JP5283962B2 - Pharmaceutical composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicine composition, to be more precise, to provide a pseudopodia formation inhibitor of a compound having dynamin functional inhibiting action, furthermore, to provide a new medicine composition such as an antitumor agent or immunosuppressive agent containing the compound or its pharmaceutically permissible salts as an effective component. <P>SOLUTION: The pseudopodia formation inhibitor for cells comprises the compound which has dynamin functional inhibiting action and is expressed by general formula (I) or its pharmaceutically permissible salts. In the formula, R<SP>1</SP>-R<SP>3</SP>are the same as or different from each other, and each of them is selected from the group consisting of hydrogen, a hydroxy group, a straight or branched non-substituted or substituted acyl group, an alkyl group, an alkenyl group, an alkynyl and an aryl group. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a novel pharmaceutical composition. More specifically, the present invention relates to a temporary pod formation inhibitor of a compound having a dynamin function inhibitory action, and further relates to a novel pharmaceutical composition such as an antitumor agent or an immunosuppressive agent containing the compound as an active ingredient.

  Actin is a globular protein with a molecular weight of about 42,000, which polymerizes under physiological conditions to form actin fibers and plays a role as a cytoskeleton. In the test tube, the generated actin fibers are in a very stable state, but the actin fibers in the cells are dynamically controlled for polymerization and depolymerization, and the state is constantly changing. For example, actin polymerization occurs immediately below the cell membrane in the leaf-shaped temporary foot of the moving cell, whereas actin depolymerization occurs in the base of the temporary foot close to the center of the cell. As a result, the monomers in the actin fibers are constantly changing.

  When a cell moves with directionality, first, a slender protrusion called a thread-like temporary foot is formed at the tip, and after searching for the directionality, the cell acquires driving force by extension of a flat membrane called a leaf-like temporary foot. Stress fibers develop at the rear of the cell, and by atrophying the rear, the cell grows forward and contracts behind. The thread-like temporary foot, the leaf-like temporary foot, and the stress fiber are all structures formed by intracellular actin polymerization. The chemoattractant signal at the cell tip is transmitted to the low molecular weight G protein, facilitating a very fast remodeling of actin fibrils. The driving force for cell movement is generated by polymerization of actin fibers by Rho family G proteins. Rho family G proteins regulate cell shape and movement by regulating actin polymerization. It consists mainly of Cdc42, Rac, and Rho, and Cdc42 controls the formation of filamentous pseudopods, Rac controls the formation of foliate pseudopods, and Rho controls the formation of stress fibers.

  There is a report on the therapeutic use of agents that modulate alpha smooth muscle actin (SMA) activity (Patent Document 1). This document discloses the use of a pharmaceutical composition containing an SMA inhibitor as an active ingredient for the prevention of tumor cell metastasis in cancer patients. As one of the causes of tumor cell metastasis, it has been shown that cells pass into and out of blood vessels.

  Endocytosis of synaptic vesicles at the nerve ending is essential for maintaining synaptic transmission, but much remains unclear about its molecular mechanism. Dynamin family isoforms are intracellular proteins that are strongly expressed in the brain, are localized in the presynaptic part of nerve cells, and function in the endocytosis of synaptic vesicles. Dynamin is known to play a part in clathrin-dependent endocytosis together with clathrin that copolymerizes and coats endocytic vesicles. This protein group is also present in non-neuronal cells, and is thought to play a role in lipoprotein metabolism via endocytosis of cell membrane receptors and sugar metabolism via sugar transport carriers.

There are reports on substances that inhibit the function of dynamin (Non-patent Document 1). Here, it is disclosed that, for some low-molecular-weight candidate compounds, dynamin was screened by confirming suppression of GTPase activity of dynamin, and Dynasore, an inhibitory substance, was obtained. In this document, it has been confirmed that dynasoa suppresses the function of endocytosis depending on dynamin. As for other actions, it has been confirmed that the action dependent on dynamin is suppressed, and it has been reported that it does not affect dynamin-independent functions. However, there is no report about the inhibitory effect of pseudopod formation. Nor is there any mention of the role that endocytosis plays in tumor cell invasion or migration.
JP 2004-167270 A Developmental Cell 10, 839-850 (2006)

  An object of the present invention is to search for a novel mechanism of a compound having a dynamin function inhibitory activity represented by the following general formula I and to provide a novel pharmaceutical composition containing the compound as an active ingredient.

