JP2007191430A - 4-phenoxyquinazoline derivative radioactive compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tumor-imaging agent containing as an active ingredient a radioactive compound which binds to an epithelial-originated growth factor receptor tyrosine kinase (EGFR-TK) as a target, and to provide an oral radiation medicine. <P>SOLUTION: The radioactive compound represented by the general formula (1) (R<SP>1</SP>is a radioactive iodine atom). The medicine contains the compound. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a novel 4-phenoxyquinazoline derivative and a medicament containing the same, such as a radiopharmaceutical for detecting a tumor disease by imaging and an internal radiotherapy drug capable of damaging tumor cells from the body.

Tyrosine kinase is an enzyme that plays an important role as an initiation factor that participates in intracellular signal transduction mechanisms such as cell differentiation and proliferation, and transmits binding signals of various growth factors into the cell. Recent studies have revealed that a part of the tyrosine kinase gene is mutated to induce canceration (Non-patent Document 1). In many cancers, increased activity is recognized regardless of the binding of various growth factors (Non-patent Documents 2 and 3). Tyrosine kinases are classified into receptor type and non-receptor type, and their inhibitors are expected as anticancer agents (Non-patent Documents 4 and 5).

Epidermal growth factor receptor tyrosine kinase (EGFR-TK), which is one of the receptor tyrosine kinases, is gastric cancer, esophageal cancer, laryngeal cancer, colon cancer, bladder cancer, ovarian cancer, thyroid gland. It is expressed in a large amount in most epithelial cancers such as cancer and brain tumor, and its activity has been confirmed to increase (Non-patent Documents 6 and 7). Therefore, EGFR-TK inhibitors are considered useful as tumor therapeutic agents.

In recent years, inhibitors targeting EGFR-TK have been developed one after another. The first generation tyrosine kinase inhibitor is a tyrophostin derivative developed by Gazit et al. Tyrophostin derivative was expected as an excellent EGFR-TK inhibitor, but PD153035 was developed after further research on structure-activity relationship (Non-patent Document 9). Currently, ZD1839 (marketed name Iressa), a derivative of PD153035, has been approved as an anticancer agent with a new mechanism of action, and clinical use has begun (Non-patent Document 10).

On the other hand, an abnormal increase in the activity of EGFR-TK is also seen in the early stage of canceration, and is considered useful for early detection of cancer. Therefore, it is considered that EGFR-TK satisfies the conditions as a target for molecular imaging agents that enable cancer characteristic diagnosis. A compound having 4-anilinoquinazoline skeleton (Patent Document 1) and a compound having 4- (phenylamino) quinazoline skeleton as an attempt to apply a drug targeting such EGFR-TK as a radioactive compound and application to an imaging agent Although there is (Patent Document 2), although in vitro EGFR-TK inhibitory activity has been studied, it has not been studied in vivo, and the stability and effectiveness in vivo are unknown. It was.
Special table 2003-500450 Special table 2004-525919 Protein Nucleic Acid Enzyme, 42, 1467-1469 (1997) Int. J. et al. Cancer, 66, 315-321 (1996). Int. J. et al. Cancer, 64, 291-297 (1996) J. et al. Med. Chem. , 38, 3482-3487 (1995) Nature, 379, 645-648 (1996). Proc. Natl. Acad. Sci. USA. , 83, 5141-5144 (1986) Methods Enzymol. 201, 347-355 (1991) Methods Enzymol. 201, 347-355 (1991) Science, 265, 1093-1095 (1994) Proc. Am. Soc. Clin. Oncol. , 19, 177a, (2000)

An object of the present invention is to provide a tumor imaging agent containing a radioactive compound that binds to EGFR-TK as a target as an active ingredient, and an internal radiotherapy agent capable of damaging cancer cells from the body.

The present inventors focused on a 4-phenoxyquinazoline derivative as a novel structure for a compound having inhibitory activity against EGFR-TK, and synthesized a radioactive iodine compound of this derivative. When the action was examined, a high inhibitory activity against EGFR-TK was observed in in vitro experiments, and cancer-specific accumulation and a high cancer-to-tissue ratio were achieved in in vivo biodistribution experiments in tumor-bearing mice.

Moreover, it became clear from the examination of several tumor cell lines from which the EGFR-TK expression level differs that the accumulation according to the EGFR-TK expression level is shown. From these results, it was found useful for imaging of tumor diseases, determination of therapeutic effects by surgery and drugs, and treatment, and the present invention was completed.

That is, the present invention relates to general formula (1)

(Wherein R ¹ represents a radioactive iodine atom), a pharmaceutical containing the same, and a method for producing the same.

