JPH07258278A - Dna polymerase alpha inhibitor containing 1-o-acylglycerol-2,3-phosphate derivative as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a DNA polymerase t inhibitor containing a specific acyl glycerolphosphate derivative as an active ingredient and useful for carcinostatic agents. CONSTITUTION:This DNA polymerase alpha inhibitor contains 1-O- acylglycerol-2,3-phosphate derivative expressed by formula I (R<1> is a 10-30C alkenyl or an alkynyl; M is H or a pair cation). Furthermore, a l-O- acylglycerol-2,3-phosphate derivative of formula II (R<2> is a 10-30C alkynyl), e.g. 1-O-[(Z)-9-hexadecenoyl]-2,3-O-isopyrideneglycerol is a new compound. The compound of formula II is obtained by reacting, e.g. 2,3-O-isopyrideneglycerol with (Z)-9-hexadecenoic acid in the presence of dicyclohexylcarbodiimide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA containing glycerophospholipid as an active ingredient, which is expected to be used as an antitumor agent.
It relates to an inhibitor of polymerase α and a novel glycerophospholipid.

[0002]

2. Description of the Related Art There is a strong demand from society for the development of excellent anticancer agents, and many anticancer agents have been developed and put into practical use. However, under the present circumstances, there is still a demand for the development of a more effective anticancer drug with less side effects, and an antitumor active compound having a novel skeleton and structure different from the known ones is currently put to practical use. There is a great possibility that an anticancer drug having superior characteristics to the existing anticancer drugs will be developed. DNA polymerase α is an enzyme that is essentially involved in cell growth, and there is a great possibility that an anticancer drug having excellent characteristics will be developed among inhibitors of DNA polymerase α. However, as a known inhibitor of DNA polymerase α, aphidicolin (W. Dalziel et al., J. Chem. Soc. Perki
n Trans. 1, 2841 (1973).) or PHYLPA (K. Mur.
akami-Murofushi et al., J. Biol. Chem., 267, 21512
(1992).) And so on. In addition, in EP-317,968-A,
1-O-oleoylglycerol-2,3-phosphate is described as a liquid electrostatic developer, but one having an alkynyl group is not disclosed, and DNA is also disclosed.
There is no description about the inhibitory activity of polymerase α.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a novel DN
It is intended to provide an inhibitor of A polymerase α.

[0004]

Means for Solving the Problems As a result of intensive studies on the inhibitor of DNA polymerase α, the present inventors have found that a specific 1-O-acylglycerol derivative strongly inhibits DNA polymerase α. The invention was completed.

That is, the present invention has the following general formula:

[0006]

[Chemical 3]

1-O-acyl represented by the formula: wherein R ¹ represents a linear or branched alkenyl group having 10 to 30 carbon atoms or an alkynyl group, and M represents a hydrogen atom or a counter cation group. The present invention relates to an inhibitor of DNA polymerase α containing a glycerol-2,3-phosphate derivative as an active ingredient.

Further, the present invention is a novel compound represented by the following general formula

[0009]

[Chemical 4]

1-O-acylglycerol-2 represented by the formula (wherein R ² represents a linear or branched alkynyl group having 10 to 30 carbon atoms, and M represents a hydrogen atom or a counter cation group). , 3-phosphate.

The substituent R ¹ in the above formula has 10 to 3 carbon atoms.
A straight or branched alkenyl group or alkynyl group having 0 to 12 carbon atoms, particularly 13 to 1 carbon atoms
5 is preferable in terms of inhibitory activity. These substituents R
Specific examples of ¹ include 8-decenyl group, 8-undecenyl group, 8
-Dodecenyl group, 8-tridecenyl group, 8-tetradecenyl group, 8-pentadecenyl group, 8-hexadecenyl group, 8-heptadecenyl group, 8-octadecenyl group, 8-icosenyl group,
8-dococenyl group, heptadeca-8,11-dienyl group, heptadeca-8,11,14-trienyl group, nonadeca-4,7,10,13-tetraenyl group, nonadeca-4,7,10,13,16- Pentaenyl group,
Henicosa-3,6,9,12,15,18-alkenyl group such as hexaenyl group, or 8-decynyl group, 8-undecynyl group,
8-dodecynyl group, 8-tridecynyl group, 8-tetradecynyl group, 8-pentadecynyl group, 8-hexadecynyl group, 8-heptadecynyl group, 8-octadecynyl group, 8-icosinyl group,
Examples thereof include alkynyl groups such as 8-docosynyl group and heptadeca-8,11-diynyl group.