As a result of investigating the mechanism of the compound represented by the following general formula I, the present inventors have found that as a novel mechanism, the formation of pseudopodia of cells is inhibited, and the present invention has been completed. Furthermore, the effect | action of the said compound with respect to tumor cell invasion and a phagocytosis which is a cellular phenomenon accompanying pseudopod formation was confirmed. As a result, it was confirmed that the compound has an antitumor effect. Moreover, the inhibitory effect was recognized with respect to the phagocytosis of a macrophage, and it also confirmed having an effect as an immunosuppressive agent.
Based on the above results, the present invention as a novel pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient was completed.

That is, this invention consists of the following.
1. A cell pseudopodiasis inhibitor comprising a compound represented by the following general formula I or a pharmaceutically acceptable salt thereof:
Formula I;
(Wherein R ^{1 to} R ³ are the same or different and each represents hydrogen, a hydroxyl group, a linear or branched, unsubstituted or substituted, saturated or unsaturated acyl group, alkyl group, alkenyl group, alkynyl group, and Selected from the group consisting of aryl groups.)
2. 3. The pseudopodiasis inhibitor according to item 2 above, wherein the compound of the general formula I is a compound represented by the following formula II:
Formula II;
3. Macrophage phagocytosis inhibitor comprising a compound represented by the following general formula I or a pharmaceutically acceptable salt thereof:
Formula I;
(Wherein R ^{1 to} R ³ are the same or different and each represents hydrogen, a hydroxyl group, a linear or branched, unsubstituted or substituted, saturated or unsaturated acyl group, alkyl group, alkenyl group, alkynyl group, and Selected from the group consisting of aryl groups.)
4). The macrophage phagocytosis inhibitor according to item 3, wherein the compound of general formula I is a compound represented by formula II below:
Formula II;
5. 5. A pharmaceutical composition comprising the temporary foot formation inhibitor or the macrophage phagocytosis inhibitor according to any one of items 1 to 4 as an active ingredient.
6). 6. The pharmaceutical composition according to item 5 above, wherein the pharmaceutical composition is an antitumor agent.
7). 6. The pharmaceutical composition according to item 5 above, wherein the pharmaceutical composition is an immunosuppressant.

The compound represented by the general formula I of the present invention exhibits a strong inhibitory effect on the formation of both temporary and lamellipodia of tumor cells in an in vitro system, and further, in vitro and in vivo. In this system, the effect of suppressing the infiltration of tumor cells into normal tissues was confirmed. Moreover, the effect which suppresses the phagocytosis of a macrophage was recognized in the in vitro system.
Thereby, the compound represented by the general formula I exhibits an effect as a pseudopodia formation inhibitor or a phagocytosis inhibitor of macrophages, and further includes the compound represented by the general formula I or a pharmaceutically acceptable salt thereof. The drug can be used as a pharmaceutical composition such as an antitumor agent or an immunosuppressant.

  In the present invention, the provisional foot formation may be any of a temporary foot formation of a thread-like temporary foot and a leaf-like temporary foot. In addition, the antitumor in the present invention means that it has an inhibitory action on all actions of tumors such as tumor formation, invasion, and metastasis.

The compound having an inhibitory effect on dynamin function of the present invention comprises a compound represented by the following general formula I or a pharmaceutically acceptable salt thereof, and has a pseudopodiasis inhibitory action and a macrophage phagocytosis inhibitory action.
Formula I:
(Wherein R ^{1 to} R ³ are the same or different and each represents hydrogen, a hydroxyl group, a linear or branched, unsubstituted or substituted, saturated or unsaturated acyl group, alkyl group, alkenyl group, alkynyl group, and Selected from the group consisting of aryl groups.)

