2,4-Diphenylaminopyrimidine derivatives, their preparation methods and applications

Abstract

The present invention discloses a 2,4-dianilinopyrimidine derivative represented by the general formula I or its optical isomer, enantiomer, diastereomer, racemate or racemic mixture, or its pharmaceutically acceptable salt or ester: Such compounds can release the signaling molecule NO while maintaining the FAK kinase inhibitory activity, ensuring the synergistic anti-tumor proliferation, invasion and metastasis effects of the FAK inhibitor and NO. In addition, the synergistic mechanism between the FAK inhibitor and NO is further explored.

Description

Technical Field

[0001] The present invention relates to the fields of medicinal chemistry and therapeutics, and relates to a 2,4-dianilinopyrimidine derivative, a preparation method and uses thereof. Background Art

[0002] Cancer metastasis is the process by which cancer cells grow away from the primary site and metastasize to distant tissues and organs to form new lesions, and is one of the main biological characteristics of malignant tumors. Tumor cells with different degrees of invasion / metastasis ability are the main obstacles to the clinical treatment of cancer. In more than 90% of cases, the metastatic spread of cancer cells is an important cause of patient death and poor prognosis. Unfortunately, for metastatic tumors, there are currently no effective clinical treatment and intervention measures.

[0003] Focal adhesion kinase (FAK) is a multifunctional key regulator of signal transduction mediated by growth factor receptors and integrins in the tumor microenvironment, and can regulate the adhesion, survival, proliferation and migration processes of tumor cells through its kinase activity and scaffold function. FAK kinase inhibitors bind to the central kinase region of FAK by selectively inserting into the ATP-binding pocket of FAK, inhibiting the phosphorylation of key sites of FAK. To date, a variety of FAK kinase inhibitors have been reported to have good in vitro and in vivo anti-tumor activities, and some have entered preclinical or clinical studies. Clinical research results show that the monotherapy of these kinase inhibitors has poor efficacy in human trials, and combination therapy can synergistically enhance the anti-cancer effects of the two drugs. Preclinical research data of a variety of FAK kinase inhibitors show that although such inhibitors have excellent ability to inhibit kinase activity, they often have no obvious effect on FAK expression. PROTAC targeting FAK can simultaneously hydrolyze FAK protein and inhibit FAK kinase activity, and has nanomolar anti-cancer activity. Administration in animal models all shows anti-cancer ability superior to that of small molecule kinase inhibitors. However, due to its low bioavailability and difficulty in drug formation, no PROTAC has entered clinical research, and the clinical trial efficacy of simultaneously inhibiting FAK kinase and expression cannot be confirmed.

[0004] NO, as a gaseous signaling molecule, is composed of L-arginine, NADPH and O 2They are generated in the human body under the action of nitric oxide synthase (NOS) and play an important role in the occurrence, development, and metastasis of tumors. During this process, high-concentration NO inhibits tumor growth through its anti-tumor mechanisms such as mediating the tumoricidal effect of macrophages by itself, generating free radicals to damage DNA, affecting cell metabolism, and inhibiting platelet aggregation. It should be noted that high-concentration NO can induce apoptosis of tumor cells by activating the expression of p53, etc., and the up-regulation of p53 has been proven to be an effective way to reduce the expression of FAK. Therefore, the introduction of NO can not only enhance the kinase activity of FAK but also has the possibility of inhibiting the expression of FAK to a certain extent. Furoxans, as an important class of NO donors, release NO in the body through enzymatic or non-enzymatic action and are often conjugated with other anti-tumor drugs or their active fragments to produce high levels of NO, and the two work synergistically to exert anti-tumor activity. On this basis, a new strategy for drug research on synergistically intervening in TNBC metastasis using NO donors and FAK inhibitors has emerged. Summary of the Invention

[0005] The purpose of the present invention is to provide a 2,4-dianilinopyrimidine derivative, which is a novel FAK kinase inhibitor containing an NO donor fragment. Such compounds can release the signaling molecule NO while maintaining the FAK kinase inhibitory activity, ensuring the synergistic anti-tumor proliferation, invasion, and metastasis effects of the FAK inhibitor and NO.

[0006] To solve the above technical problems, the technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention claims to protect the 2,4-dianilinopyrimidine derivative shown in general formula I or its optical isomers, enantiomers, diastereomers, racemates or racemic mixtures, or its pharmaceutically acceptable salts or esters:

[0008]

[0009] Wherein: L is selected from: C1-C 12 Straight-chain or branched-chain alkanes, substituted C1-C 12 Straight-chain or branched-chain alkanes, C2-C6 alkynes, substituted C2-C6 alkynes, C2-C6 alkenes, substituted C2-C6 alkenes, C3-C8 cycloalkanes, substituted C3-C8 cycloalkanes, benzene rings, substituted benzene rings;

[0010] The substituted C1-C 12The substituents in straight-chain or branched-chain alkanes, substituted C2-C6 alkynes, substituted C2-C6 alkenes, substituted C3-C8 cycloalkanes and substituted benzene rings are selected from: C1-C6 straight-chain or branched-chain alkanes, halogens, amino groups, carboxyl groups, phenyl groups, benzyl groups, phenyloxy groups, =O, haloalkyl groups, hydroxyl groups, alkoxy groups, arylalkyl groups, cycloalkyl groups, alkylamino groups.

[0011] In some embodiments, the L is selected from: C3-C12 straight-chain alkanes, substituted C3-C12 straight-chain alkanes, C2-C4 alkynes, substituted C2-C4 alkynes, C2-C4 alkenes, substituted C2-C4 alkenes, substituted C3-C6 cycloalkanes;

[0012] The number of substituents in the substituted C3-C12 straight-chain alkanes, substituted C2-C4 alkynes, substituted C2-C4 alkenes, substituted C3-C6 cycloalkanes and substituted benzene rings is 1-3, and the substituents are selected from: C1-C4 alkanes.

[0013] In some embodiments, the number of substituents in the substituted C3-C6 cycloalkanes and substituted benzene rings is 1-2, and the substitution positions of the two substituents in the substituted C3-C6 cycloalkanes and substituted benzene rings are in the meta or para positions.

[0014] In some embodiments, the L is selected from:

[0015] In some embodiments, the 2,4-dianilinopyrimidine derivatives are selected from the following compounds:

[0016] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ1)

[0017]

[0018] 4-(4-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ2)

[0019]

[0020] 4-(5-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)pentyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ3)

[0021]

[0022] 4-(6-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)hexyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ4)

[0023]

[0024] 4-(7-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)heptyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ5)

[0025]

[0026] 4-(8-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)octyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ6)

[0027]

[0028] 4-(9-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)nonyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ7)

[0029]

[0030] 4-(10-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)decyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ8)

[0031]

[0032] 4-(11-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)undecyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ9)

[0033]

[0034] 4-(12-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)dodecyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 10 )

[0035]

[0036] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)-2,2-dimethylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 11 )

[0037]

[0038] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)-2-methylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 12 )

[0039]

[0040] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 13 )

[0041]

[0042] 4-((4-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 14 )

[0043]

[0044] 4-((4-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)but-2-en-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 15 )

[0045]

[0046] 4-((4-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)cyclohexyl)methoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 16 )

[0047]

[0048] 4-((4-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)benzyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 17 )

[0049]

[0050] 4-(4-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 18 )

[0051]

[0052] 4-(3-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 19 )

[0053]

[0054] 4-(4-(2-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)ethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 20 )

[0055]

[0056] In the following pharmacological experiments, the codes of the compounds are equivalent to the compounds corresponding to the codes here.

[0057] In a second aspect, another object of the present invention is to provide a method for preparing the 2,4-dianilinopyrimidine derivative represented by general formula I of the present invention, which comprises the following steps:

[0058] A. Compound II and compound III are reacted with an acid under a solvent state and at a certain temperature to obtain compound IV;

[0059] B. Compound IV and compound V are reacted under a solvent state and with a conditional reagent to obtain compound I.

