JPH06234790A - New tetrapeptide derivative - Google Patents

Abstract

PURPOSE:To provide a new tetrapeptide derivative which can be used as an anti-cancer or anti-tumor drugs because of its cell growth-inhibitory action and antineoplastic action. CONSTITUTION:The compound of formula I (R1 to R3 is each isopropyl or R1 is H, R2 is isopropyl and R3 is sec-butyl). The compound is preferably obtained by the condensation reaction of the tripeptide fragment of formula II with the fragment of formula III in the presence of a condensation agent such as dicyclohexylcarbodiimide in an inert solvent such as THF at about 0 deg.C. The compound of formula III is used 1 to 1.0 mole per mole of the compound of formula II, while the condensation agent, in the equimolar amount. The compounds of formula II and III are new substances and can be prepared by liquid-phase condensation reaction of the constituent amino acids respectively.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel tetrapeptide amide derivative. The compound of the present invention has a cell growth inhibitory action and / or an anti-neoplastic action, and is useful as an anticancer or antitumor agent.

[0002]

BACKGROUND OF THE INVENTION Several peptides having cell growth inhibitory activity and / or anti-neoplastic activity have been isolated from the sea mollusc Aplysia vulgaris (Dolabella auricularia). Are called dolastatins 1-15. Among them, dolastatin 10 is a tetrapeptide amide having the following structural formula extracted from Indian sea mussel by Pettit et al. In 1987, and is known as a compound having the strongest cell growth inhibitory action among known compounds. (Petit, etc., Journal of the American Chemical
Society, Vol. 109, page 6883, 1987 and JP-A-2-167278).

[0003]

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[0004]

However, since all dolastatins are manufactured by extraction and isolation from marine products, there are problems in disturbance of the ecosystem due to overfishing of animals, product cost, and the like. Further, further improvement is desired in terms of biological activity.

[0005]

The present inventors have found that certain dolastatin 10 analogs in which an amino acid of dolastatin 10 is replaced with another amino acid have a higher cell growth inhibitory action and / or antineoplastic action than dolastatin 10. Found to have.

According to the present invention, however, the following general formula (I)

[0007]

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In the formula, R ₁ , R ₂ and R ₃ are the following (a) to
(A) R ₁ , R ₂ , and R ₃ each represent an isopropyl group; (b) R ₁ represents a hydrogen atom; R ₂ represents an isopropyl group; ₃
Represents an s-butyl group, (c) R ₁ represents an isopropyl group, R ₂ and R ₃ each represent an s-butyl group, (d) R ₁ represents a methyl group, and R ₂ represents an isopropyl group. Where R ₃ represents an s-butyl group, or a salt thereof.

The tetrapeptide amide derivative of the above formula (I) can be present as a salt, and examples of such salt include hydrochloride, hydrobromide, trifluoroacetate,
Examples thereof include p-toluene sulfonate and acetate.

According to the present invention, the tetrapeptide amide derivative of the above formula (I) can be obtained, for example, by a liquid phase synthesis method well known in the field of peptide chemistry (E Schläder and Kay Lübke "The Peptize", No. 1). Volume, Pages 76-136, 19
It can be produced by condensing each amino acid or peptide fragment according to (1965 Academic Press issue).

[0011]

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Wherein R ₁ , R ₂ and R ₃ have the meanings given above and a tripeptide fragment of the formula (II
I)

[0013]

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Synthesis by condensation with the fragment of formula (II) is most preferred because of the ease of synthesis of each fragment of formulas (II) and (III) and the fact that there is no fear of racemization during the condensation. It is suitable.

The reaction is generally carried out in an inert solvent such as chloroform, ethyl acetate, tetrahydrofuran (TH
F), dimethylformamide (DMF), acetonitrile or the like, in the presence of an organic base such as triethylamine, N-methylmorpholine or diisopropylethylamine (DIEA) as necessary, a condensing agent such as dicyclohexylcarbodiimide (DCC). Diphenylphosphoryl azide (DPPA), diethyl cyanophosphate (D
It can be carried out by treating with EPC), so-called BOP reagent or the like.

The reaction temperature is usually from -10 ° C to room temperature, preferably around 0 ° C, and the ratio of the compound of the formula (III), the organic base and the condensing agent to the compound of the formula (II) is as follows. It is advantageous to use at least 1 mol, preferably about 1.0 to 1.1 mol, of the compound of the formula (III) per mol of the compound of II), about 2 mol of the organic base and about 1 mol of the condensing agent. .

