The amount of the base used is usually relative to the starting compound,
It is about 0.1 to 50 equivalents, preferably about 1 to 10 equivalents.

Examples of the diazo compound in the above method include diazomethane, trimethylsilyldiazomethane, diphenyldiazomethane and the like. Examples of the sulfuric acid ester in the above method include dimethyl sulfuric acid and diethyl sulfuric acid. The amount of each of these diazo compounds and sulfates used is about 1 to 100 equivalents, preferably about 2 to 30 equivalents, relative to the starting compound.

The acetal type etherification reaction in the above method is carried out by a method known per se. For example, a raw material compound under an acid catalyst, for example, hemiacetals (e.g.,
Dihydropyran, 2-methoxypropene, ethyl vinyl ether, isopropyl vinyl ether, etc.), hemiacetals of optionally protected sugar residues (eg, optionally protected glucals, optionally protected galactals, etc.) ) And the like.
Examples of the acid used as a catalyst include haloacetic acid
(Eg, trifluoroacetic acid, etc.), hydrohalic acid (eg,
Hydrochloric acid, hydrobromic acid, etc.), sulfonic acid (eg, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, D-camphorsulfonic acid, etc.), Lewis acid (eg, zinc- Acetic acid, boron trifluoride ether complex, iodotrimethylsilane, trifluoromethanesulfonic acid trimethylsilyl ester, titanium tetrafluoride, titanium tetrachloride, stannous chloride, silver perchlorate, trifluoromethanesulfonic anhydride, etc.) To be

The glucosylation reaction in the above method is performed by a method known per se [eg, Journal of Organic Synthetic Chemistry, 50 , 378-390 (1992)]. For example, a glucosyl donor that may be protected with a raw material compound under an acid catalyst (eg, halogenated sugar, thioglycoside, 1-O-acyl sugar, carbonic acid esterified sugar, phosphoric acid esterified sugar, sugar imidate, 4- (Pentenyl thioglycoside etc.) and the like. As the acid used as the catalyst, the same acids as those mentioned above are used. The amount of these catalysts to be used may be a catalyst amount capable of promoting the etherification of the raw material compound, and is usually about 0.001 mol with respect to 1 mol of the raw material compound.
It is 1 to 10 mol, preferably about 0.001 to 5 mol.

The etherification reaction from above can be carried out in the presence of a solvent. The solvent is not particularly limited as long as it does not inhibit the reaction. For example, anhydrous formamide, dimethylformamide, amides such as N-methylpyrrolidone, sulfoxides such as dimethyl sulfoxide, aromatic amines such as pyridine, ethers such as diethyl ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, acetonitrile, etc. Nitriles, hydrocarbons such as pentane, hexane, benzene and toluene, halogenated hydrocarbons such as dichloromethane and chloroform, ketones such as acetone and ethylmethylketone, or a mixture thereof in an appropriate ratio. . The reaction temperature is appropriately selected from the range known to be usable for the reaction. Specifically, for example, usually about -100 ° C to + 100 ° C.
Is appropriately selected from the range. The reaction time is appropriately selected from the range known to require the reaction. Specifically, for example, the reaction is performed for several minutes to about 7 days.

Of the compounds of the present invention, R ² , R ³ , R ⁴ ,
The compound in which R ⁵ or R ⁶ is an esterified hydroxyl group is a compound of the general formula (I) in which the corresponding R ² , R ³ , R ⁴ , R ⁵ or R ⁶ is a hydroxyl group or a salt thereof (eg, compound (IA), compound (IB) or salts thereof, etc.)
May be subjected to an esterification reaction, for example, an acylation reaction, by a method known per se. As the acylating agent used in the present acylation reaction, for example, an organic carboxylic acid or its reactive derivative is used. Examples of the organic carboxylic acid include carboxylic acids represented by the general formula: R ⁷ —COOH (wherein R ⁷ has the same meaning as described above).

When these organic carboxylic acids are used as an acylating agent, they can be acylated by reacting with a suitable condensing agent. As the condensing agent, dicyclohexylcarbodiimide, diisopropylcarbodiimide,
Dimids such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, or the above condensing agent and pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5
Norbornene-2,3-dicarboximide, N-hydroxysuccinimide, N-hydroxyphthalimide, N
-A mixture with hydroxybenztriazole or the like is used. Alternatively, a mixture of azodicarboxylic acid esters (eg, azodicarboxylic acid diethyl ester) and trialkylphosphine (eg, triphenylphosphene, tributylphosphene) is used.

The above condensation reaction can be carried out in the presence of a solvent. The solvent can be appropriately selected from those known to be usable in the condensation reaction. For example, anhydrous formamide, dimethylformamide, amides such as N-methylpyrrolidine, sulfoxides such as dimethylsulfoxide, aromatic amines such as pyridine, halogenated hydrocarbons such as chloroform and dichloromethane,
Examples thereof include ethers such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, esters such as ethyl acetate and ethyl formate, and a mixture thereof in an appropriate ratio. The reaction temperature is appropriately selected from the range known to be applicable to the condensation reaction. Specifically, for example, it is usually appropriately selected from the range of -20 ° C to + 50 ° C. The reaction time is appropriately selected from the range known to be necessary for the condensation reaction. In particular,
For example, the reaction is performed for several minutes to about 7 days.

