JP2002265495A - Basic new dendrimer or pharmaceutically acceptable salt thereof, and neovascularization inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new dendrimer or a pharmaceutically acceptable salt thereof having neovascularization inhibitory activity. SOLUTION: This basic new dendrimer, which has neovascularization inhibitory activity, is shown by the general formula( wherein, R is a basic amino acid residue or a derivative thereof).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel dendrimer having a basic side chain or a pharmaceutically acceptable salt thereof. The compound of the present invention has an excellent angiogenesis inhibitory action.

[0002]

2. Description of the Related Art Angiogenesis has an important relationship with malignant tumors, inflammatory diseases, diabetic retinopathy and the like. Angiogenesis is an endogenous pro-angiogenic factor (VEGF or FGF)
And a suppressor (angiostatin or endostatin). Under pathological conditions, the promoter acts to induce angiogenesis. The process of angiogenesis is initiated by the secretion of angiogenesis-promoting factors, 1) digestion of basement membrane by vascular endothelial cells, 2) migration and proliferation of vascular endothelial cells, 3) vascular formation and maturation of vascular endothelial cells It is said to consist of three stages. FG which is an angiogenesis promoting factor
F induces angiogenesis by binding to a receptor on vascular endothelial cells, and it is known that binding to heparan sulfate is very important in the process (Cell, 6).
4 , 841-848, 1991). FGF is also known to have a binding domain rich in basic amino acids for binding to heparan sulfate (Science
e, 271 , 1116-1120, 1996). On the other hand, endogenous angiogenesis inhibitors that have recently attracted attention include angiostatin and endostatin. In recent years, the X-ray structure of endostatin has been elucidated, and its structure containing many basic amino acids has been elucidated. Has been suggested to inhibit angiogenesis through binding to heparan sulfate (The EMBO Journal, 17 , 1656-
1664, 1998, same magazine, 18 , 6240-624.
8, 1999). Although the detailed mechanism of action of these endogenous angiogenesis inhibitors has not been elucidated yet, they inhibit the angiogenesis and proliferation of cancer cells. Expectations are high. The molecular structure of a dendrimer is basically a branched structure, and can be synthesized by repeatedly reacting a building block serving as a branch from a core molecule. It has been reported that the structure of dendrimers becomes denser each time a building block is repeated (as the number of branch points increases), and that the structure tends to have a three-dimensional spherical structure (Angew. Chem. In).
t. Ed. Engl. , 29 , 138-175, 199
0). To date, applied researches on dendrimers include the use of dendrimers as polymeric micelle-type drug carriers (Bioconjugate Chem.,
11 , 910-917, 2000), a study in which the binding of a specific sugar to a dendrimer mimics the interaction between a sugar chain and a protein on the cell surface and prevents influenza virus infection (J. Am. Chem. So
c. , 119 , 2088-2095, 1997: International Publication WO 00/14171), and the use of lysine as a branching portion, and binding of an antigenic peptide from the tip of the branch to enhance antigenicity, thereby enhancing the antibody. Get Multip
le Antigen Peptide (MAP) S
ystem is known (Proc. Natl. Ac
ad. Sci. USA, 85 , 5409-5413, 1
988: See Japanese Patent Application Laid-Open No. 3-503535 / 1990).

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel basic dendrimer having a novel angiogenesis inhibitory activity which is useful as a prophylactic / therapeutic agent for tumors, inflammatory diseases, diabetic retinopathy and the like. It is to do.

[0004]

Means for Solving the Problems In order to achieve the above object, the present inventors have focused on the importance of basic amino acids in angiogenesis inhibition, and have developed a dendrimer type having a basic side chain as a novel angiogenesis inhibitor. We have developed angiogenesis inhibitors and completed the present invention. That is, the present invention has the following general formula:

Embedded image (Wherein, R represents a basic amino acid residue or a derivative thereof). The present invention relates to a novel peptide dendrimer having basicity represented by the formula: or a pharmaceutically acceptable salt thereof.
In the present invention, examples of the basic amino acid residue represented by R or a derivative thereof include arginine (Arg-),
Lysine (Lys-), Histidine (His-), Arg
-Arg-, Arg-Lys-, Arg-His-, H
is-Lys-, Lys-His- and the like, preferably arginine or a derivative thereof, more preferably Arg-, Arg-Arg-, Arg-Lys-, A
rg-His-. The amino group of these amino acid residues may be protected with a known and commonly used protecting group, such as an acetyl group (A
c-), succinyl group (Suc-), benzoyl group (B
z-), t-butoxycarbonyl group (Boc-), carbobenzoxy group (Cbz-), tosyl group (Ts-), dansyl group (Dns-), isovaleryl group (Iva-),
Examples include the methoxysuccinyl group (MeOSuc-), the 2-carboxybenzoyl group (2-CB-) and other terminal amino protecting groups functionally equivalent thereto. More specifically, it is a novel basic dendrimer represented by the following chemical formula (1) or a pharmaceutically acceptable salt thereof.

Embedded image Further, the present invention is a novel basic dendrimer represented by the following chemical formula (2) or a pharmaceutically acceptable salt thereof.

Embedded image The pharmaceutically acceptable salt of the compound of the present invention is not particularly limited, and examples thereof include an acid addition salt obtained by allowing a pharmaceutically acceptable acid to act. Examples of the acid include inorganic salts such as hydrochloride and sulfate, formate, trifluoroacetate, acetate, tartrate, maleate, fumarate, succinate, and metasulfonate. And the like. Further, the compound of the present invention or a pharmaceutically acceptable salt thereof may be in the form of a solvate represented by a hydrate. The amino acid constituting the compound of the present invention may be either an L-form or a D-form, and is preferably an L-form. Furthermore, the present invention is an angiogenesis inhibitor comprising at least one of these dendrimers as an active ingredient.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following general formula:

Embedded image (In the formula, R represents a basic amino acid residue or a derivative thereof.) A novel dendrimer having basicity represented by the general formula (I) can be synthesized by using a known solid phase peptide synthesis method. . For example, US Pat.
The compound can be synthesized according to the method disclosed in Japanese Patent No. 872 or No. 4410688. The obtained compound can be easily isolated and purified from the reaction mixture by a usual separation and purification means, for example, column chromatography, recrystallization and the like.

[0006] The active ingredient of the present invention is usually used in the form of a general pharmaceutical preparation. The formulation is a commonly used filler,
It is prepared using a diluent or excipient such as a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, a lubricant and the like. Various forms can be selected as the pharmaceutical preparation depending on the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections ( Liquid,
Suspensions and the like), ointments and the like. When formed into tablets, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, excipients such as silicic acid, etc.
Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, cerac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dry starch,
Disintegrators such as sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, sucrose, stearin, cocoa butter, hydrogenated oil Disintegration inhibitors such as quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, starch, lactose, kaolin, bentonite,
Adsorbents such as colloidal silicic acid, lubricating agents such as purified talc, stearates, powdered boric acid, and polyethylene glycol can be used. Further, the tablet may be a tablet coated with a usual coating, if necessary, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet or a multilayer tablet. When formed into pill form, as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, laminaran And disintegrating agents such as agar. For molding into a suppository form, for example, polyethylene glycol, cocoa butter, higher alcohol, esters of higher alcohol, gelatin, semi-synthetic glyceride and the like can be used as carriers. Capsules are prepared by mixing the compound of the present invention with the various carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules and the like according to a conventional method. When prepared as an injection, the liquid preparations, emulsions and suspensions are preferably sterilized and isotonic with blood, and when formed into these forms, diluents such as water, aqueous lactic acid, ethyl alcohol , Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. In this case, a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizer, buffer, soothing agent and the like may be used. It may be added. Further, a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent and the like and other pharmaceuticals may be contained in the pharmaceutical preparation as required. When preparing into paste, cream and gel forms, for example, white vaseline, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicone, bentonite and the like can be used as diluents.

