JP5637426B2 - Novel compounds with cell cycle inhibitory activity - Google Patents

Description

  The present invention relates to a novel compound having cell cycle inhibitory activity, a pharmaceutical composition containing the compound, and a method for producing the compound.

  As an approach for the treatment of diseases associated with abnormal cell growth such as malignant tumor (cancer), a technique for stopping the cell cycle has been proposed. Among the anticancer agents that are currently effective through such an approach, there are drugs that inhibit the function of microtubules, which play an important role in cell division.

  For example, vinorelbine tartrate (Navelbine (registered trademark) Note, Kyowa Hakko Kirin) inhibits microtubule polymerization and has already proven clinical efficacy against malignant tumors. However, since microtubules play an important role not only in cancer cells but also in normal cells, the above drugs that act on microtubule polymerization have side effects such as neuropathy with numbness of the limbs as the main symptom .

  Therefore, it has been proposed to use an inhibitor of kinesin Eg5, which is a protein involved in cell division, as a new anticancer agent through an approach to arrest the cell cycle. Since kinesin Eg5 inhibitor does not inhibit the function of microtubules, it is expected to have no side effects as the above-mentioned microtubule polymerization inhibitors.

  Terpendole E was found as a kinesin Eg5 inhibitor (Non-patent Document 1). Terpendole E stops the cell cycle of cancer cells in the M phase. However, terpendole E has the disadvantage that its cell cycle inhibitory activity is weaker than conventional cell cycle inhibitors.

Nakazawa et al. , Chemistry and Biology, 10: 131-137, 2003.

  As described above, a kinesin Eg5 inhibitor has attracted attention as an anticancer agent having a new mechanism of action. Since the cell cycle inhibitory activity of terpendole E, an Eg5 inhibitor currently specified, is not sufficient, a kinesin Eg5 inhibitor having an inhibitory activity exceeding this is desired.

  The present inventors succeeded in synthesizing novel terpendole-related compounds by manipulating the expression of genes involved in the terpendole biosynthesis system of terpendole E-producing microorganisms. A novel compound having a cell cycle inhibitory activity superior to that of Dole E was found.

Therefore, the present invention specifically has the following features.
[1] A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof.

[2] A cell cycle inhibitor comprising the compound of formula (I) defined in [1] above or a pharmaceutically acceptable salt thereof.
[3] A pharmaceutical composition comprising a compound of formula (I) as defined in [1] above or a pharmaceutically acceptable salt thereof, and an excipient.
[4] The pharmaceutical composition according to [3] above for preventing or treating a disease caused by cell proliferation.

[5] The pharmaceutical composition according to the above [4], wherein the disease caused by cell proliferation is cancer.
[6] The following steps:
(A) In Chonopikunisu microorganisms, cultured step to reduce the conversion on the expression of genes involved in the Pasuparin terpene d'biosynthesis in the 13-desoxy paxillin, and (b) the resulting microorganisms are produced A process for producing a compound of formula (I) as defined in [1] above, which comprises a step of recovering the compound.
[7] The method according to [6] above, wherein the Chonopicnicus genus microorganism is Chaunopycnis alba RK99-F33 strain (NITE P-760).
[8] genes involved in conversion to 13-desoxy paxillin from Pasuparin is paxP or TERP, the method described in the above [6] or [7].

  According to the present invention, a novel cell cycle inhibitor is provided, which has use as an anticancer agent. Therefore, the present invention provides an effective novel anticancer agent with few side effects.

It is a result of the NMR analysis of the terpendole compound produced by the P-27 strain. It is a result of the NMR analysis of the terpendole compound produced by the P-27 strain. It is a result of the NMR analysis of the terpendole compound produced by the P-27 strain. It is a result of the NMR analysis of the terpendole compound produced by the P-27 strain. It is a figure which shows the analysis of the cell cycle inhibitory activity with respect to K562 cell.

  The present invention relates to a compound represented by formula (I), or a pharmaceutically acceptable salt thereof.

  As used herein, the term “pharmaceutically acceptable salt” is used within the scope of appropriate medical judgment and in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, etc. Represents a salt suitable for Pharmaceutically acceptable salts are known in the art, e.g. M Berge et al. In Pharmaceutical Sciences, 1977, 66: 1-19. Salts can be prepared by reacting the free base with a suitable inorganic or organic acid during final isolation and purification of the compounds of the invention. Representative acid addition salts include, but are not limited to, for example, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate , Borate, butyrate, camphorsulfonate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glyceroline Acid salt, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, Lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate Oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate , Sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate and the like.

