JP2019043882A - Therapeutic effect predictors for alternative anticancer drugs - Google Patents

Abstract

To select an effective alternative anticancer agent when having resistance to various gene mutations appearing in epidermal growth factor receptors in non-small cell lung cancer cells, and thus capable of image diagnosis with minimal or no invasiveness. Provided is an agent for predicting the effect of gene mutation treatment of a radiolabeled alternative anticancer agent. An agent for predicting therapeutic effect of an alternative anticancer agent is one in which the alternative anticancer agent to be selected for drug resistance is labeled with a radioisotope, and the alternative anticancer agent to be selected for the drug resistance of an alternative anticancer agent is Those labeled with a radioisotope are preferred. The therapeutic effect prediction agent of an alternative anticancer agent is that the anticancer agent and the alternative anticancer agent are epidermal growth factor receptor tyrosine kinase inhibitors against drug resistance due to epidermal growth factor receptor gene mutation, respectively. [Selection figure] None

Description

  The present invention relates to an agent that predicts the therapeutic effect of a next available alternative anticancer agent in response to a gene mutation when gene mutation is produced to obtain drug resistance to cancer cells by administration of the anticancer agent.

  Epidermal Growth Factor Receptor (EGFR) is expressed in various cancer cells, and activation of epidermal growth factor receptor tyrosine kinase (EGFR Tyrosine Kinase: EGFR-TK) is involved in cell proliferation There is.

  Epidermal growth factor receptors are present on the cell membrane and, upon external stimuli, transmit signals necessary for cancer cells to proliferate into cells.

  Anticancer drugs that inhibit epidermal growth factor receptor tyrosine kinase are used as one of the so-called molecular target drugs that attack specific molecules involved in cancer growth and the like.

  When epidermal growth factor receptor (Epidermal Growth Factor: EGF) binds, the receptor is activated. Activated receptors migrate across cell membranes and bind to other receptors to form dimers. Upon formation of the dimer, tyrosine kinase sites in the intracellular domain utilize ATP to phosphorylate certain tyrosine residues in the receptor intracellular domain. When the tyrosine residue is phosphorylated, various proteins in the cell cause signal transduction to be activated one after another, causing gene amplification, gene alteration, or cell function or structure. As a result, carcinogenesis, cancer growth, angiogenesis, invasion, metastasis, suppression of apoptosis occur.

  At this time, when the epidermal growth factor receptor tyrosine kinase inhibitor is administered, it competitively binds to the ATP binding site of the epidermal growth factor tyrosine kinase, inhibits the epidermal growth factor autophosphorylation, and blocks the signal transduction. Can treat cancer.

  In non-small cell lung cancer patients, there are a considerable number of patients in which a mutation is found in a part of tyrosine kinase site of epidermal growth factor receptor gene. Epidermal growth factor receptor gene mutations are more common among Asians including Japanese and Europeans than among Westerners, more women than men, more non-smokers than smokers, non-small cell lung cancer patients Among them, there are many adenocarcinoma patients, and many smokers also have adenocarcinoma patients.

  Gefitinib which is a therapeutic agent for inoperable or relapsed non-small cell lung cancer (NSCLC) as a tyrosine kinase inhibitor of epidermal growth factor receptor, osimertinib (Osimertinib: AZD9291), erlotinib ( Erlotinib), afatinib (Afatinib), rosiretinib (Rociletinib) and the like are known.

  For example, among the genes encoding epidermal growth factor receptor, a mutation in which the DNA 15 base (amino acid 5 residues) encoded by exon 19 is deleted, the mutation in L858R, the glycine at position 719 encoded in exon 20 is serine, In the presence of three mutations of alanine or cysteine substitution (G719X), particularly the former two mutations, the epidermal growth factor receptor tyrosine kinase inhibitor (eg gefitinib) exhibits a tumor-reducing effect. As a result of the structural change in the ATP binding site of the epidermal growth factor receptor, this mutant epidermal growth factor receptor becomes constitutively activated without stimulation of the ligand, and is involved in the malignant transformation of the cell, The affinity to the epidermal growth factor receptor tyrosine kinase inhibitor is also enhanced, and the epidermal growth factor receptor tyrosine kinase inhibitor causes apoptosis in cancer cells and exhibits a tumor reduction effect.

  This type of mutant epidermal growth factor receptor is present in about 10% of non-small cell lung cancer and is also present in many non-smokers, women, adenocarcinomas, and Orientals. Moreover, as for the breakdown of the mutations of the ATP binding site of these epidermal growth factor receptors, the deletion mutation of exon 19 occupies 44%, the L858R occupies 41%, and the other G719X and rare mutations occupy about 15%.

  Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, is highly effective in treating non-small cell lung cancer patients with L858R mutation at the epidermal growth factor receptor target site by binding to the L858R mutant receptor Show the effect.

  However, even in cases in which gefitinib is effective, cancer cells acquire drug resistance and relapse almost without exception within several months to about 5 years, usually about 6 to 12 months. Once drug resistance is acquired, gefitinib is no longer effective. The high frequency resistance acquisition factor is a T790M secondary gene mutation, and in recent years, Osimertinib, an epidermal growth factor receptor tyrosine kinase inhibitor targeting the T790M secondary gene mutation, has been approved. Since these epidermal growth factor receptor tyrosine kinase inhibitors greatly change the therapeutic effect by gene mutation, it is essential to test gene mutation in deciding the therapeutic course.

