JP5261707B2 - Anti-telomerase or anti-tumor agent - Google Patents

Description

  The present invention relates to an anti-telomerase agent or an antitumor agent.

  Chemotherapy for cancer began in the 1940s with nitrogen mustard agents (alkylating agents) and antifolate agents (antimetabolites), and today the anticancer drug market has grown to a trillion-yen market.

  Cancer is an uncontrolled growth of cells due to DNA mutations, and in some cases a tendency to enlarge certain tumors is inherited. In a broad sense, most chemotherapeutic agents effectively target cells that divide in a short time by inhibiting cell division. Since such drugs damage cells, they are written as cytotoxic. Certain drugs cause apoptosis (the de facto “cell suicide”).

  However, treatment with anticancer drugs often results in physical rejection of the patient. With current chemotherapeutic techniques, the range of side effects occurs primarily for cells with increased body cell division. Serious side effects from anticancer drugs include, for example, loss of hair, nausea and vomiting, diarrhea or constipation, anemia, suppression of the immune system to cause infection and sepsis, bleeding, secondary cancer, cardiotoxicity, liver Toxicity, nephrotoxicity, etc. are mentioned.

  Therefore, research is currently being conducted to search for anti-tumor components from components such as traditional Chinese medicines and herbal medicines that are considered to have few side effects (Non-patent Document 1). According to this document, the Chinese traditional Chinese medicine “Kiyi's blood correcting agent” (consisting of 6 types: Ogi, Jukujiou, Toki, Jukusha, Taiso, and Rokujo) has an anti-tumor effect and its mechanism of action. One suggests that NK activity is enhanced.

  On the other hand, since apomorphine hydrochloride was first used as an emetic in 1869, it has been used as a sedative for schizophrenic patients in the first half of the 20th century and as a behavior improving drug for alcoholic and drug addicts. In Japan, it is described in the Japanese Pharmacopoeia or National Drug Concentration from the first edition of the Japanese Pharmacopoeia (announced in 1886) to the time of the revision of the seventh revised Japanese Pharmacopoeia Part 2 (1966). It has been clinically used as an emetic at a dose of 5 mg subcutaneously and an extreme dose of 20 mg subcutaneously) and as an expectorant at a low dose (water solution, 0.5-1 mg / dose).

  Apomorphine hydrochloride was recognized as a dopamine agonist in 1967 and became clinically used as an anti-parkinsonian drug. In Europe, it is currently a therapeutic drug for Parkinson's disease (subcutaneous injection; 1.5-10 mg / dose) , 2-8 times / day), etc. (Non-patent Document 2).

  Under such circumstances, since the erection effect was first reported in a clinical trial using apomorphine hydrochloride for the treatment of chronic alcoholism, the effect of apomorphine hydrochloride on erection has been studied at the animal level (Non-patent Document 3). Apomorphine hydrochloride is a dopamine (D1 / D2) receptor agonist that stimulates the D2 receptor present in the hypothalamus to activate the oxytocin pathway in the hypothalamus and hippocampus, via the midbrain, the lower erection center (sacral cord) It is transmitted to the pelvic nerve belonging to the parasympathetic nerve. Furthermore, the stimulation liberates nitric oxide (NO) from non-adrenergic and non-cholinergic nerve endings, and cGMP increases due to activation of guanylcyclase in the penile smooth muscle. As a result of relaxation of the penile deep arteries and smooth muscle smooth muscle by cGMP, blood inflow into the corpus cavernosum increases and erection is induced.

Deng Hong, Kaori Nakajima, Ma Xinling, Kenichiro Hasumi, Toshitaka Akatsuka, Haruhisa Wada, “Effects of Chinese Traditional Chinese Medicine“ Kiqi's Antihypotensive Agent ”on Immunotumor Activity”, Saitama Medical University Journal, Vol.28, No.3, July 2001, p.109-115 Andrew Lees and Kirsten Turner, “Apomorphine for Parkinson ’s Disease”, Practical Neurology 2002; 2; 280-287 Heaton JP, Morales A, Adams MA, Johnston B, el-Rashidy R., “Recovery of erectile function by the oral administration of apomorphine.”, Urology. 1995 Feb; 45 (2): 200-6.

  As described above, research is currently being conducted to search for anti-tumor components from components such as traditional Chinese medicines and herbal medicines. However, Kampo medicines and herbal medicines are composed of multiple components, and EBMs that are currently reported to be effective are not the results of studying single ingredients, and are not accepted as the result of the original double-blind test. Therefore, in order to be accurately evaluated as a single component, it is essential to study not only the monosaccharides obtained from glycosides, but also the identification of their actives and the identification of alkaloids that are less susceptible to intestinal absorption and metabolism. Therefore, it is urgently required to clarify their active action. On the other hand, at present, there is no report that apomorphine hydrochloride or its analog has antitumor properties.

  This invention is made | formed in view of the said situation, and it aims at providing the antitumor component derived from the component of a Chinese herb medicine and a crude drug. Another object of the present invention is to provide an antitumor component that is an analog of the components of Chinese herbal medicine and herbal medicine.

According to the present invention, the following general formula (I) or general formula (II)
An antitumor agent comprising a heterocyclic compound having an apomorphine skeleton or a salt thereof is provided.

Here, in the above general formulas (I) and (II), X ₁ is an —OH group or —O—CH ₃ group, and X ₂ is an —OH group or —O—CH ₃ group. X ₃ is —H group or —OH group, X ₄ is —OH group or —O—CH ₃ group, and X ₅ is —H group, —OH group, —O—CH _3. One or more functional groups selected from the group consisting of groups.