When a reactive derivative of an organic carboxylic acid is used as an acylating agent, these reactive derivatives include, for example, acid halides, acid anhydrides, active amides, active esters, active thioesters, etc., which can be produced by a conventional method. Is used. A concrete description of such a reactive derivative is as follows.

1) Acid halide: For example, acid chloride, acid bromide or the like is used. 2) Acid anhydrides: for example, symmetrical acid anhydrides, mono-C _1-6 alkyl carbonic acid mixed anhydrides, aliphatic carboxylic acids (eg, acetic acid, pivalic acid, valeric acid, isovaleric acid, trichloroacetic acid, etc.) An acid anhydride, a mixed acid anhydride containing an aromatic or an aromatic carboxylic acid (eg, benzoic acid, etc.) and the like are used. Examples of symmetrical acid anhydrides include C _1-6 alkylcarboxylic acid anhydrides such as acetic anhydride, propionic anhydride and butanoic anhydride. 3) Active amides: eg pyrazole, imidazole, 4
An amide with a substituted imidazole, dimethylpyrazole, benzotriazole or the like is used.

4) Active esters: eg methoxymethyl ester, 1-hydroxybenzotriazole ester, N-hydroxy-5-norbornene-2,3-dicarboximide ester, 4-nitrophenyl ester, 2,4-dinitrophenyl ester. In addition to esters such as trichlorophenyl ester, propargyl ester and pentachlorophenyl ester, esters with 1-hydroxy-1H-2-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide and the like are used. 5) Active thioesters such as 2-pyridylthiol,
A thioester or the like with a heterocyclic thiol such as 2-benzothiazolylthiol is used. The various reactive derivatives as described above are appropriately selected depending on the type of carboxylic acid.

As the acylating agent, a reactive derivative of sulfonic acid which can introduce an acyl sulfonate, for example,
Acid halides such as methanesulfonyl chloride, benzylsulfonyl chloride, p-toluenesulfonyl chloride,
Symmetrical anhydrides such as anhydrous methanesulfonic acid and anhydrous p-toluenesulfonic acid may be used. The acylating agent such as a carboxylic acid, a reactive derivative thereof or a reactive derivative of a sulfonic acid may be a raw material compound (eg, compound (IA), compound (I)).
For example, about 1 mol or more may be used per 1 mol of each of (B) and the like), and about 1 to 30 mol is preferable. This reaction is carried out in a solvent that does not inhibit the reaction or in the absence of a solvent. Examples of the solvent that does not inhibit the reaction include ketones such as acetone; ethers such as diethyl ether, tetrahydrofuran and dioxane; carboxylic acids such as acetic acid and propionic acid; nitriles such as acetonitrile; hydrocarbons such as benzene, toluene and xylene. Halogenated hydrocarbons such as dichloromethane, chloroform, 1,2-dichloroethane; Esters such as ethyl acetate; Amides such as dimethylformamide and dimethylacetamide; Triethylamine, tributylamine, N-
Tertiary amines such as methylmorpholine, N-methylpiperidine, N, N-dimethylaniline; pyridine, picoline,
Pyridines such as lutidine and collidine are used. These may be used alone or in combination of two or more kinds at an appropriate ratio.

The present acylation reaction proceeds more advantageously by using a catalyst capable of promoting the acylation of the raw material compound. As such a catalyst, for example, a base catalyst or an acid catalyst is used. Examples of the base catalyst include tertiary amine
[Eg, aliphatic tertiary amines such as triethylamine, pyridine, α-, β- or γ-picoline, 2,6-lutidine, 4-dimethylaminopyridine, 4- (1-pyrrolidinyl) pyridine, dimethylaniline, diethyl Aromatic tertiary amines such as aniline, etc.], alkali metal halides (eg, potassium fluoride, anhydrous lithium iodide, etc.), organic acid salts (eg, sodium acetate) and the like are used. Examples of the acid catalyst include Lewis acids (eg, anhydrous zinc chloride, anhydrous aluminum chloride (AlCl ₃ ), titanium tetrachloride (TiCl ₄ ), tin tetrachloride (SnCl ₄ ), antimony pentachloride, cobalt chloride,
Cupric chloride, boron trifluoride etherate, etc., strong inorganic acid (eg, sulfuric acid, perchloric acid, hydrogen chloride, hydrogen bromide, etc.), strong organic acid (eg, benzenesulfonic acid, p-toluenesulfonic acid, trifluoro) Acetic acid, trichloroacetic acid, camphorsulfonic acid, etc.), acidic ion exchange resin (eg, polystyrenesulfonic acid), etc. are used. Among the above catalysts, camphorsulfonic acid, pyridine, 4-dimethylaminopyridine and the like are preferable.

The amount of the catalyst used may be a catalytic amount capable of promoting the acylation of the raw material compound with a carboxylic acid, and is usually about 0.001 to 10 moles, preferably about 0.001 to 1 mole per mole of the raw material compound. It is a mole. The reaction temperature is not particularly limited, but is usually about -30 to + 100 ° C, preferably about 10 to 50 ° C. The reaction time is about several minutes to several tens hours (for example, about 5 minutes to 30 hours).