The substituent R ² in the above formula has 1 carbon atom.
A linear or branched alkynyl group of 0 to 30,
Those having 12 to 20 carbon atoms, particularly those having 13 to 15 carbon atoms are preferable from the viewpoint of inhibitory activity. Specific examples of the substituent R ² include 8-decynyl group, 8-undecynyl group, 8-dodecynyl group, 8-tridecynyl group, 8-tetradecynyl group, 8-pentadecynyl group, 8-hexadecynyl group, 8-heptadecynyl group, Examples thereof include alkynyl groups such as 8-octadecynyl group, 8-icosinyl group, 8-docosynyl group and heptadeca-8,11-diynyl group.

Further, when M in the above formula is a counter cation group, examples thereof include sodium ion, potassium ion, lithium ion and ammonium ion.

The 1-O-acylglycerol-2,3-phosphate according to the present invention can be prepared by the method described in the literature (S. Kobay).
ashi et al., Tetrahedron Letters, 34, 4047 (199
It can be synthesized according to 3).).

1-O-acylglycerol of the present invention
The 2,3-phosphate can be in either oral or parenteral dosage form. In the case of oral administration, it can also be administered by a usual method such as capsules, tablets and powders. Further, in the case of parenteral administration, it is administered in a dosage form such as an injection or a liquid preparation. Further, a sustained release agent is also effective.

To formulate the active ingredient of the present invention, a surfactant, an excipient, a coloring agent, a preservative, a coating aid and the like are appropriately used. It can also be used in combination with other drugs.

[0017]

EXAMPLES The present invention will be described in more detail below with reference to production examples, examples and test examples, but it goes without saying that the present invention is not limited to these.

Production Example 1

[0019]

[Chemical 5]

(Z) -9-hexadecenoic acid (0.631 g, 2.48)
Dimethylaminopyridine (0.028 g, 0.23 mmol), 2,3-O-isopropylideneglycerol (0.298 g, 2.25 mmol) and dicyclohexylcarbodiimide (0.464 g, 2.25 mmol) at 0 ° C. added. After stirring at room temperature for 2 hours and at 40 ° C. for 1 hour, the crystals were separated by filtration and washed with dichloromethane. The dichloromethane layers were combined and evaporated under reduced pressure, and the obtained residue was purified by silica gel chromatography (ethyl acetate / hexane) to give 1-O-[(Z) -9-hexadecenoyl] -2,
3-O-isopropylidene glycerol (0.752 g, 2.04 mm
ol, yield 90%) was obtained.

¹ H-NMR (90 MHz, CDCl ₃ ): δ = 0.88 (3H,
t, J = 7.0 Hz, CH ₃ ), 1.10-1.50 (16H, m, C (4 ') H ₂ , C
(5 ') H ₂ , C (6') H ₂ , C (7 ') H ₂ , C (12') H ₂ , C (13 ') H ₂ , C (1
4 ') H ₂ , C (15') H ₂ ), 1.38 (3H, s, isopropylidene-CH
₃ ), 1.43 (3H, s, isopropylidene-CH ₃ ), 1.57-1.77
(2H, m, C (3 ') H ₂ ), 1.87-2.17 (4H, m, C (8') H ₂ , C (1
1 ') H ₂ ), 2.35 (2H, t, J = 7.5 Hz, C (2') H ₂ ), 3.74 (1
H, dd, J = 8.6 Hz, C (3) H), 4.09 (1H, dd, J = 8.6 Hz,
C (3) H), 4.10 (1H, dd, J = 11.6 Hz, C (1) H), 4.10-4.
50 (2H, m, C (1) H, C (2) H), 5.20-5.50 (2H, m, C (9 ')
H, C (10 ') H).