  Patent Document 1 diagnoses the expression or activity of Her2 or / and EGFR in a subject by a test for detecting the expression or activity of Her2 or / and EGFR, and as a result, Her2 or / and EGFR overexpression or Her2 or / and EGFR inhibitors for administration to subjects determined to be activated are described.

  In Patent Document 2, it is determined whether a KRAS gene-derived nucleic acid or its protein is present in a blood sample collected from a subject, and the KRAS gene-derived nucleic acid or its protein in the blood sample is wild type or mutant. An EGFR inhibitor sensitivity prediction method having a step of determining whether has been described.

  In addition, in Patent Document 3, i) the level of amplification of the epidermal growth factor receptor (EGFR) gene, ii) the level of multichromosomality of the EGFR gene, iii) the level of amplification of the tyrosine kinase receptor receptor (HER2) gene And iv) therapeutic administration of an EGFR inhibitor having the step of detecting in a sample of tumor cells from a patient a level of a biomarker selected from the group consisting of polychromic levels of the HER2 gene, or Methods for selecting cancer patients that are predicted to be ineffective are described.

  Furthermore, Patent Document 4 describes a method for determining the usefulness of small molecules as functional substitutes (mimetics) for protein receptor ligands, and multidrug resistance (MDR) is an effective treatment for cancer. It is also described that it is the main obstacle.

  Since various gene mutations appear in epidermal growth factor receptor in non-small cell lung cancer cells, they can be used to select an effective alternative anticancer agent in place of a substituted anticancer agent that has already been administered and developed resistance. Biopsies are used to test for genetic mutations. Biopsies can only obtain information about the tissues collected. Multiple rounds had to be examined to select an effective alternative anticancer drug. Due to the high invasiveness of biopsies, repeated testing is burdensome for the patient.

  Unlike the methods of these patent documents, if genetic mutations can be examined by diagnostic imaging such as Positron emission tomography (PET) or Single photon computed tomography (SPECT), Because the whole body can be examined at one time and it is non-invasive, the burden on the patient is small. Moreover, since it is easy to repeat a test, it is highly useful. Conventionally, methods for examining gene mutations in these diagnostic imagings, selecting alternative anticancer agents, and predicting their therapeutic effects are not known.

  Therefore, there has been a demand for a therapeutic effect predicting agent capable of selecting an alternative anticancer agent minimally or noninvasively by a labeled drug instead of an invasive biopsy which places a burden on a patient.

International Publication No. 2003/101491 International Publication No. 2014/148557 JP, 2013-5800, A Japanese Patent Publication No. 2010-508836

  The present invention has been made to solve the above-mentioned problems, and it is possible to select an alternative anticancer agent which is effective when having resistance to a gene mutation which variously appears on epidermal growth factor receptor in non-small cell lung cancer cells. Therefore, it is an object of the present invention to provide a therapeutic effect predicting agent for minimally invasive or non-invasively image-diagnosable radiolabeled alternative anticancer agents.

  The therapeutic effect predictor of the alternative anticancer agent made to achieve the above object is one in which the alternative anticancer agent to be selected for drug resistance is labeled with a radioactive isotope.

  It is preferable that the therapeutic effect prediction agent of this alternative anticancer agent is one in which the above-mentioned alternative anticancer agent to be selected for the drug resistance of the alternative anticancer agent is labeled with a radioactive isotope.

  The therapeutic effect prediction agent of this alternative anticancer agent is that said alternative anticancer agent and said alternative anticancer agent are epidermal growth factor receptor tyrosine kinase inhibitors against drug resistance by gene mutation of epidermal growth factor receptor, respectively. .

  The therapeutic effect predictor of this alternative anticancer agent is preferably that the alternative anticancer agent is gefitinib, and the alternative anticancer agent is osimerinib, erlotinib, afatinib, or rosiretinib.

  The therapeutic effect predictor of the alternative anticancer agent is, for example, one substituted and labeled with the radioactive isotope at the aromatic ring or heteroaromatic ring of the alternative anticancer agent, or substituted with the radioactive isotope and labeled Is protected by a protective group.

  The therapeutic efficacy predictor of this alternative anticancer drug is any one wherein the radioactive isotope is selected from, for example, radioactive iodine, radioactive bromine, radioactive fluorine, radioactive technetium, radioactive thallium, radioactive gallium, radioactive indium, radioactive xenon, and radioactive carbon It contains radionuclides.

（式(Ｉ)中、Ｘは前記放射性同位体であって¹²³Ｉ、¹²⁴Ｉ、¹²⁵Ｉ、¹³¹Ｉ、⁷⁶Ｂｒ、⁷⁷Ｂｒ、及び¹⁸Ｆから選ばれる何れかの放射性核種を含んでいる）で表わされるものである。 The therapeutic effect predictor of this alternative anticancer drug is, for example, that the above-mentioned alternative anticancer drug is osimerinib, and the following chemical formula (I)

(In formula (I), X is the above-mentioned radioactive isotope and contains any radionuclide selected from ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ⁷⁶ Br, ⁷⁷ Br, and ¹⁸ F) It is represented by

  The therapeutic effect predictor of this alternative anticancer agent is used as an imaging diagnostic agent.

  The therapeutic effect prediction agent of this alternative anticancer agent is administered, for example, as an orally administered agent or an injection.

  The therapeutic effect predictor of the alternative anticancer drug of the present invention is drug resistance acquired by use of the anticancer drug, in particular, various gene mutations appear in the epidermal growth factor receptor and the use of an epidermal growth factor receptor tyrosine kinase inhibitor such as gefitinib For the drug resistance acquired by C., by examining gene mutations, it is possible to predict what gene mutations have occurred and what treatments are effective. In addition, the therapeutic effect predictor of this alternative anticancer drug can reliably predict the efficacy of the alternative anticancer drug such as osimerinib to be selected next to the gene mutation reliably, accurately and accurately in a short time. .