Y represents the following general formula (III) or general formula (IV)
It is a structural unit shown by.

  Since the anti-telomerase agent which concerns on this invention contains the heterocyclic compound which has said apomorphine skeleton, or its salt, it shows the outstanding telomerase inhibitory effect.

According to the present invention, the following general formula (I) or general formula (II)
An antitumor agent comprising a heterocyclic compound having an apomorphine skeleton or a salt thereof is provided.

Here, in the above general formulas (I) and (II), X ₁ is an —OH group or —O—CH ₃ group, and X ₂ is an —OH group or —O—CH ₃ group. X ₃ is a —H group or a —OH group, X ₄ is a —OH group or a —O—CH ₃ group, and X ₅ is a group consisting of a —H group or a —O—CH ₃ group. One or more functional groups selected from

Y represents the following general formula (III) or general formula (IV)
It is a structural unit shown by.

  Since the antitumor agent according to the present invention contains the above heterocyclic compound having an apomorphine skeleton or a salt thereof, it exhibits an excellent antitumor action.

  According to the present invention, an anti-telomerase agent derived from a natural product, which is a component extracted with hot water or ether from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia An anti-telomerase agent is provided.

  Since the anti-telomerase agent which concerns on this invention contains the component extracted with hot water or ether from said crude drug, it shows the outstanding telomerase inhibitory effect.

  According to the present invention, a natural product-derived antitumor agent, which is extracted with hot water or ether from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia An antitumor agent is provided.

  Since the antitumor agent according to the present invention contains components extracted from the above herbal medicines by hot water extraction or ether extraction, it exhibits an excellent antitumor action.

  ADVANTAGE OF THE INVENTION According to this invention, it is an anti-telomerase agent derived from a natural product, Comprising: The anti- telomerase agent containing the component extracted by ether from Koma grass is provided.

  Since the anti-telomerase agent which concerns on this invention contains the component extracted by ether from said Komakusa, it shows the outstanding telomerase inhibitory effect.

  ADVANTAGE OF THE INVENTION According to this invention, it is an anti-tumor agent derived from a natural product, Comprising: The anti-tumor agent containing the component extracted by ether from Komagusa is provided.

  Since the antitumor agent according to the present invention contains a component extracted from the above-mentioned Komakusa by ether, it exhibits an excellent antitumor action.

  Since the anti-telomerase agent according to the present invention contains a heterocyclic compound having a specific apomorphine skeleton or a salt thereof, or contains a component extracted from a specific natural product with hot water or ether, it has an excellent telomerase inhibitory action. Show.

  Since the antitumor agent according to the present invention contains a heterocyclic compound having a specific apomorphine skeleton or a salt thereof, or contains a component extracted from a specific natural product with hot water or ether, it has an excellent antitumor effect. Show.

  Hereinafter, embodiments of the present invention will be described in detail.

<Embodiment 1: Anti-telomerase agent>
The anti-telomerase agent according to this embodiment has the following general formula (I) or general formula (II)
An antitumor agent comprising a heterocyclic compound having an apomorphine skeleton or a salt thereof.

Here, in the general formula (I) and formula (II), _{X 1} is -OH group or -O-CH ₃ group, _{X 2} is an -OH group or an -O-CH ₃ group, X ₃ represents an —H group or —OH group, X ₄ represents an —OH group or —O—CH ₃ group, and X ₅ represents an —H group, —OH group, or —O—CH ₃ group. One or more functional groups selected from the group consisting of:

Y represents the following general formula (III) or general formula (IV)
It is a structural unit shown by.

  Since the anti-telomerase agent which concerns on this embodiment contains the heterocyclic compound which has said apomorphine skeleton, or its salt, it shows the outstanding telomerase inhibitory effect.

In the anti-telomerase agent according to this embodiment, the heterocyclic compound includes the following general formula (V), general formula (VI), general formula (VII), and general formula (VIII).
One or more heterocyclic compounds selected from the group consisting of

  This is because the apomorphine analogs represented by the general formula (V), the general formula (VI), the general formula (VII), and the general formula (VIII) exhibit an excellent telomerase inhibitory action in Examples described later. This is because it is proved by experimental data.

  In the anti-telomerase agent according to the present embodiment, the heterocyclic compound is a magnoflorin extracted from one or more herbal medicines selected from the group consisting of self-prevention, spicy spicy, hot pepper, yellow lotus, and magnolia. preferable. Alternatively, the anti-telomerase agent according to this embodiment is a natural product containing a component extracted with hot water or ether from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia. It may be an anti-telomerase agent.

  This is because an extraction method has been established for extracting magnoflorin represented by the general formula (V) from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia. This is because the experimental data has demonstrated that a particularly excellent telomerase inhibitory effect is exhibited in the examples.

  In the anti-telomerase agent according to the present embodiment, the heterocyclic compound is preferably dicentrin extracted from Koma grass. Alternatively, the anti-telomerase agent according to the present embodiment may be a natural product-derived anti-telomerase agent that includes a component extracted with ether from komegusa.

  This is because the dicentrin represented by the general formula (VI) has been established as an extraction method for extracting from komagrass, and in the examples described later, it has been demonstrated by experimental data that it exhibits a particularly excellent telomerase inhibitory action. Because.

In the anti-telomerase agent according to this embodiment, the effective dose of the anti-telomerase agent is preferably in the range of 1 nM to 1 × 10 ³ nM, and particularly preferably in the range of 1 nM to 1 × 10 nM. preferable.

  This is because it has been demonstrated by experimental data that the telomerase inhibitory action is particularly excellent in the examples described later if the effective dose of the anti-telomerase agent is within the above range.

