JPH06128158A - Platelet aggregation-inhibiting agent - Google Patents

Abstract

PURPOSE:To provide a platelet aggregation-inhibiting agent containing a prostaglandin E1 analogue as an active ingredient, excellent in its medicinal effect, good in durability, and not inducing the diarrhea of adverse action. CONSTITUTION:The platelet aggregation-inhibiting agent contains a compound of the formula (R is H, 1-6C alkyl) or its salt, e.g. (2E,16RS)16,20- dimethyl-2,3,13,14,18,18,19,19-octadehydro-prostaglandin E1-methyl ester as an active ingredient. The agent can be prepared in the form of tablets, granules, capsules. solution, emulsion, injection, suppositories or pessaries. The agent is administered at a dose of 0.1-100mug once to three times a day. The agent is useful for treating and preventing thrombotic diseases, and further useful for treating peripheral arterial obstruction because of its having an angiectatic action.

Description

[Detailed description of the invention]

[0001]

BACKGROUND OF THE INVENTION _1. Field of the Invention The present invention relates to a platelet aggregation inhibitor containing a prostaglandin (hereinafter abbreviated as PG) E1 analogue as an active ingredient.

[0002]

2. Description of the Related Art Platelet aggregation inhibitors are used for the treatment and prevention of thrombotic diseases such as transient ischemic attack, prevention of recurrence of cerebral infarction, unstable angina pectoris, peripheral arterial occlusion and essential thrombocythemia. used for Among various platelet aggregation inhibitors, PGE ₁ and its analogues, which are structurally similar to the platelet aggregation inhibitor of the present invention, also have a vasodilating effect and are therefore used for the treatment of peripheral arterial occlusion.

[0003]

However, these have the drawback that they are rapidly metabolized in vivo and therefore their effects do not last. In addition, when administered orally, it induces diarrhea as a side effect, so administration at a high dose was not possible and sufficient effects could not be achieved. The present invention is a conventional P
It is an object of the present invention to provide a novel platelet aggregation inhibitor that has superior efficacy, long-lasting efficacy, and reduced side effects as compared to GE type ₁ platelet aggregation inhibitors.

[0004]

SUMMARY OF THE INVENTION The present invention provides the formula

[0005]

(In the formula, R is a hydrogen atom or a
~6 alkyl groups are shown. ) is a platelet aggregation inhibitor containing a compound represented by or a salt thereof as an active ingredient. In the present invention, an alkyl group having 1 to 6 carbon atoms means a linear or branched chain, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl group, t-butyl group, n-pentyl group, isopentyl group and the like. A salt of the compound of formula (I) is
When R is a hydrogen atom compound in the formula (I), it is a salt with a metal such as sodium, potassium or aluminum or a salt with an organic amine such as trialkylamine.

Compounds of formula (I) can be readily prepared, for example, by the methods described below.

[0008]

[0009]

(In the reaction formula, R ¹ and R ² are the same or different and represent a hydroxyl-protecting group, R ³ is R
is. Here, the hydroxyl-protecting group is one commonly used in the field of prostaglandins, such as t-butyldimethylsilyl, triethylsilyl, phenyldimethylsilyl, tetrahydropyranyl, tetrahydrofuranyl, methoxymethyl group, ethoxyethyl group,
benzyl group and the like. ) That is, first, the method of Sato et al. [Journal of
Organic Chemistry (J.Org.Che
m. ), Vol. 53, p. 5590 (1988)]
0.8 to 2.0 equivalents of the organoaluminum compound of formula (III) is added to the compound of formula (II) known by
Compounds of formula (IV) are stereospecifically obtained by reacting in an inert solvent (e.g. benzene, toluene, tetrahydrofuran, diethyl ether, methylene chloride, n-hexane, etc.) at 10-30°C, preferably 0-10°C. is obtained. Here, the organoaluminum compound of formula (III) can be prepared, for example, by the method of Sato et al. [Tetrahedron Lett.
7083 (1989)].

[0011]

(In the formula, R ² has the same meaning as defined above.) 0.8 of an alkyllithium (eg, n-butyllithium, t-butyllithium, etc.) is added to the acetylene compound represented by the above formula.
-1.5 equivalents at -20 to 30°C, preferably -10 to 0
C., more preferably at ₁₀ to 30.degree. diethylaluminum halide (e.g. diethylaluminum chloride, dimethylaluminum chloride, etc.)
Prepare by adding 1.5 equivalents. It is preferable to use an inert organic solvent (eg, benzene, toluene, tetrahydrofuran, diethyl ether, methylene chloride, n-hexane, etc.) in this reaction.