  In addition to acid addition salts, the compounds of the present invention include solvates, derivatives such as intermediates, and isomers such as optical isomers.

  Derivatives include compounds containing 1 to 4 substituents on the phenyl group (for example, alkyl, hydroxy, halogen, carboxyl, amino, alkylamide, alkyloxy, etc.), compounds in which the hydroxyl group is converted to alkylcarbonyloxy, and the like. included. Halogen is fluorine, chlorine, bromine or iodine. Alkyl is saturated or unsaturated alkyl, preferably lower saturated alkyl such as methyl, ethyl, propyl, butyl and the like.

  The compounds of the present invention have strong cell cycle inhibitory activity, as demonstrated in the following examples.

A series of processes of the cell cycle is defined as follows: Beginning with the state of quiescent or resting (G ₀ phase), prepared for cell growth and chromosomal replication is performed (G ₁ phase). Then through DNA synthesis phase (the phase S), cells are made ready for splitting (G ₂ phase), followed by mitotic cell cycle complete (M phase), hereditary to disrupted daughter cells Information is carried over.

  Kinesin Eg5 is required for centrosome separation in the M phase of the cell cycle. Therefore, by inhibiting Eg5, the cell cycle can be stopped in the M phase.

  Terpendole E is the first naturally occurring kinesin Eg5 inhibitor discovered (Nakazawa et al., Chemistry and Biology, 10: 131-137, 2003). Terpendole E is classified as an indole diterpene. Indole diterpene is a general term for compounds having various structures produced by some filamentous fungi. From the analysis of three types of indole diterpene-producing bacteria (Penicillium paxilli, Aspergillus flavus, Neotyphodium llili), it is common that indole diterpenes have a common biosynthetic pathway. (Saikia et al., Mycol. Res., 112: 184-199, 2008).

  As an example, a paxillin biosynthesis system of Penicillium paxili will be described. From IPP, FPP, and indole-3-glycerol phosphate, the action of at least four enzymes, including PaxG, PaxB, PaxM and PaxC, produces the first detectable intermediate, paspaline. Paspaline is converted to 13-desoxypaxillin by PaxP (cytochrome P450 monooxygenase), and further converted to paxillin by PaxQ (cytochrome P450 monooxygenase). The above three types of filamentous fungi are believed to share this pathway. Terpendole E is presumed to be produced by C11 hydroxylation of pasparin, a common biosynthetic intermediate.

  In the terpendole E producing bacterium, Chaunopycnis alba RK99-F33 strain (NITE P-760), the present inventors have prepared paxP encoding PaxP responsible for the conversion of paspaline to 13-desoxypaxillin. By disrupting the orthologue of the gene (terP), the conversion of pasparin to 13-desoxypaxillin in the above common metabolic pathway was reduced, thereby enhancing the biosynthesis of terpendole E and its derivatives. As a result, the compound of the formula (I) of the present invention was obtained.

Chono Picnicus alba can be cultured in PDA medium (3.9% potato dextrose agar, Difco), PD medium (2.4% potato dextrose, Difco), or FDY medium (1.5% glucose, 1.5% % Soluble starch, 1% corn steep liquor, 1% dry yeast, 0.3% malt extract, 0.03% MgSO ₄ / 7H ₂ O, 0.1% KH ₂ PO ₄ , 0.1% agar, pH 6 0) at 28 ° C.

  The target compound can be recovered and purified by solvent extraction, crystallization, ion exchange chromatography, HPLC, or the like.

  As shown in the following examples, the compound of formula (I) showed cell cycle inhibitory activity far superior to terpendole E against tumor-derived cells.

  Accordingly, the present invention also relates to a cell cycle inhibitor comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

  The compound of the formula (I) of the present invention is considered to exhibit cell cycle inhibitory activity by inhibiting kinesin Eg5 like terpendole E due to its structural similarity.

  In addition, the compound of the present invention having cell cycle inhibitory activity can be used as an active ingredient in therapeutic agents for various diseases related to cell division.

  Accordingly, the present invention relates to a pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and an excipient, wherein the pharmaceutical composition preferably treats a disease caused by cell proliferation. It is for prevention or treatment. In a preferred embodiment, the disease resulting from cell proliferation is cancer.

  In the present invention, examples of the cancer include renal cell carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor Mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma , Papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, primary bronchial cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer , Bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, Melanoma, neuroblastoma, retinoblastoma, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia; chronic leukemia, Sex erythrocytes increase multi disease, lymphoma, and multiple myeloma and the like.