<Embodiment 2: Antitumor agent>
The antitumor agent according to this embodiment is represented by the following general formula (I) or general formula (II):
An antitumor agent comprising a heterocyclic compound having an apomorphine skeleton or a salt thereof.

Here, in the above general formulas (I) and (II), X ₁ is an —OH group or —O—CH ₃ group, and X ₂ is an —OH group or —O—CH ₃ group. X ₃ is a —H group or a —OH group, X ₄ is a —OH group or a —O—CH ₃ group, and X ₅ is a group consisting of a —H group or a —O—CH ₃ group. One or more functional groups selected from

Y represents the following general formula (III) or general formula (IV)
It is a structural unit shown by.

  Since the antitumor agent according to this embodiment contains the heterocyclic compound having the apomorphine skeleton or a salt thereof, it exhibits an excellent antitumor action.

In the antitumor agent according to the present embodiment, the heterocyclic compound includes the following general formula (V), general formula (VI), general formula (VII), general formula (VIII), general formula (IX), general formula (X), general formula (XI)
One or more heterocyclic compounds selected from the group consisting of

  This is because apomorphine represented by the general formula (V), the general formula (VI), the general formula (VII), the general formula (VIII), the general formula (IX), the general formula (X), or the general formula (XI). This is because it has been proved by experimental data that the analog exhibits an excellent antitumor action in Examples described later.

  In the antitumor agent according to the present embodiment, the heterocyclic compound is a magnoflorin extracted from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia. preferable. Alternatively, the antitumor agent according to the present embodiment includes a natural product containing a component extracted with hot water or ether from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia. It may be a derived antitumor agent.

  This is because an extraction method has been established for extracting magnoflorin represented by the general formula (V) from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia. This is because, in the examples, it has been demonstrated by experimental data that a particularly excellent antitumor effect is exhibited.

  In the antitumor agent according to the present embodiment, the heterocyclic compound is preferably dicentrin extracted from Koma grass. Or the antitumor agent which concerns on this embodiment may be an antitumor agent derived from a natural product containing the component ether-extracted from a Koma grass.

  This is because the extraction method for extracting dicentrin represented by the general formula (VI) from komegusa has been established, and in the examples described later, it has been demonstrated by experimental data that it exhibits particularly excellent antitumor activity. Because.

In the antitumor agent according to the present embodiment, the effective dose of the antitumor agent is preferably in the range of 1 nM to 1 × 10 ³ nM, particularly preferably in the range of 1 nM to 1 × 10 nM. preferable.

  This is because it has been proved by experimental data that the antitumor agent exhibits particularly excellent antitumor action in the examples described later if the effective dose of the antitumor agent is within the above range.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable.

  For example, in the above-described embodiment, the anti-telomerase agent or anti-tumor agent is simply an anti-telomerase agent or anti-tumor agent containing a heterocyclic compound having a specific structure of an apomorphine skeleton or a salt thereof. Ingredients that are generally acceptable in pharmaceuticals may be included.

  The anti-telomerase agent or anti-tumor agent may have any dosage form, for example, various dosage forms such as oral preparations, patches, eye drops, nasal drops, enemas, injections, etc. It can take suitably.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Background of the present invention>
FIG. 1 is a conceptual diagram for explaining the reason why the present inventor paid attention to alkaloids and telomerase. The inventor searched for an antitumor component by paying attention to alkaloids and telomerase. That is, while reports of antitumor effects with drugs mainly composed of alkaloids (direct effects can be studied) are found, the present inventor is expected to show medicinal effects with a novel mechanism of action among alkaloids. In order to find the anti-telomerase drug to be used, as shown in the examples described later, the evaluation of telomerase gene expression and activity was used as an index for examination.

  The reason why the present inventor paid attention to herbal medicine is as follows. That is, as shown in the left of FIG. 1, most of the general antitumor drugs have an effect on cell proliferation due to their cancer suppressing action, and are effective for normal cells in a short time. Therefore, general anti-tumor agents have severe side effects and are considered to have been dropped as components of herbal medicines over a long historical course. However, if it is an anti-telomerase component, as shown in the right of FIG. 1, the side effect on normal cells is relatively mild, so it is considered that it exists in general herbal medicine.

  The effect of a general antitumor drug is determined by cell death due to induction of apoptosis in a very short time such as within one week (left in FIG. 1). On the other hand, anti-telomerase drugs continue to proliferate for about one month after telomerase inhibition, depending on the strength of the inhibition. Meanwhile, telomeres shorten and behave like a state with a finite lifetime similar to normal cells. Therefore, it takes about two months to be led to apoptosis as shown in the right of FIG.

  Based on the above-mentioned idea, in order to identify an antitumor component in a crude drug, the present inventor conducted a sensitivity test with an extract of the crude drug, focusing on alkaloids, to suppress proliferation of hepatoma cells and suppress telomerase. Commonly derived components of alkaloids were found from the database. Furthermore, when this inventor examined not only this chemical substance but some alkaloids which have a structure in common with this chemical substance, the component which has a more powerful antitumor effect was identified.

  To date, the inventor has been working on the topic of oncology, especially the enzyme telomerase that is expressed in 90% of cancer tissues. We have also cloned genes that suppress this enzyme. For the first time in the world, its localization has been identified as a part of the short arm of human chromosome 10 (10p15.3) and the presence of its regulatory gene has been clarified.

