JP2002284772A - New compound and method for screening protein by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new taxol derivative and further to provide a method for screening a protein by using the derivative. SOLUTION: This new taxol derivative is represented by formula 1. The immobilized product is obtained by bonding the derivative with fine particles comprising a styrene-glycidyl methacrylate polymer through a spacer. The method for screening the protein bondable to the taxol derivative comprises bringing a cell-extract or the like into contact with the immobilized product. The taxol derivative can be used for the therapy of osteoporosis or as an anticancer agent, and the protein can be used for structural analysis for screening of a medicine or drug design as a receptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel compound or a salt thereof, an immobilized product obtained by immobilizing the compound, and a method for screening a protein using the immobilized product.

[0002]

2. Description of the Related Art In an aging society, osteoporosis and fractures are serious problems as diseases that significantly reduce quality of life. Therefore, in order to avoid the risk of osteoporosis and the risk of fracture, it is considered that the most effective countermeasure is to prevent a decrease in bone density with aging and maintain healthy bones. However, at present, there is no drug that can surely prevent bone density from decreasing. It is also well known that there is no effective therapeutic agent in the field of anticancer drugs.

[0003] Also, as one technique of drug development, a method has been proposed in which a biological receptor that catches a drug is identified, separated and purified, and structural analysis is performed using this receptor for drug screening and drug design. Have been.

[0004] Therefore, by utilizing the above-mentioned drug development technology with osteoporosis, bone fracture and cancer as target diseases, the interaction between a pharmacologically active substance and a biological receptor is analyzed, and a novel drug based on combinatorial chemistry is analyzed. There is a need for a method of creating a pharmacologically active substance and developing a more ideal drug for treating osteoporosis and bone fractures and also an anticancer drug at an early stage.

[0005]

SUMMARY OF THE INVENTION The present invention has been made from such a viewpoint, and an object of the present invention is to provide a novel compound having pharmacological activity. Another object of the present invention is to provide an immobilized product in which the novel compound is immobilized. Another object of the present invention is to provide a method for screening for a receptor protein for the compound using such an immobilized substance. The protein screened according to the present invention is used as a receptor for drug screening and structural analysis for drug design.

[0006]

SUMMARY OF THE INVENTION The present invention relates to a novel compound represented by the following formula (I) and a salt thereof.

Embedded image Wherein R ₁ is alkyl, alkenyl, alkynyl, aryl or heteroaryl, R ₂ is alkyl, aryl, heteroaryl, alkyloxy or alkylamino, R ₃ is hydrogen, alkylcarbonyl, alkyloxycarbonyl, Dialkylaminocarbonyl or alkyl.

X is a group represented by the following formula:

Embedded image In this formula, A is (CH ₂ ) n, CO (CH ₂ ) n, CONH (CH ₂ ) n, CH ₂ C
H ₂ OCH ₂ , COCH ₂ CH ₂ OCH ₂ or CONHCH ₂ CH ₂ OCH ₂ , n is 1
Represents an integer of from 6 to 6. R ₄ represents H or COR ₅ .

R ₅ is a group represented by the following formula:

Embedded image In the formula, R ₆ and R ₇ are the same or different lower alkyl groups, and n 2 is 0 or an integer of 1-4. )

[0009] The present invention also relates to an immobilized product obtained by bonding the above compound to fine particles of a styrene-glycidyl methacrylate polymer via a spacer. Furthermore, the present invention relates to a protein screening method comprising screening a protein capable of binding to the compound using the immobilized substance.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The novel compound according to the present invention is represented by the following formula (I).

Embedded image Wherein R ₁ is alkyl, alkenyl, alkynyl, aryl or heteroaryl, R ₂ is alkyl, aryl, heteroaryl, alkyloxy or alkylamino, R ₃ is hydrogen, alkylcarbonyl, alkyloxycarbonyl, In particular, R ₁ is isobutyl, isopropyl, tert-butyl, cyclopropyl, 3-pentyl,
2-methylpropenyl, 1-methylpropenyl, phenyl and the like, but R ₂ is pentyl, phenyl, 3-furyl, benzyloxy, tert-butyloxy, tert-amyloxy, isopropyloxy, adamantyloxy, 2,2-dimethylpropyl, Triamyloxy, tert-butylamino, etc.
Examples of ₃ include hydrogen, acetyl, benzoyl, butanoyl, methoxycarbonyl, N, N-dimethylaminocarbamoyl, methyl and the like.

X is a group represented by the following formula:

Embedded image In this formula, A is (CH ₂ ) n, CO (CH ₂ ) n, CONH (CH ₂ ) n, CH ₂ C
H ₂ OCH ₂ , COCH ₂ CH ₂ OCH ₂ or CONHCH ₂ CH ₂ OCH ₂ , where n is 1
Represents an integer of ~ 6. R ₄ represents H or COR ₅ .