The amount of the compound represented by the above general formula of the present invention or a salt thereof (active ingredient compound) to be contained in the above-mentioned pharmaceutical preparation is not particularly limited and may be appropriately selected over a wide range. The content is preferably 1 to 70% by weight. The administration method of the pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, the age, sex, and other conditions of the patient, the degree of the disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are administered orally. The injection is administered intravenously, alone or as a mixture with a normal replenisher of glucose and amino acid sugars, and if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. Suppositories are administered rectally. The dose of the above pharmaceutical preparation is appropriately selected depending on the usage, age of the patient, gender and other conditions, degree of disease, etc., and the amount of the compound of the present invention as an active ingredient is usually about 1 to 1000 mg / kg of body weight per day. And preferably about 10 to 100 mg, and the preparation can be administered in 1 to 4 divided doses per day.

[0008]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.
In each of the following examples, amino acids, protecting groups, active groups, and the like were used for IUPAC-IUC Commissi.
on on Biological Nomencla
It is indicated by a common abbreviation based on "ture". Examples are as follows. Arg: arginine, Gly: glycine, Fmoc: fluorenylmethoxycarbonyl, Mtr: 4-methoxy-2,3,6-trimethylbenzenesulfonyl, HOB
t: N-hydroxybenzotriazole, DIC: diisopropylcarbodiimide, DMAP: dimethylaminopyridine

[0009]

[Reference Example 1] Synthesis of Fmoc-Gly-Resin Wangresin (amino acid condensation rate 0.77 mmol / re
sin 1.0 g) 2.03 g (1.56 mmol,
1.0 eq) after swelling with dimethylformamide for 2 hours,
1.74 g of Fmoc-Gly-OH (6.24 mmo
1,4.0 eq), DMAP 381.1 mg (3.1
2 mmol, 2.0 eq) and DIC 0.97 mL
(6.24 mmol, 4.0 eq) was added to 30 mL of dimethylformamide, and the mixture was stirred at room temperature for 2 hours. After the resulting reaction solution was removed by suction, the resin was washed eight times with dimethylformamide (20-30 mL) and methanol (20-30 mL).
mL), and a ninhydrin test was performed to determine the condensation rate of the resin (the obtained solution exhibited a pale yellow color). Thereafter, the resultant was washed twice with diethyl ether (20-30 mL), dried in a desiccator, and the condensation ratio was calculated (yield: 2.49 g, yield: 100%).

[0010]

[Reference Example 2] _{(Fmoc-Lys) 4 -Lys 2} -Ly
Synthesis of s-Gly-Resin Fmoc-Gly-Resin obtained in Reference Example 1 2.4
9 g (1.56 mmol, 1.0 eq) of the Fmoc group was deprotected with a 20% piperidine / dimethylformamide solution, and washed with dimethylformamide until pH = 6. Fmoc-Lys (Fmoc) -O
H 1.85 g (3.12 mmol, 2.0 eq), H
210.8 mg of OBt (3.12 mmol, 2.0e
q), add 30 mL of dimethylformamide, and finally add D
IC 0.48 mL (3.12 mmol, 2.0 eq)
Was added and stirred at room temperature for 2 hours. The condensation rate was confirmed by a ninhydrin test. After the reaction solution was removed by suction, the resin was washed eight times with dimethylformamide (20-30 mL) and three times with methanol (20-30 mL), and a ninhydrin test was performed to determine the condensation rate of the resin (the solution was light brown yellow) Was presented). Thereafter, the above reaction was carried out at 4.0e each.
q, 8.0 eq, and the resin was washed 8 times with dimethylformamide (20-30 mL) in methanol (2
(0-30 mL), and finally washed twice with diethyl ether (20-30 mL), and dried in a desiccator to obtain 4.61 g (yield: 62.3%) of the title compound.

[0011]

Example 1 Synthesis of the following compound (1)