  The therapeutic effect predictor of the alternative anticancer agent is administered to a patient who has developed resistance to the use of the alternative anticancer agent, and the radioisotope in the radiolabeled therapeutic effect predictor of the alternative anticancer agent is administered by positron emission tomography or It can be detected and imaged by a radiation detector such as single photon emission tomography, and can be used as an imaging probe which examines a local region or whole body and makes a diagnosis based on the image. Therefore, if the therapeutic effect prediction agent of this alternative anticancer agent is used, it substitutes in a tumor tissue, for example, a cancer cell in a primary tumor of a tumor, a metastatic cancer tissue or a metastatic tissue which has metastasized, or a neovasculature by a tumor. The effectiveness of the anticancer drug can be visually confirmed.

  Since the therapeutic effect predictor of this alternative anticancer drug is one labeled with a radioactive isotope without impairing the inherent efficacy and action mechanism of the alternative anticancer drug to be selected next, the pharmacological effect of the alternative anticancer drug, for example, the epithelium on the tumor tissue It is possible to predict efficacy with radioactive isotopes while competitively binding to ATP at the ATP binding site of growth factor tyrosine kinase and having epidermal growth factor receptor tyrosine kinase inhibitory action.

  According to this therapeutic effect predictor, since the drug which is an alternative anticancer agent can be directly labeled with a radioactive isotope to predict the efficacy, the expression or activity of Her2 or / and EGFR can be diagnosed, or the KRAS gene-derived nucleic acid or There is no need for the conventional complicated steps of examining the presence of the protein or measuring the level of amplification of the EGFR gene.

  According to this therapeutic effect predicting agent, the efficacy of treatment of alternative anticancer agents can be predicted on a whole body without any invasive or non-invasive biopsies, and the burden on cancer patients is extremely low, and it is safe and necessary. It is also possible to perform repeated inspections, which is highly useful.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.

The therapeutic effect predictor of the alternative anticancer agent of the present invention is a radioisotope such as radioactive iodine ( ^123I , ^124I , ^125I , ^131I ), a radioactive alternative to be selected next to the drug resistance of the alternative anticancer agent. Bromine ( ⁷⁶ Br, ⁷⁷ Br), radioactive fluorine ( ¹⁸ F), radioactive technetium ( ^{99 m} Tc), radioactive thallium ( ²⁰¹ Tl), radioactive gallium ( ⁶⁷ Ga), radioactive indium ( ¹¹¹ In), radioactive xenon ( ¹³³ Xe) Or radioactive carbon ( ¹¹ C) radionuclide containing and labeled.

Among them, as a radioactive isotope, ¹²³ I is a nuclide for SPECT with a half life of 13.2 hours, and ⁷⁶ Br is a nuclide for PET with a half life of 16.2 hours. As their substitute nuclides, ¹²⁵ I having a long half life and easy to handle is a nuclide having a half life of 59.4 days and ⁷⁷ Br having a half life of 57.0 hours.

  The therapeutic effect predictor of the alternative anticancer drug is an epidermal growth factor receptor tyrosine kinase inhibitor in which the alternative anticancer drug has developed drug resistance due to a gene mutation of the epidermal growth factor receptor, and the alternative anticancer drug for the drug resistance is selected next It is a candidate for another epidermal growth factor receptor tyrosine kinase inhibitor. In particular, as shown by the following chemical formula, the alternative anticancer agent is gefitinib (I) and the alternative anticancer agent is osimerinib (II), erlotinib (III), afatinib (IV), or rosiretinib (V), and in particular Is preferred.

  The therapeutic effect predictor of the alternative anticancer drug does not significantly change the chemical structure of the basic skeleton so as to maintain the affinity as the alternative anticancer drug itself as a lead compound, and easily and rapidly introduces a radioactive isotope such as radioactive halogen. It is

  The therapeutic effect predictor of the alternative anticancer agent is, for example, an epidermal growth factor receptor, wherein the alternative anticancer agent is a radioisotope such as radioactive halogen introduced at the 4th to 7th positions, preferably the 5th position of the indole ring, when osimerinib is used. The ability to release radiation can be secured while maintaining the efficacy of tyrosine kinase inhibition.

  Even in the case of an alternative anticancer drug other than osimerinib, the therapeutic effect predictor of the anticancer drug may be introduced into the aromatic ring or heterocyclic ring by introducing a radioactive isotope such as radioactive halogen, or may be labeled with a halogen such as fluorine or chlorine. The radiation emission ability can be secured while maintaining the efficacy of epidermal growth factor receptor tyrosine kinase inhibition by substituting and labeling with a radioactive isotope such as

  Both such alternative anticancer agents and alternative anticancer agents are those that competitively bind ATP to the ATP binding site of tyrosine kinase of epidermal growth factor receptor by gene mutation. Because the therapeutic effect largely changes depending on the type of gene mutation of epidermal growth factor receptor, the examination of gene mutation is essential in deciding the treatment policy, but if the therapeutic effect predictor of alternative anticancer drug is used, the alternative anticancer drug is effective Indirect detection of such gene mutations such as T790M secondary gene mutations to select an optimal therapeutic strategy to select a suitable alternative anticancer drug such as, for example, Osimertinib capable of responding to T790M secondary gene mutations It is also possible to make a decision.

