JPH09268190A - Mitomycin c derivative and nonreceptor type tyrosine kinase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a mitomycin C derivative containing a highly unsaturated fatty acid residue, excellent in inhibitory action on nonreceptor type tyrosine kinase related to a cancer and an immunological disease, useful as a carcinostatic agent, a therapeutic agent for an immunological disease. SOLUTION: This mitomycin C derivative is shown by the formula (RCO is an n-3-based highly unsaturated fatty acid acyl residue; Me is methyl), is excellent in inhibitory activity against nonreceptor type tyrosine kinase related to a cancer and an immunological disease and is useful as a carcinostatic agent, a therapeutic agent for an immunological disease, etc. The compound is obtained by adding a solution of docosahexaenoic acid in ethyl acetate to a solution of diisopropylcarbodiimide in ethyl acetate at 0 deg.C, stirring at 0 deg.C for 30 minutes, adding mitomycin C and further reacting at room temperature for 24 hours.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to mitomycin C having an n-3 polyunsaturated fatty acid residue and a drug containing it as an active ingredient, particularly a non-receptor tyrosine kinase inhibitor.

[0002]

2. Description of the Related Art An inactive protein is phosphorylated into an active form in an intracellular signal transduction system until a cell receives a certain stimulus and causes reactions such as substance production, differentiation and proliferation. It is known that there are many cases where a function is exhibited. Known protein kinases involved in the reaction include cyclic AMP-dependent protein kinase, protein kinase C, calmodulin-dependent protein kinase, and receptor tyrosine kinase found in growth factor receptor. It has been known that tyrosine phosphorylation of intracellular proteins is involved in intracellular signal transduction pathways of various cytokines such as interferons and interleukins. However, since the receptors for cytokines have no kinase activity and none of the above protein kinases participates in this pathway, tyrosine kinases activated by cytokine stimulation have been unknown for a long time. By the end of 1993, a non-receptor tyrosine kinase that was activated in association with cytokine receptors was discovered, and a series of non-receptor tyrosine kinases has been identified one after another. It was revealed that these include the src gene product, which is one of the most well-known oncogenes, and the gene group products with high homology to src. Previously, it was known that abnormal expression or abnormal activation of oncogene products due to mutations in gene sequences leads to canceration, but its original role in vivo has hardly been clarified. . Recent advances in non-receptor tyrosine kinase research have led to src
It was suggested that the canceration due to the gene abnormality was due to abnormally accelerated proliferation among signals related to cell differentiation and proliferation that should be transmitted by cytokine stimulation.
Therefore, substances that inhibit these non-receptor tyrosine kinases are expected as therapeutic agents for immune diseases and cancers based on a new mechanism of action. In fact, herbimycin A (J. et al.) Specifically inhibits the tyrosine kinase activity of the src gene product.
Antibiotics, 32 , 255, 1975) have been reported to decancerize cells transformed by Rous sarcoma virus having a viral src gene (v-src) (Cancer Resea).
rch, 49 , 780-785, 1989) has not been clinically applied due to its strong toxicity. Herbimycin A also inhibits calmodulin-dependent kinase and has low selectivity. Furthermore, known protein kinase inhibitors such as H-7 and staurosporine also inhibit a plurality of types of protein kinases, which makes their clinical application difficult. That is, it is desired to develop a non-receptor type protein kinase inhibitor having higher selectivity and lower toxicity.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a substance which exhibits an excellent inhibitory activity against non-receptor tyrosine kinases which are said to be involved in cancer and immune diseases, and which is highly selective for other protein kinases. It is intended to provide a medicine containing it as an active ingredient.

[0004]

The inventors of the present invention have found that a mitomycin derivative represented by the following general formula (I) has an excellent inhibitory activity against a non-receptor tyrosine kinase, for example, a src gene product. The present invention has been completed based on the new finding.

That is, the present invention has the general formula (I)

[0006]

Embedded image

[In the formula, RCO represents an acyl residue of an n-3 highly unsaturated fatty acid.] Mitomycin C having an n-3 highly unsaturated fatty acid residue represented by the formula and an active ingredient thereof. Pharmaceuticals, especially non-receptor tyrosine kinase inhibitors are provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the acyl residue of n-3 polyunsaturated fatty acid represented by RCO includes icosapentaenoyl group, docosahexaenoyl group, docosapentaenoyl group, Examples thereof include α-linolenoyl group.

The mitomycin C derivative having a polyunsaturated fatty acid residue represented by the general formula (I) of the present invention is, for example, a dehydration condensation agent such as diisopropylcarbodiimide in a solvent of mitomycin C and the corresponding polyunsaturated fatty acid. It can be produced by reacting in the presence of.