Among the compounds of the present invention, the compound in which R ¹ is an esterified carboxyl group is a compound of the general formula (I) in which R ¹ is a carboxyl group or a salt thereof (eg, compound (IA), Compound (IB) or salt thereof)
Is produced by esterification by a method known per se. For example, a compound of the general formula (I) or a salt thereof (eg, compound (IA), compound (IB) or a salt thereof, etc.) in which R ¹ used as a raw material compound is a carboxyl group is reacted with a halide. There is a method of making it. Examples of halides used include alkyl halides (eg, methyl iodide, ethyl iodide, propyl iodide, allyl bromide, benzyl bromide, tert-butyl bromide, triphenylmethyl bromide, methoxymethyl chloride, methylthiomethyl chloride). , Tetrahydropyranyl chloride, etc.), silyl halides (eg, trimethylsilyl chloride, triethylsilyl chloride, triisopropylsilyl chloride, tert-butyldimethylsilyl chloride, tert-butyldiphenylsilyl chloride, etc.) and the like.

This reaction is usually carried out in the presence of a base. Examples of the base used include aliphatic tertiary amines such as trimethylamine, triethylamine, tripropylamine, and tri-n-butylamine, diisopropylethylamine, N-methylpiperidine, N-methylpyrrolidine,
Cyclohexyldimethylamine, tertiary amines such as N-methylmorpholine, di-n-butylamine, diisobutylamine, dialkylamine such as dicyclohexylamine, dicyclohexylamine, imidazole, pyridine, 4-dimethylaminopyridine, lutidine, diazabicyclononane, Aromatic amines such as diazabicycloundecene and γ-collidine, for example, alkali metals such as lithium, sodium and potassium, or hydroxides and carbonates such as alkaline earth metals such as calcium and magnesium. To be Further, among the above, the liquid one can be used also as a solvent.

In this reaction, a halide is usually used in an amount of about 1 to 100 mol per mol of the starting compound.
It may be used in excess unless it interferes with the reaction. When a base is used, the amount of the base used varies depending on the kind of halide and other reaction conditions, but is usually about 1 to 30 equivalents, preferably about 3 to 1 equivalent to the starting compound.
It is 0 equivalent. Alternatively, suitable condensation with the starting compound of the above general formula (I) and an alcohol represented by the general formula which may be substituted: R ⁷ —OH (wherein, R ⁷ has the same meaning as above) Esterification can be performed by reacting with an agent. Specific examples of such alcohols include lower (C _1-6 ) alcohols (eg, methanol, ethanol, isopropanol, tert-butanol, cyanomethyl alcohol, benzoylmethyl alcohol, etc.), arylalkanols [eg, lower (C _{1- 6} )
Benzyl alcohol or benzhydrols (eg, benzhydrol, p-nitro) optionally substituted by an alkyl group, a lower (C _1-6 ) alkoxy group or a halogen atom (eg, fluorine, chlorine, bromine, iodine) Benzyl alcohol, p-methoxybenzyl alcohol, 2,
4,6-trimethylbenzyl alcohol, etc.)] and the like. As the condensing agent, dicyclohexylcarbodiimide, diisopropylcarbodiimide, diimides such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, or the above condensing agent and pentachlorophenol, 2,4,5-trichlorophenol,
2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxysuccinimide, N-hydroxyphthalimide,
A mixture with N-hydroxybenztriazole or the like is used. Alternatively, a mixture of azodicarboxylic acid esters (eg, azodicarboxylic acid diethyl ester) and trialkylphosphine (eg, triphenylphosphine, tributylphosphine) is used.

Alternatively, a compound represented by the general formula (I) in which R ¹ is a carboxyl group or a salt thereof (eg, compound (IA), compound (IB) or a salt thereof, etc.) is suitable. Esterification can be carried out by reacting with the above alcohols in the presence of an acid catalyst. You may use these alcohols as a solvent. Alternatively, a compound represented by the general formula (I) in which R ¹ is a carboxyl group or a salt thereof (eg, compound (IA), compound (IB) or a salt thereof, etc.) is treated with a suitable acid catalyst. Can be esterified by reacting with lower carboxylic acid esters of the above alcohols such as formic acid ester, acetic acid ester and propionic acid ester. These lower carboxylic acid esters may be used as a solvent. Also, R
A compound represented by the general formula (I) in which ¹ is a carboxyl group or a salt thereof (eg, compound (IA), compound (I-
B) or salts thereof, etc.) under suitable acid catalyst, optionally substituted ethylenes such as 2-methylpropene,
Esterification can be carried out by reacting with 2-methylbutene, 2-ethylbutene, 2,3-dimethylbutene, 2-phenylpropene, 2-phenylstyrene and the like. When these ethylenes are gases at room temperature, the reaction is usually carried out under a closed condition. Examples of the acid catalyst used for esterification under the above acid catalyst include hydrochloric acid, hydrobromic acid,
Sulfuric acid, phosphoric acid, chloric acid, perchloric acid, bromic acid, perbromic acid,
Inorganic acids such as iodic acid and periodic acid, sulfonic acids such as p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, trichloroacetic acid, formic acid, acetic acid, etc. Examples thereof include organic carboxylic acids, Lewis acids such as aluminum chloride, zinc chloride, titanium tetrachloride, and boron trifluoride etherate.