  For example, a well-known formulation technique can be applied to make the compound of the present invention into a pharmaceutical composition. When the pharmaceutical composition of the present invention is used as an anticancer agent (that is, a cancer therapeutic agent) or other pharmaceutical agent, the administration form includes, for example, an injection, an implant, an aerosol, a suppository, a patch, and a poultice. Parenteral administration such as a lotion, liniment, ointment or eye drop, or oral administration such as a tablet, capsule, granule, powder, pill, troche or syrup. These preparations are produced by known methods using additives such as excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.

  Examples of excipients include starches such as starch, potato starch, and corn starch, lactose, crystalline cellulose, and calcium hydrogen phosphate.

  Examples of the coating agent include ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, and paraffin. Examples of the binder include polyvinyl pyrrolidone, macrogol and the same compound as the excipient. Examples of the disintegrant include the same compounds as the above excipients and chemically modified starch / celluloses such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone. Examples of the stabilizer include paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal Dehydroacetic acid; and sorbic acid. Examples of the flavoring agent include sweeteners, acidulants, and fragrances that are commonly used.

  Moreover, as a solvent for manufacturing a liquid agent, physiological saline, purified water, distilled water, ethanol, phenol, chlorocresol, or the like can be used. Examples of the surfactant or emulsifier include polysorbate 80, polyoxyl 40 stearate, lauromacrogol and the like.

  When the pharmaceutical composition of the present invention is used as an anticancer agent, the amount of the pharmaceutical composition of the present invention used on the basis of the compound of the present invention varies depending on symptoms, age, administration method and the like. For example, in the case of intraperitoneal administration, the lower limit is 0.01 mg (preferably 0.1 mg) and the upper limit is 2000 mg (preferably 500 mg, more preferably 100 mg) per day for patients (warm-blooded animals, particularly humans). Is preferably divided into several doses and administered according to the symptoms. In the case of intravenous administration, the lower limit is 0.001 mg (preferably 0.01 mg) and the upper limit is 500 mg (preferably 50 mg) per day for adults. Is desirable.

The present invention also includes the following steps:
(A) In Chonopikunisu microorganisms, step reduces the expression of genes involved in the conversion from Pasuparin terpene d'biosynthesis in the 13-desoxy paxillin (including inhibition), and (b) the resulting microbial cultures And a method for producing the compound of the above formula (I), comprising the step of recovering the produced compound.

Preferably, the Chono picnicus genus microorganism is Chaunopycnis alba RK99-F33 strain (NITE P-760). Also, preferably, the gene involved in the conversion from Pasuparin in the terpene d'biosynthesis in the 13-desoxy paxillin is paxP or TERP. The Chanopycnis alba RK99-F33 strain was established on May 20, 2009 at the Japan Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) with the accession number NITE. Deposited as P-760.

  The modification for reducing the expression of the target gene of the microorganism belonging to the genus Chonopicnicus can be carried out by using Agrobacterium-mediated genetic modification, for example, as described in detail in the following Examples. Examples of genes involved in the terpendole biosynthetic system include the Penicillium paxili paxP gene (GenBank accession number AF279808) and the paxQ gene (GenBank accession number AF279808). Orthologs of these genes in Chonopicnicus microorganisms can be easily identified by those skilled in the art by known genetic recombination methods (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). Modification of the gene includes deletion of the entire coding region of the gene, partial deletion of the coding region, insertion of a foreign gene such as a resistance gene into the coding region, replacement of the coding region with a foreign gene, or regulatory region It can be done by destroying.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[Example 1]
Production of the compounds of the invention
1-1. Strain, medium, culture and transformation In the following experimental methods, procedures not specifically described are described in the textbook of Sambrook et al. (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

A terpendole E-producing strain, Chaunopycnis alba (hereinafter also referred to as “C. alba”) RK99-F33 (NITE P-760) (Nakazawa et al., Supra) is a PDA medium (3.9). % Potato dextrose agar, Difco), PD medium (2.4% potato dextrose, Difco), or FDY medium (1.5% glucose, 1.5% soluble starch, 1% corn steep liquor, 1% dried Yeast, 0.3% malt extract, 0.03% MgSO ₄ / 7H ₂ O, 0.1% KH ₂ PO ₄ , 0.1% agar, pH 6.0). C. Aruba was transformed by Agrobacterium-mediated transformation (ATMT) (Motoyama et al., Curr. Genet., 47: 298-306, 2008). Agrobacterium tumefaciens C58 strain After co-culture with a conidial suspension of Alba strain, it was selected with 500 μg / mL hygromycin B (Wako Pure Chemical Industries). C. When cultivating the transformant of Aruba, 200 μg / mL hygromycin B was added as necessary. The plasmid was grown in E. coli DH5α strain. E. coli was cultured in LB medium and transformation was performed by standard methods (Sambrook et al., Molecular Cloning: a laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 198).