[0060] The synthetic route of the 2,4-dianilinopyrimidine derivative represented by general formula I of the present invention is as follows:

[0061]

[0062] Wherein: L is defined as above.

[0063] In some embodiments, in step A, the solvent is one or both of isopropanol and ethanol; the acid is hydrochloric acid; the reaction temperature is 90 - 100 °C;

[0064] In step B, the solvent is one or both of anhydrous N,N-dimethylformamide and N,N-dimethylacetamide; the conditional reagent is one or both of carbodiimide hydrochloride and 4-dimethylaminopyridine; the reaction temperature is room temperature.

[0065] In a third aspect, the present invention claims to protect a pharmaceutical composition, which contains a therapeutically effective amount of the said 2,4-dianilinopyrimidine derivative or its optical isomers, enantiomers, diastereomers, racemates or racemic mixtures, or its pharmaceutically acceptable salts and pharmaceutically acceptable carriers, adjuvants or vehicles.

[0066] In a fourth aspect, the application of the said 2,4-dianilinopyrimidine derivative or its solvate in the preparation of a drug for preventing and / or treating tumor diseases.

[0067] In a fifth aspect, the application of the said 2,4-dianilinopyrimidine derivative or its solvate in the preparation of an NO-donating FAK kinase inhibitor.

[0068] In a sixth aspect, the application of the said 2,4-dianilinopyrimidine derivative or its solvate in the preparation of a drug for releasing exogenous high-concentration NO for strengthening.

[0069] The beneficial effects of the present invention:

[0070] Based on the principle of drug combination, the present invention designs and synthesizes a class of NO-donating FAK kinase inhibitors for the first time. It is both a new generation of NO-donating compounds and a novel FAK kinase inhibitor. It can not only interfere with the proliferation, invasion and metastasis of cancer cells in vitro and in vivo by inhibiting FAK kinase activity, but also release exogenous high-concentration NO simultaneously to enhance the anti-cancer ability of the drug. This type of integrated prodrug molecule has high tumor specificity, enabling the specific release of FAK kinase inhibitor and NO in tumor cells, avoiding the toxic and side effects on normal cells caused by the low targeting of their single use, and solving the problems of lack of selectivity in combined drug use and possible reduction of drug efficacy. The pharmaceutical compositions containing the target compounds and their medical uses, especially in the prevention and / or treatment of diseases such as tumors, have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Shows the proliferation inhibition of MDA-MB-231 cells by the 2,4-dianilinopyrimidine derivative of the present invention;

[0072] Figure 2 Shows the binding mode of compound Ⅰ6 and FAK (PDB ID: 2JKK);

[0073] Figure 3 Shows the invasion inhibition of MDA-MB-231 cells by compound Ⅰ6 in vitro;

[0074] Figure 4 Shows the in vitro migration inhibition of MDA-MB-231 cells by compound Ⅰ6;

[0075] Figure 5 Shows the inhibition of the formation of focal adhesions (FAs) and stress fibers (SFs) by compound Ⅰ6, TAE226 and Ⅰ6(PTIO) in MDA-MB-231 cells;

[0076] Figure 6 Shows the effect of Ⅰ6 on FAK and its downstream molecules in MDA-MB-231 cells;

[0077] Figure 7 Shows the apoptosis induction of TNBC cells by compound Ⅰ6 in vitro;

[0078] Figure 8 Shows the NO release determination of the compound by the probe method and the inhibition of MDA-MB-231 cells by compound Ⅰ6, Ⅳ, Ⅴ6;

[0079] Figure 9 Shows the anti-cancer effect of compound Ⅰ6 in the MBA-MD-231 lung metastasis experimental model;

[0080] Figure 10Results of compound Ⅰ6 inhibiting triple-negative breast cancer metastasis in vivo. Detailed implementation manners

[0081] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions fall within the protection scope of the present invention.

[0082] Example 1

[0083] Synthesis of 2,4-dianilinopyrimidine derivatives

[0084] 2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetic acid (No.: Ⅳ)

[0085]

[0086] Dissolve Ⅱ (3 g, 10 mmol) and 4-aminophenylacetic acid (1.9 g, 13 mmol) in isopropanol (50 ml), add 12N HCl (30 drops) at 0 °C, stir at 95 °C for 18 h, cool the reaction solution to room temperature, filter, and wash the filter cake with isopropanol to obtain yellow solid hydrochloride Ⅳ (98%).

[0087] 1 H NMR (400 MHz, DMSO) δ 12.05 (s, 1H), 10.01 (s, 1H), 8.88 (q, J = 4.5 Hz, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.32 (s, 1H), 7.80 (dd, J = 7.9, 1.6 Hz, 1H), 7.56–7.50 (m, 2H), 7.50–7.45 (m, 1H), 7.25–7.21 (m, 2H), 7.19 (dd, J = 7.6, 1.2 Hz, 1H), 3.56 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H).

[0088] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ1)

[0089]

[0090] Compound Ⅳ (0.22 mmol) and compound Ⅴ1 (0.22 mmol) were dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added, and after stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), the organic phase was dried over anhydrous sodium carbonate, and evaporated under reduced pressure. The target compound was purified by column chromatography to obtain a white solid (53%).

[0091] 1 H NMR (500 MHz, DMSO) δ 11.61 (s, 1H), 9.46 (s, 1H), 8.76 (m, J = 6.1, 5.2 Hz, 2H), 8.22 (s, 1H), 8.04–7.98 (m, 2H), 7.91–7.84 (m, 1H), 7.76 (dd, J = 7.9, 1.6 Hz, 1H), 7.74–7.69 (m, 2H), 7.60 (m, J = 8.2 Hz, 2H), 7.52–7.45 (m, 1H), 7.19–7.15 (m, 2H), 7.13 (dd, J = 7.5, 1.2 Hz, 1H), 4.44 (t, J = 6.1 Hz, 2H), 4.17 (t, J = 6.3 Hz, 2H), 3.64 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H), 2.10 (p, J = 6.2 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 171.83, 169.20, 159.32, 159.20, 157.53, 155.91, 140.23, 139.06, 137.60, 136.63, 131.92, 130.49, 129.96, 128.78, 128.52, 127.42, 123.27, 122.25, 121.65, 121.03, 110.94, 105.75, 68.66, 61.06, 27.82, 26.80, 8.94. HRMS (ESI) calcd for C 31 H 28 ClN7O8S [M + H] + : 694.1465.

[0092] 4-(4-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ2)

[0093]

[0094] Compound Ⅳ (0.22 mmol) and compound Ⅴ2 (0.22 mmol) were dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), and the organic phase was dried over anhydrous sodium carbonate and evaporated under reduced pressure. Purification by column chromatography gave the target compound as a white solid (51%).

[0095] 1 H NMR (500 MHz, DMSO) δ 11.64 (s, 1H), 9.47 (s, 1H), 8.77 (m, J = 4.4 Hz, 2H), 8.21 (s, 1H), 8.04–7.98 (m, 2H), 7.86 (m, J = 7.5 Hz, 1H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H), 7.72 (m, J = 7.9 Hz, 2H), 7.64 (m, J = 8.2 Hz, 2H), 7.53–7.45 (m, 1H), 7.19 (m, J = 8.3 Hz, 2H), 7.14 (dd, J = 7.6 Hz, 1H), 4.40 (t, J = 6.1 Hz, 2H), 4.11 (t, J = 6.5 Hz, 2H), 3.37 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 1.80 (tt, 2H), 1.69 (tt, J = 11.6, 5.7 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 171.89, 169.38, 159.27, 158.16, 155.46, 155.01, 139.80, 139.48, 137.67, 136.55, 131.88, 130.45, 129.74, 128.76, 128.44, 127.99, 122.37, 121.90, 121.18, 120.06, 110.91, 105.48, 72.57, 65.86, 26.78, 25.05, 24.96, 15.21. HRMS (ESI) calcd for C 32 H 30 ClN7O8S [M + H] + : 708.1592.