Alternatively, a compound represented by the general formula (I) in which R ¹ is a carboxyl group or a salt thereof (eg, compound (IA), compound (IB) or a salt thereof, etc.)
Is an isourea derivative such as the above alcohols or N,
The N-dialkylformamide acetal derivative can be prepared by a method known per se [eg, Synthesis, 561 (1971)].
9) etc.] can be used for esterification. The above esterification reaction is usually carried out in a solvent that does not inhibit the reaction. Examples of the solvent include ethers such as dioxane, tetrahydrofuran, diethyl ether and diisopropyl ether, esters such as ethyl acetate and ethyl formate, chloroform, dichloromethane, 1,2-dichloroethane, 1,1,1-trichloroethane and the like. Halogenated hydrocarbons, benzene, toluene, n-hexane and other hydrocarbons, N, N-dimethylformamide, N, N-dimethylacetamide and other amides, acetonitrile and other nitriles, dimethyl sulfoxide and other sulfoxides Ordinary organic solvents are used alone or in combination of two or more. The reaction temperature is not particularly limited as long as the reaction proceeds, but is usually about -5.
It is carried out at 0 ° C to + 150 ° C, preferably about -30 ° C to + 50 ° C. The reaction time will differ depending on the starting materials used, base, reaction temperature, type of solvent, etc., but the reaction is usually completed in several tens of minutes to several tens of hours, but sometimes it may take several tens of days.

Among the compounds of the present invention, the compound in which R ¹ is an amidated carboxyl group is a compound of the general formula (I) in which R ¹ is a carboxyl group or a salt thereof (eg, compound (IA) ), Compound (IB) or a salt thereof) or a reactive derivative thereof at a carboxyl group (eg, an acid halide, an acid anhydride, an active ester, an active thioester, etc. as described above) can be represented by the general formula: HNR ⁸ It may be produced by reacting with an amine represented by R ⁹ (in the formula, each symbol has the same meaning as above) to perform amidation. For example, a compound of the general formula (I) in which R ¹ is a carboxyl group can be amidated by reacting it with the above amines in the presence of a suitable condensing agent.
As the condensing agent, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-
Dimids such as dimethylaminopropyl) carbodiimide hydrochloride or the above condensing agent and pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy- 5-norbornene-2,3
-Dicarboximide, N-hydroxysuccinimide,
A mixture with N-hydroxyphthalimide, N-hydroxybenztriazole or the like is used.

This reaction may be carried out in the presence of a base. Examples of the base used include aliphatic tertiary amines such as trimethylamine, triethylamine, tripropylamine and tri-n-butylamine, diisopropylethylamine, N-methylpiperidine, N-methylpyrrolidine, cyclohexyldimethylamine, N-methylmorpholine and the like. Tertiary amines, dialkyl amines such as di-n-butylamine, diisobutylamine and dicyclohexylamine, aromatic amines such as pyridine, lutidine and γ-collidine, alkali metals such as lithium, sodium and potassium, or alkaline earths such as calcium and magnesium. Examples thereof include hydroxides or carbonates of group metals. Further, among the above, the liquid one can be used also as a solvent.

In this amidation reaction, amines are usually used in an amount of about 1 to 100 mol per mol of the starting compound.
It can also be used in excess unless it interferes with the reaction. When a base is used, the amount of the base used varies depending on the starting compound or salt thereof, the type of the reactive derivative of carboxylic acid, and other reaction conditions, but is usually relative to the starting compound.
It is about 1 to 30 equivalents, preferably about 1 to 10 equivalents.

This amidation reaction is usually carried out in a solvent that does not inhibit the reaction. Examples of the solvent include ethers such as dioxane, tetrahydrofuran, diethyl ether and diisopropyl ether, esters such as ethyl acetate and ethyl formate, halogens such as chloroform, dichloromethane, 1,2-dichloroethane and 1,1,1-trichloroethane. Ordinary organic solvents such as derivatized hydrocarbons, hydrocarbons such as benzene, toluene and n-hexane, amides such as N, N-dimethylformamide and N, N-dimethylacetamide, and nitriles such as acetonitrile, alone or 2 Used as a mixture of two or more species. The reaction temperature is not particularly limited as long as the reaction proceeds, but usually,
About -50 ° C to + 150 ° C, preferably about -30 ° C to +5
It is carried out at 0 ° C. The reaction time will differ depending on the starting materials used, the base, the reaction temperature, the type of solvent, etc., but the reaction is usually completed in several tens of minutes to several tens of hours, but sometimes it takes several days.

The desired compound represented by the general formula (I) or a salt thereof thus obtained can be obtained by a method known per se, for example, concentration, liquid conversion, phase transfer, solvent extraction, freeze drying, crystallization, recrystallization, It can be isolated and purified by fractional distillation, chromatography and the like.

