KR102695469B1 - Composition comprising 2,4-diaminopyrimidine for preventing or treating cancer - Google Patents

Abstract

A composition for preventing or treating cancer comprising 2,4-diaminopyrimidine or a derivative thereof, wherein the 2,4-diaminopyrimidine selectively inhibits the ANO1 calcium-activated chloride ion channel, thereby being useful as a therapeutic agent for various cancers including lung cancer.

Description

Composition comprising 2,4-diaminopyrimidine for preventing or treating cancer

It relates to a composition for preventing or treating cancer containing 2,4-diaminopyrimidine.

Calcium-activated chloride channels (CaCCs) are anion channels that are activated by increases in intracellular calcium ion concentration. Endogenous CaCCs play a role in epithelial transport, regulation of neuronal and cardiac muscle cell excitability, sensory transmission, and blocking polyspermy in Xenopus oocytes.

Anoctamin 1 (ANO1) is expressed in various cell types, such as non-excitable epithelial and endothelial cells, smooth muscle cells, sensory neurons, and interstitial cells of Cajal. The wide distribution of ANO1 plays an important role in many physiological processes and has been implicated in the pathophysiology of various diseases, such as hypertension and asthma. ANO1 is overexpressed in numerous tumor cells. In fact, ANO1 (TMEM16A) is known to be located on human chromosome 11q13 and is frequently amplified in various types of malignant tumors. Since ANO1 is involved in tumorigenesis, invasion, migration, and metastasis, regulating ANO1 is very important in the development of cancer therapeutics. The conventional ANO1 current was induced by niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid [5-nitro-2-(3-phenylpropylamino)-benzoic acid NPPB], and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid [4,4 ′'-stilbenedisulfonic acid (DIDS) and other CaCC inhibitors. However, these compounds lack potency and specificity and cannot be considered as drug candidates. Recently, ANO1 inhibitors such as CaCCinh-A01, T16Ainh-A01, tannic acid, idebenone, benzbromarone, and Ani9, which were identified through high-throughput screening (HTS) of compound libraries, were found to inhibit cancer cell proliferation. However, further development is needed because the information on the druggability and drug-likeness of these compounds is in the early stage.

Meanwhile, pyrimidine is a nitrogen-containing heterocyclic compound with various pharmacological activities including anticancer, anti-inflammatory, anti-HIV, antihypertensive, antidiabetic, and antimicrobial activities. Since the pyrimidine scaffold is a component of DNA and RNA, from the perspective of major medicinal chemistry, pyrimidine derivatives have various therapeutic applications for drug discovery. In particular, many FDA-approved drugs and medicines currently under development contain pyrimidine as their core structure. Therefore, pyrimidine is continuously considered as an essential building block and special scaffold for a wide range of drug candidates.

Therefore, there is a need to develop pyrimidine derivatives with anticancer activity by inhibiting ANO1.

One aspect is to provide a calcium-activated chloride channels (CaCCs) activity inhibitor comprising 2,4-diaminopyrimidine or a derivative thereof as an active ingredient.

Another aspect is to provide a composition for preventing or treating cancer comprising 2,4-diaminopyrimidine or a derivative thereof as an active ingredient.

One aspect provides a calcium-activated chloride channels (CaCCs) activity inhibitor comprising 2,4-diaminopyrimidine of the following chemical formula 1 or a derivative thereof as an active ingredient:

[Chemical Formula 1]

Here, X and Y are the same or different from each other, and are each independently N, O or Se, or substituted phenyl amine, or substituted phenoxy,

R ₁ is alkyl having C1 to C5,

R ₂ and R ₃ are the same or different, and are each independently a C1 to C8 alcohol, a C1 to C5 alkyl ether, or a C1 to C5 aryl ether, a C1 to C5 alkoxy, morpholinyl, phenyl amine, phenoxy, or phenoxy substituted with a C1 to C5 alkyl or halide. The phenylamine or phenoxy may be substituted at the o (ortho), m (meta) and p (para)-positions.

Another aspect provides a method for inhibiting the activity of calcium-activated chloride channels (CaCCs), comprising a step of inhibiting the activity of calcium-activated chloride channels (CaCCs) in vitro by treating 2,4-diaminopyrimidine of the above chemical formula 1 or a derivative thereof.

In this specification, the term "chloride channels" refers to ion channels that regulate the electrical excitability of nerve cells or muscle cells by controlling the movement of chloride ions within the cell, and are essential ion channels involved in the secretion of water in epithelial cells by exporting chloride ions out of the cells.

In this specification, the term "calcium-activated chloride channels (CaCCs)" refers to a chloride ion channel activated by intracellular calcium (Ca ²⁺ ), which is a major ion channel that regulates secretion in epithelial cells or excitability of nerve cells. The calcium-activated chloride ion channel may be, for example, Anoctamin 1 (ANO1). The 2,4-diaminopyrimidine or a derivative thereof may represent a positional isomer having an oxygen-containing alkyl and a p-substituted diaryl ether substituent at the C2 and C4 positions of the pyrimidine. In one specific example, the R ₁ or R ₂ is , , , , , , , , , , and It may be selected from the group consisting of .

In one specific example, the 2,4-diaminopyrimidine or a derivative thereof is N4-(2-methoxyethyl)-6-methyl-N2-(4-(phenylamino)phenyl)pyrimidine-2,4-diamine, 5-((2-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-4-yl)amino)pentan-1-ol, 5-((6-methyl-2-((4-(p-tolyloxy)phenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol, 4-((4-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-2-yl)amino)butan-1-ol, etc. 2,4-diaminopyrimidine or a derivative thereof may be, for example, a compound represented by the following chemical formula 2:

[Chemical formula 2]

.

In one embodiment, it was confirmed that 2,4-diaminopyrimidine or a derivative thereof inhibits the activity of ANO1, a calcium-activated chloride ion channel, in a concentration-dependent manner without inducing the synthesis or degradation of ANO1 protein. In addition, the anticancer activity was confirmed by reducing the cell viability of lung cancer cell lines expressing ANO1 protein.

Therefore, 2,4-diaminopyrimidine or a derivative thereof according to one aspect can selectively inhibit only the physiological activity of the ANO1 chloride ion channel, and can also exhibit anticancer activity against cancer expressing the ANO1 protein, and thus can be used as an activity inhibitor of the calcium-activated chloride ion channel or a cancer treatment agent.

Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising 2,4-diaminopyrimidine of the above chemical formula 1 or a derivative thereof as an active ingredient.

In addition, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a composition comprising 2,4-diaminopyrimidine of the above chemical formula 1 or a derivative thereof.

The cancer may be, for example, breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, stomach cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's Disease, endocrine cancer, sarcoma, etc.

In addition, a pharmaceutical composition for preventing or treating a disease is provided, which contains 2,4-diaminopyrimidine of the chemical formula 1 or a derivative thereof as an active ingredient.

In addition, a method for treating a disease is provided, comprising administering to a subject in need thereof a composition comprising 2,4-diaminopyrimidine of the above chemical formula 1 or a derivative thereof:

The above diseases may be, for example, Psoriasis, Chronic Myeloproliferative Disease, Lymphoproliferative Disease, Endometrial hyperplasia, Benign Prostate Hyperplasia, etc.

The pharmaceutical composition for preventing or treating cancer according to the aspect can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods, and may contain appropriate carriers, excipients, or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or excipient or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

Solid preparations for oral administration can be prepared by mixing the 2,4-diaminopyrimidine or a derivative thereof with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerol gelatin.

The preferred dosage of the pharmaceutical composition for preventing or treating cancer according to the aspect varies depending on the patient's condition, weight, degree of disease, drug form, administration route and period, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it can be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. The administration can be administered once a day or divided into several times. However, the scope of the present invention is not limited by the above dosage.

The pharmaceutical composition for preventing or treating cancer according to the aspect can be administered to mammals such as rats, mice, livestock, and humans by various routes. All the modes of administration can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine epidural, or intracerebroventricular injection.

Another aspect provides a health functional food composition for preventing or improving cancer, comprising 2,4-diaminopyrimidine of the chemical formula 1 as an active ingredient.

In addition, a health functional food composition for preventing or improving a disease, which contains 2,4-diaminopyrimidine of the chemical formula 1 as an effective ingredient, is provided.

The specific details of the 2,4-diaminopyrimidine or its derivatives, cancer and diseases are as described above.

In the health functional food composition for improving cancer according to one aspect, when the 2,4-diaminopyrimidine or its derivative is used as an additive to the health functional food, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the effective ingredients can be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

The formulation of health functional foods can be in the form of powders, granules, pills, tablets, capsules, or in the form of general food or beverages.

There is no particular limitation on the type of the above food, and examples of foods to which the above substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense can be included.

In general, when manufacturing food or beverage, the 2,4-diaminopyrimidine or a derivative thereof can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range. Among the health functional foods according to one aspect, beverages may contain various flavoring agents or natural carbohydrates as additional ingredients, like ordinary beverages. The natural carbohydrates described above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame may be used. The proportion of the above natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food composition for preventing or improving cancer according to one aspect may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the sleep improving composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food of the present invention.

Compounds according to the present invention may be used as therapeutic agents for various cancers, including lung cancer, by selectively inhibiting the ANO1 calcium-activated chloride ion channel.

Figure 1 is a diagram illustrating a design strategy for constructing a pyrimidine library.
Figure 2 is a diagram showing the synthetic procedure of the pyrimidine library.
Figure 3a is a graph confirming the activity inhibition effect of ANO1 at a concentration of 50 μM for 19 candidate compounds.
Figure 3b is a graph confirming the activity inhibition effect of ANO1 at a concentration of 30 μM for 15 candidate compounds.
Figure 4a shows the results of evaluating the cytotoxicity of three candidate compounds in lung cancer cells.
Figure 4b shows the results of evaluating cytotoxicity in lung cancer cell lines by compounds according to one aspect.
Figure 5a is a graph confirming the concentration-dependent ANO1 activity inhibitory effect of a compound according to one aspect.
Figure 5b is a graph confirming the IC50 of compounds according to the daily aspect.
Figure 6 is a graph confirming the inhibitory effect of compounds according to one aspect on the activity of ANO1 induced by A23187.
Figure 7a shows the results of confirming the expression of ANO1 protein in lung cancer cell lines.
Figure 7b is a graph showing the cytotoxicity of compounds according to one aspect in lung cancer cell lines showing differences in the expression of ANO1.
Figure 7c shows the results of confirming the expression of ANO1 protein by compounds according to one aspect.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Establishing the theoretical basis for compound design and initial library synthesis

Recently, a series of novel ANO1 inhibitors based on pyrimidine scaffolds were identified through in-house library screening. In preliminary screening assays, two hit compounds 1 and 2 containing 2,4-disubstituted-6-methylpyrimidine scaffolds with moderate inhibitory activity against ANO1 were obtained. The hit compounds were evaluated through HTS campaigns by halide-sensitive yellow fluorescent protein (YFP) imaging technology. Based on these results, pyrimidine analogues derived from the structures of hits 1 and 2 were designed and synthesized.