For example, when the alternative anticancer agent is osimerinib and the alternative anticancer agent is a radioactive halogen substitute thereof, an indole substituted with a nonradioactive halogen at position 5 is used as the alternative anticancer agent, for example, if synthesized non-radioactive halogen substituents Oshimeruchinibu, a halogen group substituted with organotin N- chlorosuccinimide (NCS) radioactive iodide ions in the presence of ⁽¹²³ I ^- or ¹²⁵ I ^-) or radioactive bromine ion ⁽⁷⁶ Br ^- or ⁷⁷ Br ^-) is that substituting. Even if the alternative anticancer drug is another one, similarly, a non-radioactive halogen substitute is used as a starting material, a halogen-substituted alternative anticancer drug is synthesized, and finally a labeled anticancer drug is substituted by substituting a halogen group with a radioactive isotope. Can be synthesized as As a starting material, a radioactive isotope-substituted starting anticancer drug may be synthesized using a starting compound that is substituted with a radioactive isotope, and then the labeled anticancer agent may be synthesized, and then substituted or introduced into the radioactive isotope at the intermediate stage. A labeled surrogate anticancer drug may be synthesized.

When the therapeutic effect predictor of the alternative anticancer agent is a radioactive halogen-substituted osimerinib, for example, a labeled alternative anticancer agent can be synthesized as follows. Using 5-fluoro-2-nitrophenol as a raw material, its phenolic hydroxyl group is methylated, then the nitro group is reduced to an amino group, the 4-position is nitrated, the amino group is protected by a Boc group, and the fluorine group is N N, N-substitution with N, N'-trimethylethylenediamine, the nitro group is reduced to an amino group, the amino group is amidated with acroyl chloride, and Boc is deprotected to give a first intermediate. The 5-halogeno indole (eg 5-iodoindole or 5-bromoindole) is N-methylated and 2-chloropyrimidin-6-ylated with 2,4-dichloropyrimidine to give a second intermediate. The chloro group of this second intermediate is reacted with the primary amino group of the first intermediate, and the halogeno group of the indole ring is reacted with bis (tributyltin) to form a tin compound, and finally a radioactive halogen ion (eg ¹²³⁾ I ^{-, 125} I ^{-, 76} Br ^- or ⁷⁷ Br ^-) and tin reacted - by radioactive halogen exchange by combining the labeled alternative anticancer obtain the therapeutic effect prediction agent alternative anti-cancer agents.

  The therapeutic effect predicting agent of the alternative anticancer agent can be used for the use of an imaging diagnostic agent, and is administered as an orally administered agent or an injection.

  The therapeutic effect prediction agent of this alternative anticancer agent contains a radioactive isotope substituted and labeled alternative anticancer agent as an active ingredient, and, if necessary, a non-toxic inactive pharmaceutically acceptable excipient, for example, It is formulated by mixing with a solid, semi-solid or liquid diluent, dispersant, filler and carrier. Stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, flavor preservatives, medium, saline, and other medicinal effects The medicine which it has may be included as an additive.

  The dosage form of the therapeutic effect predictor of this alternative anticancer drug is, for example, elixir, capsule, granule, pill, ointment, suspension, solution, enteric agent, emulsion, plaster, suppository, powder, tablet, syrup These include agents, injections, troches, ointments, haptics, liniments, limonade agents, and lotions. It may be dissolved or suspended in a liquid medium, or may be dispersed in a solid medium.

  The therapeutic effect predicting agent of this alternative anticancer agent may be administered orally, may be administered by intravenous injection or infusion, or may be directly injected in the vicinity of a tumor tissue.

  In the therapeutic effect predictors of this alternative anticancer agent, the alternative anticancer agent which is substituted and labeled with a radioactive isotope is one that is labeled with a trace amount of a stoichiometric amount that does not show the pharmacological action of a tracer amount, and administered after purification. .

  The dose and dose of the therapeutic effect predicting agent of this alternative anticancer agent are the effectiveness of the active ingredient which is the alternative anticancer agent which is labeled and substituted with a radioactive isotope, the form and route of administration, the cancer development stage, the patient's body type and so on. It is appropriately selected according to the weight and age, and the type and amount of the therapeutic agent for other diseases to be used in combination.

  The therapeutic effect predictor of this alternative anticancer agent is used as follows. Cancer patients are administered drugs such as anti-cancer drugs, which are the first-line drugs. For example, in patients with inoperable or relapsed non-small cell lung cancer, non-small cells with structural change at the ATP binding site of epidermal growth factor receptor and local progression or metastasis of L858R mutation positive for epidermal growth factor receptor gene Gefitinib is given as the primary drug of choice for lung cancer. Gefitinib binds to the L858R mutant receptor as a molecular targeting drug, suppresses the signal transduction for proliferating cancer cells, and exhibits a high therapeutic effect, so it is highly safe and excellent in anticancer activity. However, the use of gefitinib causes additional structural changes in the ATP-binding site of the epidermal growth factor receptor, causing, for example, T790M secondary gene mutation, for several months to about five years (usually, one to one and a half years). ) Became resistant and gefitinib became ineffective. If T790M secondary gene mutation is caused, osimerinib can be used as a second line selective drug which is an alternative anticancer drug. In addition, if other secondary gene mutations are caused, erlotinib, afatinib or rosiretinib can be used as a secondary selection drug.