Embedded image(Fmoc-Lys) obtained in Reference Example 2₄-Lys₂
-Lys-Gly-Resin 100 mg (0.034m
mol) of Fmoc group in 20% piperidine / dimethylphos.
Deprotect with Lumamide solution and dilute until pH = 6.
Washing was performed with methylformamide. Fmoc-
329 mg of Arg (Mtr) -OH (16.0e
q), HOBt 73 mg (16.0 eq), dimethyl
20 mL of formamide is added and finally DIC 0.08
Add 4 mL (16.0 eq) and stir at room temperature for 2 hours
Was. The condensation rate was confirmed by a ninhydrin test. Reaction
After removing the liquid by suction, the resin was washed with dimethylformamide (20
-30 mL) eight times and methanol (20-30 mL)
After washing three times, a ninhydrin test was performed to determine the condensation rate of the resin.
Judged. If the condensation rate is low, repeat the above
A similar reaction was performed. Deprotect the Fmoc group and add diethyl
After washing twice with water (20-30 mL), the resin was
Dry in a cater. Next, add trifluoroacetic acid to the resin
13 mL, 1.8 mL of thioanisole, meta-cresol
Add 0.3 mL and stir the resin removal reaction at room temperature for 10 hours.
The reaction mixture was crystallized by adding cold ether.
Was. If a white precipitate forms, add cold ether and shake
(1000 rpm, 4 minutes) 5 times
The tide was washed to obtain a crude peptide. The obtained crude peptide
To a Sephadex G-25 column (0.1% trifluoro
Gel filtration using acetic acid solution: TFA (the same applies hereinafter)
Thereafter, freeze-drying was performed to obtain 66 mg of the crude peptide. this
The crude peptide was subjected to reverse phase high performance liquid chromatography (ODS).
-80Ts column 4.6mm x 15cm, 0.1% TF
Gradient of A water / acetonitrile containing 0.1% TFA
Solution) (retention time 8.2 minutes)
34.6 mg (Yield: 28.5%) of the title compound
Was. FT-IR (KBr): 3434.2, 1677.9,
1549.0, 1439.8, 1205.8, 113
1.9, 839.9, 800.6, 723.2 cm^-1  TOF-MS: 2221.9 [MH⁺] (Calc. 2)
220.5)

[0012]

Example 2 Synthesis of the following compound (2)

Embedded image(Fmoc-Lys) obtained in Reference Example 2₄-Lys₂
-Lys-Gly-Resin 100 mg (0.034m
mol) of Fmoc group in 20% piperidine / dimethylphos.
Deprotect with Lumamide solution and dilute until pH = 6.
Washing was performed with methylformamide. Fmoc-
329 mg of Arg (Mtr) -OH (16.0e
q), HOBt 73 mg (16.0 eq), dimethyl
20 mL of formamide is added and finally DIC 0.08
Add 4 mL (16.0 eq) and stir at room temperature for 2 hours
Was. The condensation rate was confirmed by a ninhydrin test. Reaction
After removing the liquid by suction, the resin was washed with dimethylformamide (20
-30 mL) eight times and methanol (20-30 mL)
After washing three times, a ninhydrin test was performed and the resin condensation rate was determined.
Was determined. Again, Fmoc-Arg (Mtr) -OH
 329 mg (16.0 eq), HOBt 73 mg
(16.0 eq), 20 mL of dimethylformamide
Finally, DIC 0.084 mL (16.0 eq)
Was added and stirred at room temperature for 2 hours. If the condensation rate is low,
The same reaction as above was performed once again. Fmoc group
Deprotect and twice with diethyl ether (20-30 mL)
After washing, the resin was dried in a desiccator. Then resin
TFA 13 mL, thioanisole 1.8 mL, meta
0.3 mL of cresol is added, and the resin removal reaction is performed at room temperature.
After stirring for 10 hours, cold ether was added to the reaction solution.
And crystallized. If a white precipitate forms, add cold ether
In addition, 5 rounds of vibrating (1000 rpm, 4 minutes)
The resulting peptide was washed to obtain a crude peptide. Got
The crude peptide was applied to a Sephadex G-25 column (0.1%
After gel filtration using TFA solution), freeze-dry
66 mg of the peptide were obtained. This crude peptide is reverse-phase
Body chromatography (ODS-80Ts column 4.6)
mm × 15cm, containing 0.1% TFA water / 0.1% TFA
Acetonitrile (gradient solution of acetonitrile)
Solution time of 13.4 minutes).
3 mg (yield: 15.4%) was obtained.¹ H-NMR (400 MHz, D₂O): δ 1.24
1.82 (107H, m), 3.00-3.10 (45
H, m), 3.82-4.26 (25H, m) FT-I
R (KBr): 3390.4, 1666.2, 154
9.0, 1439.8, 1204.6, 1136.1,
838.0, 802.8, 755.8, 720.5cm
^-1  TOF-MS: 3470.9 [MH⁺] (Calc. 3)
469.3)