  In the general formula I, the alkyl group, alkenyl group and alkynyl group may be a cycloalkyl group, a cycloalkenyl group and a cycloalkynyl group, respectively. As used herein, cycloalkyl means a saturated cyclic carbon chain, and cycloalkenyl and cycloalkynyl each mean a cyclic carbon chain containing at least one double or triple bond. The cycloalkyl group, cycloalkenyl group, cycloalkynyl group and aryl group may be monocyclic, polycyclic or condensed cyclic.

Specifically, R ^{1 to} R ³ are the same or different and are each preferably a hydrogen atom or a hydroxyl group, and particularly preferably a hydroxyl group.

  The cell pseudopod formation inhibitor of the present invention is a compound represented by the general formula I, for example, any one selected from the following compounds represented by the formula II, and pharmaceutically acceptable salts and solvates thereof: It may be.

As the compound selected from the above, the compound represented by the following formula II is particularly suitable. In the present invention, isomers (epimers) exist in the compounds represented by Formulas I and II. Any of these isomers and any mixtures thereof shall belong to the present invention.
Formula II:

In the present invention, the pharmaceutically acceptable salt includes the following.
Examples of basic addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts and triethylamine salts; dicyclohexylamine salts and ethanolamines. Aliphatic amine salts such as salts, diethanolamine salts, triethanolamine salts and brocaine salts; Aralkylamine salts such as N, N-dibenzylethylenediamine; and heterocyclic aromatics such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts For example, tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt Quaternary ammonium salts such as tetrabutylammonium salts; arginine; basic amino acid salts such as lysine salt and the like.

  Examples of acid addition salts include inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, hydrogencarbonates and perchlorates; for example acetates, propionates, lactates and maleates. Organic acids such as fumarate, tartrate, malate, citrate, ascorbate; sulfonic acids such as methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate Salts; for example, acidic amino acids such as aspartate and glutamate.

  In the present invention, the compound represented by Formula II is specifically a compound having a structural formula equivalent to Dynasore described in Non-Patent Document 1. Dynasore was reported in Non-Patent Document 1 as an inhibitor of Dynamin (see FIGS. 1 and 2), which is a protein that functions in cell endocytosis. Dynasoa has been reported to suppress the function of dynamin by reversibly inhibiting the GTPase activity of dynamin.

  The effect of inhibiting the formation of temporary foot in the present invention can be confirmed by a test of temporary foot formation that is generally performed. For example, it can be confirmed using U20S (derived from human osteosarcoma) or A549 cells (human non-small cell lung cancer cells) that are liable to form filamentous pseudopods due to serum stimulation.

  It is confirmed that the compound of the present invention represented by the above general formula I has an inhibitory action on the formation of both lamellipodia and lamellipodia by the above method. As mentioned in the background art section, Patent Document 1 reports the therapeutic use of a drug that modulates alpha smooth muscle actin (SMA) activity, and an SMA inhibitor for the prevention of tumor cell metastasis in tumor patients. There is a disclosure of the use of a pharmaceutical composition containing as an active ingredient. As one of the causes of tumor cell metastasis, it has been shown that cells pass into and out of blood vessels.

  Therefore, the effects of the compound of the present invention represented by the general formula I on tumor cells can be confirmed in in vitro and in vivo systems. For example, in an in vitro system, the effect on tumor cell invasion can be confirmed by confirming cell chemotaxis. In addition, in vivo systems, tumor-bearing animals are prepared by injecting tumor cells into specific sites, and the effect on tumor cell metastasis is confirmed by confirming the presence or absence of tumor cells at a site different from the injected site. Can be confirmed.

  In particular, when a tumor cell forms a temporary foot in a living body, the tumor cell may infiltrate into a normal tissue, and may further lead to metastasis of the tumor cell. The action of the compound represented by the above general formula I can prevent tumor cell infiltration and / or metastasis, reduce progression, and can be used as an antitumor agent. In the present invention, since an obvious cell infiltration inhibitory effect is recognized at a concentration of 40 μM of the above compound in the culture medium in an in vitro system, the above compound is confirmed by confirming the effect at a concentration lower than that. The dose of can be determined as appropriate. In addition, even in vivo systems, metastasis-suppressing effects were observed at 0.5 mg / 50 μl of the above compound against tumors with a diameter of about 2 to 3 mm, confirming effects at lower concentrations By doing so, the dose can be appropriately determined.