  Conventionally, it has been necessary to collect and confirm the tissue by biopsy to determine whether the alternative anticancer drug is effective or not, but according to the therapeutic effect predictor of the alternative anticancer drug of the present invention, it is necessary to perform the biopsy There is no problem, and diagnostic imaging can confirm whether or not the alternative anticancer drug is effective. The diagnostic method is as follows. The non-small cell lung cancer patients whose efficacy of the alternative anticancer agent is to be tested are administered intravenously, orally, directly to tumor tissue, and particularly preferably intravenously, for the therapeutic effect predicting agent of the alternative anticancer agent. Then, the therapeutic effect predictive agent radioactive isotope labeled osimeritinib is rapidly and systemically distributed through the blood, and the tumor tissue, for example, the cancer cell of the primary tumor of the tumor, a small cancer metastasis tissue that is metastasized Alternatively, it specifically binds to T790M secondary gene mutations on epidermal growth factor receptors of neovasculature by tumors or metastatic tissues that have metastasized. At this time, when the radioisotope radioactivity is imaged by positron emission tomography: PET or single photon emission tomography SPECT, whether the therapeutic effect predictor is accumulated in the tumor tissue, ie, the alternative anticancer agent is treated Whether or not an effect is exhibited can be visually diagnosed.

  The following is an example of preparing a therapeutic effect predicting agent of the alternative anticancer agent of the present invention and demonstrating its usefulness.

  First, Osimitinib, which is the second choice drug for gefitinib, the first choice drug, and the alternative anticancer drug that is the lead compound, and is a radioisotope labeled with radioactive isotope as the anticancer agent's therapeutic effect predictor, And synthesized.

The therapeutic effect predictor of the alternative anticancer drug is introduced without changing the basic skeleton of the chemical structure and replacing the radioactive isotope simply and quickly, as the alternative anticancer drug osimitinib itself as a lead compound and maintaining its affinity. , As a radioisotope-labeled alternative anticancer agent. As radioactive isotopes, ¹²³ I (nuclides for SPECT), ⁷⁶ Br it is preferable to use (PET nuclides), ¹²³ I, ⁷⁶ half-life as an alternative nuclide Br long handling easy ¹²⁵ I, ⁷⁷ Br The synthesis was performed using

  The synthesis of an alternative anticancer agent labeled with a radioactive isotope as a therapeutic effect predictor of the alternative anticancer agent was performed by the method shown in the following chemical reaction formulas [1] to [2]. Moreover, the therapeutic effect predictor obtained was used for the efficacy test. The details of the synthesis are as follows.

Example 1
(Compound (1): Synthesis of 4-Fluoro-2-methoxy-1-nitrobenzene (1))
Dissolve 5-fluoro-2-nitrophenol (5-fluoro-2-nitrophenol) (3.00 g, 19.1 mmol) in acetone (42.0 mL) and stir with ice-cooling under stirring with ice-cooling methyl iodide (2.37 mL, 38.2 mmol ) Was added dropwise and potassium carbonate (5.30 g, 38.2 mmol) was added. After stirring at room temperature for 50 hours, the solvent was evaporated under reduced pressure, dissolved in ethyl acetate, and washed with distilled water. The organic layer was washed with saturated brine and then dried over sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 3.00 g of a compound (1) (yield 94%).

(Compound (2): Synthesis of 4-Fluoro-2-methoxyaniline (2))
Compound (1) (1.10 g, 6.43 mmol) was dissolved in methanol (30.0 mL), and 10% Pd / C (55.0 mg) was added. After stirring under a hydrogen atmosphere at room temperature for 22 hours, the mixture was filtered through celite, and the filtrate was evaporated under reduced pressure to obtain 877 mg of compound (2) (yield: 97%).

(Compound (3): Synthesis of 4-Fluoro-2-methoxy-5-nitroaniline (3))
Compound (2) (551 mg, 3.90 mmol) is dissolved in dichloromethane (39.0 mL), concentrated sulfuric acid (1.85 mL) is added dropwise with stirring under ice-cooling, and then concentrated nitric acid (267 μL, 5.85 mmol) is added dropwise. did. After stirring for 3 hours under ice-cooling, saturated aqueous sodium bicarbonate was added until pH 8 was reached. The extract was washed with saturated aqueous sodium bicarbonate solution and then with saturated brine, and the organic layer was dried over sodium sulfate and evaporated under reduced pressure to give 675 mg of compound (3) (yield 93%).

(Compound (4): Synthesis of tert-Butyl (4-fluoro-2-methoxy-5-nitrophenyl) carbamate (4))
Compound (3) is reacted with (Boc) ₂ O in the presence of N, N-dimethyl-4-aminopyridine (N, N-dimetyl-4-aminopyridine) according to Chinese Patent Application Publication No. 104817541. Compound (4) was synthesized.

(Compound (5): Synthesis of tert-Butyl (4-((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) carbamate (5))
According to the specification of Chinese Patent Application No. 104817541, a compound (4) is reacted with N, N, N'-trimethylethylenediamine (N, N, N'-trimethylethylenediamine) in the presence of K ₂ CO ₃ to give a compound (5) Was synthesized.

(Compound (6): Synthesis of tert-Butyl (5-amino-4-((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) carbamate (6))
According to Chinese Patent Application Publication No. 104817541, compound (5) was catalytically reduced with H ₂ in the presence of Pd / C to synthesize compound (6).

(Compound (7): Synthesis of tert-Butyl (5-acrylamido-4-((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) carbamate (7))
The compound (6) was reacted with acryloyl chloride in the presence of N, N-diisopropylethylamine according to the specification of Chinese Patent Application No. 104817541, to synthesize a compound (7).

(Compound (8): Synthesis of N- (5-Amino-2-((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylate (8))
According to Chinese Patent Application Publication No. 104817541, compound (8) was synthesized by hydrolysis in the presence of trifluoroacetic acid.