Next, the biological activity of the compound represented by formula (I), particularly compound (IA) or compound (IB), will be described. Test Example 1 Microtubule Polymerization Inhibitory Activity Measurement Test The microtubule polymerization inhibitory activity of compound (IA) or compound (IB) was determined using the method of Iwasaki et al.
[Journal of Antibiotics
of Antibiotics), 40 , 66 (1987)]. The microtubules were prepared as follows. After slicing 7 pig cerebrum, 1 mM [ethylene glycol-
O, O'-bis (2-aminoethyl) -N, N, N'N'-tetraacetic acid] (EGTA), 0.5 mM MgS
100 mM containing O ₄ , 1 mM 2-mercaptoethanol
[2- (N-morpholino) ethane sulfonic acid,
Monohydrate] (MES) (pH 6.5) [hereinafter buffer A
After washing with 400 ml of buffer solution, 200 ml of buffer solution A [hereinafter referred to as buffer solution B] containing 1 mM GTP was added and homogenized with a blender.
It was centrifuged at pm at 4 ° C. for 30 minutes and the supernatant was collected. Buffer B containing the same amount of 8 M glycerol as the supernatant (hereinafter referred to as buffer C) was mixed with this supernatant, and this was mixed at 37 ° C. for 30 minutes.
After incubating for 3 minutes, it was centrifuged at 36,000 rpm and 30 ° C. for 45 minutes to collect the microtubule precipitate. 70 ml of buffer solution B was added to this and the precipitate was depolymerized by standing at 4 ° C. for 30 minutes. The operation of centrifuging again at 4 ° C. and 36,000 rpm for 1 hour and adding buffer C to the collected supernatant to recover the precipitate was repeated to collect the purified microtubule fraction. The collected microtubule fraction was diluted with buffer solution B to a concentration of 6 mg / ml, stored at -80 ° C, and used for the subsequent measurements.

Buffer B was added to the above partially purified microtubules so that the microtubule concentration was 2 mg / ml, and the same diluted solution 50 was added.
After adding 20 μl each of compound (IA) or compound (IB) diluted to 0 ml in advance to various concentrations, the degree of polymerization of microtubules at 37 ° C. was measured with a spectrophotometer at an absorbance of 400 nm over time. Measured. When the compound was not added (control), the degree of polymerization of microtubules was set to 100%, and the concentration at which the degree of polymerization was inhibited by 50% was determined. [Results] Compound (IA) and compound (IB) showed 50% inhibition of the degree of polymerization of control microtubules at 8.23 μg / ml and 15.3 μg / ml, respectively.

Test Example 2 Method for measuring squalene synthase inhibitory activity 5 μM [1-3H] farnesyl pyrophosphate (specific activity 25
μCi / mole), 1 mM reduced nicotiamide adenine dinucleotide phosphate (NADPH), 5 mM MgCl ₂ ,
6 mM glutathione, 100 mM potassium phosphate buffer (pH 7.4) and an aqueous solution (total volume 50 μl) containing the test drug compound (IA) or compound (IB) were added to the following enzyme solutions (protein amount). 0.8 μg) was added, and the mixture was reacted at 37 ° C. for 45 minutes. 150 μl of chloroform / methanol
(1: 2) Add mixed solution to stop the reaction, then 50 μl
Chloroform and 50 μl of 3N sodium hydroxide solution were added. 50 μl of a chloroform layer (lower layer) containing a reaction product containing squalene as a main component was mixed with 3 ml of a toluene liquid scintillator, and its radioactivity was measured by a liquid scintillation counter. The squalene synthase inhibitory activity was shown as the concentration (IC50, molar concentration (M)) at which the radioactivity incorporated into the chloroform layer was inhibited by 50%.

Preparation of Enzyme Solution Containing Rat Enzymes Male rat rats (6 weeks old) were exsanguinated and killed, and the livers were excised. After washing 10 g of liver with ice-cold physiological saline, 1
5 ml ice-cold buffer [100 mM potassium phosphate (pH 7.4),
It was homogenized in 15 mM nicotinamide, 2 mM MgCl ₂ ], and centrifuged at 10,000 g for 20 minutes (4 ° C.). The obtained supernatant was further centrifuged at 105,000 g for 90 minutes (4 ° C.), and the precipitate was suspended in ice-cold 100 mM potassium phosphate buffer (pH 7.4).
Centrifuge for 0 minutes (4 ° C.). Precipitate thus obtained
The (microsome fraction) was suspended in ice-cold 100 mM potassium phosphate buffer (pH 7.4) (protein concentration: about 40 mg / ml, measured by BCA protein assay kit manufactured by Pierce), and this was used as an enzyme solution.

Preparation of Enzyme Solution Containing Human Enzyme Human hepatoma cells Hep G2 (about 1 × 10 ⁹ ) obtained by culturing in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (37 ° C., in the presence of 5% CO ₂ ). cells) in 10 ml ice-cold buffer
[100 mM potassium phosphate buffer (pH 7.4), 30 mM
The cells were disrupted by sonication (twice for 30 seconds) after suspension in nicotinamide, 2.5 mM MgCl ₂ ]. From the obtained sonicate, a microsome fraction was obtained by the same operation as that for the rat enzyme. This is ice-cooled 100 mM
Suspended in potassium phosphate buffer (pH 7.4) (protein concentration about 4 m
g / ml) and used as the enzyme solution. [Results] Compound (IA) showed 50% inhibition of squalene synthase with respect to rat enzyme and human enzyme at 9.3 μg / ml and 6.9 μg / ml, respectively. On the other hand, the compound (I-
B) is 11.0 μg / rat for rat and human enzyme respectively
50% of squalene synthase at ml and 8.2 μg / ml
It showed inhibition.