Figure 1 is a schematic diagram summarizing the design and synthesis strategy for novel ANO1 blockers.

As shown in Figure 1, the structures of hit compounds 1 and 2 have an oxygen-containing alkyl and a p -substituted di-aryl ether substituent in common, but exhibit the form of positional isomers having substituents at the C2 and C4 positions.

Figure 2 is a diagram showing the synthetic procedure of the pyrimidine library.

As shown in Figure 2, a library of heterocyclic compounds containing pyrimidine scaffolds was constructed by preparing the mono-substituted regioisomers of types A and B. All 2,4-disubstituted pyrimidine derivatives were prepared via a systematic combinatorial approach in a straightforward manner. As described above, hit compounds 2 have a common p -substituted aniline moiety, but with a longer alkyl side chain compared to the carbon length of hit compound 1. Thus, 2,4-disubstituted pyrimidines were synthesized by a two-step approach using five alkyl amines with different carbon lengths and six p-substituted anilines with various chemical substituents, as shown in Table 1 below.

[Table 1]

Example 2. Synthesis of 2,4-disubstituted pyrimidines

After synthesizing a mono-substituted pyrimidine according to the two-step approach of Example 1, a di-substituted pyrimidine was synthesized.

2-1. Synthesis of mono-substituted pyrimidines

2,4-dichloro-6-methylpyrimidine (1.00 g, 6.13 mmol) was dissolved in 20 mL of ethanol (EtOH) at room temperature, to which was added 2-methoxyethylamine (0.96 mL, 11.0 mmol, 1.8 equiv) and triethylamine (1.71 mL, 12.3 mmol, 2.0 equiv). The reaction mixture was heated to 50 °C and stirred for 4 h. The resulting mixture was cooled to room temperature and the solvent was removed under reduced pressure. Then, 100 mL of ethyl acetate (EtOAc) was added to the reaction mixture, which was washed twice with 10 mL of water. The organic layer was then dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-hexane/EtOAc = 3:1 to 2:1) to obtain 4-chloro-N-(2-methoxyethyl)-6-methylpyrimidin-2-amine [4-Chloro-N-(2-methoxyethyl)-6-methylpyrimidin-2-amine] (hereinafter, Aa) (280 mg, 23%) and 2-chloro-N-(2-methoxyethyl)-6-methylpyrimidin-4-amine [2-Chloro-N-(2-methoxyethyl)-6-methylpyrimidin-4-amine] (hereinafter, Ba) (540 mg, 47%).

2-2. Synthesis of di-substituted pyrimidines

To a stirred solution of Aa (100 mg, 0.496 mmol) and 4-aminodiphenylamine (183 mg, 0.992 mmol, 2.0 equiv) in n-BuOH (4 mL) was added trimethylsilyl chloride (TMSCl) (5 drops) at room temperature. The reaction mixture was heated to reflux and stirred overnight. The resulting mixture was then cooled to room temperature and the solvent (n-BuOH) was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM/MeOH = 20:1 to 10:1) to obtain N4-(2-methoxyethyl)-6-methyl-N2-(4-phenylamino)phenyl)pyrimidine-2,4-diamine [N4-(2-methoxyethyl)-6-methyl-N2-(4-(phenylamino)phenyl)pyrimidine-2,4-diamine] (hereinafter, Aa3) (280 mg, 81%).

Example 3. Analysis of physicochemical properties

The physicochemical properties of the monosubstituted pyrimidine compounds synthesized in Example 2-1 were analyzed. Specifically, ¹ H and ¹³ C spectra were measured with JEOL-500 (JEOL, Tokyo, Japan), and the chemical shifts were expressed in ppm. High-resolution mass spectra were obtained using a Q Exactive Mass Spectrometer (Thermo Scientific, Waltham, MA, USA).

3-1. 4-Chloro-N-(2-methoxyethyl)-6-methylpyrimidin-2-amine: Aa

(1) White solid;

(2) Melting point: 40-42 ℃;

(3) ¹ H-NMR (500 MHz, CDCl ₃ )

δ 6.40 (s, 1H), 5.64 (brs, 1H), 3.57 (q,　J　= 5.3 Hz, 2H), 3.50 (t,　J　= 5.2 Hz, 2H), 3.32 (s, 3H), 2.26 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 169.6, 162.2, 161.2, 109.3, 71.2, 58.8, 41.2, 23.9;

(5) HRMS (ESI+)

202.0747 (calculated for C ₈ H ₁₂ ClN ₃ O ([M + H] ⁺ ): 202.0742).

3-2. 2-Chloro-N-(2-methoxyethyl)-6-methylpyrimidin-4-amine: Ba

(1) Colorless oil;

(2) ¹ H-NMR (500 MHz, CDCl ₃ )

δ 6.09 (s, 1H), 5.38 (brs, 1H), 3.56-3.50 (m, 4H), 3.36 (s, 3H), 2.31 (s, 3H);

(3) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 167.1, 164.1, 160.1, 103.3, 70.6, 58.8, 40.9, 23.6;

(4) HRMS (ESI+)

202.0747 (calculated for C ₈ H ₁₂ ClN ₃ O ([M + H] ⁺ ): 202.0742).

3-3. 2-((2-chloro-6-methylpyrimidin-4-yl)amino)ethan-1-ol: Ab

(1) White solid;

(2) Melting point: 130-132 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.73 (s, 1H), 6.25 (s, 1H), 4.71 (s, 1H), 3.46 (t, J = 6.0 Hz, 2H), 3.30 (brs, 2H), 2.11 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 165.4, 164.6, 159.9, 103.3, 60.2, 43.2, 23.3.

3-4. 2-((4-chloro-6-methylpyrimidin-2-yl)amino)ethan-1-ol: Bb

(1) White solid;

(2) Melting point: 100-102 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.35 (s, 1H), 6.51 (s, 1H), 4.40 (s, 1H), 3.44 (t, J = 6.3 Hz, 2H), 3.27 (brs, 2H), 2.19 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 170.1, 162.5, 160.4, 108.3, 60.2, 43.9, 23.7.

3-5. 3-((2-chloro-6-methylpyrimidin-4-yl)amino)propan-1-ol: Ac

(1) White solid;

(2) Melting point: 118-120 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.66 (t,　J　= 3.2 Hz, 1H), 6.20 (s, 1H), 4.45 (s, 1H), 3.41 (q,　J　= 5.9 Hz, 2H), 3.25-3.24 (m, 2H), 2.11 ( s, 3H), 1.60 (quint.,　J　=6.7 Hz, 2H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 165.4, 164.4, 160.0, 103.2, 58.8, 37.7, 32.3, 23.3.

3-6. 3-((4-chloro-6-methylpyrimidin-2-yl)amino)propan-1-ol: Bc

(1) White solid;

(2) Melting point: 76-78 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.44 (s, 1H), 6.49 (s, 1H), 4.41 (t,　J　= 5.2 Hz, 1H), 3.40 (q,　J　= 5.9 Hz, 2H), 3.24 (brs, 2H), 2.19 (s, 3H), 1.61 (quint.,　J　=6.7 Hz, 2H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 170.1, 162.5, 160.3, 108.1, 59.0, 38.5, 32.4, 23.7.

3-7. 4-((2-chloro-6-methylpyrimidin-4-yl)amino)butan-1-ol:　Ad

(1) White solid;

(2) Melting point: 82-84 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.68 (s, 1H), 6.19 (s, 1H), 4.38 (t,　J　= 4.9 Hz, 1H), 3.36 (q,　J　= 5.7 Hz, 2H), 3.21-3.20 (m, 2H), 2.11 ( s, 3H), 1.48 (quint., J = 7.0 Hz, 2H), 1.41 (quint., J = 6.6 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 165.4, 164.4, 160.0, 103.1, 60.9, 41.7, 30.3, 25.8, 23.3.

3-8. 4-((4-chloro-6-methylpyrimidin-2-yl)amino)butan-1-ol: Bd

(1) White solid;

(2) Melting point: 68-70 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.47 (s, 1H), 6.47 (s, 1H), 4.34 (t,　J　= 4.9 Hz, 1H), 3.36 (q,　J　= 5.9 Hz, 2H), 3.19 (brs, 2H), 2.18 (s, 3H), 1.48 (quint., J = 7.0 Hz, 2H), 1.40 (quint., J = 6.9 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 170.1, 162.6, 160.4, 108.1, 61.2, 41.0, 30.4, 25.9, 23.7.

3-9. 5-((2-chloro-6-methylpyrimidin-4-yl)amino)pentan-1-ol:　Ae

(1) White solid;

(2) Melting point: 88-90 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.67 (s, 1H), 6.19 (s, 1H), 4.32 (t,　J　= 4.9 Hz, 1H), 3.34 (q,　J　= 5.9 Hz, 2H), 3.19 (q,　J　= 5.2 Hz, 2H) , 2.11 (s, 3H), 1.45 (quint., J = 7.3 Hz, 2H), 1.39 (quint., J = 7.2 Hz, 2H), 1.45 (quint., J = 7.6 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 165.3, 164.4, 160.0, 103.1, 61.1, 41.4, 32.6, 28.9, 23.4.

3-10. 5-((4-chloro-6-methylpyrimidin-2-yl)amino)pentan-1-ol:　Be

(1) White solid;

(2) Melting point: 128-130 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 7.47 (s, 1H), 6.48 (s, 1H), 4.30 (t,　J　= 4.9 Hz, 1H), 3.34 (q,　J　= 5.7 Hz, 2H), 3.17 (brs, 2H), 2.18 (s, 3H), 1.45 (quint.,J=7.3 Hz, 2H), 1.39 (quint.,J=7.0 Hz, 2H), 1.25 (quint.,J=7.6 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 170.1, 162.6, 160.2, 108.1, 61.1, 41.1, 32.7, 29.1, 23.7, 23.2.

Example 4. Analysis of physicochemical properties

The physicochemical properties of the 2-substituted pyrimidine compound synthesized in Example 2-2 were analyzed using the same method as in Example 3.

4-1. N ² -(4-isopropoxyphenyl)-N ⁴ -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine[N ² -(4-isopropoxyphenyl)-N ⁴ -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine]:　Aa1

(1) Yield: 75%;

(2 ) Pale brown oil;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.68 (s, 1H), 7.59 (d, J = 8.6 Hz, 2H), 6.70 (s, 1H), 6.73 (dt, J = 8.6, 2.6 Hz, 2H), 5.74 (s, 1H), 4.44 ( quint.,　J　= 6.0 Hz, 1H), 3.42 (brs, 4H), 3.23 (s, 3H), 2.05 (s, 3H), 1.19 (d,　J　= 6.3 Hz, 6H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.0, 151.9, 135.2, 120.3, 116.2, 95.7, 71.1, 69.9, 58.5, 40.3, 23.8, 22.4;

(5) HRMS (ESI+)

317.1978 (calculated for C ₁₇ H ₂₅ N ₄ O ₂ ([M + H] ⁺ ): 317.1968).