  Furthermore, the compound has an action of inhibiting the phagocytosis of macrophages. Regarding the immune reaction, antigenic substances react with phagocytic cells such as macrophages and monocytes to exert cellular immunity, and in some cases, may lead to inflammation and allergic symptoms. Since these phagocytic cells are accompanied by pseudopodia formation in the phagocytic process, the action of the compound represented by the above general formula I can inhibit the formation of pseudopods of phagocytic cells and reduce undesirable immune responses. The compounds of the present invention can be used as immunosuppressants.

  As described above, the present invention extends to a pharmaceutical composition containing an effective amount of the compound represented by the above general formula I or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of the present invention can be used, for example, as an antitumor agent or an immunosuppressive agent.

  The pharmaceutical composition of the present invention comprises an effective amount of a pseudopodiasis inhibitor or a macrophage phagocytosis inhibitor comprising the compound represented by the above general formula I or a pharmaceutically acceptable salt thereof as described above, May contain an acceptable carrier. Such pharmaceutical compositions can be administered orally or parenterally. In the case of oral administration, the temporary foot formation inhibitor of the present invention is an ordinary formulation such as a solid agent such as a tablet, powder, granule, capsule, etc .; a liquid agent; an oily suspension; or a syrup or elixir. It can be used also as any dosage form of a liquid agent. When administered parenterally, the compounds of the present invention can be used as aqueous or oily suspension injections and nasal drops. In the preparation, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers and the like can be arbitrarily used.

  Examples Hereinafter, the present invention will be described in more detail with reference to examples of the action of Compound II (Dinosaur), but the present invention is not limited thereto.

(Example 1) Leaf-like temporary foot formation test 1) U20S (derived from human bone and meat species) cells with or without serum. Leaf-like temporary foot formation effect of U20S (human bone and meat species so that the number of cells becomes 3 × 10 ⁴ cells / ml. The cells were suspended in DMEM (Dulbecco's Modified Eagle's Medium, Invitrogen) medium containing 10% v / v calf serum (FBS). 0.5 ml of the cell suspension was seeded in a microculture dish having a diameter of 1.6 cm and incubated at 5% CO ₂ and 37 ± 1 ° C. The next day, incubation was performed in DMEM without FBS for 12 hours. After incubation, the cells were fluorescently stained with phalloidin, and the morphology of the cells was observed under a microscope. Cells cultured in a medium not containing FBS did not show foliar formation (see FIG. 3a).

  10% v / v FBS was added to the cell culture medium after the incubation, and the cells were further incubated for 40 minutes, and the cell morphology was observed in the same manner as described above. As a result, in the medium after the addition of FBS, foliate formation was observed in a part of the cells (see FIG. 3b).

2) U20S cell foliar pod formation effect in the presence or absence of Compound II (Dynasoa) In the same manner as in 1) above, U20S cells were treated with DMEM medium containing 10% FBS so that the number of cells was 3 × 10 ⁴ cells / ml. It was suspended in. 0.5 ml of the cell suspension was seeded into a microculture dish having a diameter of 1.6 cm and incubated at 37 ± 1 ° C. with 5% CO ₂ . The next day, incubation was performed in DMEM without FBS for 12 hours.

  Thereafter, Dynasoa was added to the cell culture solution so as to have a concentration of 240 μM, followed by further incubation for 30 minutes, and the cell morphology was observed in the same manner as in 1). As a result, no foliate formation was observed in the FBS (−) and Dynasoa (+) media (see FIG. 4 a).

  Next, FBS was added at 10% v / v and further incubated for 40 minutes, and the cell morphology was observed as in 1). As a result, in the dysoa (+) medium, no foliate formation was observed even in the case of FBS (+) (see FIG. 4b).

  After culturing the cells in the medium containing FBS and dynasoa, the cells are washed with a medium containing FBS 10% v / v and not containing dynasoa to remove dynasoa, and then in the medium of FBS (+) and dynasoa (-) And incubated for 40 minutes, and the morphology of the cells was observed as in Example 1. As a result, foliar pseudopod formation was observed in the FBS (+) and Dynasoa (−) media (see FIG. 4 c).