1-2. Genetic manipulation Aruba genomic DNA was prepared using DNeasy plant total DNA isolation kit (Qiagen). PCR amplification was performed using a thermal cycler (model TP240, Takara Shuzo) and KOD Plus (Toyobo) or Blend Taq Plus (Toyobo) DNA polymerase. For plasmid construction, KOD Plus, a proofreading DNA polymerase, was used. In normal PCR, Blend Taq Plus was used. The PCR product was cloned using Zero Blunt TOPO PCR cloning kit (Invtrogen), and the nucleotide sequence was determined as necessary. DNA sequencing was performed using BigDye Terminator ver. 3.1 Cycle sequencing kit (Amersham-Pharmacia Biotech) and 3730xl DNA analyzer (Applied Biosystems). Analysis of DNA and amino acid sequence data was performed using DNASIS-Mac software (Hitachi Software Engineering).

1-3. Preparation of an indole diterpene biosynthetic gene-modified strain C. disrupted the ortholog (terP) of the paxP gene encoding PaxP responsible for the conversion of paspaline to 13-desoxypaxillin in the above indole diterpene common metabolic pathway. The Aruba strain was prepared as follows.

  The primers shown in Table 1 were used.

  The upstream sequence of terP is defined as C.I. It was amplified from Aruba genomic DNA by PCR using XhoI-5'terP-F primer and XbaI-5'terP-R primer. The 1.0 kb amplified fragment was purified, digested with XhoI and XbaI and purified (fragment 1). Amplification of hygromycin B resistance gene expression unit from pCSN45 (Motoyama et al., Fungal Genet. Biol., 42: 200-212 (2005)) by PCR using MluI-HYG-F primer and XbaI-HYG-R primer did. The 1.6 kb amplified fragment was purified and purified after digestion with MluI and XbaI (fragment 2). The downstream sequence of terP is C.I. It was amplified from Aruba genomic DNA by PCR using MluI-3'terP-F primer and SseI-3'terP-R primer. The 1.0 kb amplified fragment was purified and subsequently digested with MluI and SseI and purified (fragment 3). The vector sequence between the right and left borders of pBI121 was amplified by PCR using pBI121 as a template and using XhoI-RB primers and SseI-LB primers. The 8.7 kb amplified fragment was purified followed by digestion with XhoI and SseI and purification (fragment 4). The terP gene disruption plasmid pBI-terP :: HPH was constructed by ligating the above four fragments. A. transformed with pBI-terP :: HPH Using the Tumefaciens strain, C.I. Aruba was transformed by the ATMT method. The terP gene disrupted body was selected by PCR using a terP-check-F primer and a terP-check-R primer that amplify most of the terP ORF. In this PCR, a 1.2 kb terP ORF sequence was amplified from the wild type strain, a 1.6 kb hygromycin B resistance gene expression unit was amplified from the terP gene disrupted strain, and both fragments were amplified in the epicic transformant. Is done. The P-27 strain was selected as the terP gene disruption strain.

  C. The full-length terP gene sequence of Aruba is shown in SEQ ID NO: 1.

1-4. Purification, HPLC analysis and structural analysis of terpendoles The above FDY medium was used as a production medium for terpendoles. The P-27 strain obtained above was cultured in FDY medium at 160 rpm and 28 ° C. for 12 days. To 1 L of culture broth, 2 volumes of acetone was added and stirred, and the supernatant was collected. After removing acetone and extracting twice with an equal volume of ethyl acetate, ethyl acetate was removed. 392 mg of extract concentrated residue was obtained. Methanol was added to the concentrated residue, the ergosterol-like crystalline precipitate was filtered off, the filtrate was divided into two portions, and divided into two portions, and preparative HPLC was performed according to a conventional method.

  HPLC was performed under the following conditions.

Preparative HPLC conditions for terpendole compounds (HPLC-1)
Waters alliance 2690 Controller, UV-VIS Detector UV702 (GL Sciences Inc.)
Reversed phase column: PEGASIL ODS 20 × 250 mm (Senshu Pak)
Developing solvent: acetonitrile-water gradient (0-10 min acetonitrile 20-80%, 10-20 min 80-100%, 20-60 min 100%), solvent flow rate: 8 mL / min, UV monitor: 281 nm

HPLC analysis method of terpendole compounds HPLC-2)
Waters 600 Controller, Waters 966 Photodiode Array Detector
Reversed phase column: 4.6 × 250 mm column ODS-3, (GL Sciences)
Developing solvent: acetonitrile-water gradient (0-10 min acetonitrile 20-80%, 10-20 min same 80-100%, 20-50 min same 100%), solvent flow rate: 1 mL / min, different from HPLC analysis 1 retention time .
EI-MS (SX-102) JEOL was used for the measurement of EI-MS (positive).