Thus, the desired tetrapeptide amide derivative of formula (I) is produced and isolated from the reaction mixture,
Purification can be performed by recrystallization, ion exchange chromatography, gel filtration, high performance liquid chromatography and the like.

The compounds of the above formulas (II) and (III) used as starting materials in the above reaction are novel compounds which have not been described in conventional literatures, and each of the constituent amino acids is It can be easily produced by condensation by a liquid phase synthesis method.

The tetrapeptide amide derivative of the formula (I) of the present invention has a higher cell growth inhibitory activity and / or antineoplastic activity than dolastatin 10, and has acute myeloid leukemia, acute lymphocytic leukemia and chronic melanoma. Tumor, adenocarcinoma of the lung, neuroblastoma, small cell lung cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer and the like.

When the compound according to the present invention is used as a drug, it may be in a solid form (eg, tablets, hard capsules, soft capsules, granules, powders, fine granules, pills, troches, etc.) depending on its use. ), A semi-solid form (for example, suppository, ointment, etc.) or a liquid form (injection, emulsion, suspension, lotion, spray, etc.). Thus, as non-toxic additives that can be used in the above-mentioned formulation, for example, starch, gelatin, glucose, lactose, fructose, maltose, magnesium carbonate,
Talc, magnesium stearate, methyl cellulose, carboxymethyl cellulose or its salt, acacia, polyethylene glycol, p-hydroxybenzoic acid alkyl ester, syrup, ethanol, propylene glycol, petrolatum, carbowax, glycerin, sodium chloride, sodium sulfite, phosphoric acid Examples include sodium and citric acid. The drug may also contain other therapeutically useful drugs.

The content of the compound of the present invention in the drug varies depending on the dosage form, but is generally 0.1 to 50% by weight in the solid and semi-solid forms, and in the liquid form. Is preferably contained in a concentration of 0.05 to 10% by weight.

The dose of the compound of the present invention can be widely varied depending on the type of warm-blooded animal including human being, the route of administration, the severity of symptoms, the diagnosis of a doctor, etc.
Generally, it can be about 0.01 to 50 mg / kg per day. However, as described above, it is, of course, possible to administer an amount smaller than the lower limit or larger than the upper limit of the above range depending on the severity of symptoms of the patient and the diagnosis of the doctor. The above dose can be administered once or several times a day.

The present invention will be further described below with reference to examples and examples.

For the structures of the compounds corresponding to the compound numbers used in Reference Examples and Examples, refer to Flow Sheets 1 and 2 below. Here, Z is a benzyloxycarbonyl group, Me is a methyl group, and But is te.
An rt-butyl group, Boc represents a tert-butoxycarbonyl group, Bzl represents a benzyl group, Ph represents a phenyl group, and R ₁ , R ₂ and R ₃ have the above-mentioned meanings.

[0025]

[Chemical 6]

[0026]

[Chemical 7]

[0027]

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Reference Example 1-A 12.6 g (50.2 mmol) of Z-valine is dissolved in 100 ml of tetrahydrofuran, 9.72 g (60 mmol) of carbonylimidazole is added thereto, and the mixture is stirred at room temperature for 4 to 5 hours.

On the other hand, 18.9 g (121 mmol) of malonic acid monomethyl ester potassium salt and 7.4 g (78 mmol) of anhydrous magnesium chloride were added to tetrahydrofuran 1
Suspend in 50 ml and stir for 6 hours while warming on a 55 ° water bath. Then, this reaction solution was ice-cooled, and the above reaction solution was poured into it all at once, and the cooling bath was immediately removed, followed by 2 hours at room temperature.
Stirring is continued for 4 to 48 hours.

A small amount of water is added to the reaction solution, a clear supernatant solution is decanted from the waxy precipitate, and this is concentrated under reduced pressure to obtain an oily substance. Ethyl acetate and ice-cooled 4N hydrochloric acid were added to the above wax-like residue and this oily substance respectively, and the mixture was shaken and dissolved, both were combined, and the layers were separated, and the aqueous layer was extracted again with ethyl acetate. The ethyl acetate layer was washed with ice-cold 2N hydrochloric acid and saturated aqueous sodium hydrogen carbonate, dried, and the solvent was evaporated to obtain 14.2 g of a pale yellow oil. Column chromatography on silica gel (eluent: ethyl acetate-n-hexane (1:
Purification in 1)) gives the desired compound 1-A as a colorless to slightly yellow oil. 14.38 g (87.5%).