3) Results From the results of 1) and 2) above, it was observed that in U20S cells, the formation of foliate pseudopods was observed by the addition of FBS, but the addition of dynasoa suppressed the formation of foliate pseudopods. In addition, when dynasoare was removed, the formation of foliate pseudopods in the cells was confirmed. Furthermore, the ratio of the cell which formed the lamellipodia to the whole cell on each condition was calculated, and the result of having calculated the lamellipodia formation rate was shown in FIG. From these results, it was confirmed that the addition of dynasoa suppresses the formation of foliate pseudopods.

(Example 2) Filamentous pseudopod formation test 1) A fibrous pseudopod formation action of A549 cells (human-derived non-small cell lung cancer cells) with or without serum As a filamentous pseudopod formation test, it is easy to form a filamentous pseudopod by serum stimulation Except for using A549 cells (human-derived non-small cell lung cancer cells), experiments were conducted and observed in the same manner as in 1) of Example 1. Cells cultured in a medium not containing FBS did not show the formation of filamentous pseudopods (see FIG. 6b).

  FBS was added at 10% v / v to the cell culture solution after the incubation, and the cells were further incubated for 45 minutes, and the cell morphology was observed in the same manner as described above. As a result, in the medium after addition of FBS, formation of filamentous temporary foot was observed in a part of the cells (see FIG. 6c).

2) A fibropodia formation action of A549 cells in the presence or absence of Compound II (Dynosa) Other than using A549 cells (human-derived non-small cell lung cancer cells) that easily form filopodia by serum stimulation The experiment was conducted by the same method as 2) of Example 1.

  Dynasoa was added to 240 μM and incubated for another 30 minutes, and the cell morphology was observed in the same manner. As a result, in the FBS (−) and Dynasoa (+) medium, no formation of filamentous pseudopods was observed (see FIG. 7a).

  Next, FBS was added at 10% v / v and further incubated for 45 minutes, and the cell morphology was observed as in Comparative Example 1. As a result, in the medium of Dynasore (+), no filiform formation was observed even in the case of FBS (+) (see FIG. 7b).

  After culturing the cells in the medium containing FBS and dynasoa, the cells are washed with a medium containing FBS 10% v / v and not containing dynasoa to remove dynasoa, and then in the medium of FBS (+) and dynasoa (-) And incubated for 45 minutes, and the cell morphology was observed as in Example 1. As a result, in the FBS (+) and Dynasore (−) media, the formation of a filamentous temporary foot was observed (see FIG. 7c).

3) Results From the results of the above 1) and 2), it was observed that in A549 cells, the formation of filopodia was observed by the addition of FBS, but the formation of filopodia was suppressed by the addition of Dynasoa. In addition, when dynasoare was removed, the formation of filamentous pseudopods in the cells was confirmed.

(Example 3) Invasion inhibitory action on cancer cells (in vitro)
The invasion effect of cancer cells was examined using a culture chamber (BD Bio Coat ^™ Matrigel ^™ Invasion Chamber 24 well: Becton Dickinson).

1) RM-9 cells (mouse prostate cancer cell line) were seeded on the upper part of the culture chamber (2.5 × 10 ⁴ cells / well), and Dynasore was added to 40 μM or 80 μM. A system in which no dynasoa was added was used as a control group. A culture solution (DMEM) containing FBS 10% v / v was added to the lower part of the chamber to induce infiltration of cancer cells in the upper part of the chamber into the lower part of the chamber.
After 22 hours, cells infiltrating the lower part of the chamber were fixed and stained, and the number of infiltrating cells was measured by microscopic observation.

2) Results The above results are shown in FIGS. Thereby, compared with the control group, when the concentration of Dynasore was 40 μM, the number of infiltrating cells was extremely reduced, and it was confirmed that Dynasore suppresses the invasion of cancer cells.

(Example 4) Inhibition of metastasis of cancer cells (in vivo)
C57 / BL6 mice were used to examine the metastatic effect of cancer cells.