4-2. N4-(2-methoxyethyl)-6-methyl-N2-(4-morpholinophenyl)pyrimidine-2,4-diamine[N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-morpholinophenyl)pyrimidine-2,4-diamine]:　Aa2

(1) Yield: 54%;

(2) dark brown solid;

(3) Melting point: 195-197 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.65 (s, 1H), 7.61 (d, J = 9.2 Hz, 2H), 6.96 (brs, 1H), 6.78 (d, J = 9.2 Hz, 2H), 5.75 (s, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.43 (m, 4H), 3.23 (d, J = 11.4 Hz, 3H), 2.94 (t, J = 4.6 Hz, 2H), 2.06 (s, 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.1, 145.8, 134.7, 120.0, 116.1, 95.7, 71.2, 66.7, 58.5, 50.0, 40.3, 23.9;

(6) HRMS (ESI+)

344.2087 (calculated for C ₁₈ H ₂₆ N ₅ O ₂ ([M + H] ⁺ ): 344.2084).

4-3. N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-phenylamino)phenyl)pyrimidine-2,4-diamine[N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-(phenylamino)phenyl)pyrimidine-2,4-diamine]:　Aa3

(1) Yield: 81%;

(2) Blue solid;

(3) Melting point: 143-145 ℃;

(4) ¹ H-NMR (500 MHz, CDCl ₃ )

δ 7.50 (d, J = 8.6 Hz, 2H), 7.21 (t, J = 7.7 Hz, 2H), 7.04-7.03 (m, 3H), 6.96 (d, J = 7.5 Hz, 2H), 6.83 (t, J = 7.5 Hz, 1H), 5.70 (s, 1H), 5.64 (s, 1H), 5.16 (brs, 1H), 3.56-3.51 (m, 4H), 3.37 (s, 3H), 2.23 (s, 3H) );　

(5) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 165.4, 163.5, 159.7, 144.6, 137.0, 134.9, 129.3, 120.5, 120.4, 119.8, 116.2, 94.4, 71.1, 58.9, 40.9, 23.8;

(6) HRMS (ESI+)

350.1981 (calculated for C ₂₀ H ₂₄ N ₅ O ([M + H] ⁺ ): 350.1971).

4-4. N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-phenoxyphenyl)pyrimidine-2,4-diamine[N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-phenoxyphenyl)pyrimidine-2,4-diamine]:　Aa4

(1) Yield: 81%;

(2) White solid;

(3) Melting point: 159-160 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.93 (s, 1H), 7.78 (d, J = 9.2 Hz, 2H), 7.28 (dd, J = 8.6, 7.5 Hz, 2H), 7.02-6.99 (m, 2H), 6.96-6.83 (m, 4H) ), 5.80 (s, 1H), 3.43 (brs, 4H), 3.22 (s, 3H), 2.08 (s, 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.0, 158.6, 149.7, 138.4, 130.3, 122.9, 120.2, 120.0, 117.8, 96.0, 71.1, 58.5, 40.3, 23.9;

(6) HRMS (ESI+)

351.1821 (calculated for C ₂₀ H ₂₃ N ₄ O ₂ ([M + H] ⁺ ): 351.1811).

4-5. N ² -(4-(4-chlorophenoxy)phenyl)-N ⁴ -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine[N ² -(4-(4-chlorophenoxy)phenyl)-N ⁴ -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine]:　Aa5

(1) Yield: 72%;

(2) White solid;

(3) Melting point: 160-161 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.97 (s, 1H), 7.50 (dt, J = 9.2, 2.3 Hz, 2H), 7.21 (dt, J = 9.2, 2.9 Hz, 2H), 7.04 (brs, 1H), 6.96-6.83 (m, 4H) ), 5.81 (s, 1H), 4.43 (brs, 4H), 3.22 (s, 3H), 2.08 (s, 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.0, 157.6, 149.2, 138.7, 130.1, 126.6, 120.2, 119.2, 96.1, 71.1, 58.4, 40.3, 23.9;

(6) HRMS (ESI+)

385.1431 (calculated for C ₂₀ H ₂₂ ClN ₄ O ₂ ([M + H] ⁺ ): 385.1422).

4-6. N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-(p-tolyloxy)phenyl)pyrimidine-2,4-diamine[N ⁴ -(2-methoxyethyl)-6-methyl-N ² -(4-(p-tolyloxy)phenyl)pyrimidine-2,4-diamine]:　Aa6

(1) Yield: 79%;

(2) White solid;

(3) Melting point: 128-129 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.90 (s, 1H), 7.75 (d, J = 9.2, 2.9 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H), 7.03 (brs, 1H), 6.84 (dt, J = 8.6, 2.6 Hz, 2H), 6.80 (dt,　J　= 8.5, 2.6 Hz, 2H), 5.79 (s, 1H), 3.43 (brs, 4H), 3.22 (s, 3H), 2.21 (s, 3H), 2.07 (s , 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.0, 156.1, 150.3, 138.0, 131.9, 130.6, 120.2, 119.5, 118.0, 96.1, 71.1, 58.4, 40.2, 23.9, 20.7;

(6) HRMS (ESI+)

365.1967 (calculated for C ₂₀ H ₂₃ N ₄ O ₂ ([M + H] ⁺ ): 365.1978).

4-7. N ⁴ -(4-isopropoxyphenyl)-N ² -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine[N ⁴ -(4-isopropoxyphenyl)-N ² -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine]:　Ba1

(1) Yield: 71%;

(2) Black solid;

(3) Melting point: 140-142 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.80 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 6.78 (dt, J = 8.6, 2.6 Hz, 2H), 6.44 (s, 1H), 5.77 (s, 1H), 4.44 ( quint.,　J　= 6.0 Hz, 1H), 3.42-3.36 (m, 4H), 3.22 (s, 3H), 2.04 (s, 3H), 1.19 (d,　J　= 6.3 Hz, 6H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.9, 162.2, 161.7, 152.8, 134.1, 121.8, 116.4, 94.6, 71.4, 69.9, 58.4, 40.7, 24.0, 22.4;

(6) HRMS (ESI+)

317.1978 (calculated for C ₁₇ H ₂₄ N ₄ O ₂ ([M + H] ⁺ ): 317.1968).

4-8. N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-morpholinophenyl)pyrimidine-2,4-diamine[N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-morpholinophenyl)pyrimidine-2,4-diamine]:　Ba2

(1) Yield: 60%;

(2) dark blue solid;

(3) Melting point: 124-126 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.80 (brs, 1H), 7.46 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 9.2 Hz, 2H), 6.44 (brs, 1H), 5.76 (s, 1H), 3.68 (t, J = 4.6 Hz, 4H), 3.41-3.36 (m, 4H), 3.22 (s, 3H), 2.98 (t, J = 4.6 Hz, 4H), 2.04 (s, 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.6, 162.2, 161.8, 146.8, 133.4, 121.5, 116.1, 94.7, 71.3, 66.7, 58.4, 49.7, 40.7, 23.9;

(6) HRMS (ESI+)

344.2087 (calculated for C ₁₈ H ₂₆ N ₅ O ₂ ([M + H] ⁺ ): 344.2077).

4-9. N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(phenylamino)phenyl)pyrimidine-2,4-diamine[N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-(phenylamino)phenyl)pyrimidine-2,4-diamine]:　Ba3

(1) Yield: 59%;

(2) Pale purple foam;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.79 (s, 1H), 7.90 (s, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.21 (dd, J = 8.6, 7.5 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 7.5 Hz, 2H), 6.70 (t, J = 7.2 Hz, 1H), 6.42 (brs, 1H), 5.76 (s, 1H), 3.45-3.45 (m, 4H), 3.21 (s, 3H), 2.04 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.5, 162.0, 161.7, 144.9, 138.0, 134.1, 129.6, 121.6, 119.2, 118.8, 116.0, 95.1, 71.3, 58.4, 40.7, 23.9;

(5) HRMS (ESI+)

350.1981 (calculated for C ₂₀ H ₂₄ N ₅ O ([M + H] ⁺ ): 350.1968).

4-10. N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-phenoxyphenyl)pyrimidine-2,4-diamine[N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-phenoxyphenyl)pyrimidine-2,4-diamine]:　Ba4

(1) Yield: 70%;

(2) White solid;

(3) Melting point: 145-146 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.06 (s, 1H), 7.70 (d, J = 8.6 Hz, 2H), 7.30 (t, J = 8.0 Hz, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.92 (d, J = 9.2 Hz, 4H), 6.54 (brs, 1H), 5.85 (s, 1H), 3.43-3.38 (m, 4H), 3.20 (s, 3H), 2.07 (s, 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 165.2, 162.2, 161.5, 158.2, 150.8, 137.3, 130.3, 123.2, 121.4, 120.0, 118.1, 95.3, 71.3, 58.4, 40.8, 24.1;

(6) HRMS (ESI+)

351.1821 (calculated for C ₂₀ H ₂₃ N ₄ O ₂ ([M + H] ⁺ ): 351.1811).

4-11. N ⁴ -(4-(4-chlorophenoxy)phenyl)-N ² -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine[N ⁴ -(4-(4-chlorophenoxy)phenyl)-N ² -(2-methoxyethyl)-6-methylpyrimidine-2,4-diamine]:　Ba5

(1) Yield: 74%;

(2) White solid;

(3) Melting point: 143-144 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.05 (s, 1H) 7.70 (d, J = 8.6 Hz, 2H), 7.34 (dt, J = 9.2, 2.9 Hz, 2H), 6.96-6.83 (m, 4H), 6.53 (s, 1H), 5.83 (s, 1H), 3.43-3.34 (m, 4H), 3.20 (s, 3H), 2.07 (s, 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 165.4, 162.3, 161.5, 157.2, 150.2, 137.8, 130.2, 126.3, 121.3, 120.2, 119.6, 95.1, 71.3, 58.4, 40.8, 24.1;

(6) HRMS (ESI+)

385.1431 (calculated for C ₂₀ H ₂₁ ClN ₄ O ₂ ([M + H] ⁺ ): 385.1422).