Next, the compound of formula (IV) is combined with 0.5 to 4 equivalents of the organocopper compound represented by formula (V) and 0.5 to 4 equivalents of chlorotrimethylsilane with an inert solvent (eg, tetrahydrofuran, diethyl). ether, methylene chloride, toluene, n-hexane, etc.) at -78 to 40°C to give the compound of formula (VI). Here, the organocopper compound of formula (V) is represented by formula I-( _CH2 ) ₃ -CH=CH- ^COOR3 (VIII) ⁽ wherein R3 has the same meaning as defined above). From the iodine compound, a known method [P. Knochel et al., Journal of Organic Chemistry, No. 53
2390 (1988)]. That is, the iodine compound of formula (VIII) is combined with 0.8 to 5 equivalents of zinc activated with, for example, 1,2-dibromomethane, chlorotrimethylsilane, iodine, etc., and an inert solvent such as tetrahydrofuran, diethyl ether,
represented by the formula ^IZn- ( _CH2 ) ₃ -CH=CH- ^COOR3 (wherein R3 has the same meaning as above) by reacting in n-hexane, n-pentane, dioxane, etc.). lead to organozinc compounds that At this time, it may be heated as necessary. Although the heating temperature depends on the boiling point of the solvent, it is usually 30
~150°C, preferably 40-80°C. The obtained organozinc compound is reacted at -50 to 10°C in the inert solvent containing copper cyanide (1 to 2.5 equivalents) and lithium chloride (2 to 5 equivalents) to obtain the formula (V)
of organic copper compounds can be obtained.

Then, the compound of formula (VI) is treated with an inorganic acid (eg, aqueous solution of hydrochloric acid) or an organic acid or its amine salt (eg, p-toluenesulfonic acid, p-toluenesulfonic acid pyridine salt, etc.) and an organic solvent. (e.g. acetone, methanol, ethanol, isopropanol,
diethyl ether or a mixed solvent thereof),
Hydrolysis at 0-40° C. stereoselectively gives compounds of formula (VII). Finally, the hydroxyl-protecting group of the compound of formula (VII) is deprotected using methods conventional in the field of prostaglandins to give the compound of formula (I) wherein R is a group other than a hydrogen atom [ compound of formula (Ia)].

The compound of the present invention [compound of formula (Ib)] wherein R is a hydrogen atom in formula (I) can be obtained by hydrolyzing the ester moiety of the compound of formula (Ia). Hydrolysis is carried out by reacting the compound of formula (Ia) in a buffer such as phosphate buffer or Tris-HCl buffer, optionally using an organic solvent (mixed with water such as acetone, methanol or ethanol). by reacting with the enzyme. Enzymes to be used include enzymes produced by microorganisms (e.g., enzymes produced by microorganisms belonging to the genus Candida and Pseudomonas), and enzymes prepared from animal organs (e.g., enzymes prepared from porcine liver and porcine pancreas).
A specific example of commercially available enzymes is lipase
VII (manufactured by Sigma, derived from microorganisms of the genus Candida), Lipase AY (manufactured by Amano Pharmaceutical Co., derived from microorganisms of the genus Candida), Lipase MF (manufactured by Amano Pharmaceutical Co., derived from microorganisms of the genus Pseudomonas), PLE-A (Amano Pharmaceutical Co., prepared from porcine liver), Esterase (Sigma, prepared from porcine liver), Lipase II (Sigma, prepared from porcine pancreas), Lipoprotein Lipase (Tokyo Kasei Kogyo Co., Ltd., prepared from porcine pancreas) and so on. The amount of the enzyme to be used may be appropriately selected according to the potency of the enzyme and the amount of the substrate [compound of formula (Ia)], but is usually 0.1 to 20 parts by weight of the substrate. The reaction temperature is 25 to 50°C, preferably 30 to
35°C.