[0096] 4-(5-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)pentyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ3)

[0097]

[0098] Compound Ⅳ (0.22 mmol) and compound Ⅴ3 (0.22 mmol) were dissolved in anhydrous DMF (2 ml). Carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), the organic phase was dried with anhydrous sodium carbonate, and evaporated under reduced pressure. The target compound was purified by column chromatography to obtain a white solid (54%).

[0099] 1 H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 9.46 (s, 1H), 8.77 (m, J = 7.2, 6.0 Hz, 2H), 8.20 (s, 1H), 8.01 (m, J = 7.2, 1.3 Hz, 2H), 7.93–7.85 (m, 1H), 7.78–7.71 (m, 3H), 7.61 (d, J = 8.2 Hz, 2H), 7.53–7.44 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.13 (dd, J = 8.1 Hz, 1H), 4.35 (t, J = 6.3 Hz, 2H), 4.07 (t, J = 6.5 Hz, 2H), 3.61 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 1.75–1.70 (m, 2H), 1.66–1.59 (m, 2H), 0.94–0.81 (m, 2H). 13 C NMR (101 MHz, DMSO) δ 171.91, 169.37, 169.21, 159.28, 158.16, 155.44, 155.01, 139.79, 139.44, 137.68, 136.57, 132.17, 132.04, 131.98, 131.87, 130.46, 129.73, 129.13, 128.75, 128.44, 128.04, 122.37, 121.88, 121.17, 120.07, 110.89, 105.56, 71.74, 64.42, 28.02, 27.47, 26.78, 22.00, 13.23. HRMS (ESI) calcd for C 33 H 32 ClN7O8S [M + H] + : 722.1753.

[0100] 4-(6-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)hexyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ4)

[0101]

[0102] Compound Ⅳ (0.22 mmol) and compound Ⅴ4 (0.22 mmol) were dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), the organic phase was dried with anhydrous sodium carbonate and evaporated under reduced pressure. The target compound was obtained as a white solid (51%) by column chromatography purification.

[0103] 1 H NMR (500 MHz, DMSO) δ 11.63 (s, 1H), 9.46 (s, 1H), 8.79–8.73 (m, 2H), 8.20 (s, 1H), 8.00 (d, J = 7.8 Hz, 2H), 7.87 (t, J = 7.5 Hz, 1H), 7.74 (dt, J = 15.4, 7.8 Hz, 3H), 7.63 (d, J = 8.0 Hz, 2H), 7.49 (t, J = 8.0 Hz, 1H), 7.16 (dd, J = 21.8, 7.5 Hz, 4H), 4.34 (t, J = 6.2 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.61 (s, 2H), 2.82 (d, J = 4.4 Hz, 3H), 1.71 (t, J = 6.7 Hz, 2H), 1.57 (q, J = 6.7 Hz, 2H), 1.33 (d, J = 6.2 Hz, 4H). 13 C NMR (126 MHz, DMSO) δ 171.91, 169.37, 159.29, 158.17, 155.45, 155.03, 139.79, 139.45, 137.71, 136.55, 131.87, 130.44, 129.72, 128.74, 128.44, 128.08, 122.37, 121.88, 121.18, 120.07, 110.87, 105.57, 71.80, 64.52, 28.45, 28.19, 25.29, 25.03, 23.08, 15.21. HRMS (ESI) calcd for C 34 H 34 ClN7O8S [M + H] +:736.1920.

[0104] 4-(7-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)heptyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ5)

[0105]

[0106] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ5 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (56%).

[0107] 1 H NMR (500 MHz, DMSO) δ 11.62 (s, 1H), 9.46 (s, 1H), 8.76 (dd, J = 9.6, 5.5 Hz, 2H), 8.20 (s, 1H), 8.03–7.97 (m, 2H), 7.92–7.85 (m, 1H), 7.82–7.70 (m, 3H), 7.61 (d, J = 8.2 Hz, 2H), 7.52–7.45 (m, 1H), 7.20–7.06 (m, 3H), 4.34 (t, J = 6.3 Hz, 2H), 4.05 (t, J = 6.5 Hz, 2H), 3.61 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H), 1.70 (h, J = 6.6 Hz, 2H), 1.57 (t, J = 6.8 Hz, 2H), 1.29 (dt, J = 8.7, 4.2 Hz, 6H). 13 C NMR (126 MHz, DMSO) δ 171.90, 169.37, 159.30, 158.16, 155.44, 155.05, 139.78, 139.44, 137.70, 136.58, 131.88, 130.47, 129.71, 128.75, 128.73, 128.45, 128.09, 122.39, 121.89, 121.18, 120.05, 110.87, 105.35, 71.87, 64.57, 28.51, 28.48, 28.19, 25.64, 25.35, 23.09, 14.77. HRMS (ESI) calcd for C 35 H36 ClN7O8S[M+H] + :750.2065.

[0108] 4-(8-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)octyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ6)

[0109]

[0110] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ6 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench the DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (53%).

[0111] 1 H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 9.47 (s, 1H), 8.76 (q, J = 6.8, 5.8 Hz, 2H), 8.21 (s, 1H), 8.04–7.97 (m, 2H), 7.93–7.84 (m, 1H), 7.74 (dt, J = 8.4, 7.1 Hz, 3H), 7.62 (d, J = 8.3 Hz, 2H), 7.53–7.44 (m, 1H), 7.18–7.10 (m, 3H), 4.34 (t, J = 6.3 Hz, 2H), 4.04 (t, J = 6.6 Hz, 2H), 3.60 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H), 1.70 (p, J = 6.5 Hz, 2H), 1.56 (q, J = 6.7 Hz, 2H), 1.33–1.23 (m, 8H). 13 C NMR (101 MHz, DMSO) δ 171.89, 169.36, 159.31, 158.15, 155.44, 155.04, 140.53, 136.57, 131.88, 130.46, 129.70, 128.73, 128.42, 127.74, 122.79, 121.87, 121.16, 120.06, 110.88, 105.55, 73.02, 64.59, 28.96, 28.86, 28.55, 28.27, 25.69, 25.37, 23.09, 15.70. HRMS (ESI) calcd for C36 H 38 ClN7O8S[M+H] + :764.2255.

[0112] 4-(9-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)nonoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ7)

[0113]

[0114] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ7 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (48%).

[0115] 1 H NMR (500 MHz, DMSO) δ 11.64 (s, 1H), 9.47 (s, 1H), 8.76 (q, J = 4.7 Hz, 2H), 8.21 (s, 1H), 8.03–7.97 (m, 2H), 7.88 (t, J = 7.5 Hz, 1H), 7.79–7.70 (m, 3H), 7.62 (d, J = 8.2 Hz, 2H), 7.52–7.45 (m, 1H), 7.20–7.07 (m, 3H), 4.34 (t, J = 6.4 Hz, 2H), 4.03 (t, J = 6.5 Hz, 2H), 3.60 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 1.70 (p, J = 6.5 Hz, 2H), 1.55 (p, J = 6.6 Hz, 2H), 1.25–1.20 (m, 10H). 1313C NMR (126 MHz, DMSO) δ 171.87, 169.36, 159.31, 158.17, 155.44, 155.05, 139.80, 139.44, 137.71, 136.56, 131.87, 130.45, 129.69, 128.72, 128.44, 128.09, 122.37, 121.88, 121.16, 120.06, 110.87, 105.55, 71.90, 64.58, 29.28, 28.99, 28.88, 28.59, 28.28, 25.75, 25.43, 22.56, 14.41. HRMS (ESI) calcd for C 37 H 40 ClN7O8S [M+H] + : 778.2437.