(Compound (9): Synthesis of 5-Iodo-1-methyl-indole (9))
Powdered potassium hydroxide (1.85 g, 32.9 mmol) was dissolved in dimethylsulfoxide (DMSO) (26.9 mL) and 5-iodoindole (2.00 g, 8.23 mmol) was added while stirring. The mixture was stirred at room temperature for 30 minutes under a nitrogen atmosphere. After dropwise addition of methyl iodide (1.03 mL, 16.5 mmol) with stirring, the mixture was stirred at room temperature for 2 hours. 10.0 mL of distilled water was added and stirred for 1 hour. After extraction with diethyl ether, it was washed with distilled water and the organic layer was dried over sodium sulfate. The organic layer was evaporated under reduced pressure to obtain 2.20 g of an orange solid (from ¹ H NMR, compound 9: water = 1: 0.51, yield 100%).

(Compound (10): Synthesis of 5-Bromo-1-methyl-indole (10))
The synthesis of compound (10) was carried out in the same manner (yield: 96%) except that 5-Iodoindole in the synthesis of compound (9) was replaced with 5-bromoindole (5-Bromoindole).

(Compound (11): Synthesis of 3- (2-Chloropyrimidin-4-yl) -5-iodo-1-methyl-indole (11))
2,4-Dichloropyrimidine (1.47 g, 9.88 mmol) was dissolved in ethylene glycol dimethyl ether (7.60 mL), powdered aluminum chloride (659 mg, 4.94 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Compound (9) (1.27 g, 4.94 mmol) was added and stirred at 80 ° C. for 4 hours. After cooling to room temperature, 3.00 mL of distilled water was added dropwise while stirring. The mixture was extracted with ethyl acetate, washed with distilled water and saturated brine, and the organic layer was dried over sodium sulfate. The organic layer was evaporated under reduced pressure, redissolved in a small amount of dichloromethane, diethyl ether was gradually added, and the resulting crystals were collected to obtain 1.00 g of compound (11) (yield 55%).

(Compound (12): Synthesis of 5-Bromo-3- (2-chloropyrimidin-4-yl) -1-methyl-indole (12))
The synthesis of compound (12) was performed in the same manner (yield: 82%) except that compound (9) in the synthesis of compound (11) was replaced with compound (10).

(Compound (13): N- (2-((2- (Dimethylamino) ethyl) (methyl) amino) -5-((4- (5-iodo-1-methyl-indol-3-yl) pyrimidin-2 -yl) amino) -4-methoxyphenyl) acrylate (13) Synthesis)
The trifluoroacetate salt (1.20 g, 1.07 mmol) of the compound (8) was dissolved in 1-butanol (10.0 mL), and the compound 11 (395 mg, 1.07 mmol) and p-toluenesulfonic acid monohydrate (407 mg) were dissolved. , 2.14 mmol) was added. Stir at 100 ° C. for 4 hours. The mixture was cooled to room temperature, saturated aqueous sodium bicarbonate was added until pH 8 was reached, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried over sodium sulfate and evaporated under reduced pressure. Thereafter, the residue was subjected to silica gel chromatography using dichloromethane: methanol (10: 1) as an elution solvent to obtain 196 mg of a compound (13) (yield: 29%).
¹ H NMR (400 MHz, CDCl ₃ ) δ 2.12 to 2.41 (m, 8 H), 2.71 (s, 3 H), 2.82 to 2.99 (m, 2 H), 3.89 (s, 3 H), 3.98 (s) , 3 H), 5.65-5. 76 (m, 1 H), 6.22-6. 55 (m, 2 H), 6. 80 (s, 1 H), 7.12 (d, J = 5.6 Hz, 1 H), 7.17 (d, J = 8.8 Hz, 1 H), 7.53 (d, J = 8.4 Hz, 1 H), 7.74 (s, 1 H), 8. 37 (s, 1 H), 8. 40 (d, J = 5.2 Hz, 1 H), 9.10 (s, 1 H), 9. 82 (s, 1 H), 10. 17 (s, 1 H).
MS (ESI +) (calcd for C 28 H 33 IN 7 O 2 [M + H] +): m / z = 626.2 found: 626.3.

(Compound (13): N- (5-((4- (5-Bromo-1-methyl-indol-3-yl) pyrimidin-2-yl) amino) -2-((2- (dimethylamino) ethyl) Synthesis of (methyl) amino) -4-methoxyphenyl) acrylate (14)
Compound (8) (20.0 mg, 68.0 μmol) is dissolved in 1-butanol (680 μL), compound (12) (22.0 mg, 68.0 μmol) and p-toluenesulfonic acid monohydrate (25.9 mg, 136 μmol) Added. Stir at 100 ° C. for 4 hours. The mixture was cooled to room temperature, saturated aqueous sodium bicarbonate was added until pH 8 was reached, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried over sodium sulfate and evaporated under reduced pressure. Thereafter, the residue was subjected to silica gel chromatography using dichloromethane: methanol (10: 1) as an elution solvent to obtain 19.7 mg of a compound (13) (yield 50%).
¹ H NMR (400 MHz, CDCl ₃ ) δ 2.27 (s, 6 H), 2.30 (t, J = 5.6 Hz, 2 H), 2.70 (s, 3 H), 2. 90 (t, J = 5.6 Hz, 2 H), 3.89 (s, 3 H), 3.97 (s, 3 H), 5.70 (dd, J = 8.4, 2.8 Hz, 1 H), 6.34-6.50 (m, 2 H), 6.80 (s, 1 H), 7.11 (d, J = 4.8 Hz, 1 H), 7.24 (d, J = 8.8 Hz, 1 H), 7.35 (d, J = 8.8 Hz, 1 H), 7.74 (s, 1 H), 8.17 (s , 1 H), 8.39 (d, J = 4.8 Hz, 1 H), 9.08 (s, 1 H), 9.81 (s, 1 H), 10.15 (s, 1 H).
MS (ESI +) (calcd for C 28 H 33 BrN 7 O 2 [M + H] +): 578.2, 580.2 found: m / z = 578.3, 580.3.