The platelet aggregation inhibitors of the present invention can be administered orally or parenterally (eg, intravenously, intrarectally, intravaginally). As dosage forms for oral administration, for example, solid preparations such as tablets, granules and capsules, and liquid preparations such as solutions, fat emulsions and liposome suspensions can be used. When used as this oral preparation, α,
It can also be formulated by forming an inclusion compound with β- or γ-cyclodextrin, methylated cyclodextrin, or the like. As preparations for intravenous administration, aqueous or non-aqueous solutions, emulsifiers, suspensions, solid preparations dissolved in a solvent for injection immediately before use, and the like can be used. In addition, dosage forms such as suppositories can be used as preparations for rectal administration, and pessaries and the like can be used as preparations for intravaginal administration.
The dosage is 0.1-100 μg, which is administered 1-3 times daily.
Administer in divided doses.

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to provide a platelet aggregation inhibitor having excellent efficacy and sustained efficacy. Moreover, the platelet aggregation inhibitor of the present invention is a conventional PG
It hardly induces diarrhea, which is the most serious problem with systemic platelet aggregation inhibitors, at doses that exhibit definite pharmacological effects.

The effects of the present invention will be specifically described below by way of test examples. Test Example 1 [Rabbit Platelet Aggregation Inhibition Test] New Zealand white rabbits weighing 2.5 to 4.0 kg were used in the test as 4 animals per group. Under ether anesthesia, 9 volumes of blood per 1 volume of 3.2% sodium citrate solution were collected from the common carotid artery of this rabbit. The collected blood was centrifuged at 1100 rpm for 15 minutes, and the upper layer was platelet-rich plasma (PRP).

Measurement of platelet aggregation was carried out by Born's method (Na
194, 927, 1962)
was carried out according to 1 μl of various concentrations of the test drug dissolved in ethanol was added to 275 μl of PRP and incubated at 37° C. for 10 minutes.
After 3 minutes under stirring at 00 rpm, 25 μl of an aggregating agent [adenosine diphosphate (ADP) final concentration 5 μM] was added, and the maximum aggregation rate (within 5 minutes after inducing platelet aggregation) was measured using a platelet aggregometer (aggregometer). (maximum change in light transmittance) was measured.

Aggregation-inhibiting activity was calculated by calculating the aggregation-inhibiting rate against aggregation when ethanol was used instead of the test drug solution, and the IC ₅₀ value was determined from the dose-response curve.

The results are shown in Table 1. The compound numbers in the table are the compound numbers shown in Production Examples below. In addition, _IC50
Values are shown as mean values.

[0022]

[Table 1]

Test Example 2 [Human Platelet Aggregation Inhibition Test] Blood was collected from a human and immediately mixed with 1/10 volume of 3.8% sodium citrate aqueous solution. After centrifugation at 180×g for 15 minutes at room temperature, PRP was obtained from the upper layer.

Measurement of platelet aggregation was carried out by Born's method (Na
194, 927, 1962)
was carried out according to Using an aggregometer, PRP
100 μl and 5 μl of test drug solutions of various concentrations dissolved in ethanol were added, and stirred at 37° C. and 1000 rpm.
After incubating for 1 minute, platelet aggregation was induced by adding 5 µl of ADP, and the maximum aggregation rate was determined.

Aggregation inhibitory activity was calculated by calculating the inhibitory rate against aggregation when ethanol was used in place of the test drug solution, determining IC ₅₀ from the dose-response curve, and simultaneously measuring PGE ₁ . It was evaluated as relative activity to _IC50 .

As a result, when the IC ₅₀ value of PGE ₁ was set to 1, the relative activity of Compound 2 prepared in Preparation Example 2 was 10%.
was 3.

[0027]