  In the second preparative HPLC, there was a problem that the peak of ergosterol-like substance overlapped with the eluted fraction, and preparative HPLC was performed again. As a result of analyzing each fraction by HPLC analysis (HPLC-2), as shown in Table 2, two main terpendole compound peaks were observed. The two peaks had few impurities, and each fraction was concentrated, and after measuring the weight, the physical properties could be measured.

  The structure of the compound was determined by NMR.

  The results are shown in FIGS. 1-1 to 1-4.

  Table 2 shows the names of the substances obtained from the weight and structural analysis of the obtained substances. In addition, information such as the estimated molecular formula is shown in Table 3.

  Terpendole E was mass-produced from the P-27 strain. In addition, the compound corresponding to the second peak was found to be a novel compound 11-ketopasparin.

[Example 2]
Biological activity of new terpendole analogues
1. Cell cycle inhibitory activity of terpendoles K562 cells, which are human promyelocytic leukemia cells, were seeded in a 96-well plate at 10 ⁵ cells / 500 μL / well and dissolved in DMSO (2.3 μM or 23 μM terpendole E). 2.3 μM or 23 μM 11-ketopasparin, 1 μM vinblastine, or DMSO control) was added to the indicated final concentrations and incubated for 24 hours. Cells were washed with PBS, intracellular nucleic acid was stained with propidium iodide, and the amount of fluorescence was measured by flow cytometry (Tamura et al., Oncogene, 28: 107-116, 2009).

The results are shown in FIG. 4C peaks are cells in the stage M, 2C peaks represent cells _G 0 ~G ₂ phase. In vinblastine with known cell cycle inhibitory activity and newly identified 11-ketopasparin, many cells were arrested in the M phase. From this result, it was shown that 11-ketopasparin has a very strong cell cycle inhibitory activity compared with terpendole E.

2. Cytotoxicity Human promyelocytic leukemia cells in which K562 cells terpene Doll compound (1.5 × 10 ⁴ cells / mL), human fibrosarcoma-derived HT1080 cells (0.3 × 10 ⁴ cells / mL), and the human cervix Cancer-derived HeLa cells (0.6 × 10 ⁴ cells / mL) were seeded in 96-well plates at the indicated densities at 200 μL / well. HT1080 cells and HeLa cells were cultured overnight at 37 ° C., 5% CO ₂ . After the cells adhered, 0.2 μL of a test compound dissolved in DMSO was added and incubated for 48 hours. 20 μL of WST-8 solution (Nacalai Tesque, live cell measurement reagent SF) was added, and the absorbance at 450 nm was measured with a plate reader (Ishida et al., Chem. Biol., 11: 367-377, 2004).

  The results are shown in Table 4.

  For all cell types, 11-ketopasparin showed an IC50 value less than half that of terpendole E. Therefore, the newly identified 11-ketopasparin was significantly more cytotoxic than terpendole E against tumor-derived cells.

  From the above, it was shown that 11-ketopasparin newly identified is promising as an anticancer agent.

[Example 3]
Chemical synthesis of 11-ketopasparin 11-ketopasparin could be prepared by oxidizing terpendole E obtained by known methods (Nakazawa et al., 2003, supra) with Dess-Martin reagent. (Data not shown).

  According to the present invention, a novel compound having cell growth inhibitory activity is provided, and the compound is useful for the treatment of diseases associated with cell proliferation. Therefore, the present invention has applicability in the medical and health science fields.

SEQ ID NOs: 2-11: Primers

Claims (6)

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
  A cell cycle inhibitor comprising a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof.   A pharmaceutical composition comprising a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof, and an excipient.   The pharmaceutical composition according to claim 3, for preventing or treating a disease caused by cell proliferation.   The pharmaceutical composition according to claim 4, wherein the disease caused by cell proliferation is cancer. The following steps:
(A) reducing the expression of a paxP gene or a terP gene in a terpendole biosynthesis system in Chaunopycnis alba strain RK99-F33 (NITE P-760) , and (b) culturing the obtained microorganism And producing the compound of formula (I) as defined in claim 1, comprising the step of recovering the compound produced.