Test Example 3 Cell Growth Inhibition Test HeLa cells were suspended in a medium at a concentration of 2 × 10 ⁴ cells / ml, and 0.1 ml was dispensed into each well of a 96-well flat bottom plate (Nunc). . For HeLa cells, a medium prepared by adding 10% of fetal bovine serum (Wittucker Bioproduct) to Eagle's minimum essential medium (Whittucker Bioproduct, USA) was used. After culturing the above plate in a carbon dioxide incubator set at 37 ° C. and 5% CO ₂ for 1 day, various concentrations of compound (IA) or compound (IB) were previously prepared.
Phosphate physiological saline in which 10 μl was dissolved was added to each well into which the cell suspension was dispensed, in an amount of 10 μl. After culturing for 3 days after addition, tetrazolium salt MTT (Sigma, USA)
25 μl of a solution of PBS in 5 mg / ml
l was added and kept warm in the carbon dioxide incubator. After 4 hours, 0.1 ml of a solution prepared by adding 10% SDS to 0.01 N hydrochloric acid was added to each well and kept warm in the carbon dioxide gas incubator overnight at 620 nm.
Absorbance at TiterTech Multiscan Absorbance Meter
(Flow Co., USA). PBS only 10
Using the absorbance when μl was added as a control, the inhibition rate (%) of the cell growth of compound (IA) and compound (IB) at each concentration was calculated. [Results] Compound (IA) and compound (IB) caused proliferation of HeLa cells at 7.5 μg / ml and 29. respectively.
There was 50% inhibition at 5 μg / ml.

Test Example 4 In Vivo ( In Vivo ) Mouse Lung Cancer Metastasis Inhibition Test The inhibitory activity of Lewis lung cancer cells against 8-week-old Crj: C57BL / 6 female mice against lung metastasis by compound (IA) was determined by the following method. It was measured. First, Lewis lung cancer cells were subcutaneously transplanted into mice by a trocar, and five of them were used as group A and the compound (I-
A) was administered subcutaneously at a dose of 30 mg / kg. Another group of 5 animals (group B) was intraperitoneally administered with the compound (IA) at a dose of 100 mg / kg on each of the 5th day and the 6th day. The metastasis-suppressing activity was measured by removing the lungs 18 days after the administration of the cancer cells and counting the cancer cell colonies formed in the lung tissue. The metastasis inhibition rate was 100 colonies in the lungs of 10 untreated control groups.
%, The number of cancer cell colonies in the lung tissue of group A or group B was counted and expressed as a percentage of the control group. [Results] Lung cancer metastasis inhibition rates of 34% and 51% were shown in groups A and B, respectively.

As shown in the above test examples, the compound represented by the general formula (I) of the present invention or a salt thereof, particularly the compound (IA) and the compound (IB) or a salt thereof are microtubules. It exhibits polymerization inhibitory activity, squalene synthase inhibitory activity, cell growth inhibitory activity and metastasis inhibitory activity. Moreover, these compounds of the present invention have low toxicity. For example, the compound (I
-A) 100 mg / kg of Crj: C57BL / 6 female mouse was intraperitoneally administered once a day twice, but no death was observed.

The microtubule polymerization inhibitor, tumor metastasis inhibitor or squalene synthase inhibitor containing the compound represented by the general formula (I) or a salt thereof of the present invention as an active ingredient is a mammal
(Example, rat, mouse, cow, horse, monkey, human etc.), is useful as a microtubule polymerization inhibitor, a metastasis inhibitor or a squalene synthase inhibitor, for example, the treatment of mammalian tumors or It can be used for prevention of metastasis or prevention or treatment of ischemic heart disease and prevention or treatment of coronary atherosclerosis. The drug can be obtained by mixing the compound represented by the general formula (I) or a salt thereof with a pharmacologically acceptable carrier by a method known per se. This drug is a parenteral drug such as injection, drip, and external preparation.
(Eg, nasal formulation, transdermal formulation, etc.), suppository (eg, rectal suppository, vaginal suppository, etc.), or as an oral agent such as capsules, tablets, syrups, powders and granules or others. Can be administered parenterally or orally.

These preparations can be produced by a method known per se which is generally used in the preparation process. For example, the compound represented by the general formula (I) of the present invention or a salt thereof is a dispersant (eg, Tween 80 (manufactured by Atlas Powder Co., USA), HCO60 (manufactured by Nikko Chemicals) polyethylene glycol, carboxymethyl cellulose, Sodium alginate, etc.), preservatives (eg, methylparaben, benzyl alcohol, chlorobutanol, etc.), isotonic agents (eg, sodium chloride, glycerin, sorbitol, glucose, etc.) with aqueous injections, olive oil, sesame oil, peanut, etc. An injection can be prepared by dissolving, suspending or emulsifying in oil, vegetable oil such as cottonseed oil, corn oil or the like, propylene glycol or the like to form an oily injection.

For example, to prepare an external preparation, the compound represented by the general formula (I) of the present invention or a salt thereof is prepared as a solid, semi-solid or liquid external preparation according to a method known per se. You can For example, as the solid, the compound represented by the general formula (I) or a salt thereof is used as it is, or an excipient (eg, glycol, mannitol, starch, microcrystalline cellulose, etc.), a thickener (eg, , Natural rubber,
Cellulose derivative, acrylic acid polymer, etc.) are added and mixed to obtain a powdery composition. The above-mentioned liquid form is almost the same as the case of the injection, and is an oily or aqueous suspension. In the case of semi-solid form, an aqueous or oily gel or ointment form is preferable. In addition, all of these are pH regulators (eg, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride, etc.), etc. May be added.