[Α] _D ²⁶ -22.3 ° (c = 1.00, MeOH) ¹ H-NMR (CDCl ₃ , δ) 0.82 (3H, d, J = 6.8 Hz), 1.0
3 (3H, d, J = 6.8 Hz), 2.0 to 2.4 (1H, m), 3.54 (2
H, s), 3.72 (3H, s), 4.2 to 4.6 (1H, m), 5.11 (2
H, s), 5.1 to 5.5 (1H, m), 7.34 (5H, s) Perform Reference Example 1-B in exactly the same manner as Reference Example 1-A,
Compound 1-B was obtained as an oil.

[0032]

[Table 1]

Reference Example 2-A 9.26 g of compound 1-A obtained in Reference Example 1-A (30.
16 mmol) was dissolved in 170 ml of methanol, and -7
2.28 g sodium borohydride with stirring at 8 °
Charge (60.00 mmol) at once. After cooling and stirring for 6 hours, ice-cooled 1N hydrochloric acid was gradually added. When it was confirmed that the solution became acidic, it was concentrated under reduced pressure, and the precipitated oily matter was extracted with ethyl acetate. The ethyl acetate layer was washed with saturated aqueous sodium hydrogen carbonate and dried, and the solvent was evaporated to give 9.27 g of crystals.
Is obtained. Recrystallization from isopropyl ether gives the target compound 2-A as colorless needle crystals with a melting point of 81 °. 7.52 g (80.7%).

[Α] _D ²⁸ + 9.6 ° (c = 1.00, MeOH) Calculated value as C ₁₆ H ₂₃ NO ₅ C = 62.12% H = 7.49% N = 4.53% Actual value C = 62.16% H = 7.51% N = 4.70% ¹ H-NMR (CDCl ₃ , δ) 0.87 (3H, d, J = 6.5 Hz), 0.9
5 (3H, d, J = 6.5 Hz), 1.9 to 2.35 (1H, m), 2.4 to 2.6
(2H, m), 3.18 (1H, br, d), 3.69 (3H, s), 4.45
~ 4.80 (1H, m), 5.10 (2H, s), 7.34 (5H, s) Perform Reference Example 2-B in exactly the same manner as Reference Example 2-A,
Compound 2-B was obtained.

[0035]

[Table 2]

Reference Example 3-A 8.63 g of the compound 2-A obtained in Reference Example 2-A (27.
(93 mmol) is dissolved in 90 ml of dimethylformamide, 32.5 g (140.1 mmol) of silver oxide and 42 ml of methyl iodide are added, and the mixture is stirred for 5 hours in a 35 ° water bath. After filtration, the silver oxide is washed with dimethylformamide, and the washings are combined and concentrated under reduced pressure at 50 ° or less. The residue was thoroughly extracted with ethyl acetate, the ethyl acetate layer was washed with 5% sodium thiosulfate and saturated aqueous sodium hydrogen carbonate, dried and the solvent was distilled off to obtain 8.75 g of a yellow oil. Column chromatography on silica gel (eluent: benzene-ethyl acetate (5:
Purification in 1)) gives the desired compound 3-A as a pale yellow oil. 6.39 g (67.9%).

[Α] _D ²⁶ -20.7 ° (c = 1.00, MeOH) ¹ H-NMR (CDCl ₃ , δ) 0.8-1.15 (6H, m), 1.8-2.2
(1H, m), 2.4 to 2.6 (2H, m), 2.80 (3H, s), 3.31,
3.38 (3H, s), 3.65, 3.66 (3H, s), 5.13 (2H,
s), 7.33 (5H, s) Performed Reference Example 3-B in exactly the same manner as Reference Example 3-A,
Compound 3-B was obtained as an oil.

[0038]

[Table 3]

Reference Example 4-A 5.71 g of compound 3-A obtained in Reference Example 3-A (16.
94 mmol) was dissolved in 60 ml of dioxane, 18.5 ml (23.5 mmol) of 1N sodium hydroxide was added, and the mixture was stirred at room temperature for 2 to 3 hours. 20% citric acid is added to the reaction solution to adjust the pH to 4.0, and the mixture is concentrated under reduced pressure, and the precipitated oily matter is extracted with ethyl acetate. The ethyl acetate layer is washed with saturated saline, dried, and the solvent is distilled off to leave a colorless to slightly yellow oily substance.