(Compound (15):
N- (2-((2- (Dimethylamino) ethyl) (methyl) amino) -4-methoxy-5-((4- (1-methyl-5- (tributylstannyl) -indol-3-yl) pyrimidin-2 Compound 13 (89.4 mg, 143 μmol) was dissolved in anhydrous dioxane (7.15 mL), and bis (tributyltin) (182 mg, 314 μmol) and bis (triphenylphosphine) were dissolved. ) Palladium (II) dichloride (20.1 mg, 28.6 μmol) was added. The mixture was stirred at 60 ° C. for 6 hours under a nitrogen atmosphere. The solvent was distilled off under reduced pressure, the residue was redissolved in methanol, and purified by reverse phase HPLC to obtain 7.20 mg of compound (15) (yield: 6.4%). Reversed-phase HPLC is a gradient method using a Cosmosil 5C _18- MS (20 × 250 mm) column and converting methanol: water = 95: 5 to 0.05 in 20 minutes into 100: 0 using triethylamine: It carried out on the conditions of flow volume 18 mL / min.
¹ H NMR (400 MHz, CDCl ₃ ) δ 0.90 (t, J = 7.2 Hz, 9 H), 1.12 (t, J = 8.4 Hz, 6 H), 1.31-1.43 (m, 6 H), 1.52-1.66 (m, 6 H), 2.23-2. 30 (m, 8 H), 2.71 (s, 3 H), 2. 89 (t, J = 6.0 Hz, 2 H), 3. 89 (s, 3 H), 3.99 (s, 3 H), 5.70 (dd, J = 9.6, 1.6 Hz, 1 H), 6.31-6.51 (m, 2 H), 6.80 (s, 1 H), 7.20 (d, J = 5.6 Hz, 1 H), 7.34 ( d, J = 8.0 Hz, 1 H, 7. 40 (d, J = 8.0 Hz, 1 H), 7.73 (s, 1 H), 8. 14 (s, 1 H), 8. 40 (d, J = 5.2 Hz, 1 H), 9.09 (s, 1 H), 9.86 (s, 1 H), 10.18 (s, 1 H).
MS (ESI +) (calcd for C 40 H 59 N 7 O 2 Sn [M + H] +): m / z = 790.4 found: 790.5.

(Compound (16):
N- (2-((2- (Dimethylamino) ethyl) (methyl) amino) -4-methoxy-5-((4- (1-methyl-5- ( ¹²⁵ iodo) -indol-3-yl) pyrimidin- Synthesis of 2-yl) amino) phenyl) acrylamide (16) Compound (15) (50.0 μg, 63.4 nmol) is dissolved in acetonitrile (5 μL), and acetonitrile containing 1% of acetic acid (10 μL) is added. After adding [ ¹²⁵ I] NaI solution (2 μL), NCS / acetonitrile (1 mg / mL, 15 μL) was added. After stirring for 15 min at 80 ° C., the reaction was stopped by adding NaHSO ₃ / H ₂ O (1 mg / mL, 15 μL). By purification by reverse phase HPLC, radioisotope-labeled compound (16) was synthesized as a ¹²⁵ I-labeled alternative anticancer agent therapeutic effect predictor.
Reversed phase HPLC analysis and purification of the ¹²⁵ I-substituted therapeutic efficacy predictor using a Cosmosil 5C ₁₈ -MS-II (4.6 x 150 mm) column, mobile phase 0.05% triethylamine containing methanol and 0.05% triethylamine The reaction was carried out at a flow rate of 1.0 mL / min and a temperature of 40 ° C. in a gradient method of 0 to 20 min with water containing triethylamine at 80:20 to 100: 0, and the radiochemical yield was 43.6%. The radiochemical purity was 98.1%. Also, ¹²⁵ I-labeled compound (16) and ¹²⁵ I unlabeled compound of I substituents compared to analysis by reverse phase HPLC (13), each single elution peak is detected at the same retention time, compound It was confirmed to be (16).

In addition, it is possible to synthesize | combine similarly the compound (17) of a < ⁷⁷ > Br label | marker.

(Stability test)
As physical property evaluation of ¹²⁵ I therapeutic effect predicted agent, compound labeled (16), a 0.1M phosphate buffer (pH 7.4) with ¹²⁵ I-labeled therapeutic effect prediction agent Eppendorf tube was added ethanol 4μL of 46μL In addition, the stability in buffer was evaluated by incubating at 37 ° C. and analyzing after 24 hours. After 24 hours, 83.5 ± 3.6% was observed as unchanged.