In addition, the present inventor has already started research on telomerase inhibitors and cancer inhibitors. Specifically, the present inventor has attempted to identify an unknown antitumor component and an antitelomerase component for alkaloids and glycosides for the past two years. As a result, for the alkaloids, the substance in the process of artificial synthesis of berberine that has been reported as an antitumor component in the past, and that three types of common structures have a telomerase inhibitory action. Clarified as described later. In addition, the present inventor calculated IC ₅₀ for these four types (magnoflorin, dicentrin, michepressin, and glaucine), and determined the growth inhibitory action and telomerase expression inhibitory action by molecular biological techniques and clinical pharmacological techniques. Analyzing the mechanism of action using the drug, we are promoting the realization of drug discovery.

<Selection of candidate compounds>
Based on the above-mentioned idea, the present inventor has studied a lot about the liver cancer cell inhibitory effect of the extract liquid decocted with herbal extract powder, A cytostatic effect was observed. FIG. 2 is a diagram showing a partial extract of alkaloid components found from the database that are commonly contained in extracts in crude drugs that have induced antitumor effects. In addition, in FIG. 2, the molecular weight of each component, a development code, etc. are shown collectively.

  Then, as shown in FIG. 2, the present inventor extracted a part of the alkaloid components found from the database, which are commonly contained in the crude drug extracts that induced the antitumor effect, and (+)-isocordine. Nine kinds of candidate anti-tumor agents consisting of HCl, isocordine, (+)-boldine, magnofluorine, dicentrine, glaucine, bulocacapine HCl, dehydry dihydroxytaximine, and michepressine. From this, the inventor conducted a drug sensitivity test and examined antitumor components as shown in Examples described later.

<Outline of experimental method>
The present inventor examined the expression and activity of the telomerase gene by the real-time RT-PCR method, the telomerase activity measurement, the senescence induction detection method, and the apoptosis detection method using the drug sensitivity test for the above-mentioned nine kinds of candidate compounds.

  In addition, the present inventor has decocted these nine alkaloids, filtered and purified, and added to the cell line, so that cell proliferation and gene expression effects in vitro can be achieved in cell biology and molecular tumors. And pharmacologically determined. Furthermore, the present inventor tried to identify the components pharmacochemically for a drug that showed an effective anticancer effect.

<Chemical structure of candidate compound>
FIG. 3 is a diagram showing the chemical structures of four candidate compounds exhibiting telomerase inhibitory action and antitumor action. FIG. 3 (a) shows the chemical structure of Magnoflorine (magnoflorin, development code: T05827). FIG. 3 (b) shows the chemical structure of Dicentrine (Dicentrine, development code: T30626). FIG. 3 (c) shows the chemical structure of Michepressine (Michepressin, development code: L29150). FIG. 3 (d) shows the chemical structure of (+)-Glaucine ((+)-Glaucine, development code: T00858). FIG. 3 (e) shows the chemical structure of the basic skeleton of Apomorphine (also known as apomorphine). These four candidate compounds all exhibited a telomerase inhibitory action and an antitumor action, as shown in Examples described later.

  FIG. 4 is a diagram showing the chemical structures of three kinds of candidate compounds showing antitumor action and a positive control compound having antitumor action. FIG. 4 (f) shows the chemical structure of Berberine, which is a positive control compound for antitumor activity. FIG. 4 (g) shows the chemical structure of (+)-boldine ((+)-bolzine, development code: T22430). FIG. 4 (h) shows the chemical structure of Bulbocapnine HCl (Vrubocacapnin hydrochloride, development code: T22399). FIG. 4 (i) shows the chemical structure of (+)-isocordyne-HCl (hydrochloric acid (+)-isocollidine, development code: T15391). These three candidate compounds and the positive control compound for antitumor activity all showed antitumor activity, but did not induce telomerase suppression except for berberine.

<Changes in morphology of human liver cancer cell lines>
FIG. 5 is a diagram showing the state of morphological change and aging induction of human liver cancer cell lines by 1 nM magnoflorin. The present inventor conducted an experiment to examine the effect of suppressing the morphological change of the human liver cancer cell line for the above-mentioned magnoflorin. Specifically, 1 nM magnoflorin was added to untreated HLF Cells (human hepatoma cell line) to examine morphological changes and cancer suppression effects. Further, staining was performed using Sigma's Scenens cells staining kit, and the state of aging induction by β-gal staining was observed.

  As a result, as shown in FIG. 5 (a), the swelling of HLF Cells after 30 days after addition of 1 nM magnoflorin was observed to be clearly swollen compared with the untreated state, and β-gal staining was observed. Aging induction was also observed. This shows that the addition of 1 nM magnoflorin produces an antitumor effect against HLF Cells.

  FIG. 6 is a figure which shows the morphological change of the human liver cancer cell line by 1 nM michepressin. The present inventor conducted an experiment to examine the effect of suppressing the morphological change of the human hepatoma cell line for the above-mentioned michepressin. Specifically, 1 nM michepressin was added to untreated HLF Cells (human hepatoma cell line) to examine morphological changes.

  As a result, as shown in FIG. 6, the swelling of HLF Cells after 30 days after adding 1 nM michepressin was observed to be clearly swollen compared to the untreated state. From this, it can be seen that the addition of 1 nM michepressin produces an antitumor effect against HLF Cells.

  FIG. 7 is a diagram showing the morphological change of a human liver cancer cell line by 1 nM (+)-glaucin. The present inventor conducted an experiment to examine the effect of suppressing the morphological change of the human hepatoma cell line for the above (+)-glaucine. Specifically, 1 nM (+)-glaucine was added to untreated HLF Cells (human hepatoma cell line) to examine morphological changes.

  As a result, as shown in FIG. 7, the swelling of HLF Cells after 30 days after adding 1 nM (+)-glaucin was observed in comparison with the untreated state. From this, it can be seen that the addition of 1 nM (+)-glaucin produces an antitumor effect against HLF Cells.