This was dissolved in 50 ml of dichloromethane, 0.5 ml of concentrated sulfuric acid was added, and 25 m of isobutene was added in a pressure bottle.
1 and room temperature for 48 to 96 hours. The reaction solution is poured into saturated aqueous sodium hydrogen carbonate, nitrogen gas is blown into the solution to remove isobutene and most of the dichloromethane, and the precipitated oily substance is extracted with ethyl acetate. The ethyl acetate layer is washed with saturated aqueous sodium hydrogen carbonate and dried. The solvent was distilled off to leave a yellow oil (6.
17 g) is purified by silica gel chromatography (eluent: benzene-ethyl acetate (10: 1)) to obtain 4.83 g (75.2%) of the desired compound 4-A as a colorless to slightly yellow oily substance.

[Α] _D ²⁷ -17.8 ° (c = 1.00, MeOH) ¹ H-NMR (CDCl ₃ , δ) 0.8 to 1.1 (6H, m), 1.45 (9
H, s), 1.75 to 2.25 (1H, m), 2.25 to 2.5 (2H, m),
2.81 (3H, s), 3.31, 3.39 (3H, s), 3.7 to 4.05 (2H,
m), 5.13 (2H, s), 7.33 (5H, s) Perform Reference Example 4-B in exactly the same manner as Reference Example 4-A,
Compound 4-B was obtained as an oil.

[0042]

[Table 4]

Reference Example 5-A 1.14 g (3.0%) of compound 4-A obtained in Reference Example 4-A
1 mmol) of t-butanol / water (9: 1) 20 ml
0.1 g of 5% palladium carbon is added, and the mixture is stirred under a hydrogen stream for 2 hours. After the reaction, the catalyst is separated by filtration, washed, and the filtrate is concentrated under reduced pressure. The remaining oily substance is dissolved in 30 ml of benzene, concentrated again under reduced pressure, and this operation is repeated once more. The obtained oily substance was 0.83 g (3.31 g) of Z-valine.
(6 mmol) and dissolved in 10 ml of acetonitrile, and 0.66 g (3.20 mmol) of DCC is added under stirring with ice cooling. Soon crystals precipitate. 0 ° for at least 3 hours
After that, the mixture is allowed to melt in ice, and stirring is continued overnight. Then, the reaction solution is diluted with ethyl acetate, the crystals are separated by filtration and washed with ethyl acetate. The filtrate was concentrated under reduced pressure and the syrup-like residue was dissolved in ethyl acetate. If any insoluble matter was filtered off, the ethyl acetate solution was washed with ice-cold 2N hydrochloric acid and saturated aqueous sodium hydrogen carbonate, dried, and evaporated to remove the solvent. 1.55 g of product are obtained. The target compound 5-A was purified by silica gel column chromatography (eluent: benzene-ethyl acetate (5: 1)).
06 g (73.6%) are obtained as a colorless oil.

[Α] _D ²⁵ -32.9 ° (c = 1.00, MeOH) ¹ H-NMR (CDCl ₃ , δ) 0.75 to 1.1 (12 H, m), 1.46 (9
H, s), 2.25 to 2.45 (2H, m), 2.97 (3H, s), 3.35
(3H, s), 3.7 to 4.0 (1H, m), 4.3 to 4.7 (2H, m),
5.09 (2H, s), 5.48 (1H, br, d), 7.32 (5H, s) The following compounds were obtained in exactly the same manner as in Reference Example 5-A.

[0045]

[Table 5]

Reference Example 6-A 0.72 g (1.5%) of the compound 5-A obtained in Reference Example 5-A
1 mmol) of t-butanol / water (9: 1) 15 ml
, 100% of 5% palladium carbon is added, and the mixture is stirred under a hydrogen stream for 2 hours. After the reaction, the catalyst is separated by filtration, washed, and the filtrate is concentrated under reduced pressure. The oily residue is dissolved in 30 ml of benzene and concentrated again under reduced pressure, and this operation is repeated once more.
The obtained oily substance was dissolved in 6 ml of dimethylformamide, 0.26 g (1.79 mmol) of N, N-dimethylvaline and 0.30 g (1.84 mmol) of DEPC were added, and the mixture was stirred at room temperature until a uniform solution was obtained. After stirring, the mixture was cooled with ice, and 0.19 g (1.88 mmol) of triethylamine was added.
Was dissolved in 1 ml of dimethylformamide and added dropwise over 4 minutes. Thereafter, the mixture is allowed to melt for at least 4 hours at 0 ° and stirred overnight, and the transparent reaction solution is diluted with ethyl acetate. The ethyl acetate solution is thoroughly washed with saturated aqueous sodium hydrogen carbonate and dried. 0.75 g of pale brown oily substance remained after distilling off the solvent
Was purified by silica gel chromatography (eluent: ethyl acetate / hexane (1: 1)) to obtain 0.55 g (77.5%) of crystals 6-A having a melting point of 122 °.