For example, to obtain a suppository, an oily or aqueous solid, semi-solid or liquid suppository is prepared from the compound represented by the general formula (I) of the present invention or a salt thereof according to a method known per se. Can be Examples of the oily base used in the above composition include glycerides of higher fatty acids [eg, cacao butter, witepsols (manufactured by Dynamite Nobel), etc.], or vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.) and the like. . Examples of the aqueous base include polyethylene glycols, propylene glycol,
Examples of the aqueous gel base include natural rubbers, cellulose derivatives, vinyl polymers, acrylic acid polymers and the like.

For example, in order to prepare a preparation for oral administration, the compound represented by the general formula (I) of the present invention or a salt thereof can be prepared by, for example, an excipient (eg, lactose, sucrose, starch, etc.) according to a method known per se. ), Disintegrants (eg, starch, calcium carbonate, etc.), binders (eg, starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, etc.) or lubricants (eg, talc, magnesium stearate, polyethylene glycol) 60
00 etc.) and the like, followed by compression molding, and if necessary, coating by a method known per se for the purpose of taste masking, direct solubility or sustainability, to give an oral administration preparation. Examples of the coating agent include hydroxypropyl methylcellulose, ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, Tween 80, Pluronic F
68, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit
(Rohm Co., West Germany, methacrylic acid / acrylic acid copolymer) and pigments such as titanium oxide and red iron oxide are used.

When the compound represented by the general formula (I) or a salt thereof is used in humans, the dose may vary depending on the type and degree of the target disease, the age of the patient, etc., but the general formula (I) is usually used. As the content of the compound represented by
It is preferable that about 1 mg to 40 mg, and especially about 2 mg to 30 mg per adult person (body weight 50 kg) is used for treating the disease. These formulations can be administered in 1 to 3 divided doses per day. When the compound represented by the general formula (I) or a salt thereof is parenterally administered as an injection subcutaneously, intravenously or intramuscularly, the dose is about 0.5.
To 50 mg / kg / day, preferably 1 to 50 mg / kg
/ Day.

[0087]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In addition, unless otherwise specified, the percentage (%) in the medium represents the weight / volume percentage. Further, the mixing ratio of the solvent is expressed as a volume ratio unless otherwise specified. ^{The 13} C nuclear magnetic resonance (NMR) spectrum was measured with a (75 MHz) type spectrometer using tetramethylsilane as an internal standard, and all δ values are shown in ppm. In the examples, Q means quaternary carbon. Example 1 Production of Compound (IA) and Compound (IB) Dextrin 50 g, glucose 5 g, raw soybean powder 35 g,
Two strains of Streptomyces roseoglyceus AL-28015, which had been cultured for 7 days at 28 ° C. on a slant medium consisting of 7 g of calcium carbonate, 20 g of agar and 1 liter of water, were used.
% Glucose, 3% Maltose, 1.0% Raw Soybean Flour, 0.3% Corn Steep Liquor, 0.5% Peptone, 0.5%
40 ml containing calcium carbonate and 0.3% sodium chloride
To a seed medium (pH 7.0) and cultivated in a 200 ml Erlenmeyer flask at 28 ° C. for 48 hours on a rotary shaker to obtain a seed culture. 1 ml of the resulting seed culture was added to 40 ml of 0.5% glucose, 5.0% dextrin in a 200 ml flask.
It was transplanted to a fermentation medium (pH 7.0) containing 3.5% raw soybean flour and 0.7% calcium carbonate, and cultured at 28 ° C. for 5 days on a shaker.

The obtained culture solution (4.0 liter) was adjusted to pH 3.
Correct to 0 and add ethyl acetate (4.0 liters) to 30
Extract by stirring for a minute, and then use High Flow-Parcel (Johns-
Mannville, USA) was added and filtered. The obtained organic layer (3.6 liter) was washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure and dried to an oil (2
7.9 g) was obtained. A portion of this (24.0 g) was dissolved in a mixture of methanol and ethyl acetate (200 ml), and silica gel (50
g) was added, the solvent was removed and adsorbed on silica gel. This was subjected to column chromatography (450 g) on silica gel 60 (A Merck, Germany), and chloroform: methanol: acetic acid (98: 1: 1, 2.0 liters then 97: 2 :).
After washing with 1, 1.0 liter), the active fraction containing the compound (IA) was washed with chloroform: methanol: acetic acid (97: 2: 1,
It was eluted with 1.0 liter). Further, after washing with chloroform: methanol: acetic acid (96: 3: 1, 1.0 liter), the active fraction containing the compound (IB) was washed with chloroform:
Methanol: acetic acid (96: 3: 1, 1.0 liter, then 9
Elution was carried out at 5: 4: 1, 1.2 liters, then 92: 7: 1, 0.5 liters). The eluted fractions were concentrated under reduced pressure to obtain a crude powder of compound (IA) (1.64 g) and a crude powder of compound (IB) (1.13 g). Compound (I-
The crude powder of A) was mixed with ethyl acetate and methanol (20 ml).
, Silica gel (6.0 g) was added, the solvent was removed, and the product was adsorbed on silica gel. This was subjected to silica gel 60 column chromatography (44 g) and dichloromethane:
After washing with methanol: acetic acid (98: 1: 1, 800 ml), the active fraction containing compound (IA) was diluted with dichloromethane: methanol: acetic acid (98: 1: 1, 200 ml, then 96: 3:
It was eluted with 1, 100 ml). The eluted fractions were concentrated to dryness, dissolved in ethyl acetate (200 ml), washed with 0.2N hydrochloric acid and water, dried over anhydrous sodium sulfate and concentrated, then crystallized from ethyl acetate-hexane to give the compound ( I-
Yellow crystals of A) (408 mg) were obtained. The crude powder of compound (IB) was dissolved in methanol (10 ml) and concentrated, and then dissolved in ethyl acetate (200 ml), 0.2 N hydrochloric acid and water (1 each).
It was washed with (00 ml). The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated, and then crystallized from ethyl acetate-hexane to give a yellow crystal (1.12) of compound (IB).
g) was obtained.