EXAMPLES The present invention will now be described in more detail with reference to Production Examples and Examples. Production Example 1 (2E,16RS)-16,20-dimethyl-2,3,
13,14,18,18,19,19-octadehydro
-PGE1 methyl ester (compound 1) (1) (3S,4RS)-3-(t-butyldimethylsiloxy)-4-methylnona-1,6-diyne (1.65
g) was dissolved in 19.2 ml of benzene, n-butyllithium (2.5 M, hexane solution, 2.3 ml) was added at 0° C., and the mixture was stirred at the same temperature for 30 minutes. Diethylaluminum chloride (0.94 M, hexane solution, 7.2 ml) was added to this solution at 0° C., and the mixture was heated to room temperature and stirred for 30 minutes. To this solution at room temperature was added (4R)-2-(N,N-diethylamino)methyl-4-(t-butyldimethylsiloxy)cyclopent-2-en-1-one (0.25 M,
Benzene solution, 19.2 ml) was added and stirred for 15 minutes. The reaction mixture was hexane (46.5 ml)-saturated ammonium chloride aqueous solution (46.5 ml)-hydrochloric acid aqueous solution (3M,
13.5 ml) with stirring, the organic layer was separated and washed with saturated aqueous sodium bicarbonate solution (10 ml).
The residue obtained after drying and concentrating the obtained organic layer was purified by silica gel column chromatography (developing solvent: hexane:ethyl acetate = 50:1) (3R, 4R).
-2-methylene-3-[(3'S,4'RS)-3'-
(t-butyldimethylsiloxy)-4'-methylnona-
2.09 g of 1',6'-diynyl]-4-(t-butyldimethylsiloxy)cyclopentan-1-one are obtained. ¹ H-NMR (CDCl ₃ , 300 MHz) δppm:
0.09, 0.10 and 0.12 (3s, 12H),
0.88(s, 18H), 1.02 and 1.03(2
d, J=6.8Hz and 6.8Hz, 3H), 1.1
0 (t, J=7.3Hz, 3H), 1.73-1.91
(m, 1H), 2.00-2.39 (m, 4H), 2.
32 (dd, J=7.4Hz, 17.9Hz, 1H),
2.70 (dd, J = 6.4Hz, 17.9Hz, 1
H), 3.53 (d, J=6.5Hz, 1H), 4.2
1 to 4.30 (m, 1H), 4.37 and 4.47
(2d, J = 4.1Hz, 6.3Hz, 1H), 5.5
4 (d, J = 2.7Hz, 1H), 6.13 (d, J =
3.0Hz, 1H) IR (neat): 2960, 2934, 2862, 2
364, 1738, 1649, 1473, 1363, 1
255,1123,1079,837,777 cm ^-1

(2) (4E)-5-carbomethoxypent-4-enylzinc(II) iodide (0.8M tetrahydrofuran solution, 2.50ml) at -70°C
2.50 ml of a tetrahydrofuran solution of copper (I) cyanide/lithium dichloride (436 mg) was added to the solution and
Stirred for 5 minutes. To this solution was added the compound (489 mg) obtained in the above (1) and 0.5% of chlorotrimethylsilane at -70°C.
3.5 ml of 23 ml of diethyl ether solution was added, and the temperature was raised to room temperature over about 2 hours while stirring.

15 of saturated ammonium chloride aqueous solution was added to the reaction solution.
ml was added and extracted with hexane. The organic layer was washed with saturated brine, dried and concentrated, and the residue obtained was washed with ether.
Dissolve in 5.0 mL of isopropyl alcohol (1:4) and add p-toluenesulfonic acid pyridine salt (12.6 mL).
g, 0.050 mmol) was added, and the mixture was stirred at room temperature for 12 hours. After extraction with 20 ml of hexane and 10 ml of saturated aqueous sodium bicarbonate solution to the reaction mixture, the organic layer was dried and concentrated.
3:2) over (2E,16RS)-16,20-dimethyl-2,3,13,14,18,18,19,19-
Octadehydro _- PGE1 methyl ester 11,1
5-bis(t-butyldimethylsilyl ether) 423
mg was obtained. Subsequently, this compound (423 mg, 0.6
86 mmol) was dissolved in acetonitrile (23 ml) and 50% hydrofluoric acid aqueous solution (5.1 ml) was added at 0°C. After stirring for 90 minutes at 0° C., the reaction was treated with ethyl acetate (40 ml) and saturated aqueous sodium bicarbonate (150 ml).
ml). Extract with ethyl acetate, wash with saturated aqueous sodium bicarbonate solution and saturated brine, dry and concentrate. The residue obtained is purified by silica gel column chromatography (developing solvent: hexane:ethyl acetate = 1:1). 240 mg of the title compound were obtained.

¹ H-NMR (CDCl ₃ , 300 MHz)
δ ppm: 0.90 to 1.16 (m, 6H), 1.30
~1.99 (m, 7H), 2.04 ~ 2.37 (m, 8
H), 2.63 (t, J=10.0 Hz, 1H), 2.
75 (dd, J=7.2Hz, 18.5Hz, 1H),
3.72 (s, 3H), 4.27-4.38 (m, 1
H), 4.42 and 4.46 (2d, J=6.6Hz
and J = 4.4 Hz, 1H), 5.82 (d, J = 1
5.7Hz, 1H), 6.95 (dt, J = 7.4H
z, 15.7 Hz, 1 H) IR (neat): 3390, 2910, 2230, 1
730, 1690, 1650, 1430, 1270, 1
010 cm ^-1