4-12. N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-(p-tolyloxy)phenyl)pyrimidine-2,4-diamine[N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-(p-tolyloxy)phenyl)pyrimidine-2,4-diamine]: Ba6

(1) Yield: 77%;

(2) Gray solid;

(3) Melting point: 140-142 ℃;

(4) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.96 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 6.86 (dt, J = 9.2, 2.9 Hz, 2H), 6.82 (dt, J = 8.6, 2.9 Hz, 2H), 6.48 (s, 1H), 5.80 (s, 1H), 3.42-3.29 (m, 4H), 3.20 (s, 3H), 2.23 (s, 3H), 2.05 (s) , 3H);　

(5) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 162.7, 161.5, 155.8, 151.4, 137.0, 132.3, 130.7, 121.3, 119.4, 118.4, 71.3, 58.4, 40.8, 23.0, 20.7;

(6) HRMS (ESI+)

365.1978 (calculated for C ₂₁ H ₂₄ N ₄ O ₂ ([M + H] ⁺ ): 365.1969).

4-13. 2-((2-(4-isopropoxyphenyl)amino)-6-methylpyrimidin-4-yl)amino)ethan-1-ol: Ab1

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.95 (s, 1H), 8.71 (s,1H), 7.61 (d,　J　= 9.5 Hz, 2H), 7.00 (brs, 1H), 6.74 (d,　J　= 8.6 Hz, 2H), 5.76 (s, 1H), 4.72 (s, 1H), 4.42 (heptet,　J　= 6.0 Hz, 1H), 3.52 (t,　J　= 5.5 Hz, 2H), 3.33 (brs, 2H), 2.07 (s, 3H), 1.18 ( d,　J　=5.8 Hz, 6H);　

(3) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 163.8, 159.8, 152.0, 135.1, 120.4, 116.3, 95.7, 69.9, 60.3, 43.5, 23.7, 22.4.

4-14. 2-((6-methyl-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)ethan-1-ol: Ab2

(1) Purple solid;

(2) Melting point: 206-208 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.10 (s, 1H), 8.94 (s, 1H), 7.38 (d,　J　= 8.0 Hz, 2H), 6.91 (d,　J　= 8.1 Hz, 2H), 5.99 (s, 1H), 4.89 (brs, 1H), 3.69 (brs, 4H), 3.52 (brs, 2H), 3.39 (brs, 2H), 3.03 (brs, 4H), 2.18 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.3, 153.0, 152.0, 148.4, 129.4, 122.3, 115.9, 96.9, 66.6, 59.4, 49.1, 44.1, 18.9.

4-15. 2-((6-methyl-2-((4-phenylamino)phenyl)amino)pyrimidin-4-yl)amino)ethan-1-ol: Ab3

(1) Purple solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.25 (s, 1H), 9.14 (s, 1H), 8.26 (s, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.17 (t, J = 7.8 Hz, 2H), 7.06 (t, J = 8.6 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.75 (t, J = 7.2 Hz, 1H), 6.02 (s, 1H), 4.94 (brs, 1H), 3.53 (t, J　= 5.5 Hz, 2H), 3.40 (q,　J　= 5.4 Hz, 2H), 2.19 (s, 3H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.3, 152.7, 151.4, 144.0, 140.6, 129.6, 122.5, 119.9, 117.7, 116.9, 96.9, 59.4, 44.2, 18.7.

4-16. 2-((6-methyl-2-((4-phenoxyphenyl)amino)pyrimidin-4-yl)amino)ethan-1-ol:　Ab4

(1) White solid;

(2) Melting point: 245-247 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.30 (s, 1H), 8.97 (s, 1H), 7.64 (d, J = 6.3 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.09 (t, J = 6.9 Hz, 1H) , 7.00 (d,　J　= 8.1 Hz, 2H), 6.97 (d,　J　= 7.5 Hz, 2H), 6.04 (s, 1H), 4.87 (brs, 1H), 3.52 (brs, 2H), 3.40 (brs, 2H), 2.21 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.3, 157.5, 153.3, 153.1, 133.4, 130.5, 123.8, 122.8, 119.8, 118.8, 97.3, 59.4, 44.2, 19.0.

4-17. 2-((2-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-4-yl)amino)ethan-1-ol:　Ab5

(1) White solid;

(2) Melting point: 244-246 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.96 (s, 1H), 7.80 (d, J = 9.2 Hz, 2H), 7.33 (dt, J = 9.2, 2.9 Hz, 2H), 7.01 (brs, 1H), 6.90 (d, J = 8.6 Hz, 4H), 5.79 (s, 1H), 4.68 (brs, 1H), 3.51 (t,　J　=5.7 Hz, 2H), 4.33 (brs, 2H), 2.08 (s, 3H);　

(4) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 163.8, 159.9, 157.6, 149.2, 138.7, 130.1, 126.5, 120.2, 120.2, 119.3, 96.1, 60.3, 43.6, 23.8.

4-18. 2-((6-methyl-2-((4-(p-tolyloxy)phenyl)amino)pyrimidin-4-yl)amino)ethan-1-ol:　Ab6

(1) Gray solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.91 (s, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 7.01 (brs, 1H), 6.85 (d, J = 9.2 Hz, 2H) , 6.80 (d,　J　= 8.0 Hz, 2H), 5.80 (s, 1H), 4.71 (brs, 1H), 3.52 (t,　J　= 5.7 Hz, 2H), 3.34 (brs, 2H), 2.21 (s, 3H), 2.08 (s, 3H);

(3) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 163.8, 159.9, 156.1, 150.3, 138.0, 132.0, 130.6, 120.2, 119.5, 118.0, 96.3, 60.3, 43.4, 23.8, 20.6.

4-19. 2-((4-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-2-yl)amino)ethan-1-ol:　Bb1

(1) Gray solid;

(2) Melting point: 214-216 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.53 (s, 1H), 7.54 (m, 3H), 6.89 (d, J = 9.2 Hz, 2H), 6.05 (s, 1H), 4.89 (s, 1H), 4.45 (heptet, J = 6.0 Hz, 1H), 3.52 (t, J = 5.5 Hz, 2H), 3.39 (q, J = 5.7 Hz, 2H), 2.23 (s, 3H), 1.22 (d, J = 5.8 Hz, 6H);　

(4) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 160.9, 154.9, 154.7, 152.5, 131.3, 123.2, 116.2, 96.9, 69.9, 59.5, 44.0, 22.3, 18.8.

4-20. 2-((4-methyl-6-((4-morpholinophenyl)amino)pyrimidin-2-yl)amino)ethan-1-ol:　Bb2

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.75 (s, 1H), 7.72 (s, 1H), 7.57 (d,　J　= 8.1 Hz, 2H), 6.91 (d,　J　= 8.6 Hz, 2H), 6.13 (s, 1H), 4.90 (brs, 1H), 3.69 (t,　J　= 4.6 Hz, 4H), 3.52 (t,　J　= 5.7 Hz, 2H), 3.40 (q,　J　= 5.2 Hz, 2H), 3.05 (m, 4H), 2.21 (s, 3H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 160.8, 154.9, 152.2, 148.3, 130.4, 122.5, 115.6, 97.1, 66.5, 59.5, 49.0, 43.8, 18.8.

4-21. N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-(phenylamino)phenyl)pyrimidine-2,4-diamine[N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-(phenylamino)phenyl)pyrimidine-2,4-diamine]:　Bb3

(1) Blue solid;

(2) Melting point: 228-230 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.63 (s, 1H), 8.22 (s, 1H), 7.66-7.53 (m, 2H), 7.19 (t,　J　= 7.2 Hz, 2H), 7.05 (t,　J　= 9.2 Hz, 2H), 7.03 ( d,　J　= 8.0 Hz, 2H), 6.77 (t,　J　= 7.2 Hz, 1H), 6.08 (s, 1H), 4.89 (brs, 1H), 3.52 (t,　J　= 5.7 Hz, 2H), 3.40 ( q,　J　= 5.3 Hz, 2H), 2.22 (s, 3H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 160.6, 154.9, 152.0, 143.9, 140.7, 130.8, 129.6, 122.8, 120.1, 117.3, 117.1, 96.9, 59.5, 43.8, 18.8.

4-22. 2-((4-methyl-6-((4-phenoxyphenyl)amino)pyrimidin-2-yl)amino)ethan-1-ol:　Bb4

(1) Gray solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.36 (s, 1H), 9.07 (s, 1H), 7.54 (d, J = 9.2 Hz, 2H), 7.35 (t, J = 8.0 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H) , 7.01 (d, J = 9.2 Hz, 2H), 6.99 (d, J = 9.2 Hz, 2H), 6.05 (s, 1H), 4.80 (brs, 1H), 3.52 (t, J = 5.4 Hz, 2H) , 3.40 (q,　J　=5.4 Hz, 2H), 2.22 (s, 3H);

(3) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 163.2, 157.4, 153.4, 152.7, 151.7, 133.2, 130.5, 123.9, 123.0, 119.8, 118.9, 97.4, 59.4, 44.2, 18.8.

4-23. 2-((4-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-2-yl)amino)ethan-1-ol:　Bb5

(1) Gray solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.11 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.34 (dt, J = 9.2, 2.9 Hz, 2H), 6.96-6.92 (m, 4H), 6.52 (s, 1H), 5.85 (s, 1H), 4.64 (brs, 1H), 3.49 (t, J = 6.0 Hz, 2H), 3.31 (q, J = 6.1 Hz, 2H), 2.07 (s, 3H);　

(3) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 164.9, 162.2, 161.5, 157.2, 150.3, 137.7, 130.2, 126.9, 121.4, 120.2, 119.6, 95.1, 60.7, 44.1, 24.0.

4-24. N2-(2-methoxyethyl)-6-methyl-N4-(4-(p-tolyloxy)phenyl)pyrimidine-2,4-diamine[N ² -(2-methoxyethyl)-6-methyl-N ⁴ -(4-(p-tolyloxy)phenyl)pyrimidine-2,4-diamine]:　Bb6

(1) Gray solid;

(2) Melting point: 108-110 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.77 (s, 1H), 7.69 (brs, 3H), 7.16 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 6.89 (dt, J = 8.6 Hz, 2H) , 6.13 (s, 1H), 4.88 (brs, 1H), 3.51 (t,　J　= 5.5 Hz, 2H), 3.40 (q,　J　= 5.2 Hz, 2H), 2.49 (s, 6H);　

(4) ¹³ C-NMR (125 MHz, CDCl ₃ )

δ 161.3, 154.9, 154.7, 154.2, 152.9, 133.8, 133.2, 130.9, 123.2, 119.3, 118.9, 97.1, 59.5, 43.8, 20.7, 18.9.