The dose of the mitomycin C derivative having a polyunsaturated fatty acid residue of the present invention depends on age, sex, body weight,
Generally, 1 depending on the symptom or the administration form.
It is about 1 to 100 mg per day, which can be taken once or divided into several times.

The inhibitor of the present invention can be administered orally or parenterally. As the orally-administered agent, solid preparations such as powder, granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be used. In addition, injections can be prepared as parenteral administration agents. These preparations are prepared in a conventional manner by adding a pharmaceutically acceptable production auxiliary to the active ingredient. Further, it is also possible to prepare a sustained-release preparation by a known technique.

In order to produce a solid preparation for oral administration, the active ingredient and excipients such as lactose, starch, crystalline cellulose, calcium lactose, magnesium aluminometasilicate, and silicic acid anhydride are mixed to prepare a powder. Alternatively, if necessary, a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, etc., a disintegrating agent such as carboxymethylcellulose, carboxymethylcellulose calcium, etc. are added to wet or dry granulate to obtain granules. To produce tablets, these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc. These granules or tablets are coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid, methyl methacrylate copolymer and the like, and enteric-coated preparations, or coated with ethylcellulose, carnauba wax, hydrogenated oil, etc. You can also. To produce capsules, powders or granules can be filled in hard capsules, or the active ingredient can be dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to give soft capsules. .

To prepare a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol and glycerin are dissolved in water to prepare a transparent syrup, and essential oil, ethanol and the like are added to form an elixir. Alternatively, gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose, etc. may be added to prepare an emulsion or suspension. These liquid preparations may optionally contain a flavoring agent,
You may add a coloring agent, a preservative, etc.

In order to produce an injection, the active ingredient is optionally added with hydrochloric acid, sodium hydroxide, lactose, sodium lactate,
Dissolve in distilled water for injection together with a pH adjuster such as sodium monohydrogen phosphate and sodium dihydrogen phosphate, and an isotonic agent such as sodium chloride and glucose, and aseptically filter and fill into ampoules, or mannitol, dextrin , Cyclodextrin, gelatin and the like may be added and freeze-dried under vacuum to give a dissolvable injection before use. In addition, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like may be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

These formulations can be produced by a known production method, for example, the method described in the Japanese Pharmacopoeia, 10th Edition General Rules for Preparation or a method with appropriate modification.

[0016]

EXAMPLES The present invention will be described in more detail with reference to synthesis examples and test examples.

Synthesis Example 1 Synthesis of 1 α-docosahexaenoylmitomycin C derivative Diisopropylcarbodiimide (19 mg, 0.15 mmol) in ethyl acetate (1.0 mL) was added to docosahexaenoic acid (49 m).
g, 0.15 mmol) in ethyl acetate (1.0 mL) was added at 0 ° C. and the mixture was stirred at 0 ° C. for 30 min, then mitomycin C (10 mg,
0.03 mmol was added, and the mixture was further stirred at room temperature for 24 hours (termination of the reaction was confirmed by TLC). The precipitated crystals were filtered off and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel chromatography (chloroform: acetone = 8/1), 1α-
Docosahexaenoylmitomycin C (21 mg, 0.03 m
mol) was obtained.

¹ H NMR (CDCl ₃ ) 400 MHz δ 0.97 (3H, t,
J = 7.5 Hz, CH ₃ ), 1.78 (3H, s, 6-Me), 2.08 (2H,
m, CH ₂ ), 2.30-2.52 (4H, m, CH ₂ CH ₂ ), 2.76-2.90
(10H, m, CH ₂ x 10), 3.20 (3H, s, 9a-OMe), 3.22 (1
H, dd, J = 1.8, 4.5 Hz, 2-H), 3.50 (1H, d, 4.5 Hz,
1-H), 3.52 (1H, 1H, dd, J = 1.8, 13.4 Hz, 3-H), 3.
69 (1H, dd, J = 4.7, 10.9 Hz, 9-H), 4.07 (1H, dd,
J = 10.9, 10.9 Hz, 10-H), 4.46 (1H, d, J = 13.4 H
z, 3-H), 4.83 (2H, br, CONH ₂ ), 4.86 (1H, dd, J =
4.7, 10.9 Hz, 10-H), 5.26-5.43 (14H, m, CH = CH, 7
-NH ₂ ) ¹³ C NMR (CDCl ₃ ) δ 7.90, 14.29, 20.57, 22.48, 25.5
5, 25.59, 25.64, 36.40, 39.55, 41.97, 42.36, 48.6
2, 49.78, 62.01, 105.06, 105.46, 110.42, 127.04, 1
27.82, 127.91, 128.00, 128.11, 128.30, 128.37, 12
8.60, 129.51, 132.07, 147.29, 154.06, 156.41, 175.
75, 178.46, 183.01 IR (neat) 3441, 3333, 3013, 2964, 1709, 1651, 156
0, 1442, 1385, 1340, 1230, 1145, 1062, 709 cm ^-1 Mass 644 (M ⁺ )