Production Example 2

[0023]

[Chemical 6]

A solution of 1-O-[(Z) -9-hexadecenoyl] -2,3-O-isopyrideneglycerol (0.752 g, 2.04 mmol) obtained in Preparation Example 1 in dry methanol (10
mL) pyridinium p-toluenesulfonate (0.1 g,
0.4 mmol) was added and the mixture was heated under reflux for 5 hours. After cooling to room temperature, water was added to the reaction solution and extracted with ether. The ether layer was washed with water and dried over anhydrous sodium sulfate. The residue obtained by concentration under reduced pressure was purified by silica gel chromatography (ethyl acetate / hexane) to give 1-O-[(Z) -9.
-Hexadecenoyl] glycerol (0.300 g, 0.91 mmo
l, yield 45%) was obtained.

¹ H-NMR (90 MHz, CDCl ₃ ): δ = 0.90 (3H,
t, J = 6.0 Hz, CH ₃ ), 1.08-1.78 (18H, m, C (3 ') H ₂ , C
(4 ') H ₂ , C (5') H ₂ , C (6 ') H ₂ , C (7') H ₂ , C (12 ') H ₂ , C (1
3 ') H ₂ ,, C (14') H ₂ ,, C (15 ') H ₂ ), 1.88-2.28 (4H, m, C
(8 ') H ₂ , C (11') H ₂ ), 2.38 (2H, t, J = 7.0 Hz, C (2 ')
H ₂ ), 3.59-4.29 (5H, m, C (1) H ₂ , C (2) H, C (3) H ₂ ), 5.2
0-5.60 (2H, m, C (9 ') H, C (10') H).

Production Example 3

[0027]

[Chemical 7]

Under an argon atmosphere, triazole (0.227
g, 3.29 mmol) in tetrahydrofuran (8 mL), and at 0 ° C. phosphorus oxychloride (0.10 mL, 1.10 mmol),
Triethylamine (0.71 mL, 5.11 mmol) was added, and the mixture was stirred for further 5 minutes to prepare phosphoryl tristriazolide. 1 obtained in Production Example 2 was added to the above reaction solution at 0 ° C.
-O-[(Z) -9-hexadecenoyl] glycerol (0.3
A solution of 00 g, 0.91 mmol) in tetrahydrofuran (5.5 m
L) was added and the mixture was stirred at room temperature for 20 minutes, then the reaction solution was poured into ice-cooled 2% hydrochloric acid (50 mL) and extracted with ether. The ether layer was dried over anhydrous sodium sulfate. Separately, sodium hydride (60% mineral oil, 0.073 g, 0.91 mmol) was washed with pentane to remove the mineral oil, and the suspension in ether (1.5 mL) was added to the above ether solution. It was extracted with distilled water (8 mL) and the aqueous layer was freeze-dried to give 1-O-[(Z)-
A sodium salt of 9-hexadecenoyl] glycerol-2,3-phosphate ((I), 0.293 g, 0.71 mmol, yield 78%) was obtained.

¹ H-NMR (400 MHz, CDCl ₃ / CD ₃ OD): δ = 0.89
(3H, t, J = 7.0 Hz, CH ₃ ), 1.24-1.40 (16H, m, C (4 ')
_{H 2, C (5 ')} H 2, C (6') H 2, C (7 ') H 2, C (12') H 2, C (13 ') H 2,
C (14 ') H ₂ , C (15') H ₂ ), 1.55-1.67 (2H, m, C (8 ') H ₂ ),
1.98-2.06 (2H, m, C (11 ') H ₂ ), 2.35 (2H, t, J = 7.6 H
z, C (2 ') H ₂ ), 3.97 (1H, ddd, J = 6.9, 9.1, 9.1 Hz, C
(3) H), 4.20 (1H, dd, J = 5.2, 11.7 Hz, C (1) H), 4.26
(1H, dd, J = 6.1, 11.7Hz, C (1) H), 4.27 (1H, ddd, J
= 6.2, 9.1, 12.4 Hz, C (3) H), 4.58 (1H, ddddd, J =
5.2, 6.0, 6.1, 6.2, 6.9 Hz, C (2) H), 5.30-5.40 (2H,
m, C (9 ') H, C (10') H).