R ₅ is a group represented by the following formula:

Embedded image In the formula, R ₆ and R ₇ are the same or different lower alkyl groups, and n 2 is 0 or an integer of 1-4. )

In the formula (I), examples of the alkyl group represented by R ₁ and R ₂ include straight or branched ones having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, t-pentyl, hexyl and the like.

Salts of compound (I) include metal salts such as alkali metal salts (eg, sodium or potassium salts) and alkaline earth metal salts (eg, calcium salts or magnesium salts), ammonium salts, and organic bases. With salts (eg, trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine or
Salts with N, N dibenzylethylenediamine), salts with organic acids (eg, salts with acetic acid, maleic acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid, formic acid, toluenesulfonic acid or trifluoroacetic acid), inorganic acids (Eg, salts with hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid),
Salts with amino acids (eg, salts with arginine, aspartic acid or glutamic acid) and the like. Compound
(I) may contain one or more stereoisomers due to asymmetric carbon atoms and double bonds, and such isomers and mixtures thereof are also included in the scope of the present invention.

Compound (I) and a salt thereof can be produced by the following method. Method 1 For example, in compound (I), compound (Ia) in which R ₅ is an alkyl group substituted with bromine and A is CONHCH ₂ CH ₂ OCH ₂
(Wherein R ₁ , R ₂ , R ₃ and R ₆ are the same as above)
It can be produced by a method represented by the following reaction formula.

[0016]

Embedded image

As the starting compound (II), a compound commercially available as taxol can be used, or Ojima et al.
l., Bioorg. Med. Chem. Lett., 4, 2631-2934, 1994,
Ojimaet al., J. Med.Chem., 39, 3889-3896, 1996, O
jima et al., Tetrahedron Lett., 34, 4149-4152, 199
3, Georg et al., Bioorg.Med.Chem. Lett., 4, 1825
-1830, 1994, Georg et al., Bioorg. Med. Chem. Let.
t., 4, 335-338, 1994; Kant et al., Tetrahedron Let
t., 35, 5543-5546, 1994 and Ojima et al., Tetrahe
dron, 48, 6985-7012, 1992 and the like. In this reaction formula, CbzCl
Represents carbobenzoxycyclolide and CDI represents carbonyldiimidazole.

The compound (I) of the present invention and a salt thereof have an effect of increasing bone density and are useful not only as a preventive for fractures and as a therapeutic agent for osteoporosis, but also as an anticancer agent.

The compound (I) binds to fine particles (hereinafter, referred to as “SG particles”) made of a styrene-glycidyl methacrylate polymer via a spacer to form the immobilized product of the present invention.

The SG particles are not particularly limited in polymerization state, morphology and the like, but are preferably those which can be used as a stationary phase in ordinary liquid chromatography. For example, a polymer obtained by polymerizing styrene and glycidyl methacrylate and having a glycidyl group on the surface of the polymer is used. The size of the SG particles is, for example, 0.05 to 0.5 μm
The degree is preferred.

The spacer has one end bonded to the SG particle and the other end bonded to the compound (I). If the spacer connects the SG particle to the compound (I) via an appropriate distance, , Its structure, form and the like are not particularly limited. For example, the spacer may be an SG particle and / or a compound.
Before bonding to (I), it is preferable that both ends thereof have the same or different functional groups such as an amino group, a thiol group, a carboxy group, and an epoxy group. Examples of the structure of the spacer that reacts / bonds with the glycidyl group of the SG particles include the following compounds, for example. They bind to compound (I) under very mild conditions.

[0022]

Embedded image

The method for screening a protein of the present invention comprises:
It is carried out by screening a protein capable of binding to the compound (I) using the above-mentioned immobilized substance. Specifically, for example, by mixing the above-mentioned fixing compound in a cell extract containing a protein, stirring if necessary, separating the immobilized product, eluting the protein from the immobilized product, and dissociating it. Done. Thus, a specific protein (eg, receptor) containing a site acting on compound (I)
Can be screened. The specific bond between the immobilized substance and the protein may be any of a chemical bond such as a hydrogen bond, and a chemical or physical adsorption.

The obtained protein can be optionally purified by a known method such as chromatography, the gene encoding the protein can be cloned, and the amino acid sequence and the nucleotide sequence of the gene can be determined. By producing a large amount of a peptide having a partial sequence thereof or a recombinant microorganism, it can be used for drug screening, structural analysis for drug design, and the like. That is, by searching for a compound having an affinity for the protein as a receptor obtained as described above, the compound has an effect of increasing bone density, and is useful not only as an agent for preventing fractures or treating osteoporosis but also as an anticancer agent. Compounds exhibiting efficacy can be found.