  FIG. 8 is a graph showing the results of MTT assay for human liver cancer cell lines. In order to examine the optimal concentration of drug sensitivity, the present inventor examined the cell proliferation of a human hepatoma cell line by MTT assay in the case where the above seven candidate compounds and the negative control were added. In addition, the cell proliferation of the human hepatoma cell line by MTT assay was measured with the microplate reader using MTT Cell Count Kit of Nacalai tesque. The horizontal axis of this graph indicates the concentration (nM) of the added candidate compound. The vertical axis of this graph represents the color development of the formazan dye produced in the cells during cell proliferation, counted by absorbance. In this graph, each average value when N number = 6 two days after the addition of each compound is shown.

As a result, as shown in FIG. 8, in any of the seven types of candidate compounds, the cell proliferation index is low at an addition amount in the range of 1 nM to 1 × 10 ³ nM, particularly in the range of 1 nM to 1 × 10 nM. The cell growth index was low with the added amount of. On the other hand, for human liver cancer cell lines that were not treated with any compound, which is a negative control, the index of cell proliferation was not particularly changed. Therefore, in any of the seven types of candidate compounds, the drug sensitivity is improved at an addition amount in the range of 1 nM to 1 × 10 ³ nM, and the drug sensitivity is particularly improved at an addition amount in the range of 1 nM to 1 × 10 nM. It turns out that it becomes the highest.

  FIG. 9 is a graph of a growth curve with and without the addition of a candidate compound for an antitumor agent to a human liver cancer cell line. The present inventor examined the growth curve of a human liver cancer cell line in order to examine the effect of adding a candidate compound for an antitumor agent to the human liver cancer cell line.

  FIG. 9 (a) shows four types of antitumor tumors, T05827 (magnoflorin), T30626 (descentrin), L29150 (michepressin), and T00858 ((+)-grausin) when no candidate antitumor agent compound is added. It is a graph of the cell growth curve of a human hepatoma cell line at the time of adding 1 nM of the candidate compound of the agent. The horizontal axis of this graph indicates the number of days after addition of the candidate compound, and the vertical axis of this graph indicates the logarithm of the number of cells.

  From the results shown in FIG. 9A, when 1 nM of candidate compounds of four types of antitumor agents T05827 (magnoflorin), T30626 (discentrin), L29150 (michepressin), and T00858 ((+)-glaucine) are added. It is clear that the cell growth of the liver cancer cell line is suppressed. This shows that these four kinds of candidate compounds for antitumor agents have an antitumor effect.

  In addition, when 1 nM of candidate compounds of three types of antitumor agents T05827 (magnoflorin), T30626 (descentrin), and T00858 ((+)-glaucine) is added, the effect of suppressing cell proliferation is particularly excellent. . This shows that these three types of candidate compounds for antitumor agents have particularly excellent antitumor effects.

  Further, FIG. 9 (b) is a diagram showing the form of a human hepatoma cell line when no candidate compound of an antitumor agent is added. FIG. 9 (c) is a diagram showing the morphological change of human hepatoma cell line by 1 nM magnoflorin. FIG. 9 (d) is a diagram showing the morphological change of human hepatoma cell line by 1 nM dicentrin.

  According to these figures, a clear swelling of the human hepatoma cell line is observed with the addition of 1 nM magnoflorin. From this, the antitumor effect with respect to a human hepatoma cell line is recognized by addition of 1 nM magnoflorin. Moreover, according to these figures, the clear whitening (death) of a human hepatoma cell line is recognized by the addition of 1 nM descentrin. From this, the antitumor effect with respect to a human hepatoma cell line is recognized by addition of 1 nM descentrin.

FIG. 10 is a graph showing the telomerase inhibitory action when four types of candidate compounds for antitumor agents are added. The present inventor conducted an experiment to examine suppression of hTERT gene mRNA expression when 10 M of four types of antitumor drug candidate compounds were added. This experiment was carried out by quantifying hTERT mRNA by the One Step Real-Time RT-PCR method. More specifically, quantification was carried out with a light cycler using Roche's SYBRGreen I and QIAGEN's OneStep RT-PCR kits. At that time, RNA control is to draw a calibration line using one copy to 10 ⁷ copies of hTERTmRNA, was quantified.

  The horizontal axis of this graph represents the type of the sample, “attached” indicates the measurement result of the cells fixed to the support, and “floating” indicates the measurement result of the cells detached in the culture medium. The vertical axis of this graph indicates the copy number of hTERT gene mRNA.

  As can be seen from this graph, it is apparent when 10 nM of four types of candidate compounds for antitumor agents, T052727 (magnoflorin), T30626 (discentrin), L29150 (michepressin), and T00858 ((+)-glaucine) are added. In addition, the copy number of hTERT gene mRNA is decreased. This indicates that when 10 nM of these antitumor agent candidate compounds is added to human hepatoma cells, a telomerase inhibitory action is obtained.

  FIG. 11 is a diagram showing a telomerase activity assay in human liver cancer cells. The present inventor confirmed that human hepatoma cells in the case where 10 nM of candidate compounds of four kinds of antitumor agents T05827 (magnoflorin), T30626 (discentrin), L29150 (michepressin), and T00858 ((+)-glaucine) were added. Telomerase activity assay was performed. More specifically, the activity was measured using TeloChaser (Telomerase assay kit by stretch PCR method) manufactured by TOYOBO.