1) Preparation of orthotopic prostate cancer model mice C57 / BL6 mice are anesthetized with pentobarbital, 5000 RM-9 cells (mouse prostate cancer cell line) are locally injected into the left side of the mouse prostate lobe, and orthotopic prostate cancer Was made.
2) Administration of dynasoa After 7 days (cancer diameter, about 2 to 3 mm), 0.5 mg / 50 μl of dynasoa (1 μl of DMSO + 49 μl of PBS) was locally injected into prostate cancer so as to spread as far as possible into the tumor mass (7 Animals). The control group received 50 μl vehicle (DMSO 1 μl + PBS 49 μl) (5 animals).
3) Analysis of metastasis of cancer cells to lymph nodes Mice were sacrificed 10 days after administration of Dynasore or vehicle, and the weight of prostate cancer and the number of enlarged retroperitoneal / pelvic lymph nodes were measured.
4) The above results are shown in FIGS. Thereby, it was confirmed that Dynasore suppresses the metastasis | transition to the lymph node of a cancer cell compared with a control group.

(Example 5) Phagocytic activity test 1) Measurement of phagocytic activity of RAW264.7 (derived from mouse macrophages) RAW264.7 cells have a phosphatidylserine receptor on the cell surface and activate this receptor. Then, it has been reported that phagocytosis is induced. Utilizing this property, stimulation-dependent phagocytic activity by phosphatidylserine addition in RAW264.7 cells was measured as shown below. RAW264.7 cells suspended in DMEM (Dulbecco's Modified Eagle's Medium, Invitrogen) medium containing 10% v / v calf serum (FBS, manufactured by Invitrogen) is collagen type I so as to become 7 × 10 ⁴ cells. It was seeded on a 3.5 cm (diameter) culture dish (Corning) containing four coated cover glasses (diameter 12 mm, manufactured by Asahi Glass). Thereafter, the cells were incubated for 24 hours at 5% CO ₂ and 37 ± 1 ° C. Artificial lipid membrane (30% (v / v) phosphatidylserine, 60% (v / v) phosphatidylcholine, 10%) containing 30% (v / v) phosphatidylserine in streptavidinized polystyrene beads (diameter 2 μm, manufactured by Polyscience) (v / v) biotinylated phosphatidylethanolamine). The beads were suspended at 0.0004% (weight ratio) in DMEM without FBS, and 2 ml of the suspension was added to the cultured RAW264.7 cells. In addition, the cells were incubated for 1 hour. After incubation, the cells were washed with phosphate buffered saline to remove beads that were not attached or taken up by the cells. The beads that were attached to the cells but not taken up were labeled with rhodamine-avidin. Thereafter, the incorporated beads were measured by fluorescence and differential interference observation. Cells that had not been dynasoa treated took up beads. (See FIG. 11a).

2) phagocytic activity of RAW264.7 cells in the presence or absence of Compound II (Dinosaur) As in 1) above, RAW264.7 so that the number of cells is 7 × 10 ⁴ cells / 3.5 cm (diameter) culture dish. The cells were suspended and seeded in DMEM medium containing 10% FBS. Thereafter, the cells were incubated for 24 hours at 5% CO ₂ and 37 ± 1 ° C. Thereafter, the cell culture medium was exchanged with DMEM without FBS to which Dynasoa was added to 40 μM, and further incubated for 30 minutes, and phagocytic activity was measured in the same manner as in 1). In the case of dynasoar treatment, dynasoare was also added to the bead suspension so as to be 40 μM.

  The polystyrene beads attached to the cells or taken into the cells were observed with a differential interference microscope (left of FIG. 11a). Beads that only adhered to the cells but were not taken up into the cells were labeled with avidin-rhodamine (red) (in FIG. 11a). In order to make it easier to see the incorporated beads, the left and middle figures are shown on the right in FIG. 11a. As a result, it was confirmed that the phagocytic activity of the cells treated with Dynasore was significantly reduced compared to that of the cells not treated (see FIG. 11a).

3) Results From the results of 1) and 2) above, in RAW264.7 cells, uptake of polystyrene beads was observed, but suppression of phagocytic activity was observed by the addition of Dynasore. Furthermore, the phagocytic activity of many cells was measured under these conditions, and the results of calculating the amount of beads taken up per cell are shown in FIG. 11b. From these results, it was confirmed that phagocytic activity was suppressed by the addition of dynasoar.