[0025]

EXAMPLES Examples of the compound of the present invention and the immobilized product will be specifically described below.

Example 1 (1) 7- (2- (2-azidoethoxy) -ethyl) -carbamoyltaxol (azidotaxol) Imidazole taxol was obtained from Bio. Med. Chem. Lett 199.
It was synthesized with reference to 8, 8, 113-116. Imidazole taxol (400 mg, 0.422 mmol) and 2-azidoethoxy-aminoethane (200 mg) in 2-propanol (5 ml)
The solution was refluxed for 5 hours. The solvent was filtered under reduced pressure, and purified by column chromatography (developing solvent: 100% ethyl acetate) to obtain the title compound (381 mg).

1H NMR 1.78-2.38 (m, 15H), 2.60-2.68
(m, 1H), 3.38 (t, 4H, J = 4.9Hz), 3.55-3.66 (m, 4H),
3.92 (d, 1H, J = 8.5Hz), 4.17 (d, 1H, J = 8.5Hz), 4.2
9 (d, 1H, J = 8.3Hz), 4.79 (d, 1H, J = 2.3Hz), 4.93
(d, 1H, J = 7.5Hz), 5.01 (s, 1H), 5.44 (dd, J = 6.9,
9.8Hz), 5.66 (d, 1H, J = 7.0Hz), 5.80 (dd, 1H, J = 2.
6,8.9Hz), 6.16 (t, 1H, J = 8.3Hz), 6.38 (s, 1H), 7.16
-7.58 (m, 12H), 7.76 (d, 2H, J = 7.3Hz), 8.10 (d, 2H,
J = 7.3Hz).

(2) 7- (2- (2-aminoethoxy) -ethyl)
-Carbamoyltaxol (aminotaxol) Azidotaxol (107 mg) was dissolved in a mixed solution of methanol (5 ml) and ethyl acetate (5 ml), and 5% palladium carbon (20 mg) was added. After stirring at room temperature under 1 atm of hydrogen for 3 hours, palladium was removed by celite filtration. The solvent was removed under reduced pressure to obtain the title compound (97mg).

1H NMR 1.80-2.70 (m, 16H), 3.60 (t, 2
H, J = 4.6 Hz), 3.35 (m, 2H), 3.56-3.66 (m, 4H),
3.94 (d, 1H, J = 7.0 Hz), 4.24 (d, 1H, J = 8.2 H
z), 4.32 (d, 1H, J = 8.2 Hz), 4.79 (d, 1H, J = 2.9
Hz), 4.99 (d, 1H, J = 8.6 Hz), 5.45-5.51 (m, 1H),
5.71 (d, 1H, J = 7.0 Hz), 5.75 (d, 1H, J = 3.0 H
z), 6.18 (t, 1H, J = 8.6 Hz), 6.42 (s, 1H), 7.31-7.
56 (m, 1H), 7.64 (t, 1H, J = 7.5 Hz), 7.81 (d, 2H,
J = 7.3 Hz), 8.13 (d, 2H, J = 7.2 Hz). ESI-MS; 98
4.5 (M + 1)

(3) 7- (2- (2-bromoacetamido) -ethoxy) -ethyl) -carbamoyltaxol (bromoacetyltaxol) Azidotaxol (97.3 mg) is mixed with methanol (3 ml) and ethyl acetate (3 ml). The solution was dissolved, and 5% palladium carbon (20 mg) was added. After stirring at room temperature under 1 atm of hydrogen for 3 hours, palladium was removed by celite filtration. The solvent was removed under reduced pressure to obtain an amino derivative (97 mg). The obtained compound was diluted with a methanol solution (5 ml), triethylamine (0.2 ml) and an excess amount of bromoacetylsuccinimide (100 ml) were added at room temperature, and the mixture was stirred for 1 hour. The resulting mixture was diluted with ethyl acetate and added to a saturated aqueous ammonium chloride solution. The aqueous solution was extracted with ethyl acetate, and the obtained organic layer was washed with saturated sodium chloride and dried over magnesium sulfate. After removing the solvent under reduced pressure, the residue was purified by column chromatography (developing solvent: 5% methanol: chloroform) to obtain the title compound (86 mg).

1H NMR 1.69-2.69 (m, 16H), 3.16-3.70
(m, 8H), 3.88-4.00 (m, 2H), 4.18 (d, 1H, 8.6 Hz),
4.31 (d, 1H, J = 8.6 Hz), 4.80 (s, 1H), 4.93-5.00
(m, 2H), 5.46 (dd, 1H, J = 3.3, 7.3 Hz), 5.66 (d,
1H, J = 6.6 Hz), 5.81 (dd, 1H, J = 2.4, 9.0 Hz), 6.
17-7.64 (m, 10H), 7.77 (d, 2H, J = 7.2 Hz), 8.10
(d, 2H, J = 7.2 Hz).