[0116] 4-(10-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)decoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ8)

[0117]

[0118] Compound Ⅳ (0.22 mmol) and compound Ⅴ8 (0.22 mmol) were dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added, and the mixture was stirred at room temperature for 1 - 6 h. Then, DMF was quenched with water, and the mixture was extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), the organic phase was dried over anhydrous sodium carbonate, and evaporated under reduced pressure. The target compound was obtained as a white solid (51%) by column chromatography purification.

[0119] 11H NMR (500 MHz, DMSO) δ 11.65 (s, 1H), 9.47 (s, 1H), 8.77 (q, J = 4.6 Hz, 2H), 8.21 (s, 1H), 8.03–7.97 (m, 2H), 7.92–7.85 (m, 1H), 7.79–7.70 (m, 3H), 7.62 (d, J = 8.2 Hz, 2H), 7.52–7.44 (m, 1H), 7.19–7.11 (m, 3H), 4.34 (t, J = 6.3 Hz, 2H), 4.03 (t, J = 6.5 Hz, 2H), 3.59 (s, 2H), 2.81 (d, J = 4.4 Hz, 3H), 1.74–1.65 (m, 2H), 1.54 (t, J = 6.8 Hz, 2H), 1.24–1.20 (m, 12H). 13 13C NMR (126 MHz, DMSO) δ 171.86, 169.36, 159.32, 158.18, 155.44, 155.04, 139.81, 139.44, 137.72, 136.57, 131.87, 130.46, 129.67, 128.72, 128.44, 128.09, 122.37, 121.88, 121.15, 120.06, 110.88, 105.55, 71.90, 64.57, 29.33, 29.31, 29.09, 28.95, 28.59, 28.30, 25.79, 25.43, 22.57, 14.41. HRMS (ESI) calcd for C 38 H 42 ClN7O8S [M+H] + : 792.2585.

[0120] 4-(11-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)undecyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ9)

[0121]

[0122] Compound Ⅳ (0.22 mmol) and compound Ⅴ9 (0.22 mmol) were dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), the organic phase was dried over anhydrous sodium carbonate, and evaporated under reduced pressure. The target compound was obtained as a white solid (49%) by purification by column chromatography.

[0123] 1 1H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 9.47 (s, 1H), 8.80–8.73 (m, 2H), 8.22 (d, J = 1.2 Hz, 1H), 8.03–7.97 (m, 2H), 7.89 (t, J = 7.4 Hz, 1H), 7.75 (q, J = 7.4 Hz, 3H), 7.62 (d, J = 8.2 Hz, 2H), 7.48 (t, J = 7.9 Hz, 1H), 7.17–7.03 (m, 4H), 4.35 (t, J = 6.3 Hz, 2H), 4.02 (t, J = 6.5 Hz, 2H), 3.59 (s, 2H), 2.81 (d, J = 4.4 Hz, 3H), 1.69 (q, J = 6.8 Hz, 2H), 1.53 (d, J = 7.5 Hz, 2H), 1.22 (d, J = 12.4 Hz, 14H). 13 13C NMR (101 MHz, DMSO) δ 171.94, 169.36, 159.53, 159.33, 158.17, 155.43, 139.79, 139.43, 137.70, 136.58, 131.88, 130.47, 129.67, 128.73, 128.44, 128.10, 123.92, 122.38, 121.14, 120.05, 110.27, 105.52, 72.09, 64.55, 29.42, 29.39, 29.34, 29.12, 28.96, 28.59, 28.30, 26.78, 25.78, 25.45. HRMS (ESI) calcd for C 39 H 44 ClN7O8S [M+H] + : 806.2747.

[0124] 4-(12-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)dodecyloxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 10 )

[0125]

[0126] Compound Ⅳ (0.22 mmol) and compound Ⅴ 10(0.22 mmol) was dissolved in anhydrous DMF (2 ml), and carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), and the organic phase was dried with anhydrous sodium carbonate and evaporated under reduced pressure. Purification by column chromatography gave the target compound as a white solid (46%).

[0127] 1 H NMR (400 MHz, DMSO) δ 11.66 (s, 1H), 9.48 (s, 1H), 8.80–8.73 (m, 2H), 8.22 (s, 1H), 8.04–7.97 (m, 2H), 7.89 (td, J = 7.3, 1.3 Hz, 1H), 7.80–7.70 (m, 3H), 7.63 (d, J = 1.8 Hz, 1H), 7.53–7.44 (m, 1H), 7.17–7.06 (m, 3H), 4.35 (t, J = 6.3 Hz, 2H), 4.02 (t, J = 6.5 Hz, 3H), 3.59 (s, 2H), 2.81 (d, J = 4.5 Hz, 4H), 1.71 (t, J = 7.1 Hz, 2H), 1.53 (t, J = 6.8 Hz, 3H), 1.22–1.15 (m, 16H). 13 C NMR (101 MHz, DMSO) δ 171.85, 169.36, 159.33, 157.77, 155.43, 155.05, 139.81, 139.44, 137.70, 136.57, 131.87, 130.46, 129.67, 128.73, 128.44, 128.08, 124.83, 122.37, 120.03, 119.47, 110.88, 105.53, 71.90, 64.54, 29.47, 29.43, 29.39, 29.11, 28.98, 28.58, 28.30, 26.78, 25.78, 25.44, 22.58, 14.42. HRMS (ESI) calcd for C 40 H 46 ClN7O8S [M + H] + : 820.2885.

[0128] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)-2,2-dimethylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 11 )

[0129]

[0130] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ 11 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (59%).

[0131] 1 H NMR (500 MHz, DMSO) δ 11.64 (s, 1H), 9.46 (s, 1H), 8.78 (dd, J = 20.0, 6.3 Hz, 2H), 8.22 (s, 1H), 8.01–7.96 (m, 2H), 7.90–7.83 (m, 1H), 7.78 (dd, J = 7.9, 1.5 Hz, 1H), 7.75–7.67 (m, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.51–7.44 (m, 1H), 7.18–7.09 (m, 3H), 4.12 (s, 2H), 3.94 (s, 2H), 3.63 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 0.97 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 171.67, 169.36, 159.25, 158.10, 155.43, 155.02, 140.35, 139.09, 137.77, 136.61, 132.24, 130.57, 129.70, 128.54, 128.46, 127.83, 122.37, 121.88, 121.17, 119.77, 110.67, 105.58, 75.16, 68.63, 29.87, 29.52, 25.95, 20.82, 8.21. HRMS (ESI) calcd for C 33 H 32 ClN7O8S [M + H] + : 722.1798.

[0132] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)-2-methylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 12 )

[0133]

[0134] Dissolve Compound Ⅳ (0.22 mmol) and Compound Ⅴ 12 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench the DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (51%).

[0135] 1 H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 9.47 (s, 1H), 8.77 (q, J = 4.5 Hz, 2H), 8.22 (s, 1H), 8.04–7.97 (m, 2H), 7.87 (t, J = 7.5 Hz, 1H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H), 7.74–7.69 (m, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.53–7.44 (m, 1H), 7.19–7.08 (m, 3H), 4.30 (d, J = 5.8 Hz, 2H), 4.06 (d, J = 6.1 Hz, 2H), 3.64 (s, 2H), 2.82 (d, J = 4.4 Hz, 3H), 2.31 (dp, J = 13.8, 7.4, 6.9 Hz, 1H), 0.98 (d, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 171.79, 169.37, 159.24, 158.13, 155.44, 154.63, 139.79, 139.51, 137.70, 136.59, 131.88, 130.51, 129.72, 128.69, 128.45, 127.86, 122.38, 121.89, 121.19, 119.91, 111.25, 105.58, 72.40, 65.40, 29.27, 25.88, 23.18, 21.12. HRMS (ESI) calcd for C 32 H 30 ClN7O8S [M + H] + : 708.1664.