(Distribution coefficient measurement)
^As evaluation of physical properties of compound (16), which is a therapeutic effect predictor of ¹²⁵ I labeling, therapeutic effect predictor of ¹²⁵ I labeling in a test tube to which 3 mL of 1-octanol and 0.1 M phosphate buffer (pH 7.4) were added Was added, and after vortexing for 1 minute, the operation of standing at room temperature for 15 minutes was repeated three times, and centrifuged at 1,000 g for 5 minutes. A 2.4 mL portion of the 1-octanol layer was collected and added to a test tube with 2.4 mL of 0.1 M phosphate buffer (pH 7.4). After vortexing for 1 minute, the procedure of standing at room temperature for 15 minutes was repeated three times, and centrifuged at 1,000 g for 5 minutes. The distribution coefficient was determined by collecting 2 mL each from each layer and measuring the radioactivity. The 1-octanol / water partition coefficient log P value was 2.64 ± 0.11. As a result, it was found that the present labeled compound (16) had sufficient lipid solubility for cell membrane permeation.

In a 6-well plate, use NSCLC cell line H1975 (L858R, T790M double-mutant EGFR) in DMEM / F12 medium with 10% FBS, so that the number of cells is 5 × 10 ⁵ / well, under CO ₂ 5% conditions 24 The culture was carried out at 37 ° C. for a time. Compound (16) dissolved in DMSO was added to serum-free DMEM / F12 medium to a DMSO concentration of 1% to adjust the medium with compound (16). The cells were washed with PBS and then incubated for 15, 30, 60 and 120 minutes using medium containing compound (16). After incubation, the medium was removed, washed twice with PBS / 1% DMSO / 0.1% Tween 80, the cells were lysed with 1 M NaOH, the cells were collected, and the radioactivity was measured. Thereafter, the amount of protein contained in the cell lysate was quantified with a protein assay bicinchoninic acid kit (nacalai tesque), and the amount of uptake was corrected. The% dose / μg protein reached a plateau at 30 minutes of incubation and the value was 0.824 ± 0.11. As a result, it was found that the compound (16) was rapidly taken up into cells.

(Tumor accumulation of compound (16) in cancer transplanted mice)
NSCLC cell line H3255 (L858R mutant EGFR) is cultured under 10% FBS-containing DMEM / F12 medium under CO ₂ 5% conditions, and the number of cells is 1 × 10 ⁷ / animal: 4 nude mice (4 weeks old, female) , BALB / c Slc-nu / nu (12-17 g; Japan SLC)) were implanted subcutaneously in the right back. Thirteen days later, NSCLC cell line H1975 (L858R, T790M double-mutant EGFR) was implanted at a cell number of 5 × 10 ⁶ / mouse into the left dorsal subcutaneous. Nine days later, a saline solution (74 kBq, 100 μL) containing 10% ethanol and 1% Tween 80 of compound (16) was administered to the tail vein. As a result of sacrificing 1 hour after administration, excising each tumor and measuring each weight and radioactivity,% dose / g was 3.47 ± 0.62 in H1975 and 2.38 ± 0.61 in H3255. As a result, it was found that the compound (16) had higher accumulation in H1975 (L858R, T790M double-mutant EGFR) in vivo than H3255 (L858R mutant EGFR).

  The therapeutic efficacy predictor of the alternative anticancer drug of the present invention is against the treatment with an anticancer drug which has acquired drug resistance in response to genetic mutation of epidermal growth factor receptor on tumor tissue in non-small cell lung cancer patients by using gefitinib, The effectiveness of alternative anticancer agents that can be used for the next alternative treatment can be predicted with certainty. By using the therapeutic effect predicting agent of this alternative anticancer drug, it is possible to quickly diagnose whether or not the drug is effective as an alternative therapeutic drug for a patient who has developed tolerance, and to decide on a sure course of treatment.

Claims (9)

  An agent for predicting the therapeutic effect of an alternative anticancer agent, wherein the alternative anticancer agent to be selected for drug resistance is one labeled with a radioactive isotope.   The agent for predicting the therapeutic effect of the alternative anticancer agent according to claim 1, wherein the alternative anticancer agent to be selected for drug resistance of the substituted anticancer agent is one labeled with a radioactive isotope.   3. The alternative anticancer agent according to claim 2, wherein the alternative anticancer agent and the alternative anticancer agent are each an epidermal growth factor receptor tyrosine kinase inhibitor against drug resistance due to a gene mutation of an epidermal growth factor receptor. Therapeutic effect predictor.   The agent for predicting the therapeutic effect of the alternative anticancer agent according to any one of claims 2 to 3, wherein the alternative anticancer agent is gefitinib and the alternative anticancer agent is ocimertinib, erlotinib, afatinib, or rosiretinib.   The alternative anticancer agent is one labeled with the radioactive isotope substituted at the aromatic ring or heteroaromatic ring, or one substituted with the radioactive isotope protected by a protective group The therapeutic effect predictor of the alternative anticancer agent according to claim 4, characterized in that   The radioactive isotope is characterized in that it contains any radioactive nuclide selected from radioactive iodine, radioactive bromine, radioactive fluorine, radioactive technetium, radioactive thallium, radioactive gallium, radioactive indium, radioactive xenon, and radioactive carbon. The therapeutic effect predictor of the alternative anticancer agent according to any one of claims 1 to 5. The above-mentioned alternative anticancer drug is osimerinib, which has the following chemical formula (I)

(In formula (I), X is the above-mentioned radioactive isotope and contains any radionuclide selected from ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ⁷⁶ Br, ⁷⁷ Br, and ¹⁸ F) The agent for predicting the therapeutic effect of the alternative anticancer agent according to any one of claims 1 to 6, which is represented by   It is a diagnostic imaging agent, The therapeutic effect predictor of the alternative anticancer agent in any one of the Claims 1-7 characterized by the above-mentioned.   The agent for predicting the therapeutic effect of the alternative anticancer agent according to any one of claims 1 to 8, which is an orally administered agent or an injection.