[Α] _D ²⁷ -51.0 ° (c = 1.00, MeOH) ¹ H-NMR (CDCl ₃ , δ) 0.7 to 1.15 (18 H, m), 1.46 (9
H, s), 2.25 (6H, s), 3.02 (3H, s), 3.35 (3H,
s), 3.7 to 4.0 (1H, m), 4.3 to 4.6 (1H, m), 4.65 to
4.9 (1H, m), 6.86 (1H, br, d) The following compounds were obtained in exactly the same manner as in Reference Example 6-A.

[0048]

[Table 6]

Reference Example 7 Production of Compound 8 30.5 mg (0.106 mmol) of carboxylic acid obtained by treating known compound 7 with hydrogen in the presence of palladium carbon was dissolved in 1 ml of acetonitrile, and BOP reagent 5 was added.
1.6 mg (1.1 eq) and phenethylamine 14.
1 mg (1.1 eq) was added, and 20.6 mg (1.5 equivalents) of diisopropylethylamine was added dropwise under ice cooling. After stirring overnight at room temperature, the reaction solution is concentrated under reduced pressure. This was dissolved in dichloromethane, washed with 10% citric acid water, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The crude product is prepared using dichloromethane-methanol (10: 1) as a developing solvent.
Purified by TLC, 38.3 mg (9
2.5%) as an oil.

[0050] _{^{[α] 26 D -21.6 ° (}} c = 1.02, MeOH) MS 358,317 1 H-NMR (CDCl 3, δ) 1.19 (3H, d, J = 7.0 Hz), 1.4
8 (9H, s), 3.37 (3H, s), 7.1 to 7.4 (5H, m) Example 1 Compound 6-A (30.7 mg, 0.065 mmol) was dissolved in dichloromethane (0.3 ml), and trifluoroacetic acid (0.3 ml) was added under ice cooling. After stirring at room temperature for 1 hour, the solvent is distilled off under reduced pressure, and the residue is dried under reduced pressure. On the other hand, 25.4 mg (0.065 mmol) of compound 8 was added to 2N under ice cooling.
Dissolve in hydrogen chloride / ethyl acetate and stir at room temperature for 1 hour.
The solvent was distilled off under reduced pressure and the residue was dried to give dimethylformamide (0.1%).
Dissolve in 5 ml, add to the above tripeptidecarboxylic acid, and under ice cooling, add 14.5 mg (1.0 equivalent) of 95% DEPC and 40 μl (4 equivalent) of triethylamine.
After stirring under ice cooling for 1 hour, the mixture is stirred at room temperature overnight.

The solvent is distilled off under reduced pressure, the residue is dissolved in dichloromethane, washed with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. After distilling off the solvent, fractionation was carried out by preparative TLC using dichloromethane-methanol (10: 1) as a developing solvent, and the target fraction was further eluted with hexane: dichloromethane: methanol (2: 7.5: 2.5). Purified by Sephadex LH-20 chromatography. 36.6 mg (81.9%) of the target compound 9-A was obtained as an amorphous powder.

[0052] _{^{[α] 26 D -44.3 ° (}} c = 0.31, MeOH) MS 687,644 1 H-NMR (CDCl 2, δ) 2.39 (6H, s), 3.04 (3H,
s), 3.32 (3H, s), 3.35 (3H, s), 6.44 (1H, m),
6.9 to 7.1 (1H, m), 7.23 (5H, m) In the same manner as in Example 1, the following compounds were obtained.

[0053]

[Table 7]

Claims (1)

[Claims] 1. A compound of the general formula In the formula, R ₁ , R ₂ and R ₃ represent any one of the following (a) to (d): (a) R ₁ , R ₂ and R ₃ each represent an isopropyl group, b) R ₁ represents a hydrogen atom, R ₂ represents an isopropyl group, R ₃ represents an s-butyl group, (c) R ₁ represents an isopropyl group, and R ₂ and R ₃ each represent an s-butyl group. (D) R ₁ represents a methyl group, R ₂ represents an isopropyl group, and R ₃ represents an s-butyl group.