4-25. 3-((2-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-4-yl)amino)propan-1-ol:　Ac1

(1) Gray solid;

(2) Melting point: 156-158 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.19 (s, 1H), 9.1 (s, 1H), 7.42 (d,　J　= 9.6 Hz, 2H), 6.88 (d,　J　= 9.2 Hz, 2H), 5.98 (s, 1H), 4.54 (s, 1H), 4.52 (heptet,　J　= 6.0 Hz, 1H), 3.41 (t,　J　= 6.0 Hz, 2H), 3.36 (q,　J　= 6.3 Hz, 2H), 2.19 (s, 2H), 1.65 (quint. ,　J　= 6.6 Hz, 2H), 1.21 (d, J = 6.3 Hz, 6H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 161.2, 155.6, 154.6, 131.6, 123.2, 116.2, 96.2, 69.9, 58.8, 38.6, 32.3, 22.3, 19.4.

4-26. 2-((6-methyl-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)propan-1-ol:　Ac2

(1) Black solid;

(2) Melting point: 135-137 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.04 (s, 1H), 7.54 (d, J = 7.5 Hz, 2H), 6.82 (d, J = 8.0 Hz, 2H), 5.77 (s, 1H), 4.47 (s, 1H), 3.68 (brs, 4H), 3.43 (s, 2H), 3.33 (s, 2H), 2.97 (s, 3H), 2.08 (s, 3H), 1.64 (quint., J = 6.6 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.6, 146.5, 133.2, 120.7, 116.1, 95.6, 66.7, 58.9, 49.8, 38.0, 32.6, 22.3.

4-27. 2-((6-methyl-2-((4-phenylamino)phenyl)amino)pyrimidin-4-yl)amino)propan-1-ol:　Ac3

(1) Blue solid;

(2) Melting point: 113-115 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.59 (s, 1H), 8.25 (s, 1H), 8.04 (s, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.14 (t, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.71 (t, J = 7.2 Hz, 2H), 5.89 (s, 1H), 4.53 (s, 1H), 3.44 (d, J = 6.3 Hz, 2H), 3.36-3.33 ( m, 2H), 2.14 (s, 3H), 1.66 (quint.,　J　=6.6 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.3, 155.8, 144.7, 138.9, 131.9, 129.6, 121.7, 119.4, 118.4, 116.2, 96.5, 58.8, 38.3, 32.4, 20.6.

4-28. 2-((6-methyl-2-((4-phenoxyphenyl)amino)pyrimidin-4-yl)amino)propan-1-ol:　Ac4

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.94 (s, 1H), 7.81 (dt, J = 9.2, 2.6 Hz, 2H), 7.28 (m, 2H), 7.00 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 9.2 Hz, 4H), 5.76 (s, 1H), 4.47 (brs, 1H), 3.45 (t,　J　= 6.3 Hz, 2H), 3.32 (brs, 2H), 2.08 (s, 3H), 1.67 (quint.,　J　= 6.7 Hz, 2H);

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 159.9, 158.6, 149.6, 138.4, 130.3, 122.8, 120.2, 120.1, 117.7, 95.8, 59.1, 37.9, 32.8, 23.8.

4-29. 2-((2-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-4-yl)amino)propan-1-ol:　Ac5

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.96 (s, 1H), 7.83 (dt, J = 9.2, 2.6 Hz, 2H), 7.32 (dt, J = 8.6, 2.9 Hz, 2H), 6.88 (dt, J = 9.2, 2.6 Hz, 2H), 6.98 (brs, 1H), 6.92-6.88 (m, 4H), 5.76 (s, 1H), 4.47 (s, 1H), 3.46 (t,　J　= 6.0 Hz, 2H), 3.32 (brs, 2H), 2.08 (s, 3H), 1.67 (quint.,　J　= 6.6 Hz, 2H);

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 159.9, 157.6, 149.1, 138.8, 130.1, 126.5, 120.2, 120.2, 119.2, 96.6, 59.1, 37.8, 32.8, 23.8.

4-30. 2-((6-methyl-2-((4-(p-tolyloxy)phenyl)amino)pyrimidin-4-yl)amino)propan-1-ol:　Ac6

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.37 (s, 1H), 9.05 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 9.2 Hz, 2H) , 6.86 (t,　J　= 8.0 Hz, 2H), 6.01 (s, 1H), 4.54 (s, 1H), 3.41 (t,　J　= 6.0 Hz, 2H), 3.38 (q,　J　= 6.3 Hz, 2H) , 2.23 (s, 3H), 2.20 (s, 1H), 1.66 (quint.,　J　=6.6 Hz, 2H);

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 155.0, 153.7, 152.9, 151.9, 133.1, 132.9, 130.9, 122.7, 119.3, 118.9, 97.2, 58.6, 38.5, 32.0, 20.7, 18.9.

4-31. 2-((4-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-2-yl)amino)propan-1-ol:　Bc1

(1) Gray solid;

(2) Melting point: 176-178 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.25 (s, 1H), 7.52 (s, 1H), 6.86 (d, J = 9.2 Hz, 2H), 5.97 (s, 1H), 4.57 (s, 1H), 4.53 (heptet, J = 6.0 Hz, 2H), 3.45 (t, J = 6.0 Hz, 2H), 3.36 (q, J = 5.8 Hz, 2H), 2.18 (s, 2H), 1.66 (quint., J = 6.5 Hz, 2H), 1.22 (d ,　J　=6.5 Hz, 6H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 161.2, 155.6, 154.6, 131.6, 123.2, 116.2, 96.2, 69.9, 58.8, 38.6, 32.3, 22.3, 19.4.

4-32. 2-((4-methyl-6-((4-morpholinophenyl)amino)pyrimidin-2-yl)amino)propan-1-ol:　Bc2

(1) Gray solid;

(2) Melting point: 162-166 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.19 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 9.2 Hz, 2H), 5.77 (s, 1H), 4.54 (s, 1H), 3.69 (t, J = 4.6 Hz, 4H), 3.42 (t, J = 6.0 Hz, 2H), 3.27 (q, J = 6.3 Hz, 2H), 2.99 (t, J = 4.6 Hz, 2H), 2.06 (s, 1H) , 1.63 (quint.,　J　=6.3 Hz, 6H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 161.2, 155.6, 154.6, 131.6, 123.2, 116.2, 96.2, 69.9, 58.8, 38.6, 32.3, 22.3, 19.4.

4-33. 2-((4-methyl-6-((4-phenylamino)phenyl)amino)pyrimidin-2-yl)amino)propan-1-ol:　Bc3

(1) Blue solid;

(2) Melting point: 104-108 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.77 (s, 1H), 8.05 (s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.15 (t, J = 7.7 Hz, 2H), 7.01 (t, J = 8.6 Hz, 2H) , 6.98 (t, J = 8.0 Hz, 2H), 6.72 (t, J = 7.2 Hz, 2H), 5.90 (s, 1H), 4.59 (s, 1H), 3.44 (t, J = 6.0 Hz, 4H) , 3.32 (q,　J　=5.7 Hz, 2H), 2.01 (s, 3H), 1.65 (quint.,J=6.3 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 172.7, 161.4, 158.7, 144.5, 139.0, 132.3, 129.6, 122.2, 119.5, 118.3, 116.3, 95.3, 59.0, 38.6, 32.7, 21.6.

4-34. 2-((4-methyl-6-((4-phenoxyphenyl)amino)pyrimidin-2-yl)amino)propan-1-ol:　Bc4

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.10 (s, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.31 (td, J = 9.2, 2.3 Hz, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.93-6.91 ( m, 4H), 6.63 (brs, 1H), 5.83 (s, 1H), 4.44 (s, 1H), 3.43 (t,　J　= 6.3 Hz, 2H), 3.28 (q,　J　= 6.9 Hz, 2H), 2.06 (s, 3H), 1.64 (quint.,　J　= 6.4 Hz, 2H);

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.7, 162.2, 161.5, 158.2, 150.7, 137.3, 130.4, 123.2, 121.4, 120.0, 118.1, 95.0, 59.2, 38.5, 33.1, 23.9.

4-35. 2-((4-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-2-yl)amino)propan-1-ol:　Bc5

(1) White solid;

(2) Melting point: 142-144 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.10 (s, 1H), 7.73 (d, J = 9.2 Hz, 2H), 7.34 (dt, J = 9.2, 2.9 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.93 (dt, J = 9.2, 2.3 Hz, 2H), 6.62 (brs, 1H), 5.82 (s, 1H), 4.41 (brs, 1H), 3.43 (t, J = 6.3 Hz, 2H), 3.29 (q, J = 6.5 Hz, 2H), 2.06 (s, 3H), 1.64 (quint.,　J　=6.3 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.9, 162.2, 161.5, 157.3, 150.2, 137.7, 130.2, 126.8, 121.3, 120.2, 119.6, 94.7, 59.2, 49.1, 38.4, 33.1, 23.8.

4-36. 3-((4-methyl-6-((4-(p-tolyloxy)phenyl)amino)pyrimidin-2-yl)amino)propan-1-ol:　Bc6

(1) White solid;

(2) Melting point: 136-138 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.03 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 6.88 (dt, J = 9.2, 2.6 Hz, 2H), 6.82 (dt, J = 8.6, 2.3 Hz, 2H), 6.59 (brs, 1H), 5.81 (s, 1H), 4.43 (s, 1H), 3.43 (t, J = 6.3 Hz, 2H), 3.28 (q, J = 6.5 Hz, 2H), 2.22 (s, 3H), 2.06 (s, 3H), 1.64 (quint.,　J　=6.4 Hz, 2H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.9, 162.3, 161.5, 155.8, 151.4, 137.0, 132.3, 130.7, 121.4, 119.5, 118.3, 94.6, 59.2, 38.5, 33.1, 24.0, 20.7.

4-37. 4-((2-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-4-yl)amino)butan-1-ol:　Ad1

(1) Gray solid;

(2) Melting point: 170-172 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.19 (s, 1H), 9.04 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.99 (s, 1H), 4.51 (heptet, J = 5.9 Hz, 1H), 4.46 (s, 1H), 3.36 (t, J = 6.0 Hz, 2H), 3.29 (m, 2H), 2.18 (s, 3H), 1.53 (quint., J = 7.0 Hz) , 2H), 1.41 (quint.,　J　= 6.7 Hz, 2H), 1.20 (quint.,J=5.8 Hz, 6H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 154.5, 153.1, 151.9, 130.5, 122.7, 116.4, 96.9, 69.9, 60.8, 41.1, 30.4, 25.6, 22.3, 18.9.

4-38. 4-((6-methyl-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)butan-1-ol:　Ad2

(1) Black solid;

(2) Melting point: 160-162 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.95 (s, 1H), 8.70 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.1 Hz, 2H), 5.92 (s, 1H), 4.44 (s, 1H), 3.69 (brs, 4H), 3.37 (t,　J　= 6.3 Hz, 2H), 3.30 (s, 2H), 3.02 (brs, 4H), 2.17 (s, 3H), 1.53 (quint.,　J　= 7.2 Hz, 2H), 1.42 (quint.,　J　= 7.2 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.1, 153.8, 148.1, 130.1, 122.1, 115.9, 96.7, 66.6, 49.2, 41.0, 30.4, 25.7, 19.4.