[0136] 4-(3-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)butoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 13 )

[0137]

[0138] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ 13 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (53%).

[0139] 1 H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 9.46 (s, 1H), 8.77 (q, J = 5.7, 5.1 Hz, 2H), 8.22 (s, 1H), 8.02–7.94 (m, 2H), 7.92–7.82 (m, 1H), 7.77 (dd, J = 7.9, 1.5 Hz, 1H), 7.74–7.69 (m, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.54–7.44 (m, 1H), 7.18–7.08 (m, 3H), 4.18–4.07 (m, 4H), 3.95 (s, 2H), 3.62 (s, 2H), 3.08–3.00 (m, 2H), 2.82 (d, J = 4.5 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 171.62, 169.37, 159.22, 158.10, 155.43, 155.01, 139.80, 139.52, 137.83, 136.60, 131.86, 129.93, 129.66, 128.49, 128.44, 127.81, 122.36, 121.88, 121.18, 119.73, 110.64, 105.58, 74.64, 68.70, 67.31, 28.83, 26.78, 11.24. HRMS (ESI) calcd for C 32 H 30 ClN7O8S [M + H] + : 708.1649.

[0140] 4-((4-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 14 )

[0141]

[0142] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ 14 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench the DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (62%).

[0143] 1 H NMR (500 MHz, DMSO) δ 11.62 (s, 1H), 9.48 (s, 1H), 8.76 (d, J = 4.7 Hz, 1H), 8.22 (s, 1H), 8.04–7.99 (m, 2H), 7.89 (t, J = 7.6 Hz, 1H), 7.79–7.71 (m, 3H), 7.63 (d, J = 8.2 Hz, 2H), 7.54–7.47 (m, 1H), 7.21–7.13 (m, 3H), 5.25 (s, J = 1.8 Hz, 2H), 4.87 (s, J = 1.8 Hz, 2H), 3.70 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 171.21, 169.37, 158.48, 158.16, 155.46, 155.06, 147.54, 147.33, 139.76, 139.58, 137.42, 136.70, 131.92, 130.52, 129.84, 128.83, 128.45, 127.48, 122.44, 121.91, 120.09, 119.47, 111.16, 105.60, 85.07, 76.93, 67.46, 59.53, 29.51, 26.78. HRMS (ESI) calcd for C 32 H 26 ClN7O8S [M + H] + : 704.1313.

[0144] 4-((4-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)but-2-en-1-yl)oxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 15 )

[0145]

[0146] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ 15 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (64%).

[0147] 1 H NMR (400 MHz, DMSO) δ 11.62 (s, 1H), 9.47 (s, 1H), 8.81–8.72 (m, 2H), 8.22 (s, 1H), 8.05–7.98 (m, 2H), 7.93–7.84 (m, 1H), 7.76 (d, J = 7.4 Hz, 2H), 7.75–7.65 (m, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.54–7.45 (m, 1H), 7.25–7.10 (m, 3H), 5.94–5.80 (m, 1H), 5.06 (d, J = 4.8 Hz, 2H), 4.77–4.71 (m, 2H), 4.20–4.08 (m, 1H), 3.65 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 171.27, 170.52, 158.99, 158.16, 155.45, 155.07, 139.76, 139.49, 137.56, 136.62, 132.18, 132.06, 131.90, 130.45, 129.79, 128.78, 128.44, 127.80, 122.42, 121.89, 121.20, 120.07, 111.14, 105.58, 66.94, 60.67, 28.81, 27.58. HRMS (ESI) calcd for C 32 H 28 ClN7O8S [M + H] + : 706.1504.

[0148] 4-((4-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)cyclohexyl)methoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 16 )

[0149]

[0150] Dissolve compound Ⅳ (0.22 mmol) and compound Ⅴ 16 (0.22 mmol) in anhydrous DMF (2 ml), add carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol), stir at room temperature for 1 - 6 h, quench the DMF with water, then extract with DCM (30 ml × 3), wash with saturated salt (30 ml × 2), dry the organic phase with anhydrous sodium carbonate, and evaporate under reduced pressure. Purify by column chromatography to obtain the target compound as a white solid (57%).

[0151] 1 H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 9.48 (d, J = 2.9 Hz, 1H), 8.77 (q, J = 6.1, 5.3 Hz, 2H), 8.22 (s, 1H), 8.04–7.96 (m, 2H), 7.87 (dd, J = 8.7, 6.5 Hz, 1H), 7.74 (td, J = 8.5, 8.0, 5.2 Hz, 3H), 7.63 (dd, J = 8.6, 3.1 Hz, 2H), 7.49 (td, J = 8.2, 3.2 Hz, 1H), 7.22–7.15 (m, 3H), 4.28 (d, J = 6.9 Hz, 1H), 4.18 (d, J = 5.6 Hz, 2H), 4.01 (d, J = 7.2 Hz, 1H), 3.89 (d, J = 6.4 Hz, 2H), 3.63 (s, 2H), 2.82 (d, J = 4.4 Hz, 3H), 1.75–1.67 (m, 2H), 1.50–1.39 (m, 2H), 0.99 (dt, J = 13.6, 8.7 Hz, 2H), 0.82 (dd, J = 26.3, 7.9 Hz, 2H). 1313C NMR (101 MHz, DMSO) δ 172.74, 168.94, 158.86, 158.15, 155.44, 155.26, 153.21, 139.78, 139.44, 137.73, 136.56, 131.36, 130.48, 129.74, 129.11, 128.46, 127.67, 122.40, 121.52, 121.00, 119.45, 110.81, 105.72, 75.97, 72.84, 69.15, 66.29, 30.58, 28.50, 28.05, 26.79, 25.07, 24.57. HRMS (ESI) calcd for C 36 H 36 ClN7O8S [M+H] + : 762.2107.

[0152] 4 - ((4 - ((2 - (4 - ((5 - chloro - 4 - ((2 - (methylcarbamoyl)phenyl)amino)pyrimidin - 2 - yl)amino)phenyl)acetoxy)methyl)benzyl)oxy) - 3 - (phenylsulfonyl)-1,2,5 - oxadiazole 2 - oxide (No.: Ⅰ 17 )

[0153]

[0154] Compound Ⅳ (0.22 mmol) and compound Ⅴ 17 (0.22 mmol) were dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4 - dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), and the organic phase was dried over anhydrous sodium carbonate and evaporated under reduced pressure. The target compound was purified by column chromatography to give a white solid (57%).

[0155] 11H NMR (500 MHz, DMSO) δ 11.62 (s, 1H), 9.48 (s, 1H), 8.76 (q, J = 6.0, 5.2 Hz, 2H), 8.22 (s, 1H), 8.01–7.96 (m, 2H), 7.87 (t, J = 7.5 Hz, 1H), 7.76 (dd, J = 7.8, 1.6 Hz, 1H), 7.71 (t, J = 7.9 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 7.1 Hz, 1H), 7.46–7.40 (m, 4H), 7.21 (d, J = 8.4 Hz, 2H), 7.17–7.11 (m, 1H), 5.47 (s, 2H), 5.17 (s, 2H), 3.71 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H). 13 13C NMR (126 MHz, DMSO) δ 171.75, 169.37, 159.11, 158.15, 155.46, 155.03, 139.75, 139.50, 137.59, 137.33, 136.60, 134.47, 131.90, 130.48, 129.84, 128.86, 128.75, 128.44, 127.86, 124.83, 124.61, 122.43, 121.92, 121.23, 120.07, 119.47, 111.02, 105.57, 72.58, 65.83, 31.66, 30.62. HRMS (ESI) calcd for C 36 H 30 ClN7O8S [M+H] + : 756.1754.