Formulation Example 1 Using the compound (IA) and the compound (IB) obtained in Example 1, all the ingredients of the formulation shown below were mixed and filled in a gelatin capsule to obtain one capsule. Hit,
Capsules each containing 30 mg of compound (IA) or compound (IB) were prepared. Compound (IA) or compound (IB) 30 mg Lactose 100 mg Corn starch 40 mg Magnesium stearate 10 mg Total 180 mg

Formulation Example 2 Compound (IA) or compound (IB) obtained in Example 1 and magnesium stearate were granulated with an aqueous solution of soluble starch, dried, and then mixed with lactose and corn starch. The mixture was compression-molded to produce tablets having the formulations shown below. Compound (IA) or compound (IB) 30 mg Lactose 65 mg Corn starch 30 mg Soluble starch 35 mg Magnesium stearate 20 mg Total 180 mg

Formulation Example 3 Compound (IA) or compound (I) obtained in Example 1
-B) (500 mg) was dissolved in methanol (40 ml), sodium carbonate (163 mg, 1.1 eq) and water (30 ml) were added,
Stir for 10 minutes at room temperature. Then, the methanol is removed and freeze-dried to give compound (IA) or compound (I).
-B) the sodium salt (550 mg) was obtained. The obtained sodium salt of compound (IA) or compound (IB) was dissolved in physiological saline containing 30% (W / V) polyethylene glycol 400 to give compound (IA) or compound (I-). Prepare a 0.05% solution of the sodium salt of B),
After sterile filtration, 30 ml was dispensed into each vial. Intravenous injection preparations containing 15 mg of the sodium salt of compound (IA) or compound (IB) per vial were prepared.

[0092]

According to the present invention, microtubule polymerization inhibitory activity,
Novel pentacyclic compound having squalene synthase inhibitory activity and cell growth inhibitory activity, production method thereof, novel microbial strain used in the production, and microtubule polymerization inhibitor containing the same, squalene synthase inhibitor and tumor metastasis Inhibitors are provided. The compound of the present invention has low toxicity and can be safely administered.

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption (UV) spectrum of compound (IA).

FIG. 2 shows an infrared absorption (IR) spectrum of compound (IA).

FIG. 3 shows ¹³ C nuclear magnetic resonance (NM) of compound (IA).
R) shows the spectrum.

FIG. 4 shows an ultraviolet absorption (UV) spectrum of compound (IB).

FIG. 5 shows an infrared absorption (IR) spectrum of the compound (IB).

FIG. 6: ¹³ C nuclear magnetic resonance (NM) of compound (IB)
R) shows the spectrum.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C07C 235/84 7106-4H C12N 1/20 A 8828-4B C12P 7/50 8114-4B // ( C12N 1/20 C12R 1: 465) (C12P 7/50 C12R 1: 465)

Claims (10)

[Claims] 1. A compound represented by the general formula (I): (In the formula, R ¹ represents a carboxyl group which may be esterified or amidated, and R ² , R ³ , R ⁴ , R ⁵ and R ⁶ represent a hydroxyl group which may be etherified or esterified. ) The compound or its salt represented by these. 2. The compound according to claim 1, wherein R ¹ is a carboxyl group. 3. Formula (IA): embedded image The compound according to claim 1, which is represented by: 4. Formula (IB): embedded image The compound according to claim 1, which is represented by: 5. A bacterium belonging to the genus Streptomyces and having the formula (I-
A compound having the ability to produce the compound represented by A) and / or the compound represented by the formula (IB) is cultured in a medium, and the compound represented by the formula (IA) and / or the formula (IA). The compound represented by IB) is produced and accumulated in the medium,
A method for producing a compound represented by the formula (IA) and / or a compound represented by the formula (IB) or a salt thereof, which comprises collecting the compound. 6. The method according to claim 5, wherein the microorganism is a microorganism belonging to Streptomyces roseoglyceus. 7. The microorganism Streptomyces roseoglyceus AL-28015. 8. A microtubule polymerization inhibitor comprising the compound according to claim 1 or a salt thereof. 9. A squalene synthase inhibitor comprising the compound according to claim 1 or a salt thereof. 10. A tumor metastasis inhibitor comprising the compound according to claim 1 or a salt thereof.