In other words, the present invention
(1) A radioactive compound represented by the general formula (1) (wherein R ¹ represents a radioactive iodine atom),
(2) The above radioactive compound wherein the radioactive iodine atom is selected from I-123, I-124, I-125 and I-131,
(3) A medicament containing the radioactive compound according to any one of (1) and (2) above,
(4) The medicament according to (3) above, which is an imaging agent for diagnostic imaging,
(5) The medicament according to (4) above, which is an imaging agent for diagnostic imaging of a tumor disease region,
(6) The medicament according to (5) above, which is an imaging agent for diagnostic imaging of a tumor disease region for single photon tomography (SPECT),
(7) The medicament according to claim 5, which is an imaging agent for diagnostic imaging of a tumor disease region for positron emission tomography (PET),
(8) The medicament according to (3) above, which is an internal radiotherapy drug,
(9) General formula (2)
(Wherein R ² represents an iodine atom, a trialkyltin group or a trialkylsilyl group), a 4-phenoxyquinazoline derivative represented by:
(10) reacting an alkali metal radioiodide with a 4-phenoxyquinazoline derivative represented by the general formula (2) (wherein R represents an iodine atom, a trialkyltin group or a trialkylsilyl group). A method for producing a radioactive iodine compound represented by the general formula (1), wherein R ¹ represents a radioactive iodine atom,
It is.

The radioactive compound (1) of the present invention having a 4-phenoxyquinazoline skeleton is useful for tumor diagnostic imaging and treatment targeting EGFR-TK.

In the general formula (1), as the radioactive iodine atom, I-123 suitable for tissue imaging using a SPECT apparatus and I-131 suitable for internal radiotherapy as well as imaging are desirable. I-125, which is widely used in Japan, is a positron emitting nuclide, but I-124 that can be supplied without installing a small cyclotron in a medical facility can also be used.

The radioactive compound (1) of the present invention is produced, for example, according to the following reaction formula.

(In the formula, R ¹ represents a radioactive iodine atom, and R ² represents an iodine atom, a trialkyltin group or a trialkylsilyl group.)

That is, the radioactive compound represented by the general formula (1) is produced by reacting the 4-phenoxyquinazoline derivative represented by the general formula (2) with an alkali metal radioactive iodide.

In the general formula (2), the trialkyltin group represented by R ² includes a tri (C1-C6 alkyl) tin group, and a tributyltin group is particularly preferable. Examples of the trialkylsilyl group include a tri (C1-C6 alkyl) silyl group, and a trimethylsilyl group is particularly preferable.

The compound of the general formula (2) can be produced, for example, according to the following reaction formula.

(In the formula, R ^2a represents an iodine atom; R ^2b represents a trialkyltin group or a trialkylsilyl group.)

That is, the compound (4) can be obtained by reacting morpholine (3) with 1-bromo-3-chloropropane. In addition, methionine is reacted with 6,7-diethoxyquinazolin-4 (3H) -one (5) to give compound (6), followed by acetyl protection of the 6-position hydroxyl group to give compound (7). can get. Further, the compound (7) is chlorinated to obtain the compound (8), followed by phenoxylation at the 4-position in the presence of 3-iodophenol to obtain the compound (9). Subsequently, compound (2a) is obtained by reacting compound (9) with compound (4). The compound (2b) is obtained by trialkyltinating or trialkylsilylating the obtained compound (2a).

Examples of the alkali metal radioiodide used for the radioiodination of the compound (2) include sodium compounds of radioiodine and potassium compounds of radioiodine.

The reaction between the compound (2) and the alkali metal radioactive iodide is different depending on whether the compound (2) is the compound (2a) or the compound (2b). That is, in the reaction between the compound (2a) and the alkali metal radioactive iodide, non-radioactive iodine atoms are converted to radioactive iodine atoms by heating under acidic conditions. The reaction between the compound (2b) and the alkali metal radioactive iodide is carried out by reacting under acidic conditions and further with an oxidizing agent. As the oxidizing agent, chloramine-T, hydrogen peroxide solution, peracetic acid or the like is used.

When the obtained radioactive compound (1) is used as a radiopharmaceutical, it is desirable to purify unreacted radioiodine ions and insoluble impurities by a membrane filter, columns packed with various packing materials, HPLC, or the like.

The thus obtained radioactive compound (1) of the present invention has an excellent EGFR-TK inhibitory activity and accumulates specifically in an EGFR-expressing tumor, so that it is useful as a tumor therapeutic agent or a tumor diagnostic agent. . For example, 7-ethoxy-4- (3- [ ¹²⁵ I] iodophenoxy) -6- (3-morpholinopropoxy) quinazoline (hereinafter abbreviated as [ ¹²⁵ I] -PYK. This non-radioactive iodine form is abbreviated as PYK). Is administered from a tail vein to a tumor-bearing model mouse transplanted with cancer cells with enhanced EGFR-TK, [ ¹²⁵ I] PYK accumulates 4.4% in the cancer 1 hour after administration, and then 6 hours after administration. Later, 3.6%, 1.7% 12 hours after administration, 1.5% 24 hours after administration, high uptake into cancer and subsequent gradual disappearance were confirmed. As a result, the cancer-to-tissue ratio, which is an index of image contrast, increased, and the [ ^125I ] PYK cancer-to-blood, muscle ratio was 1 hour after administration. 6.8, 4.6, administration 2 After 5 hours, it showed good values of 57.0 and 45.5, while the ratio of cancer to tissue for major tissues was low at 1 hour after administration, but at 24 hours after administration. The cancer-to-liver, cancer-to-kidney, and cancer-to-lung ratios were 3.0, 4.3, and 8.5, respectively, and [ ¹²⁵ I] PYK is the intended purpose. It succeeded in increasing the cancer accumulation amount and maintaining the cancer accumulation for a long time, and was confirmed to have basic properties as a cancer diagnostic agent.