[0156] 4-(4-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 18 )

[0157]

[0158] Compound Ⅳ (0.22 mmol) and Compound Ⅴ 18(0.22 mmol) was dissolved in anhydrous DMF (2 ml), and carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), and the organic phase was dried over anhydrous sodium carbonate and evaporated under reduced pressure. The target compound was obtained as a white solid (56%) by column chromatography purification.

[0159] 1 H NMR (400 MHz, DMSO) δ 11.62 (s, 1H), 9.47 (s, 1H), 8.76 (t, J = 6.0 Hz, 2H), 8.23 (s, 1H), 8.07–8.00 (m, 2H), 7.91 (q, J = 8.5, 7.9 Hz, 1H), 7.80–7.73 (m, 3H), 7.62 (d, J = 8.2 Hz, 2H), 7.49 (t, J = 3.4 Hz, 1H), 7.48–7.40 (m, 4H), 7.19 (d, J = 8.6 Hz, 2H), 7.14 (t, J = 7.5 Hz, 1H), 5.16 (s, 2H), 3.71 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 171.75, 169.37, 158.88, 158.16, 155.46, 155.06, 152.72, 139.75, 139.50, 137.37, 136.73, 135.10, 132.06, 131.90, 130.50, 130.19, 129.83, 129.13, 129.02, 128.44, 127.84, 122.44, 121.92, 121.22, 120.21, 120.07, 111.74, 108.19, 65.82, 28.83, 26.79. HRMS (ESI) calcd for C 35 H 28 ClN7O8S [M + H] + : 742.1510.

[0160] 4-(3-((2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 19 )

[0161]

[0162] Compound Ⅳ (0.22 mmol) and compound Ⅴ19 (0.22 mmol) was dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), the organic phase was dried with anhydrous sodium carbonate, and evaporated under reduced pressure. The target compound was obtained as a white solid (57%) by column chromatography purification.

[0163] 1 H NMR (400 MHz, DMSO) δ 11.62 (s, 1H), 9.47 (s, 1H), 8.77 (q, J = 5.9, 5.3 Hz, 2H), 8.22 (d, J = 4.4 Hz, 1H), 8.08–8.02 (m, 1H), 7.94–7.87 (m, 1H), 7.80–7.71 (m, 3H), 7.65–7.59 (m, 2H), 7.53–7.27 (m, 5H), 7.20 (d, J = 8.5 Hz, 2H), 7.16–7.10 (m, 1H), 5.16 (s, 2H), 3.72 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 172.35, 169.38, 158.79, 158.16, 155.46, 154.83, 153.13, 139.76, 139.52, 139.18, 137.38, 136.71, 131.50, 130.73, 130.48, 129.84, 129.01, 128.44, 127.77, 126.26, 122.41, 121.91, 120.98, 120.07, 119.67, 119.31, 110.37, 106.03, 66.80, 30.61, 27.34. HRMS (ESI) calcd for C 35 H 28 ClN7O8S [M + H] + : 742.1476.

[0164] 4-(4-(2-(2-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetoxy)ethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (No.: Ⅰ 20 )

[0165]

[0166] Compound Ⅳ (0.22 mmol) and compound Ⅴ20 (0.22 mmol) was dissolved in anhydrous DMF (2 ml), carbodiimide hydrochloride (0.44 mmol) and 4-dimethylaminopyridine (0.05 mmol) were added. After stirring at room temperature for 1 - 6 h, the DMF was quenched with water, then extracted with DCM (30 ml × 3), washed with saturated salt (30 ml × 2), and the organic phase was dried over anhydrous sodium carbonate and evaporated under reduced pressure. The target compound was purified by column chromatography to obtain a white solid (61%).

[0167] 1 H NMR (500 MHz, DMSO) δ 11.62 (s, 1H), 9.45 (s, 1H), 8.76 (q, J = 5.0 Hz, 2H), 8.20 (s, 1H), 8.05 (d, J = 7.8 Hz, 2H), 7.91 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.3 Hz, 3H), 7.61 (d, J = 8.1 Hz, 2H), 7.48 (t, J = 7.9 Hz, 1H), 7.31 (m, 4H), 7.14 (t, J = 7.8 Hz, 3H), 4.27 (t, J = 6.6 Hz, 2H), 3.60 (s, 2H), 2.93 (q, J = 6.7, 5.5 Hz, 2H), 2.82 (d, J = 4.4 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 171.74, 169.37, 158.99, 158.17, 155.46, 155.02, 151.19, 139.78, 139.44, 137.44, 137.03, 136.70, 131.90, 130.97, 130.50, 129.77, 128.99, 128.44, 127.90, 122.40, 121.89, 121.20, 120.13, 120.07, 111.64, 105.58, 65.01, 31.66, 30.62, 26.78. HRMS (ESI) calcd for C 36 H 30 ClN7O8S [M + H] + : 756.1649.

[0168] Example 2

[0169] Compound Ⅰ1 - Ⅰ of the present invention 20 Study on the in vitro anti - proliferative effect on triple - negative breast cancer cell MDA - MB - 231

[0170] The anti - proliferative activity of MDA - MB - 231 cells was tested by the MTT method. TAE226 was used as a positive control. At the final density (5 × 10 4Cells / mL), the cells were seeded into 96-well plates. After 24 h, the cells were treated with 5 μM of the test compound and incubated at 37 °C for 72 h at different incubation concentrations. Then, 20 μL of MTT (5 mg / mL) was added to each well and incubation continued for 4 h. Finally, the culture medium was poured out, 150 μL of DMSO was added to each well to dissolve the purple crystalline formazan, and the absorbance was measured at 570 nm. All experiments were performed three times and the results were recorded as the mean. Inhibition rate (%) = [(negative control group - experimental group) / negative control group] × 100%.

[0171] Using TAE226 as a positive control, the inhibitory effects of compounds 8a-t on MDA-MB-231 cells were detected by the MTT method to evaluate their in vitro anti-triple-negative breast cancer activity. Most of the tested compounds had effective anti-proliferative activity against MDA-MB-231 cells, and the growth inhibition rate exceeded 80% at a concentration of 5 μM. Generally, when the linker (R group) was a straight-chain aliphatic hydrocarbon, the compounds were more effective than those with increased steric hindrance or increased unsaturation. In addition, compounds I1-I6 had a straight-chain aliphatic hydrocarbon R group with a carbon number less than 8; compared with the corresponding long-chain aliphatic hydrocarbon moiety containing derivatives (I7-I 10 ), C8 usually showed higher inhibitory activity. Among them, compounds I3, I5, I6 and I 12 had a proliferation inhibition rate of more than 92% against MDA-MB-231 cells, higher than that of TAE226 and 16 other compounds ( Figure 1 ).

[0172] Example 3

[0173] The anti-proliferative effects of the compounds I3, I5, I6 and I of the present invention 12 on different TNBC cells and normal cells MCF-10A were studied

[0174] The MTT method was used to detect the proliferation inhibitory effects of compounds I3, I5, I6 and I 12 on MDA-MB-231, MDA-MB-157, and MDA-MB-453 cells. TAE226 and 9a were used as positive controls. The cells were seeded at a final density of (5×10 4Cells were seeded at a density of 50 cells / mL in 96-well plates. At the end of 24 h, the cells were treated with test compounds at different concentrations and incubated at 37 °C for 72 h. Then, 20 μL of MTT (5 mg / mL) was added to each well and incubation was continued for 4 h. Finally, the medium was poured out, 150 μL of DMSO was added to each well to dissolve the purple crystal formazan, and the absorbance was read at 570 nm by spectrophotometry. All experiments were repeated three times and the results were recorded as the mean. Inhibition rate (%) = [(negative control group - experimental group) / negative control group] × 100%. The IC

[0175] values were calculated using GraphPad Prism software. Four of the most active compounds, I3, I5, I6, and I 12 were selected and their IC 50 values against different TNBC cells (MDA-MB-453, MDA-MB-157, and MDA-MB-231) were determined and compared with TAE226 (Table 1). These compounds were observed to have stronger inhibitory activity against the three cancer cells than TAE226 and the structurally similar compound 9a. The structure of 9a is as follows:

[0176]

[0177] As a result, compound I6 was the most active, especially against MDA-MB-231 (IC 50 = 0.122 μM), which was approximately 8 times that of the positive drug TAE226.