  The gel electrophoresis photograph shown in this figure is the result of the telomerase activity assay. In this figure, PC represents HMc-Li7 cell line derived from human hepatoma. As can be seen from this figure, the inhibitory effect on telomerase activity was stronger when 10 nM of michepressin, glaucine and descentrin was added than when 10 nM of magnoflorin was added. However, as can be seen from this figure, even magnoflorin is suppressed to a level of about 1/10 of the activity of general cancer cells. Therefore, it is clear that all of magnoflorin, michepressin, glaucin, and dicentrin show a strong telomerase inhibitory action on human hepatoma cells.

  FIG. 12 shows the state of aging induction when 1 nM descentrin is added to human hepatoma cells. The present inventor performed β-galactosidase staining to examine the state of aging induction when 10 nM descentrin was added to human hepatoma cells. FIG. 12 (a) is a photograph after 30 days of human hepatoma cells treated with 10 nM dicentrin. FIG. 12 (b) is a photograph of human hepatoma cells after 30 days when no compound is added. In addition, all were dye | stained using Sigma's Sensent cells staining kit. As can be seen from these figures, when 10 nM descentrin is added to human hepatoma cells, it is clear that aging induction has occurred. As a result, it is clear that an antitumor effect is obtained when 10 nM descentrin is added to human hepatoma cells.

  FIG. 13 is a graph showing the state of apoptosis induction when four types of anti-tumor agent candidate compounds are added to human hepatoma cells. The present inventor conducted an assay for caspase 3 activity in order to examine apoptosis induction when 10 nM of four types of antitumor drug candidate compounds were added to human hepatoma cells. More specifically, the caspase 3 activity assay was measured with a microplate reader using Promega's Caspase-Glo 3/7 Assay kit.

  In addition, this graph has shown the result one month after adding 10M of candidate compounds of four types of antitumor agents to human hepatoma cells. The horizontal axis of this graph indicates the type of candidate compound added. The vertical axis of this graph represents an index (RLU) of caspase 3 activity indicated by Promega's Caspase-Glo 3/7 Assay kit. In this graph, Z-VAD is a pancaspase inhibitor (pancaspace inhibitor). As can be seen from these figures, when 4 types of 10 nM anti-tumor agents are added to human hepatoma cells, it is clear that apoptosis is induced. As a result, it is clear that when 4 types of 10 nM antitumor agents are added to human hepatoma cells, an antitumor effect can be obtained in any case.

  FIG. 14 is a graph showing the state of apoptosis induction when four types of candidate compounds for antitumor agents are added to human hepatoma cells. The present inventor conducted a DNA fragmentation assay in order to examine apoptosis induction when 1 nM of four types of candidate compounds for antitumor agents was added to human hepatoma cells. More specifically, DNA extracted from the treated cells was electrophoresed with 1% agarose using Quick Vision DNA Ladder Detection Kit of Biovision.

  This graph shows the results 1 month and 2 months after adding 1 nM of four types of antitumor candidate compounds to human hepatoma cells. The horizontal axis of this graph indicates the type of candidate compound added. The vertical axis of this graph is this graph, where 1M is a monthly later, after the continuous treatment by chemical (one month after continuous treatment with the compound), and 2M is 2 months later after the treatment by chemicals (2 months after continuous treatment with the compound), and C is an HLF cell line derived from human hepatoma (HLF cell line derived from human liver cancer) . As can be seen from these figures, when 4 types of 10 nM anti-tumor agents are added to human hepatoma cells, it is clear that apoptosis is induced. As a result, it is clear that when 4 types of 10 nM antitumor agents are added to human hepatoma cells, an antitumor effect can be obtained in any case.

  FIG. 15 is a graph and a table showing cell cycle-specific effects when (+)-glaucin is added to human hepatoma cells. The present inventor examined the cell cycle specific effect when 10 nM of (+)-glaucine was added to human hepatoma cells. More specifically, as a representative example of alkaloids acting in a cell cycle specific manner, (+)-Glaucine was measured using a GE Guava EasyCyte using a dedicated reagent from GE. The measurement results are summarized in FIGS. 15 (a) to 15 (c). As can be seen from these figures, when 10 nM of (+)-glaucine was added to human hepatoma cells, the cells were concentrated in the preG1 (subG1) phase, and cell growth suppression was induced. Therefore, when 10 nM of (+)-glaucine is added to human hepatoma cells, it is clear that the antitumor action is exerted.

  FIG. 16 is a diagram showing candidate compounds for antitumor agents that showed anti-telomerase activity. This figure is a diagram summarizing the compounds showing anti-telomerase action among the candidate compounds of anti-tumor agents corresponding to FIG. From the above-mentioned various experimental results, the alkaloid component in the herbal medicine that induced the antitumor effect was Magnoflorine (magnoflorin, development code: T05827), Dicentrine (discentrin, development code: T30626), Michelpressine (michepressin, development code: L29150) ), (+)-Glaucine ((+)-Glaucine, development code: T00858), (+)-boldine ((+)-Bolzine, development code: T22430), Bulbocapnine HCl (Bruvocacapnin hydrochloride, development code: T22399), Seven types of (+)-isocordyne-HCl (hydrochloric acid (+)-isocollidine, development code: T15391).

Here, in any of the above-mentioned seven types of compounds showing antitumor activity, the effective dose for showing antitumor activity in human hepatoma cells may be in the range of 1 nM to 1 × 10 ³ nM. A range of 1 nM to 1 × 10 nM is particularly preferable. This is because the above-mentioned various experimental data demonstrate that suitable antitumor properties are exhibited within these ranges.