When the radioactive compound (1) of the present invention is used as a radiopharmaceutical, as a pharmaceutical carrier, a stabilizer such as ascorbic acid; a pH adjusting agent such as acid or base; a buffer such as phosphate buffer; An isotonic agent such as a liquid can be used.

The radiopharmaceutical of the present invention is optimally administered by intravenous injection, but can be administered by other general parenteral means. The dose is appropriately determined depending on the patient's weight, age, sex, and various conditions such as a measuring instrument represented by a SPECT apparatus. Generally, in the case of diagnostic agents, the radioactivity of I-123 ranges from 37 MBq to 370 MBq, and in the case of therapeutic agents, the radioactivity of I-131 ranges from 37 MBq to 3700 MBq.

EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Example 1 (Synthesis of PYK)
Synthesis of 3-morpholinopropyl chloride (4) 1-Bromo-3-chloropropane (3.6 g, 22.9 mmol) was added to benzene (10 ml, anhydrous) followed by morpholine (3, 3.0 g, 34.4 mmol). ) Was slowly added dropwise. Thereafter, the mixture was stirred at room temperature for 1 hour and then refluxed (120 ° C.) for 3 hours. After cooling, the precipitated crystals were filtered, and the crystals were washed with methyl-t-butyl ether (MTBE). The organic layer was extracted with 3M hydrochloric acid (20 ml), then dissolved in alkali with 40% aqueous sodium hydroxide solution, and extracted with MTBE (30 ml). The organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was separated and purified by silica gel column chromatography using methanol as an eluting solvent to obtain compound (4) as a colorless liquid.

Yield; 42.8% MS; m / z 163 found 163
¹ H-NMR (CDCl ₃ ); 3.71 (t, J = 6.0 Hz, 4H, morpholino), 3.61 (t, J = 7.3 Hz, 2H, propyl), 2.48 (t, J = 7.3 Hz, 2H, propyl), 2.44 (t, J = 6.0 Hz, 4H, morpholino), 1.95 (m, J = 7.3 Hz, 2H, propyl)

Synthesis of 7-ethoxy-6-hydroxyquinazolin-4 (3H) -one (6) Compound (5) 6,7-diethoxyquinazolin-4 (3H) -one (9.2 g, 39.4 mmol) ) Was dissolved little by little in methanesulfonic acid (60 ml). Then, D, L-methionine (7.05 g, 47.3 mmol) was added and refluxed (100 ° C.) for 4 hours. Thereafter, the reaction solution was cooled to room temperature, ice-cold water (200 ml) was added, and neutralized with a 40% aqueous sodium hydroxide solution in an ice bath. The precipitated crystals were collected by filtration, washed with water, further washed with a small amount of methanol, and dried to obtain compound (6). This was used in the next experiment without purification.

Synthesis of 6-acetoxy-7-ethoxyquinazolin-4 (3H) -one (7) Compound (6) (7.3 g, 35.4 mmol) was added to pyridine (6 ml) and acetic anhydride (40 ml) was added. ) And refluxed (100 ° C.) for 3 hours. The reaction solution was cooled to room temperature, poured into ice-cold water (150 ml), and the precipitated crystals were collected by filtration. Recrystallization from methanol gave Compound (7).

Yield; 61.8% m. p. 258-260 ° C MS; m / z 248 found; 248
¹ H-NMR (DMSO); 8.07 (s, 1 H, aromatics), 7.73 (s, 1 H, aromatics), 7.24 (s, 1 H, aromatics), 4.19 (q, J = 7 .6 Hz, 2H, CH ₃ CH ₂ O), 2.30 (s, 3H, CH ₃ CO), 1.33 (t, J = 7.6 Hz, 3H, CH ₃ CH ₂ O)

Synthesis of 6-acetoxy-4-chloro-7- ethoxyquinazoline (8) Compound (7) (2.0 g, 8.06 mmol) was added to phosphoryl chloride (30 ml) and refluxed (120C) for 3 hours. did. The reaction solution was cooled to room temperature, poured into ice-cold water (200 ml) in an ice bath, and extracted with chloroform. The organic layer was washed twice with 1M aqueous sodium hydroxide solution (200 ml), and the organic layer was dried over sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was separated and purified by silica gel column chromatography using ethyl acetate as an elution solvent to obtain Compound (8).