[0178] Table 1: Inhibitory effects of compounds on three triple-negative breast cancer cells MDA-MB-453, MDA-MB-157, MDA-MB-231, and normal cells MCF10A

[0179]

[0180] Example 4

[0181] Study on the inhibitory activity of the compounds I3, I5, I6, and I of the present invention against FAK kinase 12

[0182] ​ADP-glo kinase assay to screen FAK inhibitors. 5 μL reaction solution included 2.6 ng FAK, 0.4 μg / μL peptide substrate poly (4:1 glutamate, tyrosine), 25 μM ATP and the specified final concentration of test compound or negative control DMSO. The assay was performed in a 384-well plate according to the manufacturer's instructions. Briefly, the reaction solution was incubated at room temperature for 1 h, and then 5 μL ADP-glo reagent was added and incubated at room temperature for 40 min to stop the kinase reaction and deplete unconsumed ATP. Finally, 10 μL kinase detection reagent was added to the well and incubated for 30 min to convert ADP into ATP and generate luminescent signals. The signal was measured using a microplate reader (Flexstation 3). IC was calculated using Prism Graphpad software 50 value.

[0183] Table 2: Inhibitory activity of compounds against FAK kinase

[0184]

[0185] Description of compounds Ⅰ3, Ⅰ5, Ⅰ6 and Ⅰ 12 The inhibitory activity of FAK kinase is not completely consistent with its inhibitory effect on tumors.

[0186] Example 5

[0187] Molecular docking study of compound Ⅰ6 of the present invention

[0188] The docking study was completed using MOE 2018.01 software. The ligands were converted into 3D structures using Chem3D. The crystal structure of the FAK kinase domain bound by TAE226 was downloaded (PDB: 2JKQ). We defined the entire FAK enzyme as a receptor and selected the binding site of TAE226 as a site sphere. 3D images and visualization were prepared by OSS PyMol. The results are shown in Figure 2 shown.

[0189] To obtain detailed information on the binding mode of 8f in the FAK catalytic kinase domain, we performed molecular docking studies using the co-crystal structure (PDB: 2JKQ) in the Molecular Operating Environment (2019 version). Docking analysis showed that compound Ⅰ6 exactly occupied the position of the ATP binding pocket, and the binding mode of Ⅰ6 with FAK was similar to that of TAE226. The key interaction between Ⅰ6 and FAK was due to the bidentate bond formed between the 2-amino group on the 2,4-diaminopyrimidine core of Ⅰ6 and the Cys502 residue on the kinase hinge. In addition, the hydrogen bonds formed between the methylcarbamoyl part of the aromatic functional group of Ⅰ6 and the Asp564 residue and Asp550 of the DFG motif on the FAK activation loop also contributed to the binding of Ⅰ6 to the FAK kinase domain.

[0190] Example 6

[0191] Study on the anti - proliferative effects of compound Ⅴ6, Ⅳ and Ⅴ6 + Ⅳ(1:1) of the present invention on MDA - MB - 231 cells

[0192] The MTT method was used to detect compounds Ⅰ3, Ⅰ5, Ⅰ6 and Ⅰ 12 for their inhibitory effects on the proliferation of MDA - MB - 231, MDA - MB - 157, and MDA - MB - 453 cells. TAE226 was used as a positive control. Cells were seeded in 96 - well plates at a final density of (5×10 4 cells / mL). At the end of 24 h, the cells were treated with different concentrations of the test compounds and incubated at 37 °C for 72 h. Then, 20 μL of MTT (5 mg / mL) was added to each well and incubation continued for 4 h. Finally, the culture medium was poured out, 150 μL of DMSO was added to each well to dissolve the purple - blue crystalline formazan, and the absorbance was read at 570 nm by spectrophotometry. All experiments were repeated three times, and the results were recorded as the mean. Inhibition rate (%) = [(negative control group - experimental group) / negative control group]×100%. The IC 50 value was calculated using GraphPad Prism software.

[0193] Since compound Ⅰ6 consists of a TAE226 moiety (Ⅳ) and a benzenesulfonylfurazan moiety (Ⅴ6) ( Figure 3 ), we examined their inhibitory activities against TNBC cells in vitro. The IC 50 value of Ⅰ6 against MDA - MB - 231 cells (0.126 μM) was significantly lower than that of the individual compounds Ⅳ (1.95 μM), Ⅴ6 (1.51 μM), and the combination of Ⅳ and Ⅴ6 at equimolar doses (IC 50 = 1.22 μM). These results suggest that the anti - proliferative activity of Ⅰ6 may be due to the synergistic effect of the FAK kinase inhibitory activity generated by the TAE226 moiety and the NO generated by the benzenesulfonylfurazan moiety.

[0194] Example 7

[0195] Compounds Ⅰ3, Ⅰ5, Ⅰ6 and Ⅰ of the present invention 12 Study on the ability of generating intracellular NO

[0196] The cell digestion and counting method was used to prepare a cell suspension with a concentration of 5×10 4 cells / mL and seeded in 6 - well plates. According to the grouping settings, Ⅰ6 (1, 2, 4 μM), Ⅰ3 (4 μM), Ⅰ5 (4 μM) and Ⅰ 12(4 μM), and a negative control group was set. After 48 h, the cells were digested with 0.25% trypsin and collected. The cells were washed once with PBS (centrifuged at 1000 RPM for 5 min) and the cell concentration was adjusted to 1×10 6 / ml. DAF-FM DA was diluted 1:1000 with serum-free medium to a final concentration of 5 μM / L. The cells were collected, suspended in the diluted DAF-FM DA, and incubated in an incubator at 37 °C for 20 min. The cells were washed 3 times with serum-free cell culture medium to completely remove the DAF-FM DA that did not enter the cells. Flow cytometry was used to detect intracellular NO (Ex = 495 nm; Em = 515 nm).

[0197] To evaluate the intracellular NO generation ability of these compounds and explore their relationship with the in vitro proliferation of MDA-MB-231 cells, the NO release level was first detected using the DAF-FM DA fluorescent probe. As Figure 4 shown in A, compound I6 released NO in a dose-dependent manner. In addition, compared with the negative control group and the positive control group, the compounds containing NO donors produced different levels of NO intracellularly. The most active compound I6 released the highest concentration of NO, while the less active compounds produced relatively lower concentrations of NO( Figure 4 B). In addition, pretreatment of the cells with cPTIO would reduce the anti-proliferative activity of compound I6( Figure 4 C). The above results indicate that the effectiveness and selectivity of the anti-proliferative activity depend at least in part on the release of NO.

[0198] Example 8

[0199] Effect of compound I6 of the present invention on the invasion of MDA-MB-231 cells.

[0200] The inhibitory effect of 1, 2, and 4 μM compound I6 on the motility of MDA-MB-231 cells was detected by Transwell assay. Briefly, the cell density was adjusted to 1×10 5 cells / mL using incomplete medium. 100 μL of cell suspension was added to the Transwell chamber, and 500 μL of medium containing 20% FBS was added to the lower chamber. The tissue culture plate was incubated in a 37.5% CO2 incubator for 48 h. Then, the cells that passed through the Matrigel-coated chamber were stained with 0.1% crystal violet. After rinsing with PBS and drying, 3 images magnified 200 times were randomly taken. All experiments were repeated three times.