  Also. As shown in this figure, from the above-mentioned various experimental results, alkaloid components in herbal medicines that induced the anti-telomerase effect were Magnofluorine (magnoflorin, development code: T05825), Dicentrine (Descentrin, development code: T30626), There are four types: Michelpressine (Michepressin, development code: L29150), (+)-Glaucine ((+)-Glaucine, development code: T00858).

  And although antitelomerase action was recognized by these four types, from the viewpoint of antitumor action, the order of the most powerful component is Dicentrine (Descentrin, development code: T30626) = (+)-Glaucine ( (+)-Glaucine, development code: T00858) = Magnoflorine (magnoflorin, development code: T05827)> Michepressine (Michepressin, development code: L29150). However, the anti-tumor action of these four types of compounds is not necessarily based only on the anti-telomerase action. Since the four types of compounds show somewhat different forms of cell death, they may have antitumor effects other than antitelomerase activity.

Here, in any of the above-mentioned four types of compounds showing antitumor action and antitelomerase action, the effective dosage for showing antitumor and antitelomerase action in human hepatoma cells is 1 nM to 1 × 10 ^3. It is preferably in the range of nM, particularly preferably in the range of 1 nM to 1 × 10 nM. This is because the above-described various experimental data demonstrate that suitable antitumor and antitelomerase properties are within these ranges.

<Extraction method of anti-tumor / anti-telomerase compound derived from natural product>
FIG. 17 is a flowchart for explaining a method for extracting magnoflorin and dicentrin, which are anti-tumor / anti-telomerase compounds derived from natural products. As shown in this figure, since magnoflorin and dicentrin are difficult to chemically synthesize, it is preferable to extract by hot water extraction or ether extraction as shown in this flowchart.

FIG. 17 left is a flowchart for explaining the ether extraction method of magnoflorin. Thus, in order to obtain magnoflorin, first, the chopped cut piece is extracted with methanol to recover the methanol phase. Then, the methanol phase is applied to a Diaion HP20 column, the water elution part is discarded, and the 50% methanol elution part is recovered. Further, 50% methanol eluate is separated by 0.1N HCl ethyl acetate, the ethyl acetate layer is discarded, and the aqueous phase is recovered. Then, the aqueous phase is separated by 1N NH ₃ aq / CHCl ₃ , the CHCl ₃ phase is discarded, and the aqueous phase is recovered. Furthermore, the aqueous phase is subjected to ODS column chromatography, and the solvent is switched from water to 20% CH ₃ CN and eluted to obtain separated magnoflorin.

The right side of FIG. 17 is a flowchart for explaining the ether extraction method of dicentrin. Thus, in order to obtain Dicentrin, first, Komagaku, which is a natural monument, is obtained through a legal route, and then Komagakusa is extracted with methanol to recover the methanol phase. Then, the methanol phase is separated with 2.5% tartaric acid aq / Et ₂ O, the Et ₂ O phase is discarded, and the aqueous phase is recovered. Further, the aqueous phase is separated with 2.5% K ₂ CO ₃ aq / Et ₂ O, the Et ₂ O phase is discarded, and the aqueous phase is recovered. The phases are then separated with 0.5 M NaClO ₃ / CHCl ₃ , the aqueous phase is discarded and the CHCl ₃ phase is recovered. Then, the CHCl ₃ phase is adsorbed on SiO ₂ , and the solvent is switched to CHCl 3: MeOH: NH 4 OH (9: 2.5: 0.5) → CHCl 3: MeOH: H 2 O (8: 2: 0.2) and eluted. To obtain the separated descentrin.

  FIG. 18 is a conceptual diagram for schematically illustrating a method for obtaining four types of compounds exhibiting antitumor action and antitelomerase action on human liver cancer cells. The method for extracting magnoflorin and dicentrin has been mentioned in part in the above description, but Magnoflorine (magnoflorin, development code: 4 types of compounds that exhibit antitumor and antitelomerase activity in human liver cancer cells. To obtain T05827), Dicentrine (Dicentrine, development code: T30626), Michelpressine (Michepressin, development code: L29150), (+)-Glacine ((+)-Glaucine, development code: T00858) Various methods shown can be used.

  For example, in order to obtain magnoflorin, ether extraction or hot water extraction may be performed not only from self-prevention but also from any of herbal ingredients: self-prevention, spicy, spicy spicy, yellow lotus, and magnolia. However, it is preferable to separate by chromatography or the like for purification thereafter. On the other hand, no effective method has been developed for dicentrin other than the method of extracting ether or hot water from Koma grass. Further, (+)-glaucine and michepressin can be obtained by general chemical synthesis.

<Future issues>
The present inventor intends to examine a number of carcinoma-derived cell lines, administer an effective amount to a tumor-bearing mouse, and examine whether there is an in vivo tumorigenic inhibitory effect and a metastatic inhibitory effect. In addition, since it is difficult to artificially synthesize some of the drug candidate compounds having a telomerase inhibitory effect, it is planned to use those obtained by purifying components from natural products by component extraction and chromatography.

  In the future, the present inventor will investigate 1) the binding sites and binding modes to the target protein using drug discovery software (DS modeling) for 4 types of anti-tumor components, and 2) the common skeleton. In addition, we plan to discover new derivatives based on the binding site information of the target protein.

  More specifically, as shown in FIG. Search for points of action by DS and develop derivative substances based on 2.1. Then, the compounds thus listed as candidate compounds are chemically synthesized. 1. MTT assay (determining the optimal concentration IC50), 2. Growth curve, 3. Telomerase inhibitory effect (mRNA, activity) 4. Telomere length, Aging induction (β-gal staining), 6. Apoptosis (early, middle, late) (caspase activity, cell cycle, mitochondrial membrane potential, DNA fragmentation) is studied. And 1. 1. Sensitivity in each carcinoma by cancer cell panel. 2. tumor suppressive effect in in vivo tumor-transplanted mice; In vivo pharmacokinetic parameters The objective is to confirm the antitumor effect and antitelomerase effect, consider the minimum toxic concentration, etc., develop it as a drug discovery, and obtain approval from the Ministry of Health, Labor and Welfare.