[0013]

[Test Example 1] Angiogenesis inhibitory activity test This test was conducted using ADVANCES IN CANCER.
ESEARCH, 43 , 175-203, the fertilized chicken egg chorioallantoic membrane (Chorio-lantoicmembr)
ance, CAM for short). After aseptically removing about 2 mL of egg white from a 3-day-old fertilized egg with a syringe needle, the shell of the air chamber side of the egg is broken with forceps and the diameter is about 2 cm.
And put a stainless steel cap over the hole.
Incubated at ℃. After 24 hours, 0.9% NaC
10 μL of a solution of each of the compounds (1) and (2) synthesized in Examples 1 and 2 dissolved in a constant concentration in 1/1% methylcellulose was dropped into a silicon ring placed on the chorioallantoic membrane, The cap was put on again, the eggs were incubated at 37 ° C. for 24 hours, and further incubated at 40 ° C. for 48 hours. At that time, the compound (1),
The angiogenesis inhibitory effect of (2) was observed, and the results are shown in Table 1. From these results, the compound of the present invention (1),
(2) was confirmed to inhibit angiogenesis in a concentration-dependent manner at 10 μg-1000 μg / CAM.

[0014]

[Table 1]

[0015]

[Test example 2] Binding to heparin using a heparin agarose column Prepacked heparin agarose (2.5 mL, 750-
1000 μg heparin / mL gel, SIG
(Manufactured by MA) was washed with 5 mmol phosphate buffer (pH = 7.4) containing 2 mol NaCl (5 times the volume of the column), and then the column was washed with 5 mmol phosphate buffer (pH = 7.4) (column). 10 times).
0.5 m of solution containing each of the present compounds (1) and (2)
L (400 μg / mL) was applied to the column, and p
The column was washed with a 5 mmol phosphate buffer of H = 7.4 (10 times the volume of the column). 0-2000mmo
The compounds of the present invention (1) and (2) were eluted with a phosphate buffer containing 1NaCl, and the NaCl concentration when eluted from the column was determined using the BCA method. The results are shown in Table 2, and it was confirmed that each of the compounds (1) and (2) of the present invention had strong heparin binding ability.

[0016]

[Table 2]

[0017]

EFFECT OF THE INVENTION It has been confirmed that the novel basic dendrimer of the present invention or a pharmaceutically acceptable salt thereof has excellent angiogenesis inhibitory activity and heparin binding ability. Treatment and prevention of diseases associated with
For example, solid tumors, tumor metastases, benign tumors (eg, hemangiomas, acoustic schwannoma, neurofibromas, trachoma and purulent granuloma), vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis , Psoriasis, diabetic retinopathy and other ocular vascular diseases (eg post lens fibroplasia, macular degeneration, corneal transplant rejection, neovascular glaucoma),
It is useful for treating or preventing osteoporosis and the like.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) A61P 29/00 A61P 35/00 35/00 A61K 37/02 F term (reference) 4C084 AA02 AA07 DC50 NA14 ZA332 ZA362 ZB112 ZB262 ZC352 4H045 AA10 AA30 BA17 EA28 FA32

Claims (6)

[Claims] 1. The following general formula: (In the formula, R represents a basic amino acid residue or a derivative thereof.) A novel dendrimer having basicity represented by the formula: or a pharmaceutically acceptable salt thereof. 2. The novel basic dendrimer having basicity according to claim 1, wherein R is arginine or a derivative thereof, or a pharmaceutically acceptable salt thereof. 3. R is Arg-, Arg-Arg-, Ar
The novel basic dendrimer having basicity according to claim 1, which is g-Lys- or Arg-His-, or a pharmaceutically acceptable salt thereof. 4. A novel basic dendrimer represented by the following chemical formula (1) or a pharmaceutically acceptable salt thereof. Embedded image 5. A novel basic dendrimer represented by the following chemical formula (2) or a pharmaceutically acceptable salt thereof. Embedded image 6. An angiogenesis inhibitor comprising at least one of the dendrimers represented by claims 1 to 5 as an active ingredient.