Yield; 89.6% m. p. 110-114 ° C. MS; m / z 266 found 266
¹ H-NMR (CDCl ₃ ); 8.93 (s, 1 H, aromatics), 7.88 (s, 1 H, aromatics), 7.40 (s, 1 H, aromatics), 4.25 (q, J = 7.6 Hz, 2H, CH ₃ CH ₂ O), 2.39 (s, 3H, CH ₃ CO), 1.50 (t, J = 7.6 Hz, 3H, CH ₃ CH ₂ O)

7-Ethoxy-6-hydroxy-4- (3-iodophenoxy) quinazoline (9)
After 3-iodophenol (5.0 g, 22.7 mmol) was heated (50 ° C.) and dissolved, potassium hydroxide (0.42 g, 7.49 mmol) was added and completely dissolved. Next, compound (8) (0.56 g, 2.10 mmol) was added over about 10 minutes and refluxed (90 ° C.) overnight. The reaction product was extracted with chloroform, the organic layer was washed twice with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was separated and purified by silica gel column chromatography using ethyl acetate as an elution solvent to obtain compound (9).

Yield; 71.2% m. p. 214-216 ° C HRMS; m / z 407.9971 found 407.978
¹ H-NMR (DMSO); 10.30 (s, 1 H, OH), 8.48 (s, 1 H, aromatics), 7.73 (t, 1 H, aromatics), 7.68 (d, 1 H, aromatics) ), 7.48 (s, 1H, aromatics), 7.30 (m, 3H, aromatics), 4.25 (q, J = 7.6 Hz, 2H, CH ₃ CH ₂ O) 1.44 (t, J = 7.6 Hz, 3H, CH ₃ CH ₂ O)

Synthesis of 7-ethoxy-4- (3-iodophenoxy) -6- (3-morpholinopropoxy) quinazoline (2a) [PYK] Compound (9) (0.60 g, 1.47 mmol) was converted to dimethyl To formamide (DMF, anhydrous, 15 ml) was added followed by potassium carbonate (1.0 g, 7.24 mmol). Thereafter, morpholinopropyl chloride (compound (4), 0.30 g, 1.83 mmol) was added, and the mixture was refluxed (90 ° C.) for 3.5 hours. The reaction solution was cooled to room temperature, ice-cold water (50 ml) was added, and the mixture was extracted with ethyl acetate (50 ml × 1, 20 ml × 2). The organic layer was dried over sodium sulfate and the solvent was distilled off under reduced pressure. The residue was separated by silica gel column chromatography using ethyl acetate as an elution solvent, and then recrystallized from 2-propanol to obtain compound (2a).

Yield; 38.1% m. p. 135-137 ° C. HRMS; m / z 535.0968 found 535.0966 ¹ H-NMR (CDCl ₃ ); 8.54 (s, 1 H, aromatics), 7.59 (t, 1 H, aromatics), 7. 56 (d, 1H, aromatics), 7.43 (s, 1H, aromatics), 7.18 (m, 3H, aromatics), 4.20 (t, J = 8.0 Hz, 2H, propyl), 4. 17 (q, J = 7.6 Hz, 2H, CH ₃ CH ₂ O), 3.66 (t, J = 5.0 Hz, 4H, morpholino), 2.53 (t, J = 8.0 Hz, 2H, propyl), 2.43 (t, J = 5.0 Hz, 4H, morpholino), 2.06 (m, J = 8.0 Hz, 2H, propyl), 1.49 (t, J = 7.6 Hz, 3H, CH ₃ CH ₂ O)

Example 2 (Synthesis of 7-ethoxy-6- (3-morpholinopropoxy) -4- (3-tributylstannylphenoxy) quinazoline (2b))
Compound (2a) (0.15 g, 0.28 mmol), bis (tributyltin) (0.49 g, 0.84 mmol), and tetrakis (triphenylphosphine) -palladium (0.02 g, 0.01 mmol) in anhydrous toluene (25 ml) and refluxed (140 ° C.) overnight. After cooling, the mixture was filtered using celite, and the filtrate was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography using chloroform / methanol (15/1 v / v) as an elution solvent to obtain compound (2b) as a yellow oil.

Yield; 35.8% HRMS; m / z 699.3058 found 699.3054
¹ H-NMR (CDCl ₃ ); 8.60 (s, 1 H, aromatics), 7.60 (s, 1 H, aromatics), 7.49-7.15 (m, 5 H, aromatics), 4.27 ( t, J = 8.0 Hz, 2H, propyl), 4.25 (q, J = 7.6 Hz, 2H, CH ₃ CH ₂ O), 3.75 (t, J = 5.0 Hz, 4H, morpholino) 2.63 (t, J = 8.0 Hz, 2H, propyl), 2.54 (t, J = 5.0 Hz, 4H, morpholino), 2.15 (m, J = 8.0 Hz, 2H, propyl). ), 1.55 (t, J = 7.6 Hz, 3H, CH ₃ CH ₂ O), 1.42-0.86 (m, 27H, Bu ₃ )