4-39. 4-((6-methyl-2-((4-(phenylamino)phenyl)amino)pyrimidin-4-yl)amino)butan-1-ol:　Ad3

(1) dark blue solid;

(2) Melting point: 222-224 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.08 (s, 1H), 8.99 (s, 1H), 8.23 (s, 1H), 7.41 (d, J = 6.3 Hz, 2H), 7.15 (t, J = 7.2 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 7.5 Hz, 2H), 6.73 (t, J = 6.9 Hz, 1H), 5.99 (s, 1H), 4.50 (s, 1H), 3.38 (t, J = 5.8 Hz, 2H), 3.30 (brs, 2H), 2.16 (s, 3H), 1.53 (quint.,J=6.3 Hz, 2H), 1.43 (quint.,J=6.9 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 153.3, 152.4, 144.2, 140.2, 130.2, 129.6, 122.3, 119.7, 117.9, 116.6, 96.8, 60.8, 41.1, 30.4, 25.7, 19.2.

4-40. 4-((6-methyl-2-((4-phenoxyphenyl)amino)pyrimidin-4-yl)amino)butan-1-ol:　Ad4

(1) White solid;

(2) Melting point: 156-158 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.41 (s, 1H), 9.10 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.07 (d, J = 7.2 Hz, 2H) , 7.01 (d, J = 8.6 Hz, 2H), 6.95 (t, J = 7.5 Hz, 1H), 6.02 (s, 1H), 4.43 (s, 1H), 3.35 (t, J = 6.6 Hz, 2H) , 3.30 (q,　J　=6.6 Hz, 2H), 2.21 (s, 3H), 1.53 (quint., J = 7.3 Hz, 2H), 1.41 (quint., J = 6.9 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 157.5, 153.1, 153.0, 151.9, 133.4, 130.5, 123.7, 122.8, 119.9, 118.6, 97.2, 60.8, 41.2, 30.4, 25.6, 18.9.

4-41. 4-((2-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-4-yl)amino)butan-1-ol: Ad5

(1) Gray solid;

(2) Melting point: 142-144 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.42 (s, 1H), 9.08 (s, 1H), 7.57 (d, J = 9.2 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H) , 6.98 (d,　J　= 8.6 Hz, 2H), 6.02 (s, 1H), 4.43 (s, 1H), 3.34 (t,　J　= 6.3 Hz, 2H), 3.32 (q,　J　= 6.3 Hz, 2H) , 2.21 (s, 3H), 1.53 (quint.,　J　= 7.3 Hz, 2H), 1.41 (quint.,J=6.9 Hz, 2H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 156.6, 152.9, 152.7, 151.9, 133.8, 130.3, 127.4, 122.9, 120.2, 120.2, 97.3, 60.8, 41.2, 30.4, 25.6, 18.9.

4-42. 4-((6-methyl-2-((4-(p-tolyloxy)phenyl)amino)pyrimidin-4-yl)amino)butan-1-ol:　Ad6

(1) White solid;

(2) Melting point: 122-124 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.32 (s, 1H), 9.03 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.14 (t, J = 7.5 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H) , 6.86 (d,　J　= 7.5 Hz, 2H), 6.00 (s, 1H), 4.42 (s, 1H), 3.34-3.32 (m, 4H), 2.24 (s, 3H), 2.21 (s, 3H), 1.53 (quint.,J=6.9 Hz, 2H), 1.41 (m, 2H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 155.0, 153.9, 152.9, 151.9, 133.0, 130.9, 123.0, 119.3, 118.9, 97.2, 60.8, 41.2, 30.3, 25.6, 20.7, 18.8.

4-43. 4-((4-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-2-yl)amino)butan-1-ol:　Bd1

(1) Purple solid;

(2) Melting point: 153-155 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.74 (s, 1H), 7.92 (s, 1H), 7.58 (brs, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.10 (s, 1H), 4.54 (heptet, J = 6.1 Hz, 2H), 4.45 (s, 1H), 3.38 (t,　J　= 6.3 Hz, 2H), 3.32 (q,　J　= 6.3 Hz, 2H), 2.21 (s, 3H), 1.55 (quint.,　J　= 7.3 Hz) , 2H), 1.43 (quint.,　J　= 7.0 Hz, 2H), 1.21 (d,　J　=5.7 Hz, 6H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 161.0, 154.8, 152.6, 131.3, 127.1, 123.3, 116.2, 96.6, 69.9, 60.8, 41.2, 30.2, 25.9, 22.3, 18.9.

4-44. 4-((4-methyl-6-((4-morpholinophenyl)amino)pyrimidin-2-yl)amino)butan-1-ol:　Bd2

(1) Pink solid;

(2) Melting point: 208-210 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.77 (s, 1H), 7.94 (s, 1H), 7.57 (brs, 2H), 6.90 (d,　J　= 9.2 Hz, 2H), 6.11 (s, 1H), 4.47 (s, 1H), 3.68 ( t,　J　= 4.6 Hz, 4H), 3.37 (t,　J　= 6.3 Hz, 2H), 3.32 (q,　J　= 6.1 Hz, 2H), 3.03 (t,　J　= 4.3 Hz, 2H), 2.20 (s, 3H), 1.53 (quint.,　J　= 7.2 Hz, 2H), 1.43 (quint.,J=6.9 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 160.7, 154.9, 152.2, 148.5, 130.5, 122.5, 115.6, 96.7, 66.5, 60.5, 49.0, 41.1, 30.2, 25.9, 18.9.

4-45. 4-((4-methyl-6-((4-phenylamino)phenyl)amino)pyrimidin-2-yl)amino)butan-1-ol: Bd3

(1) dark blue solid;

(2) Melting point: 192-194 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.77 (s, 1H), 8.25 (s, 1H), 7.93 (brs, 2H), 7.17 (t, J = 7.4 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.76 (t, J = 7.2 Hz, 1H), 6.13 (s, 1H), 4.47 (s, 1H), 3.38 (t, J = 6.0 Hz, 2H), 3.32 (q, J = 5.8 Hz, 2H), 2.21 (s, 3H), 1.55 (quint.,J=7.0 Hz, 2H), 1.44 (quint.,J=6.7 Hz, 2H);

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 160.7, 154.8, 152.2, 143.9, 140.6, 130.8, 129.6, 122.8, 120.0, 117.4, 116.9, 96.8, 60.8, 41.2, 30.3, 25.9, 18.8.

4-46. 4-((4-methyl-6-((4-phenoxyphenyl)amino)pyrimidin-2-yl)amino)butan-1-ol:　Bd4

(1) White solid;

(2) Melting point: 140-142 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.12 (s, 1H), 7.70 (d, J = 9.2 Hz, 2H), 7.31 (t, J = 8.0 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.92 (d,　J　= 8.0 Hz, 2H), 6.71 (brs, 1H), 5.82 (s, 1H), 4.36 (brs, 1H), 3.37 (t,　J　= 6.6 Hz, 2H) , 3.21 (q,　J　=6.7 Hz, 2H), 2.06 (s, 1H), 1.51 (quint.,J=7.2 Hz, 2H), 1.42 (quint.,J=7.0 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.5, 161.9, 161.5, 158.2 150.7, 137.3, 130.4, 123.2, 121.4, 120.0, 118.0, 94.7, 61.1, 41.2, 30.6, 26.5, 23.8.

4-47. 4-((4-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-2-yl)amino)butan-1-ol:　Bd5

(1) White solid;

(2) Melting point: 164-166 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 10.41 (s, 1H), 9.10 (s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.07 (d, J = 7.2 Hz, 2H) , 7.01 (d, J = 8.6 Hz, 2H), 6.95 (t, J = 7.5 Hz, 1H), 6.02 (s, 1H), 4.43 (s, 1H), 3.35 (t, J = 6.6 Hz, 2H) , 3.30 (q,　J　=6.6 Hz, 2H), 2.21 (s, 3H), 1.53 (quint., J = 7.3 Hz, 2H), 1.41 (quint., J = 6.9 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.0, 157.5, 153.1, 153.0, 151.9, 133.4, 130.5, 123.7, 122.8, 119.9, 118.6, 97.2, 60.8, 41.2, 30.4, 25.6, 18.9.

4-48. 4-((4-methyl-6-((4-(p-tolyloxy)phenyl)amino)pyrimidin-2-yl)amino)butan-1-ol:　Bd6

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.22 (s, 1H), 7.69 (d, J = 9.2 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 9.2 Hz, 2H), 6.82 (d, J = 9.2 Hz, 2H) 8.6 Hz, 2H), 6.74 (s, 1H), 5.84 (s, 1H), 4.34 (brs, 1H), 3.38 (t,　J　= 6.3 Hz, 2H), 3.23 (q,　J　= 6.7 Hz, 2H) , 2.21 (s, 3H), 2.07 (s, 3H), 1.52 (quint.,　J　= 7.2 Hz, 2H), 1.43 (quint.,　J　= 7.0 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.8, 161.5, 155.7, 151.5, 136.8, 132.3, 130.7, 121.5, 119.5, 118.3, 94.7, 61.2, 41.2, 30.6, 26.5, 23.2, 20.7.

4-49. 5-((2-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-4-yl)amino)pentan-1-ol:　Ae1

(1) Gray solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.71 (s, 1H), 7.61 (dt, J = 8.6, 3.5 Hz, 2H), 7.03 (s, 1H), 6.74 (dt, J = 9.2, 3.5 Hz, 2H), 5.70 (s, 1H), 4.44 (heptet,　J　= 6.0 Hz, 1H), 4.34 (s, 1H), 3.35-3.32 (m, 4H), 2.05 (s, 3H), 1.49 (quint.,　J　= 7.3 Hz, 2H), 1.41 ( quint.,　J　= 7.0 Hz, 2H), 1.31 (quint.,　J　= 7.2 Hz, 2H), 1.19 (d,　J　= 6.3 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.6, 159.7, 152.0, 135.1, 120.4, 116.2, 69.9, 61.2, 40.5, 32.8, 29.5, 23.7, 22.4.

4-50. 5-((6-methyl-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol:　Ae2

(1) Gray solid;

(2) Melting point: 148-150 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.71 (s, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.09 (s, 1H), 6.80 (d, J = 9.2 Hz, 2H), 5.70 (s, 1H), 4.34 (brs, 1H), 3.68 (t,　J　= 4.6 Hz, 4H), 3.35 (brs, 2H), 3.23 (brs, 2H), 2.96 (t,　J　= 4.6 Hz, 4H), 2.06 (s, 3H), 1.49 ( quint.,　J　= 7.2 Hz, 2H), 1.41 (quint.,　J　= 7.0 Hz, 2H), 1.30 (quint.,　J　= 7.3 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.6, 159.5, 146.0, 134.3, 120.2, 116.1, 95.8, 66.7, 61.2, 49.9, 40.3, 32.8, 29.5, 23.7, 23.4.