As described in detail above, even under conditions where the cells recognize the formation of pseudopods, in the presence of the compound of the present invention, a strong inhibitory effect on the formation of any of the pseudopods of the filamentous and lamellipodia. It was confirmed that
Phenomenon such as cell migration is caused by the formation of a filamentous pseudopodia, which is a long actin fiber protrusion-like structure at the tip of the cell, and the directionality is investigated. This is because the actin fibers that are constructed and formed just below the membrane of the lamellipodia push the cell membrane and move the cell. It is said that tumor cells infiltrate and the like based on such an action. Alpha smooth muscle actin (SMA) inhibitors have also been reported to suppress tumor cell metastasis.

  As a result of actually investigating the effect on tumor cell invasion and metastasis, the compounds of the present invention each showed an inhibitory effect. From this result, the use as a pharmaceutical composition having an antitumor effect is expected. Furthermore, the pseudopodia formation inhibitor of the present invention is also considered to suppress the phagocytic function of cells. In addition, since it has been confirmed that the phagocytic function of macrophages is suppressed, it is expected to be used as a pharmaceutical composition having an immunosuppressive effect by suppressing the phagocytic ability of immune cells in cellular immune reactions and the like. Use as an inflammatory agent or antiallergic agent is also expected.

It is a schematic diagram explaining the function of dynamin. Dynamin acts on the uptake of substances in the cell, ie endocytosis. It is a schematic diagram of the structure of dynamin which is an endocytosis functional protein. It is a figure which shows the lamellipodia formation effect by the presence or absence of serum. (1 of Example 1) It is a figure which shows the foliate formation effect of the cell by the presence or absence of dynasoa and serum. (Example 1-2) It is a figure which shows the result of having measured the leaf-like temporary leg formation rate on each condition. Example 1 It is a figure which shows the filamentous temporary foot formation effect by the presence or absence of serum. (Example 2) It is a figure which shows the filamentous temporary foot formation effect of the cell by the presence or absence of dynasoa and serum. (Example 2-2) It is a figure which shows the test result of infiltration suppression effect (in vitro). (Example 3) It is a figure which shows the test result of infiltration suppression effect (in vitro). (Example 3) It is a figure which shows the test result of the metastasis inhibitory effect (in vivo) of a cancer cell. (Example 4) Effect of dynasore on phagocytosis of mouse-derived macrophage cell line (RAW264.7) (photo) (Example 5) Effect of dynasore on phagocytosis of mouse-derived macrophage cell line (RAW264.7) (graph) (Example 5)

Claims (6)

A cell pseudopodiasis inhibitor comprising a compound represented by the following general formula I or a pharmaceutically acceptable salt thereof:
Formula I;
(Wherein R ^{1 to} R ³ are the same or different and each represents hydrogen, a hydroxyl group, a linear or branched, unsubstituted or substituted, saturated or unsaturated acyl group, alkyl group, alkenyl group, alkynyl group, and Selected from the group consisting of aryl groups.) The compounds of general formula I, pseudopodia formation inhibitor according to claim 1 which is a compound represented by the following formula II:
Formula II;
Macrophage phagocytosis inhibitor comprising a compound represented by the following general formula I or a pharmaceutically acceptable salt thereof:
Formula I;
(Wherein R ^{1 to} R ³ are the same or different and each represents hydrogen, a hydroxyl group, a linear or branched, unsubstituted or substituted, saturated or unsaturated acyl group, alkyl group, alkenyl group, alkynyl group, and Selected from the group consisting of aryl groups.) The phagocytosis inhibitor of macrophages according to claim 3, wherein the compound of the general formula I is a compound represented by the following formula II:
Formula II;
The antitumor agent which contains the temporary foot formation inhibitor of any one of Claims 1-4, or the phagocytosis inhibitor of a macrophage as an active ingredient . The immunosuppressive agent which contains the temporary foot formation inhibitor of any one of Claims 1-4, or the macrophage phagocytosis inhibitor as an active ingredient .