Production Example 2 (2E,16RS)-16,20-dimethyl-2,3,
13,14,18,1,8,19,19-octaldehyde
Rho PGE 1 (Compound 2) Lipase VII 2.40 g was dissolved in 111 ml of phosphate buffer (10 mM, pH 7.0) and 12.4 ml of 5
0% (v/v) acetone-dissolved in water (2E, 16R
S)-16,20-dimethyl-2,3,13,14,1
240 mg of 8,18,19,19-octadehydro _- PGE1 methyl ester (compound obtained in Preparation 1)
(0.618 mmol) was added and stirred at 30° C. for 5 hours. The reaction solution was extracted three times with 50 ml of ethyl acetate, and the organic layer was washed with 30 ml of saturated brine, dried over anhydrous magnesium sulfate and concentrated. The obtained crude product was purified by silica gel column chromatography (developing solvent: ethyl acetate) to obtain 185 mg of the title compound. ¹ H-NMR (CDCl ₃ , 300 MHz) δppm:
0.90-1.32 (m, 6H), 1.35-2.01
(m, 7H), 2.04-2.50 (m, 8H), 2.
63 (t, J = 10.0 Hz, 1H), 2.75 (d
d, J=7.2Hz, 18.5Hz, 1H), 4.27
~4.37(m, 1H), 4.41 and 4.45(2
d, J = 6.5 Hz and J = 4.5 Hz, 1H),
5.82 (d, J=15.7Hz, 1H), 7.04
(dt, J = 15.7Hz, 7.3Hz, 1H)

Example 1 (Tablet) Compound 2 5 mg and α-cyclodextrin 500 mg
was dissolved in 80 ml of distilled water and lyophilized. This freeze-dried product, 80 g of crystalline cellulose, 48.5 g of lactose and 10 g of carboxymethylcellulose calcium are mixed, and 10 g of hydroxypropylcellulose is mixed with 1 part of purified water.
00 ml as a binder and granulated using a fluid bed granulator. After adding and mixing 1 g of magnesium stearate to this granulated product, tablets each weighing 150 mg were produced to obtain tablets containing 5 μg of compound 2 per tablet.

Example 2 (Capsule) Compound 2 5 mg and γ-cyclodextrin 500 mg
was dissolved in 80 ml of distilled water and lyophilized. This freeze-dried product, 50 g of crystalline cellulose, 77.5 g of potato starch, and 10 g of low-substituted hydroxypropyl cellulose were mixed, and 1 part of hydroxypropyl cellulose was mixed.
Granulation was carried out using a fluidized bed granulator using a liquid obtained by dissolving 0 g in 100 ml of purified water as a binder. Hardened oil 1 to this granule
After adding and mixing g and 1 g of magnesium stearate, 150 mg was filled into No. 3 capsules to obtain capsules containing 5 μg of compound 2 per capsule.

Example 3 (fine granules) 5 mg of compound 2 and 500 mg of β-cyclodextrin
was dissolved in 80 ml of distilled water and lyophilized. After mixing 659.5 g of this freeze-dried product, corn starch and 300 g of mannitol, 40 g of hydroxypropylmethylcellulose dissolved in 600 ml of purified water was used as a binder and granulated using a fluidized bed granulator. Fine granules containing 25 μg were obtained.

[0035]

──────────────────────────────────────────────────── ──── continuation of the front page (72) Inventor Kazuya Kameo Made in Taisho, 3-24-1 Takada, Toshima-ku, Tokyo Yaku Co., Ltd. (72) Inventor Toru Tanami Made in Taisho, 3-24-1 Takada, Toshima-ku, Tokyo Yaku Co., Ltd. (72) Inventor Ken Muto Made in Taisho, 3-24-1 Takada, Toshima-ku, Tokyo Yaku Co., Ltd. (72) Inventor Naoya Ono Made in Taisho, 3-24-1 Takada, Toshima-ku, Tokyo Yaku Co., Ltd. (72) Inventor Jun Goto Made in Taisho, 3-24-1 Takada, Toshima-ku, Tokyo Yaku Co., Ltd.

Claims (1)

[Claims] [Claim 1] Formula (In the formula, R represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.) A platelet aggregation inhibitor comprising a compound represented by or a salt thereof as an active ingredient.