Test Method for Inhibiting Protein Kinase Activity The test is performed by Fukasawa et al. (Analytical Biochemistry, 13, 1
957-1964, 1993). That is, cAMP dependence
Protein Kinase, Protein Kinase C, Calmoji
Urin-dependent kinase, EGF receptor tyrosine kinase
The NIH-3T3 cells containing the
A receptor protein tyrosine kinase was introduced. this
Cells with 5 mM MgCl_Two, And antipine, leupepti
1mM Hepes buffer containing 25 μg / ml each of Peptin A and Pepstein A
The mixture was reacted for 10 minutes on ice in (pH 7.4). Further at room temperature
Crush cells by agitating for 2 minutes using a Tex mixer
Then, add 20 mM Hepes buffer and centrifuge at 500 xg for 5 minutes.
To settle the nuclei. The supernatant was collected and the protein concentration was 2.5m.
10mM MgCl to g / ml_Two / 0.1mM Na_ThreeVO_Four / 10 mM β
-Glycerophosphate / 1 mM NaF / 20 mM Hepes (pH 7.4)
Diluted. Add 20 μl of this supernatant to a final concentration of 1 μM phorbol.
12-myristate-13-acetate, 10 μM CaCl _Two, 20μ
Add dimethyl sulfoxide solution of M cAMP and test substance.
Add [γ-³²Add P] -ATP (12.5 μM, 10 μCi)
The enzyme reaction started. After reaction at 25 ℃ for 15 minutes, 4 times concentration
Sample buffer for SDS-polyacrylamide gel electrophoresis
Stop the reaction by adding 10 μl of the solution and add 9% of the reaction mixture.
(W / v) SDS-polyacrylamide gel electrophoresis,
Detects phosphorylated proteins by autoradiography
Was. Corresponds to the protein that is the substrate of each protein kinase
Evaluate protein kinase activity based on the intensity of the band
did. In Table 1, complete inhibition of phosphorylation of substrate proteins
++ if there is a partial inhibition, + if partial inhibition, no inhibition
Indicates the result with-.

Toxicity test for mouse leukemia cell P388 The cells were subcultured in a 96-well plate and cultured for 24 hours, then a test substance was added and further cultured for 48 hours. Viable cells were colorimetrically determined by sulforhodamine B staining, and the number of cells was measured.
When the solvent dimethyl sulfoxide was added in place of the test substance, the cell number was set to 100%, and the concentration of the test substance at which the cell number reached 50% was determined as IC ₅₀ .

Test Example 1 Docosahexaenoylmitomycin C (DHA-MMC) was used as a test substance, and the protein kinase inhibitory activity was measured according to the above test method. The results are shown in Table 1.

Comparative Test Example 1 Herbimycin A was used as a test substance, and the protein kinase inhibitory activity was measured according to the above test method. The results are shown in Table 1.

Comparative Test Example 2 Mitomycin C was used as a test substance, and the protein kinase inhibitory activity was measured according to the above test method. The results are shown in Table 1.

[0024]

[Table 1] Table 1 Protein kinase inhibitory activity ─────────────────────────── Test substance concentration Inhibitory effect μg / ml PTK PKA PKC CAMK EGFR ─────────────────────────── DHA-MMC 100 ++ + + + + 31.6 +--+-10 +----─ ────────────────────────── Mitomycin C 100-----───────────────── ─────────── Herbimycin A 100 ++--++-31.6 +--+ 10 +---────────────────── ───────── PTK = Non-receptor protein tyrosine kinase, PKA = cAMP
-Dependent protein kinase, PKC = protein kinase C, CAMK = calmodulin-dependent kinase, EGFR = EGF receptor tyrosine kinase

Test Example 2 Docosahexaenoylmitomycin C (DHA-MMC) was used as a test substance, and P388 cytotoxicity was measured according to the above test method. Table 2 shows the results.