Example 3 (synthesis of [ ¹²⁵ I] PYK)
Compound (2b) (50 μl, 0.5 mg / ml ethanol solution) in 0.1 mL hydrochloric acid (25 μl), [ ¹²⁵ I] sodium iodide (1.0 μl, 3.7 MBq), 30 w / v% excess in a sealed vial Hydrogen oxide water (10 μl) was added in order and allowed to react at room temperature for 15 minutes. The target product was separated and purified by HPLC, and it was confirmed that the target product was the target labeled product by completely matching the HPLC retention times of the corresponding unlabeled products of various compounds. In addition, the radiochemical purity of all the labeled bodies was 95% or more, and the specific activity was about 74 TBq / mmol.
Elution solvent: Methanol / 0.01M citric acid aqueous solution (55/45 v / v)
HPLC retention time; 15.5 minutes Labeling rate: 97.5%

Example 4 (Measurement of the ability of PYK to inhibit EGFR-TK phosphorylation)
EGFR-TK phosphorylation inhibitory ability was measured about the synthesized new derivative. The measurement was performed using a measurement kit manufactured by Promega and biotinylated EGFR-TK phosphorylated peptide as a substrate and [γ-32P] ATP. The inhibitory activity was determined by measuring the phosphorylation ability in the presence of a drug at a concentration of 10 μM to 100 pM, and measuring the EGFR-TK phosphorylation inhibition ability obtained as a 50% inhibitory concentration (IC50) value. At the same time, comparison was made using PD153035, which is a representative EGFR-TK selective inhibitor.

PTK assay 5X buffer (5.0 μl), PTK biotinylated peptide substrate (2.5 μl), 2.5 mM sodium vanadate (2.5 μl), ATP mixed solution {0.5 mM ATP, [γ-32P] ATP ( 7.4 kBq)} (2.5 μl) and a 10% DMSO solution (7.5 μl) of the test substance to make a total volume of 20 μl, and incubated at 30 ° C. for 5 minutes. Subsequently, EGFR PTK buffer solution (40 mM imidazole hydrochloride, 40 mM β-glycerophosphate, 0.5 mg / ml BSA) (5.0 μl) was added and incubated at 30 ° C. for 15 minutes. Subsequently, 7.5 M guanidine hydrochloride (13 μl) was added and centrifuged at 14000 × g for 10 seconds. 13 μl of the reaction solution was spotted on a SAM ² (registered trademark) membrane, washed with 2M aqueous sodium chloride solution (200 ml) for 30 seconds, once, 3 minutes, twice, and then with 2M aqueous sodium chloride solution containing 1% phosphoric acid (200 ml) ) For 3 minutes twice, and finally washed once with ion-exchanged water (100 ml) for 30 seconds. After drying the SAM ² (registered trademark) membrane, 10 ml of Clearsol I scintillator (manufactured by Nacalai Tesque) was added, and the radioactivity was measured with a liquid scintillation counter. As a control, the radioactivity when a 10% DMSO solution was added instead of the test substance was measured. The radioactivity when the test compound was added was calculated as a percentage of the control, and the concentration showing 50% of the control was taken as the IC ₅₀ value (Table 1).

PYK showed high EGFR-TK phosphorylation inhibitory activity comparable to PD153035 used as a control. Further, from this result, it was confirmed that PYK has an excellent EGFR-TK phosphorylation inhibitory activity and has potential as a novel agent for diagnosing EGFR-TK activity for SPECT.

Example 5 (Investigation of binding affinity of [ ¹²⁵ I] PYK to A431 membrane fraction)
Preparation of A-431 cancer cell membrane fraction A-431 cancer cells were cultured until they reached confluence, and then collected from the dish by trypsin treatment, and then the cell solution was centrifuged (400 × g, 5 min, room temperature). Next, 2 ml of PBS (−) was added to the precipitate under ice cooling, and the cells were disrupted with a syringe. Thereafter, an equal amount of 0.2 mM phosphate / 0.32 M sucrose buffer (pH 7.4) was added, and the mixture was centrifuged again (800 × g, 10 min, 4 ° C.) to obtain a supernatant. The supernatant was subjected to ultracentrifugation (75000 × g, 15 min, 4 ° C.), 25 mM HEPES buffer (pH 7.4) was added to the resulting precipitate, and the mixture was incubated at 25 ° C. for 15 minutes. Ultracentrifugation (75000 × g, 15 min, 4 ° C.) was performed again, and the precipitate was used as a cell membrane fraction, which was suspended in 25 mM HEPES buffer (pH 7.4). The amount of protein was measured by the Raleigh method using BSA as a standard sample.

The binding affinity A-431 cancer cell membrane fraction of [ ¹²⁵ I] PYK to EGFR-TK was suspended in 25 mM HEPES buffer, and incubated at 25 ° C. for 10 minutes after addition of EGF. Subsequently, DMSO was added to measure the total amount of binding, and 100 μl of PD153035 (final concentration 10 μM) was added to measure the amount of non-specific binding. Further, 50 μl of [ ¹²⁵ I] PYK in DMSO and 50 μl of unlabeled PYK prepared to a final concentration of 1 nM to 200 nM were added to make a total volume of 1 ml, and incubated at 25 ° C. for 60 minutes. Immediately after completion of the reaction, the reaction solution was suction filtered using a glass filter (GF / B, manufactured by Whatman) immersed in a 0.5% polyethyleneimine solution for 30 minutes or more, and 0.5% Triton X-100 0.2 mM phosphorus. The filter and tube were washed with 3 ml of acid buffer (pH 7.2). The radioactivity of the filter is measured with a γ counter to determine the total binding amount and non-specific binding amount of [ ¹²⁵ I] PYK and tyrosine kinase, and the difference between the total binding amount and the non-specific binding amount is determined as the specific binding amount. did. From the obtained specific binding amount curve, Kd value and Bmax value were calculated by Scatchard analysis (FIG. 1).