Production Example 4

[0031]

[Chemical 8]

By the same method as in Production Example 1, 9-hexadecynoic acid (0.671 g, 2.66 mmol), 2,3-O-isopropylideneglycerol (0.319 g, 2.41 mmol), dicyclohexylcarbodiimide (0.498 g, 2.41 mmol). ) And dimethylaminopyridine (0.030 g, 0.24 mmol) are reacted to give 1-O- (9-hexadecinoyl) -2,3-O-isopyrideneglycerol (0.768 g, 2.10 mmol, 87% yield).
Got

¹ H-NMR (90 MHz, CDCl ₃ ): δ = 0.90 (3H,
t, J = 6.0 Hz, CH ₃ ), 1.03-1.95 (24H, m, C (3 ') H ₂ , C
(4 ') H ₂ , C (5') H ₂ , C (6 ') H ₂ , C (7') H ₂ , C (12 ') H ₂ , C (1
3 ') H ₂ , C (14') H ₂ , C (15 ') H ₂ , and isopropylidene
-CH ₃ x2), 2.00-2.27 (4H, m, C (8 ') H ₂ , C (11') H ₂ ),
2.37 (2H, t, J = 7.0 Hz, C (2 ') H ₂ ), 3.76 (1H, dd, J =
8.6Hz, C (3) H), 4.00-4.45 (4H, m, C (1) H ₂ , C (2) H, C
(3) H).

Production Example 5

[0035]

[Chemical 9]

1-O- (9-hexadecinoyl) -2,3-O-isopyrideneglycerol (0.120 g, 0.33 mmol) obtained in Preparation Example 4 was dried in methanol (5 mL).
Pyridinium p-toluenesulfonate (0.016 g,
0.065 mmol) was added and the mixture was heated under reflux for 3 hours. By the same method as in Production Example 2, 1-O- (9-hexadecinoyl) glycerol (0.061 g, 0.19 mmol, yield 58%) was obtained.

¹ H-NMR (90 MHz, CDCl ₃ ): δ = 0.90 (3H,
t, J = 6.5 Hz, CH ₃ ), 1.15-1.95 (18H, m, C (3 ') H ₂ , C
(4 ') H ₂ , C (5') H ₂ , C (6 ') H ₂ , C (7') H ₂ , C (12 ') H ₂ , C (1
3 ') H ₂ ,, C (14') H ₂ ,, C (15 ') H ₂ ), 2.20-2.27 (4H, m, C
(8 ') H ₂ , C (11') H ₂ ), 2.38 (2H, t, J = 7.0 Hz, C (2 ')
H ₂ ), 3.45-4.47 (5H, m, C (1) H ₂ , C (2) H, C (3) H ₂ ).

Example 1

[0039]

[Chemical 10]

By the same method as in Production Example 3, triazole (0.047 g, 0.68 mmol) and phosphorus oxychloride (0.02 m
L, 0.23 mmol), triethylamine (0.15 mL, 10.5 mm
ol) was used to prepare a solution of phosphoryl triazolide in tetrahydrofuran (2 mL). Production Example 5 in the above solution at 0 ° C.
A tetrahydrofuran solution (1 mL) of the 1-O- (9-hexadecinoyl) glycerol (0.061 g, 0.19 mmol) obtained in 1. was added, and the mixture was reacted at room temperature for 20 minutes. In the same manner as in Production Example 3, sodium salt of 1-O- (9-hexadecinoyl) glycerol-2,3-phosphate ((I
I), 0.040 g, 0.10 mmol, yield 52%).