[0031]

Example 2 Synthesis of Latex Particles Containing Thiol SGEGDE particles were synthesized according to the document of JP-A-10-195099. The SGEGDE (styrene-glycidyl methacrylate polymer obtained by adding ammonia and reacting with ethylene glycol diglycidyl ether) particles (5.07% (w
/ v)) aqueous solution (400ml), 2M cysteamine hydrochloride aqueous solution
0.5ml, 4M aqueous sodium hydroxide solution 0.5ml, and 1ml
Was added, and the mixture was heated to 70 ° C. and reacted for 24 hours. After the reaction,
After the particles were precipitated by centrifugation, the supernatant was removed. Again, it was dispersed in 0.5 ml of water and 0.1 ml of mercaptoethanol and reacted at room temperature for 3 hours. The particles obtained were again precipitated by means of a centrifuge, the supernatant was removed and again dispersed in water. After repeating this washing operation three times, the solvent was replaced with dioxane. The obtained thiol latex was precipitated by a centrifugal separator, dispersed in 0.5 ml of a 50 mM bromoacetyltaxol in dioxane solution, added with 0.1 ml of diisopropylethylamine, and reacted at room temperature for 72 hours. The obtained taxol-immobilized particles were washed with dioxane to obtain target taxol-immobilized particles.

Determination of Immobilized Taxol The obtained particles (1 mg) were dispersed in a mixed solution of methanol (1 ml) and triethylamine (0.1 ml) and reacted for 24 hours. After the reaction, the amount of the immobilized taxol was determined by quantifying the cut methyl ester portion of the taxol side chain by HPLC under the following conditions. As a result, the fixed amount of taxol was 3 to 10 nmol / mg fine particles.

Analysis conditions Column Symmetry column C18 5m 4.6 mm I.DX 50 manufactured by Waters
mm Mobile phase 55% 10 mM Ammonium acetate 45% MeOH Flow rate 1 ml / min Detection UV (254 nm) Retention time 4.6 min

[0034]

According to the present invention, a novel compound is provided. This novel compound is used as an active ingredient of an anticancer drug because it prevents a decrease in bone density, is used as a therapeutic agent for osteoporosis and fractures, and has an anticancer effect. Further, since it has a large number of functional groups, it can be bound to fine particles made of an ethylene-glycidyl methacrylate polymer via a spacer to form an immobilized product, and a protein that can bind to this compound can be screened using this compound. This protein is used as a receptor for drug efficacy screening and structural analysis for drug design.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroyuki Koji One of 632 Mifuku, Oni-cho, Tata-gun, Shizuoka Asahi Kasei Co., Ltd. (72) Inventor Yasuyuki Takahashi 1 Asahi Kasei Corporation F-term (reference) 2G045 AA40 DA36 FB06 4C048 TT08 UU01 XX01 XX04

Claims (4)

[Claims] 1. A compound represented by the following formula (I) or a salt thereof. Embedded image Wherein R ₁ is alkyl, alkenyl, alkynyl, aryl or heteroaryl, R ₂ is alkyl, aryl, heteroaryl, alkyloxy or alkylamino, R ₃ is hydrogen, alkylcarbonyl, alkyloxycarbonyl, X is a dialkylaminocarbonyl or alkyl, and X is a group represented by the following formula: In this formula, A is (CH ₂ ) n, CO (CH ₂ ) n, CONH (CH ₂ ) n, CH ₂ C
H ₂ OCH ₂ , COCH ₂ CH ₂ OCH ₂ or CONHCH ₂ CH ₂ OCH ₂ , n is 1
Represents an integer of from 6 to 6. R ₄ represents H or COR ₅ . R ₅ is a group represented by the following formula. Embedded image In the formula, R ₆ and R ₇ are the same or different lower alkyl groups, and n 2 is 0 or an integer of 1-4. ) 2. R ₁ is selected from isobutyl, isopropyl, tert-butyl, cyclopropyl, 3-pentyl, 2-methylpropenyl, 1-methylpropenyl or phenyl,
R ₂ is pentyl, phenyl, 3-furyl, benzyloxy,
Tertiary butyloxy, tertiary amyloxy, isopropyloxy, adamantyloxy, 2,2-dimethylpropyl or tertiary butylamino, wherein R ₃ is hydrogen, acetyl, benzoyl, butanoyl, methoxycarbonyl, N,
The compound according to claim 1, which is selected from N-dimethylaminocarbamoyl or methyl, or a salt thereof. 3. An immobilized product obtained by bonding the compound according to claim 1 to fine particles of a styrene-glycidyl methacrylate polymer via a spacer. 4. A method for screening a protein, comprising screening a protein capable of binding to the compound according to claim 1 using the immobilized product according to claim 3.