The Kd value of PYK for the A-431 cell membrane fraction was 51.3 nM, the Bmax value was 27.0 pmol / mg protein, and it was confirmed that PYK showed high affinity for EGFR-TK. Furthermore, the Hill coefficient, which is important for knowing the crossover property of the binding site, was 1.02, and it was confirmed that there was one PYK binding site and there was no interaction with other binding sites. In addition, since the EGFR-TK selective inhibitor PD153035 used in this experiment exhibits inhibitory activity by acting on the ATP binding site of tyrosine kinase existing in the cell membrane, PYK is also bound to the same site. it was thought.

Example 6 (Change in distribution of [ ¹²⁵ I] PYK in the body)
Regarding the detailed biodistribution of [ ¹²⁵ I] PYK and EGFR-TK specificity, the body distribution was examined using cancer-bearing mice at 1, 6, 12, and 24 hours after administration. In this experiment, A-431 cancer cells, which are well expressed as EGFR-TK and are often used as cancer-bearing model mice, were used.

Preparation of A-431 cell-bearing model mouse A-431 cancer cells were cultured in a DMEM culture solution containing 5% FCS and penicillin-streptomycin mixed solution at 37 ° C. in the presence of 5% CO ₂ according to a conventional method. The medium was changed once every two days, and after culturing until confluent, it was used for the experiment. Those that reached a confluent state were trypsinized and cells were collected. The cells were suspended at the optimal concentration in the medium used during the culture, and the cells were prepared so as to be 1 × 10 ⁷ cells per 200 μl. 200 μl of the obtained cell suspension was injected subcutaneously into the thigh of a 4-week-old BALB / c-nu male nude mouse (20 to 25 g) and reared for 2 weeks.

Accumulation of [ ¹²⁵ I] PYK in A431 cancer tissue A-431 cancer cell-bearing mice [ ¹²⁵ I] PYK was administered from the tail vein and sacrificed 1, 6, 12, 24 hours later, and cancer and organs Were collected, and the weight and radioactivity were measured and compared. From the measurement results, the radioactivity per gram of each organ was calculated as a percentage of the total dose (% dose / g tissue), and the accumulation of [ ¹²⁵ I] PYK in various cancers was compared (Table 2).

[ ¹²⁵ I] PYK accumulates 4.4% in the cancer 1 hour after administration, then 3.6% 6 hours after administration, 1.7% 12 hours after administration, 1. 24 hours after administration 1. A high uptake into cancer and early gradual disappearance after 5% were confirmed. In addition, disappearance earlier than cancer was observed from other tissues. As a result, the cancer-to-tissue ratio, which is an index of image contrast, increased. [ ¹²⁵ I] PYK cancer-to-blood and muscle ratios showed good values of 6.8 and 4.6 at 1 hour after administration, and 57.0 and 45.5 at 24 hours after administration (FIG. 2A). ). On the other hand, the cancer-to-tissue ratio to the main tissue was low at 1 hour after administration, but the cancer-to-liver, cancer-to-kidney, and cancer-to-lung ratios were 3.0, 4 and 24 hours after administration, respectively. .3 and 8.5 were good values (FIG. 2B).

Example 7 (in vivo cancer accumulation amount correlates with EGFR-TK expression level and EGFR-TK phosphorylation activity)
Culture of various cancer cells Various cancer cells were added at 1.0 × 10 ⁶ in a 75 cm ² dish and cultured at 37 ° C. in the presence of 5% CO ₂ by a conventional method. Medium is A-431, A-375, C-6 cancer cells are DMEM, Du-145, Me-180, PC-3, NALM-6 cancer cells are RPMI1640, A-431 is 5% FCS, other cells are What added 10% FCS and the penicillin-streptomycin mixed solution was used.

Preparation of various cancer cell membrane fractions (for adherent cells)
Various cancer cells cultured until they reached confluence were collected from the dish by trypsin treatment, and then the cell solution was centrifuged (400 × g, 5 min, room temperature). Next, 2 mL of PBS (−) was added to the precipitate under ice cooling, and the cells were crushed with a syringe. Thereafter, an equal volume of 0.2 mM phosphate / 0.32 M sucrose buffer (pH 7.4) was added, and a supernatant was obtained by centrifugation again (800 × g, 10 min, 4 ° C.). The supernatant was subjected to ultracentrifugation (75000 × g, 15 min, 4 ° C.), 25 mM HEPES buffer (pH 7.4) was added to the resulting precipitate, and the mixture was incubated at 25 ° C. for 15 minutes. Ultracentrifugation (75000 × g, 15 min, 4 ° C.) was performed again, and the precipitate was used as a cell membrane fraction, which was suspended in 25 mM HEPES buffer (pH 7.4). The amount of protein was measured by the Raleigh method using BSA as a standard sample.
(For floating cells)
The cells were collected from the dish without trypsin treatment, and the same operation as that for the adherent cells was performed.