4-51. 5-((6-methyl-2-((4-(phenylamino)phenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol [5-((6-methyl-2-((4-(phenylamino)phenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol]: Ae3

(1) dark gray solid;

(2) Melting point: 238-240 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.32 (s, 1H), 8.02 (s, 2H), 7.53 (s, 2H), 7.13 (s, 2H), 6.97-6.95 (m, 4H), 6.68 (s, 1H), 5.83 (s, 1H) ), 4.39 (brs, 1H), 3.34-3.25 (m 4H), 2.11 (s, 3H), 1.50 (s, 2H), 1.30 (peak splitting, 2H), 1.19 (s, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.4, 157.4, 145.0, 137.6, 133.5, 129.5, 124.7, 120.7, 119.0, 118.8, 115.8, 96.0, 61.1, 40.9, 32.7, 29.2, 23.6, 21.6.

4-52. 5-((6-methyl-2-((4-phenoxyphenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol: Ae4

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.94 (s, 1H), 7.79 (dt, J = 9.2, 2.6 Hz, 2H), 7.29 (t, J = 8.0 Hz, 2H), 7.04 (brs, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.90-6.88 (m, 4H), 5.75 (s, 1H), 4.34 (brs, 1H), 3.35 (t,　J　= 6.3 Hz, 2H), 3.24 (brs, 2H), 2.08 (s, 3H) ), 1.50 (quint.,　J　= 7.5 Hz, 2H), 1.40 (quint.,　J　= 7.0 Hz, 2H), 1.31 (quint.,　J　= 7.3 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 159.9, 158.6, 149.6, 138.3, 130.3, 122.9, 120.2, 120.0, 117.7, 96.3, 61.2, 49.0, 40.7, 32.8, 29.4, 23.7, 23.7.

4-53. 5-((2-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-4-yl)amino)pentan-1-ol:　Ae5

(1) Gray solid;

(2) Melting point: 146-148 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.94 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 7.32 (dt, J = 8.6, 2.6 Hz, 2H), 6.98 (s, 1H), 6.90 (d, J = 9.2 Hz, 2H), 6.90 (d,　J　= 8.6 Hz, 2H), 5.74 (s, 1H), 4.34 (brs, 1H), 4.35 (t,　J　= 6.3 Hz, 2H), 3.24 (brs, 2H), 2.07 ( s, 3H), 1.50 (quint.,　J　=7.3 Hz, 2H), 1.40 (quint.,J=6.9 Hz, 2H), 1.31 (quint.,J=7.3 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.0, 157.6, 149.1, 138.8, 130.1, 126.5, 120.2, 120.1, 119.2, 96.0, 61.2, 49.0, 40.7, 32.8, 29.5, 23.8, 23.7.

4-54. 5-((6-methyl-2-((4-(p-tolyloxy)phenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol: Ae6

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.90 (s, 1H), 7.78 (dt, J = 9.2, 2.6 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 7.00 (s, 1H), 6.85 (dt, J = 9.2, 2.6 Hz, 2H), 6.79 (dt, J = 8.6, 2.6 Hz, 2H), 5.74 (s, 1H), 4.36 (brs, 1H), 3.36 (t, J = 6.3 Hz, 2H), 3.27 (brs, 2H) ), 2.20 (s, 3H), 2.07 (s, 3H), 1.50 (quint.,J=7.3 Hz, 2H), 1.41 (quint.,J=6.9 Hz, 2H), 1.31 (quint.,J=7.3 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 159.9, 156.2, 150.3, 138.0, 131.9, 130.6, 120.2, 119.5, 117.9, 96.3, 61.2, 49.1, 40.8, 32.8, 29.5, 23.7, 23.7, 20 .6.

4-55. 5-((4-((4-isopropoxyphenyl)amino)-6-methylpyrimidin-2-yl)amino)pentan-1-ol:　Be1

(1) Black solid;

(2) Melting point: 138-140 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.67 (s, 1H), 7.62 (dt, J = 8.6, 2.6 Hz, 2H), 6.96 (s, 1H), 6.73 (dt, J = 9.2, 2.6 Hz, 2H), 5.70 (s, 1H), 4.43 (heptet,　J　= 5.0 Hz, 1H), 4.35 (t,　J　= 6.6 Hz, 2H), 3.23 (brs, 2H), 2.05 (s, 1H), 1.49 (quint.,　J　= 7.3 Hz, 2H) , 1.42 (quint.,　J　= 7.0 Hz, 2H), 1.31 (quint., J = 7.6 Hz, 2H), 1.18 (d, J = 5.8 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.7, 160.0, 151.9, 135.2, 120.3, 116.2, 95.7, 69.9, 61.2, 49.1, 32.8, 29.5, 23.7, 22.4.

4-56. 5-((4-methyl-6-((4-morpholinophenyl)amino)pyrimidin-2-yl)amino)pentan-1-ol:　Be2

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.07 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 9.2 Hz, 2H), 6.74 (s, 1H), 5.77 (s, 1H), 4.35 (brs, 1H), 3.68 (t, J = 4.6 Hz, 4H), 3.35 (t, J = 6.3 Hz, 2H), 3.20 (q, J = 6.9 Hz, 2H), 2.99 (t, J = 4.6 Hz, 2H) , 2.05 (s, 3H), 1.48 (quint.,　J　= 7.3 Hz, 2H), 1.41 (quint.,J=7.0 Hz, 2H), 1.29 (quint.,J=7.5 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 161.6, 146.9, 142.9, 142.7, 121.5, 118.0, 116.0, 115.3, 66.8, 66.6, 61.2, 51.1, 49.6, 49.1, 41.3, 32.9, 29.7, 23.6.

4-57. 2-((4-methyl-6-((4-(phenylamino)phenyl)amino)pyrimidin-2-yl)amino)propan-1-ol:　Be3

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 8.73 (s, 1H), 7.80 (s, 1H), 7.62 (d,　J　= 9.2 Hz, 2H), 7.11 (t,　J　= 7.7 Hz, 2H), 6.99 (brs, 1H), 6.93 (dt, J = 8.6, 2.6 Hz, 2H), 6.90 (dd, J = 8.6, 1.2 Hz, 2H), 6.66 (t, J = 7.5 Hz, 1H), 5.70 (s, 1H), 4.33 (t, J = 5.2 Hz, 1H), 3.35 (q,　J　= 5.9 Hz, 2H), 3.23 (brs, 2H), 2.06 (s, 3H), 1.50 (quint.,　J　= 7.5 Hz, 2H), 1.41 (quint.,　J　= 6.9 Hz, 2H), 1.30 (quint. ,　J　= 7.6 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 163.6, 145.5, 136.5, 135.6, 129.5, 120.0, 119.4, 118.6, 115.3, 104.0, 61.2, 32.8, 29.5, 23.7.

4-58. 5-((4-methyl-6-((4-phenoxyphenyl)amino)pyrimidin-2-yl)amino)pentan-1-ol:　Be4

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.16 (s, 1H), 7.71 (d, J = 9.2 Hz, 2H), 7.30 (t, J = 8.0 Hz, 2H), 7.04 (t, J = 7.2 Hz, 2H), 6.92-6.91 (m, 4H), 6.75 (brs, 1H), 5.82 (s, 1H), 4.32 (s, 1H), 3.33 (t,　J　= 6.3 Hz, 2H), 3.20 (q,　J　= 6.9 Hz, 2H), 2.07 ( s, 3H), 1.48 (quint.,　J　=7.3 Hz, 2H), 1.39 (quint.,J=6.9 Hz, 2H), 1.28 (quint.,J=7.5 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.3, 161.9, 161.5, 158.2, 150.8, 137.2, 130.4, 123.2, 121.4, 120.0, 118.1, 94.7, 61.2, 49.1, 41.3, 32.8, 29.6, 23.6.

4-59. 5-((4-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-2-yl)amino)pentan-1-ol:　Be5

(1) White solid;

(2) Melting point: 136-138 ℃;

(3) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.16 (s, 1H), 7.73 (d, J = 9.2 Hz, 2H), 7.33 (dt, J = 9.2, 2.9 Hz, 2H), 6.95-6.90 (m, 4H), 6.75 (s, 1H), 5.83 (s, 1H), 4.28 (s, 1H), 3.34 (t,　J　=6.3 Hz, 2H), 3.21 (q,　J　=6.7 Hz, 2H), 2.07 (s, 3H), 1.49 (quint., J　= 7.3 Hz, 2H), 1.39 (quint.,　J　= 6.9 Hz, 2H), 1.29 (quint.,　J　= 7.3 Hz, 2H);　

(4) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 164.6, 162.0, 161.5, 157.2, 150.3, 137.7, 130.2, 126.9, 121.3, 120.2, 119.6, 94.9, 61.2, 41.3, 32.9, 29.6, 23.6.

4-60. 5-((4-methyl-6-((4-(p-tolyloxy)phenyl)amino)pyrimidin-2-yl)amino)pentan-1-ol:　Be6

(1) White solid;

(2) ¹ H-NMR (500 MHz, DMSO-d ₆ )

δ 9.44 (s, 1H), 7.68 (d, J = 9.2 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 6.92 (s, 1H), 6.88 (d, J = 8.6 Hz, 2H) , 6.83 (d,　J　= 8.6 Hz, 2H), 5.86 (s, 1H), 4.32 (brs, 1H), 3.33 (t,　J　= 6.3 Hz, 2H), 3.21 (q,　J　= 6.7 Hz, 2H) , 2.22 (s, 3H), 2.08 (s, 3H), 1.48 (quint.,J=7.5 Hz, 2H), 1.39 (quint.,J=7.1 Hz, 2H), 1.28 (quint.,J=7.8 Hz, 2H);　

(3) ¹³ C-NMR (125 MHz, DMSO-d ₆ )

δ 161.5, 155.6, 151.9, 136.7, 132.4, 130.7, 121.7, 119.3, 118.5, 95.1, 61.2, 41.3, 32.8, 29.5, 23.6, 22.5, 20.7.