¹ H-NMR (400 MHz, CDCl ₃ / CD ₃ OD): δ = 0.85
(3H, t, J = 7.1 Hz, CH ₃ ), 1.20-1.48 (16H, m, C (4 ')
_{H 2, C (5 ')} H 2, C (6') H 2, C (7 ') H 2, C (12') H 2, C (13 ') H 2,
C (14 ') H ₂ , C (15') H ₂ ), 1.53-1.62 (2H, m, C (3 ') H ₂ ),
2.09 (4H, t, J = 6.9 Hz, C (8 ') H ₂ , C (11') H ₂ ), 2.31
(2H, t, J = 7.7 Hz, C (2 ') H ₂ ), 3.92 (1H, ddd, J = 6.
9,9.1, 9.1 Hz, C (3) H), 4.15 (1H, dd, J = 5.3, 11.7
Hz, C (1) H), 4.18-4.27 (2H, m, C (1) H, C (3) H), 4.53
(1H, ddddd, J = 5.3, 6.0, 6.0, 6.0, 6.9 Hz, C (2) H).

Test Example 1

Bovine thymus-derived DNA polymerase α [Bioc
Biophys. Acta, 950, 263 (1988).] (5 ng), activated DNA (2 μg), deoxyribonucleotide triphosphate (20 μM, containing 0.4 μCi of ³ H-dTTP), 2-mercapto Ethanol (2 mM), bovine serum albumin (400 μ
g / mL), 10% glycerol, magnesium chloride (10 m
M) and tris hydrochloride (50 mM (pH 7.5)) were mixed to make the reaction solution 50 μL, and the 1-O-obtained in Production Example 3 was obtained.
[(Z) -9-hexadecenoyl] glycerol-2,3-
The sodium salt of phosphate (ene-PHYLPA) was added at each concentration, and the mixture was incubated at 37 ° C for 60 minutes for reaction. After the reaction, spot this on a circular filter paper (Whattman 3MM), fix with 10% trichloroacetic acid for 15 minutes, wash, and then with 5% trichloroacetic acid.
Washing was repeated 3 times for 15 minutes, and after further washing with 99% ethanol, the filter paper was dried. The radioactivity on the filter paper was measured using a liquid scintillation counter (Packard, Tri-carb3255) in a toluene scintillator,
The synthesized DNA was quantified. The activity of DNA polymerase α when the test compound was added is shown in Table 1 as a relative ratio when the test compound was not added.

[0044]

[Table 1] Table 1 ────────────────────── Amount of compound (I) added DNA polymerase α activity ─────────── ──────────── No additive 100 5 μg / mL 82 10 μg / mL 69 20 μg / mL 41 40 μg / mL 11 ──────────────── ───────

Test Example 2

It was obtained in Example 1 in the same manner as in Test Example 1 except that sodium salt of 1-O- (9-hexadecinoyl) glycerol-2,3-phosphate (yne-PHYLPA) was used as a test compound. The inhibitory activity of 1-O- (9-hexadecinoyl) glycerol-2,3-phosphate sodium salt (yne-PHYLPA) against polymerase α was determined. DNA when a test compound is added to Table 2
The activity of polymerase α was expressed as a relative ratio when no test compound was added.

[0047]

[Table 2] Table 2 ────────────────────── Amount of compound (II) added DNA polymerase α activity ─────────── ──────────── No additive 100 5 μg / mL 97 10 μg / mL 87 20 μg / mL 57 40 μg / mL 21 ─────────────── ───────

[0048]

INDUSTRIAL APPLICABILITY 1-O-acylglycerol of the present invention
An inhibitor of DNA polymerase α containing 2,3-phosphate as an active ingredient has excellent inhibitory activity and is expected to be useful as an anticancer agent.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kimiko Murofushi 2-1-1 Otsuka, Bunkyo-ku, Tokyo Ochanomizumeko University Faculty of Science

Claims (2)

[Claims] 1. The following general formula: (In the formula, R ¹ represents a linear or branched alkenyl group having 10 to 30 carbon atoms or an alkynyl group, and M represents a hydrogen atom or a counter cation group).
An inhibitor of DNA polymerase α, which comprises an acylglycerol-2,3-phosphate derivative as an active ingredient. 2. The following general formula: (In the formula, R ² represents a linear or branched alkynyl group having 10 to 30 carbon atoms, and M represents a hydrogen atom or a counter cation group.) 1-O-acylglycerol-2,3- Phosphate derivative.