Measurement of EGFR-TK expression level in various cancer cells Various cancer cell membrane fractions were suspended in 25 mM HEPES buffer, and incubated at 25C for 10 minutes after addition of EGF. Subsequently, DMSO was added to measure the total amount of binding, and 100 μl of PD153035 (final concentration 10 μM) was added to measure the amount of non-specific binding. Further, 50 μl of [ ¹²⁵ I] PYK in DMSO and 50 μl of unlabeled PYK prepared to a final concentration of 1 nM to 200 nM were added to make a total volume of 1 ml, and incubated at 25 ° C. for 60 minutes. Immediately after completion of the reaction, the reaction solution was suction filtered using a glass filter (GF / B, manufactured by Whatman) immersed in a 0.5% polyethyleneimine solution for 30 minutes or more, and 0.5% Triton X-100 0.2 mM phosphorus. The filter and tube were washed with 3 ml of acid buffer (pH 7.2). The radioactivity of the filter is measured with a γ counter to determine the total binding amount and non-specific binding amount of [ ¹²⁵ I] PYK and tyrosine kinase, and the difference between the total binding amount and the non-specific binding amount is determined as the specific binding amount. did. From the obtained specific binding curve, Kd value and Bmax value were calculated by Scatchard analysis (Table 3).

Measurement of EGFR-TK phosphorylation activity in various cancer cells PTK assay 5 × buffer (5.0 μl), PTK biotinylated peptide substrate (2.5 μl), 2.5 mM sodium vanadate (2.5 μl), ATP mixed solution {0.5 mM ATP, [γ-32P] ATP (18.5 kBq)} (2.5 μ) is added, 1% DMSO solution is used as a control, and EGFR-TK selection is used to measure nonspecific phosphorylation. Inhibitor PD153035 (final concentration 10 μM) 7.5 μl was added and incubated at 30 ° C. for 5 minutes. Subsequently, 1 μg of various cancer cell membrane fractions were added and incubated at 30 ° C. for 15 minutes. Thereafter, 7.5M guanidine hydrochloride (13 μl) was added and centrifuged at 14000 × g for 10 seconds. 13 μl of the reaction solution was spotted on a SAM ² (registered trademark) membrane, washed with 2M aqueous sodium chloride solution (200 ml) for 30 seconds, once, 3 minutes, twice, and then with 2M aqueous sodium chloride solution containing 1% phosphoric acid (200 ml). ) For 3 minutes twice, and finally washed once with ion-exchanged water (100 ml) for 30 seconds. After drying the SAM ² (registered trademark) membrane, 10 ml of Clearsol I scintillator (manufactured by Nacalai Tesque) was added, and the radioactivity was measured with a liquid scintillation counter. The value obtained by subtracting the radioactivity when PD153035 was pretreated from the control was defined as the specific phosphorylation activity of EGFR-TK, and converted into units to calculate the EGFR-TK phosphorylation activity in various cancer cells (Table 4). . One unit was the amount of enzyme for converting 1 pmol of a phosphate group into Angiotensin II at 30 ° C. for 1 minute.

Preparation of various cancer cell-bearing model mice Various cancer cells were cultured by the above-described method, and those that reached a confluent state were trypsinized to collect cells. The cells were suspended at an optimal concentration in the medium used for culturing various cancer cells. Cells were prepared at 1 × 10 ⁷ cells per 200 μl of various cancer cell suspensions. 200 μl of the obtained cell suspension was injected subcutaneously into the thigh of a 4-week-old BALB / c-nu male nude mouse (20-25 g), and A-375 cancer cells were transferred to C-6, PC for 2 weeks. -3 cancer cells were bred for 3 weeks, and Me-180 and Du-145 cancer cells were bred for 4 weeks.

Accumulation of [ ¹²⁵ I] PYK in various cancers [ ¹²⁵ I] PYK was administered to various cancer cell-bearing mice from the tail vein, and sacrificed 24 hours later, and the cancer and each organ were collected and weighed. In addition, the radioactivity was measured and compared. From the measurement results, the radioactivity per gram of each organ was calculated as a percentage of the total dose (% dose / g tissue), and the accumulation of [ ¹²⁵ I] PYK in various cancers was compared (Table 5).

[ ¹²⁵ I] PYK showed no significant difference in accumulation in major organs in various tumor-bearing model mice. On the other hand, the amount of [ ¹²⁵ I] PYK accumulated in cancer that is the target tissue is A-431, 1.51%, Du-145, Me-180, PC-3, A-375, and C-6, respectively. 1.14%, 0.70%, 0.43%, 0.39%, 0.11% and varied depending on the cancer.