Example 5. Screening of ANO1 bioactivity modulators

5-1. 1st screening

To identify ANO1 inhibitors, a cell-based HTS assay was performed using the heterocyclic library containing 60 pyrimidine derivatives synthesized in Example 2. First, YFP-H148Q/I152L/F46L and Fischer rat thyroid (FRT) cells stably expressing ANO1 were cultured at 20,000 cells/well in 96-well microplates in Dulbecco's Modified Eagle Medium (DMEM) and Ham's F12 medium (1:1 ratio) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 mg/mL G418, and 0.1 mg/mL hygromycin B for 24 h. Cells were washed twice with 100 μL of 1xPBS and treated with 100 μM of pyrimidine candidate compounds in 100 μL of NaCl aqueous solution for 20 min. All candidate compounds used here were prepared using DMSO. After incubation, 100 μL of NaI aqueous solution containing 200 μM ATP, known as an agonist of P2Y2 receptor that activates calcium-dependent iodide influx, was injected at 1 s to stimulate ANO1-mediated ^{I -} influx. Fluorescence was measured continuously every 0.2 s for 6 s. The ANO1 inhibitory effect of the candidate compounds was confirmed by the decrease in YFP fluorescence due to I ^- influx. The analysis was performed using a SpectraMax i3x multi-microplate reader (Molecular Devices, San Jose, CA, USA) equipped with a 488 nm excitation and 520 nm emission filter. Nineteen candidate compounds showing more than 70% inhibition response at 100 μM concentration were selected.

5-2. Second screening

For the 19 candidate compounds selected through the first screening of Example 5-1 above, a second screening was performed at a concentration of 50 μM using the same method as Example 5-1 above.

Figure 3a is a graph confirming the activity inhibition effect of ANO1 at a concentration of 50 μM for 19 candidate compounds.

As a result, as shown in Fig. 3a, it was confirmed that 15 candidate compounds, excluding Be1, Ac3, Bc3, and Bb3, inhibited the physiological activity of ANO1 at a concentration of 50 μM.

5-3. 3rd screening

For the 15 candidate compounds selected through the second screening of Example 5-2 above, a third screening was performed at a concentration of 30 μM using the same method as Example 5-1 above.

Figure 3b is a graph confirming the activity inhibition effect of ANO1 at a concentration of 30 μM for 15 candidate compounds.

As a result, as shown in Fig. 3b, it was confirmed that compounds Aa3, Ae5, Ae6, and Bd5 inhibited the physiological activity of ANO1 at a concentration of 30 30 μM.

Example 6. Evaluation of cytotoxicity of ANO1 bioactive regulator

The cytotoxicity of four compounds (Aa3, Bd5, Ae5, and Ae6) selected through the above Example 5 was confirmed. First, human lung adenocarcinoma A549 cell line and human large-cell lung carcinoma NCI-H460 cell line were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 ㎍/mL streptomycin. The cells were seeded at a density of 10,000 cells per well in a 96-well microplate and cultured for 24 hours. Each well was treated with the compounds at concentrations of 5 μM, 10 μM, and 20 μM and incubated at 37°C in a humidified atmosphere of 5% CO ₂ . Afterwards, each cell was fixed and the cytotoxicity of the compound was evaluated using crystal violet staining.

Figure 4a shows the results of evaluating the cytotoxicity of three candidate compounds (Bd5, Ae5, and Ae6) in lung cancer cells.

Figure 4b shows the results of evaluating the cytotoxicity of one candidate compound (Aa3) in lung cancer cells.

As a result, as shown in Fig. 4a, it was confirmed that the Bd5, Ae5, and Ae6 compounds exhibited cytotoxicity at a concentration of 5 μM. On the other hand, as shown in Fig. 4b, it was confirmed that the Aa3 compound did not exhibit cytotoxicity at concentrations of 5 to 10 μM, and exhibited cytotoxicity at a concentration of 20 μM. In particular, it was confirmed that the Aa3 compound had a more excellent anti-proliferative effect in NCI-H460 cells in which the expression of ANO1 was relatively high.

Based on the above results, three compounds showing cytotoxicity at low concentrations (5 μM) were excluded from the candidates, and Aa3 was finally selected as the ANO1 bioactivity modulator.

Example 6. Confirmation of the possibility of use as a selective inhibitor for ANO1

6-1. Confirmation of concentration-dependent ANO1 inhibition

To confirm the concentration-dependent ANO1 inhibitory effect of the Aa3 compound selected as the final compound, HTS analysis was performed using the same method as in Example 5-1.

Figure 5a is a graph confirming the concentration-dependent ANO1 activity inhibitory effect of a compound according to one aspect.

Figure 5b is a graph confirming the IC50 of compounds according to the daily aspect.

As a result, as shown in Fig. 5a, it was confirmed that Aa3, which was selected as the final compound, inhibited the physiological activity of ANO1 in a concentration-dependent manner. In addition, as shown in Fig. 5b, it was confirmed that the Aa3 compound exhibited an IC50 value at a concentration of 32 μM and maintained IC100 at a concentration of about 75 to 100 μM.

6-2. Confirmation of the active inhibition pathway of ANO1

Inhibition of ANO1 activity can be achieved through indirect inhibition that inhibits Ca ²⁺ released from intracellular endoplasmic reticulum through ligand and GPCR pathway activation, and direct inhibition of ANO1. The ANO1 activity inhibition pathway of the Aa3 compound selected as the final compound was confirmed. Specifically, the FRT-YFP-ANO1 cells of Example 5-1 were pretreated with Aa3 in a concentration-dependent manner for 20 minutes. Thereafter, ANO1 was activated by treating with 20 μM of A23187, a Ca ²⁺ ion transporter. The A23187 does not induce secretion and release of Ca 2+ from intracellular endoplasmic reticulum, but induces intracellular uptake of Ca ²⁺ ions existing outside the cell. Thereafter, HTS analysis was performed in the same manner as in Example 5-1.

Figure 6 is a graph confirming the inhibitory effect of compounds according to one aspect on the activity of ANO1 induced by A23187.

As a result, as shown in Fig. 6, it was confirmed that Aa3, which was selected as the final compound, directly inhibited the physiological activity of ANO1 in a concentration-dependent manner. Specifically, it was confirmed that A23187 directly blocks the activity of ANO1 without inhibiting the release of ^{Ca2 +} from GPCR or the endoplasmic reticulum, which are indirect stimulation pathways, by absorbing Ca2+ ions into cells.

That is, Aa3 is known to directly act on ANO1 to block the biological activity of ANO1 without blocking the pathway that increases intracellular Ca2 ⁺ through ligand-receptor activation.

Example 7. Confirmation of anticancer activity by ANO1 regulation

7-1. Confirmation of cytotoxicity of Aa3 compound by difference in ANO1 expression

Since ANO1 has been reported to be highly expressed in many human tumors, biochemical inhibition of ANO1 activity and induction of proteolysis using selective inhibitors suggest that it may be therapeutically exploited to kill tumor cells. Previous studies have shown that inhibitors such as CaCCinh-A01, T16Ainh-A01, idebenone, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), Ani9, and luteolin are known to inhibit the proliferation of ANO1-overexpressing cancer cells. In order to confirm the biological effect of Aa3 selected in Example 5, we examined whether Aa3 could affect the proliferation of A549 and NCI-H460 cells with different expression levels of ANO1.

First, the endogenous ANO1 expression of human lung adenocarcinoma A549 cell line and human large-cell lung carcinoma NCI-H460 cell line was analyzed by immunoblotting. In addition, cell viability was analyzed using the same method as in Example 6.

Figure 7a shows the results of confirming the expression of ANO1 protein in lung cancer cell lines.

As a result, as shown in Fig. 7a, it was confirmed that ANO1 was expressed in A549 and NCI-H460 cell lines. Specifically, it was confirmed that the expression level of ANO1 was relatively high in the NCI-H460 cell line compared to the A549 cell line.

Figure 7b is a graph showing the cytotoxicity of compounds according to one aspect in lung cancer cell lines showing differences in the expression of ANO1.

As a result, as shown in Fig. 7b, Aa3 not only significantly reduced cell viability in a dose-dependent manner in both NCI-H460 and A549 cells, but also significantly lower cell viability was observed in NCI-H460 cells overexpressing ANO1 compared to A549 cells.

That is, Aa3 reduced the survival rates of both types of lung cancer cells, but showed higher anti-proliferation in NCI-H460 cells with relatively high ANO1 expression, indicating that it may exhibit better anticancer effects in lung cancer cells with positive expression of ANO1.

7-2. Confirmation of the effect of Aa3 compound on ANO1 protein expression

The mechanism of action of ANO1 inhibitors includes a method of inhibiting the activity of ANO1 by inducing the synthesis inhibition or protein degradation of ANO1 protein, and a method of selectively inhibiting only the physiological activity of the ANO1 chloride ion channel without inducing the synthesis inhibition or degradation of ANO1 protein. Therefore, the mechanism of action of ANO1 inhibitors was confirmed by confirming the effect of Aa3 compound on ANO1 protein expression. Specifically, according to the results of Example 7-1, the NCI-H460 cell line with relatively high ANO1 expression was treated with the Aa3 compound at concentrations of 10 μM and 20 μM for 24 and 48 hours, respectively, and the amount of ANO1 protein expression was confirmed through Western blotting.

Figure 7c shows the results of confirming the expression of ANO1 protein by compounds according to one aspect.

As a result, as shown in Fig. 7c, there was almost no change in the expression level of ANO1 protein in NCI-H460 regardless of the treatment concentration and time of Aa3, confirming that it did not induce inhibition of synthesis or degradation of ANO1 protein.

That is, compounds according to one aspect selectively inhibit only the physiological activity of the ANO1 chloride ion channel, thereby inhibiting the activity of ANO1, and thus can be used as a therapeutic agent for various cancers, including lung cancer.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (10)

A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following chemical formula 1 or chemical formula 2 as an active ingredient;
[Chemical Formula 1]

[Chemical formula 2]

Here, R ¹ is , , , , and , R ² is , , , , , and is selected from the group consisting of
In claim 1, the compound is N4-(2-methoxyethyl)-6-methyl-N2-(4-(phenylamino)phenyl)pyrimidine-2,4-diamine, 5-((2-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-4-yl)amino)pentan-1-ol, A pharmaceutical composition for preventing or treating cancer, wherein the composition is selected from the group consisting of 5-((6-methyl-2-((4-(p-tolyloxy)phenyl)amino)pyrimidin-4-yl)amino)pentan-1-ol, and 4-((4-((4-(4-chlorophenoxy)phenyl)amino)-6-methylpyrimidin-2-yl)amino)butan-1-ol. A pharmaceutical composition for preventing or treating cancer, wherein the compound according to claim 1 is a compound represented by the following chemical formula 3:
[Chemical Formula 3]
.
delete delete A pharmaceutical composition for preventing or treating cancer, which inhibits the activation of anoctamamine 1 according to claim 1.
delete A pharmaceutical composition for preventing or treating cancer, which selectively inhibits anoctamamine 1 according to claim 1. A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, wherein the cancer is selected from the group consisting of breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, stomach cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's Disease, endocrine cancer, and sarcoma. A health functional food composition for preventing or improving cancer, comprising a compound represented by the following chemical formula 1 or chemical formula 2 as an active ingredient;
[Chemical Formula 1]

[Chemical formula 2]

Here, R ¹ is , , , , and , R ² is , , , , , and is selected from the group consisting of