[0026]

[Table 2] Table 2 P388 cytotoxicity ────────────────── Test substance IC ₅₀ (μg / ml) ─────────────── ──── DHA-MMC 21.0 Mitomycin C 1.9 ──────────────────

[0027]

The compounds of the present invention have excellent inhibitory activity against non-receptor tyrosine kinases,
In particular, it can be expected to be used as an anticancer agent and a therapeutic agent for immune diseases.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 27, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction contents]

¹ H NMR (CDCl ₃ ) 400 MHz δ 0.97 (3H, t,
J = 7.5 Hz, CH ₃ ), 1.78 (3H, s, 6-Me), 2.08 (2H,
m, CH ₂ ), 2.30-2.52 (4H, m, CH ₂ CH ₂ ), 2.76-2.90
(10H, m, CH ₂ x 5 ), 3.20 (3H, s, 9a-OMe), 3.22 (1H,
dd, J = 1.8, 4.5 Hz, 2-H), 3.50 (1H, d, 4.5 Hz, 1
-H), 3.52 (1H , d d, J = 1.8, 13.4 Hz, 3-H), 3.69 (1
H, dd, J = 4.7, 10.9 Hz, 9-H), 4.07 (1H, dd, J = 1
0.9, 10.9 Hz, 10-H), 4.46 (1H, d, J = 13.4 Hz, 3-
H), 4.83 (2H, br, CONH ₂ ), 4.86 (1H, dd, J = 4.7, 1
0.9 Hz, 10-H), 5.26-5.43 (14H, m, CH = CH, 7-NH ₂ ) ¹³ C NMR (CDCl ₃ ) δ 7.90, 14.29, 20.57, 22.48, 25.5
5, 25.59, 25.64, 36.40, 39.55, 41.97, 42.36, 48.6
2, 49.78, 62.01, 105.06, 105.46, 110.42, 127.04, 1
27.82, 127.91, 128.00, 128.11, 128.30, 128.37, 12
8.60, 129.51, 132.07, 147.29, 154.06, 156.41, 175.
75, 178.46, 183.01 IR (neat) 3441, 3333, 3013, 2964, 1709, 1651, 156
0, 1442, 1385, 1340, 1230, 1145, 1062, 709 cm ^-1 Mass 644 (M ⁺ )

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction contents]

Test Method for Inhibiting Protein Kinase Activity The test is performed by Fukasawa et al. (Analytical Biochemistry, 13, 1
957-1964, 1993). That is, cAMP dependence
Protein Kinase, Protein Kinase C, Calmoji
Urin-dependent kinase, EGF receptor tyrosine kinase
The NIH-3T3 cells containing the
A receptor protein tyrosine kinase was introduced. this
Cells with 5 mM MgCl_Two, And antipine, leupepti
PeptaH1mM Hepes buffer containing 25 μg / ml each
The mixture was reacted for 10 minutes on ice in (pH 7.4). Further at room temperature
Crush cells by agitating for 2 minutes using a Tex mixer
Then, add 20 mM Hepes buffer and centrifuge at 500 xg for 5 minutes.
To settle the nuclei. The supernatant was collected and the protein concentration was 2.5m.
10mM MgCl to g / ml_Two / 0.1mM Na_ThreeVO_Four / 10 mM β
-Glycerophosphate / 1 mM NaF / 20 mM Hepes (pH 7.4)
Diluted. Add 20 μl of this supernatant to a final concentration of 1 μM phorbol.
12-myristate-13-acetate, 10 μM CaCl _Two, 20μ
Add dimethyl sulfoxide solution of M cAMP and test substance.
Add [γ-³²Add P] -ATP (12.5 μM, 10 μCi)
The enzyme reaction started. After reaction at 25 ℃ for 15 minutes, 4 times concentration
Sample buffer for SDS-polyacrylamide gel electrophoresis
Stop the reaction by adding 10 μl of the solution and add 9% of the reaction mixture.
(W / v) SDS-polyacrylamide gel electrophoresis,
Detects phosphorylated proteins by autoradiography
Was. Corresponds to the protein that is the substrate of each protein kinase
Evaluate protein kinase activity based on the intensity of the band
did. In Table 1, complete inhibition of phosphorylation of substrate proteins
++ if there is a partial inhibition, + if partial inhibition, no inhibition
Indicates the result with-.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // C07M 7:00

Claims (3)

[Claims] 1. A compound of the general formula (In the formula, RCO represents an acyl residue of an n-3 highly unsaturated fatty acid.) A mitomycin C derivative having a highly unsaturated fatty acid residue. 2. A medicament containing the mitomycin C derivative having a polyunsaturated fatty acid residue according to claim 1 as an active ingredient. 3. A non-receptor tyrosine kinase inhibitor containing the mitomycin C derivative having a polyunsaturated fatty acid residue according to claim 1 as an active ingredient.