Correlation between [ ^125I ] PYK cancer accumulation amount and EGFR-TK phosphorylation activity in various cancer- bearing model mice Next, EGFR-TK phosphorylation activity and EGFR-TK expression level of various cancers The correlation between the Bmax value and the amount of cancer accumulation of [ ¹²⁵ I] PYK in vivo was examined (FIG. 3).
The amount of cancer accumulation of [ ¹²⁵ I] PYK was found to be highly correlated with the Bmax value (R ² = 0.89), and a good value was also obtained for the EGFR-TK phosphorylation activity (R ² = 0). 72). Therefore, [ ¹²⁵ I] PYK is considered to be applicable to various cancers expressing EGFR-TK even in vivo, and EGFR-TK phosphorylation activity can be estimated from the amount of cancer accumulation of [ ¹²⁵ I] PYK. This suggested the possibility as a molecular imaging agent targeting EGFR-TK.

Example 8 (autoradiogram of [ ¹²⁵ I] PYK)
Autoradiograms were prepared at 1, 6, 12, and 24 hours after administration using A-431 cancer-bearing model mice with the highest cancer accumulation and cancer-to-tissue ratio, and [ ^125I ] PYK images were evaluated. went.
200 μl of A-431 cancer cell suspension (1 × 10 ⁷ cells) was subcutaneously injected into the back of 4-week-old BALB / c-nu male nude mice (20-25 g) and reared for 2 weeks. [ ¹²⁵ I] PYK was administered from the tail vein (1.1 MBq / 200 μl) and sacrificed after 1, 6, 12, and 24 hours, and the trunk was frozen in dry ice / hexane. After 1 hour, the torso was longitudinally cut along the spine, fixed to the base with o, c, t compound, and frozen at about −30 ° C. for 12 hours. Thereafter, a frozen section of 30 μm was prepared using a microtome and attached to a slide glass. The obtained sections were adhered to the film, exposed to light in a dark room for 1 week, developed for 5 minutes with a developing solution, developed for 2 minutes with a developing stop solution, and treated with a fixing solution for 10 minutes. Grams were obtained (Figure 4).

In autoradiograms 1 and 6 hours after [ ¹²⁵ I] PYK administration, clear [ ¹²⁵ I] PYK accumulation in cancer was confirmed, but high accumulation was also observed in major tissues. However, 12 hours after administration, disappearance of [ ¹²⁵ I] PYK was observed from the non-target tissue to such an extent that an organ could be discriminated, and 24 hours after administration, cancer that was the target tissue of [ ¹²⁵ I] PYK. High accumulation in other organs, especially in organs of metabolism such as liver and kidney, and large volume organs such as lung and stomach, which are found in many diagnostic imaging drugs, are not recognized. I was able to get it.

As described above, [ ¹²⁵ I] PYK exhibited high cancer accumulation and rapid disappearance from non-target tissues in various cancers examined, and a good cancer-to-tissue ratio was obtained. Furthermore, clear images were obtained from the autoradiogram of the A-431 cancer-bearing model mouse, suggesting that [ ¹²⁵ I] PYK can be applied to clinical practice sufficiently.

It is a figure which shows the experimental result which examined the binding characteristic of [< ¹²⁵ >] PYK with respect to the membrane fraction EGFR-TK of A431 cancer cell. In the distribution of [ ¹²⁵ I] PYK in tumor-bearing mice, the ratio of tumor to blood or surrounding organ accumulation (% dose / g tissue) was calculated as the cancer-to-organ ratio, and shows the change over time. . It is a figure which shows the experimental result which examined the correlation with the cancer accumulation amount of [ ^125I ] PYK in vivo, and the Bmax value which is EGFR-TK phosphorylation activity and EGFR-TK expression level of various cancer cells. It is the figure (photograph) which showed the autoradiogram of [ ^125I ] PYK in A-431 cancer bearing model mouse over time. In the figure, the arrow indicates a tumor.

Claims (10)

General formula (1)
(Wherein R ¹ represents a radioactive iodine atom). The radioactive compound according to claim 1, wherein the radioactive iodine atom is selected from I-123, I-124, I-125 and I-131. The pharmaceutical containing the radioactive compound of any one of Claims 1-2. The medicament according to claim 3, which is an imaging agent for diagnostic imaging. The medicament according to claim 4, which is an imaging agent for diagnostic imaging of a tumor disease region. The medicament according to claim 5, which is an imaging agent for diagnostic imaging of a tumor disease region for single photon tomography (SPECT). The medicament according to claim 5, which is an imaging agent for diagnostic imaging of a tumor disease region for positron emission tomography (PET). The medicament according to claim 3, which is an internal radiotherapy drug. General formula (2)
(Wherein R ² represents an iodine atom, a trialkyltin group or a trialkylsilyl group). A 4-phenoxyquinazoline derivative represented by: General formula (2)
(Wherein R ² represents an iodine atom, a trialkyltin group or a trialkylsilyl group). A general formula (characterized by reacting an alkali metal radioiodide with a 4-phenoxyquinazoline derivative represented by the formula ( 1)
(Wherein R ¹ represents a radioactive iodine atom).