  And by performing the above various studies, the present inventors have found that the heterocyclic compound having an apomorphine skeleton represented by the above general formula (I) or (II) has any antitumor activity. I think that will become clear. However, this point is assumed by those skilled in the art of medicine, pharmacy, biochemistry, molecular biology, and the like based on the various experimental data already conducted. It is.

<Benefit effect obtained from the above embodiment>
In recent years, the clinical application of herbs has been actively carried out, and reports have been made in various medical fields at international conferences. However, basic research reports on single ingredients such as those contained in herbal medicines or active ingredients in the human body are not sufficient. The herbal medicine-derived components account for a significant proportion of the antitumor drugs currently in clinical application, so isolation of new components is an unknown and promising area. In other words, by examining the presence or absence of anti-tumor effects using telomerase-related gene expression as an index and creating related derivatives, it will become a new topic in the drug discovery scene and provide options for cancer patients Connected.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

  For example, in the above examples, experimental data was shown only for some of the heterocyclic compounds having an apomorphine skeleton represented by the above general formula (I) or general formula (II), but only those compounds were shown. Is not intended to limit the antitumor activity or antitelomerase activity. Those skilled in the art such as medicine, pharmacy, biochemistry, molecular biology and the like will naturally assume such activity based on the various experimental data already conducted.

  Moreover, in the said Example, although the human liver cancer cell was used, it is not the meaning which limits the object of said anti-tumor agent or anti-telomerase agent to human liver cancer. For example, the above anti-telomerase agent or anti-tumor agent can be used for various tumors other than liver cancer, or can be used for various tumors of mammals other than humans. In this way, these anti-telomerase agents or anti-tumor agents can be suitably used as pharmaceuticals for mammals including humans.

  Regarding the marketability of the present invention, the possibility of avoidance by other technologies, etc., especially the four compounds showing anti-telomerase action have unique action mechanisms as anti-tumor drugs, and therefore their clinical application due to their novelty. Be expected. In addition, regarding the chemical substance represented by the general formula (I) or the general formula (II), which is currently focused on, it is considered that the same effect can be obtained with respect to the basic structure, side chain, and derivative substance. In the future, it will be possible to develop more effective antitumor drugs by clarifying the interaction of the tumor-specific protein with the active site.

It is a conceptual diagram for demonstrating the reason which this inventor paid attention to alkaloid and telomerase. It is a figure which extracts and shows a part of the component of the alkaloid discovered from the database that it is commonly contained in the extract in the crude drug which induced the antitumor effect. It is a figure which shows the chemical structure of four types of candidate compounds which show a telomerase inhibitory action and an antitumor action, and an apomorphine basic skeleton. It is a figure which shows the chemical structure of three types of candidate compounds which show antitumor action, and the positive control compound of antitumor action. It is a figure which shows the state of the morphological change and aging induction of a human liver cancer cell line by 1 nM magnoflorin. It is a figure which shows the morphological change of the human liver cancer cell line by 1 nM michepressin. It is a figure which shows the morphological change of the human liver cancer cell line by 1 nM (+)-glaucine. It is a graph which shows the result of the MTT assay with respect to a human liver cancer cell line. It is a graph of the growth curve when not adding the candidate compound of an antitumor agent with respect to a human liver cancer cell strain. It is a graph which shows the telomerase inhibitory effect at the time of adding the candidate compound of four types of antitumor agents. It is a figure which shows the telomerase activity assay in a human liver cancer cell. It is a figure which shows the state of aging induction at the time of adding 10 nM descentrin to a human hepatoma cell. It is a graph which shows the state of apoptosis induction at the time of adding the candidate compound of four types of antitumor agents to a human hepatoma cell. It is a graph which shows the state of apoptosis induction at the time of adding the candidate compound of four types of antitumor agents to a human hepatoma cell. It is a graph and a table | surface which show a cell cycle specific effect | action when (+)-glaucine is added to a human hepatoma cell. It is the figure which showed the candidate compound of the antitumor agent which showed the antitelomerase action. It is a flowchart for demonstrating the extraction method of magnoflorin and a dicentrin which are antitumor and antitelomerase compounds derived from a natural product. It is a conceptual diagram for demonstrating the method for obtaining four types of compounds which show an antitumor action and an antitelomerase action to a human hepatoma cell.

Claims (6)

An anti-telomerase agent comprising at least one heterocyclic compound selected from the group consisting of the following general formulas (V) and (VII), or a salt thereof.
In the anti-telomerase agent according to claim 1,
The heterocyclic compound is magnoflorin extracted from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia.
Anti-telomerase agent. In the anti-telomerase agent according to claim 1 or 2,
The effective dosage of the anti-telomerase agent is in the range of 1-1 × 10 ³ nM,
Anti-telomerase agent. An antitumor agent comprising one or more heterocyclic compounds selected from the group consisting of the following general formulas (V) and (VII), or a salt thereof.
The antitumor agent according to claim 4,
The heterocyclic compound is magnoflorin extracted from one or more herbal medicines selected from the group consisting of self-prevention, spicy, spicy spicy, yellow lotus, and magnolia.
Antitumor agent. The antitumor agent according to claim 4 or 5,
The effective dose of the anti-tumor agent is in the range of 1 to 1 × 10 ³ nM,
Antitumor agent.