[0201] To develop a wide range of novel FAK inhibitors as therapeutic agents for TNBC, it is valuable to study the effect of I6 on cell migration. To determine the in vitro anti-metastatic effect of I6, a cell migration assay was first performed using MDA-MB-231 cells. We again selected TAE226 as the positive control, and the results showed that I6 inhibited the invasion of MDA-MB-231 cells in a dose-dependent manner ( Figure 5 A,B), and I6 was stronger than TAE226 at the same concentration. Meanwhile, I3, I5, and I 12 also had a significantly stronger inhibitory effect on cell invasion than TAE226 at the same concentration. The inhibitory effects of I1, I2, I4, I 13 , I 14 , I 15 , I 16 , I 17 , I 20 were slightly worse than those of I6.

[0202] Example 9

[0203] Compound I6 of the present invention inhibits the migration of MDA-MB-231 cells.

[0204] The scratch assay was used to detect the inhibitory effect of compound I6 on cell migration. MDA-MB-231 cells were cultured in a 6-well plate at 37 °C until the concentration reached approximately 80%. Fresh medium containing different concentrations of compound I6 (1, 2, 4 μM) was added. A wound line was created in the monolayer of cells and washed with PBS to remove cell debris. Images were observed under a microscope after 48 h. Each experiment was performed at least 3 times.

[0205] To evaluate the inhibitory effect of compound I6 on the migration of MDA-MB-231 cells, a cell scratch assay was used. As Figure 6 shown, 48 h after scratching the cell monolayer, the untreated MDA-MB-231 cells filled most of the scratched area, while I6 significantly inhibited the migration of MDA-MB-231 cells in a dose-dependent manner. The migration rate of I6 was 10.43% at 8 μM, which was much lower than that of TAE226. Meanwhile, I3, I5, and I 12 also had a significantly stronger inhibitory effect on cell migration than TAE226 at the same concentration. The inhibitory effects of I1, I2, I4, I 13 , I 14 , I 15 , I 16 , I 17 , I 20 were slightly worse than those of I6.

[0206] Example 10

[0207] Compound Ⅰ6 of the present invention inhibits the formation of focal adhesions (FAs) and stress fibers (SFs) in MDA-MB-231 cells.

[0208] MDA-MB-231 cells were plated in a 12-well plate containing sterile 12 mm Φ coverslips at a density of 1×10 5 cells / well and left in a CO2 incubator overnight for adhesion. After incubating the cells with test compound Ⅰ6 for 2 h, MDA-MB-231 cell samples (cell suspensions or round coverslips) were immersed in 4% paraformaldehyde fixative for 30 min or overnight to enhance cell permeability, and then immersed 3 times in PBS for 3 min each time. 1% BSA (50 - 100 μl) was added and incubated at room temperature for 20 minutes. After washing the cells 3 times with PBS, the cells on the coverslips were incubated with anti-vinculin antibody in an immunohistochemistry humid chamber for 2 h. The cells were washed 3 times again with PBS, and then incubated with Alexa Fluor 488-labeled anti-vinculin antibody in the darkroom for 1 h and DAPI for 30 min for F-actin and nuclear staining, respectively. Then, the coverslips were mounted onto glass slides with fluorescence mounting medium. Image analysis was performed on 5 expression regions under an Olympus microscope.

[0209] Compound Ⅰ6 was selected to determine possible downstream effectors (such as focal adhesions (FAs) and stress fibers (SFs), etc.) to understand the potential invasion mechanism of these compounds. After treatment with indicated doses of Ⅰ6 and TAE226, MDA-MB-231 cells were stained with anti-vinculin antibody to detect focal adhesions (FAs, red), DAPI to detect nuclei (blue), and FITC-conjugated phalloidin to detect F-actin stress fibers (SFs, green). MDA-MB-231 cells shrank, the number of cell FAs decreased, and the organization of actin filaments was disrupted ( Figure 7 ), significantly stronger than the TAE226 treatment group. However, quantitative analysis showed that the inhibitory effect of the PTIO treatment group was lower than that of the control group. These data clearly indicate that the inhibition of cell motility by Ⅰ6 is mediated by the disruption of focal adhesions, and NO plays an essential role in this process. The mechanisms of other compounds are the same.

[0210] Example 11

[0211] Compound Ⅰ6 of the present invention induces the autophosphorylation of FAK and its downstream signals in MDA-MB-231 cells.

[0212] The protein expression levels in MDA-MB-231 cells were detected by Western blot. The cells were seeded into cell culture dishes and incubated at 37 °C for 24 h, and then 1, 2, 4 μM of I6 and 4 μM of I6 (cPTIO) were added and incubated for 48 h. FAK, p-FAK (ABCAM), AKT (ABCAM), p-AKT (CST), SRC (ABCAM), p53 (PROTEINTECH), MMP2 (ABCAM), MMP9 (CST) and ACTIN (ABCAM) were taken out respectively, placed in a constant temperature metal bath and boiled for 3 - 5 min, and then poured out after centrifugation. Protein samples and protein markers were added to the wells on the electrophoresis gel in the required order using a pipette or sample needle. The membrane was immersed in the prepared primary antibody solution (antibody dilution ratio 1:1000) and incubated overnight. The primary antibody was recovered, and the membrane was washed with 20 mL of TBST at room temperature for 10 min and repeated 3 times. The secondary antibody was selected according to the primary antibody. The secondary antibody was prepared with 5% skim milk TBST solution at 1:5000, and the membrane was soaked in the secondary antibody solution and incubated at room temperature for about 1 h. The film was placed in the imager, the parameters were set, the exposure was started, the brightness and contrast were adjusted, and the image was saved.

[0213] To further understand the molecular mechanism of the anti-TNBC activity of I6, we used TAE226 as a control and detected the regulatory effect of I6 on the FAK-mediated signaling pathway in MDA-MB-231 cells by western blotting ( Figure 8)。We observed that although I6 did not alter the expression level of FAK in MDA-MB-231 cells, I6 effectively inhibited the autophosphorylation of Y397 in a dose-dependent manner. Importantly, at the same dose, the regulatory effect of I6 on Y397 autophosphorylation was significantly stronger than that of TAE226 and was attenuated by pretreatment with carboxy-PTIO. These results suggest that the effective inhibition of Y397 autophosphorylation by I6 is at least partially due to the inhibition of integrin expression by the high levels of NO generated after I6 treatment. Although I6 has a weaker inhibitory effect on the FAK kinase than TAE226, it has a better regulatory effect on the autophosphorylation of Y397, which may be because in addition to the autophosphorylation of Y397, FAK can also be activated by multiple factors (such as specific growth factors and other tyrosine phosphorylation sites). In addition, we focused on the effect of I6 on p53 expression, which is related to the kinase-independent function of FAK. In fact, the NO generated by I6 treatment is beneficial for enhancing p53 expression. TAE226 can upregulate p53 expression due to the regulation of the feedback mechanism between FAK and p53. Western blot results showed that I6 effectively blocked the FAK-mediated signaling pathway by reducing the relative expression levels of p-AKT, MMP-2, and MMP-9 in a dose-dependent manner, and the effect was better than that of TAE226. In sharp contrast, pretreatment with carboxy PTIO significantly attenuated the inhibitory effect of I6. Therefore, compared with TAE226, I6 has a more obvious effect on the effector targets in the FAK signaling pathway, which may be due to the synergistic effect of the TAE226 scaffold and the high levels of NO generated by furoxan, inhibiting the kinase catalytic function and non-kinase function of FAK, ultimately leading to anti-TNBC activity.

[0214] Example 12

[0215] Compound I6 of the present invention induces apoptosis in MDA-MB-231 cells.

[0216] MDA-MB-231 cells in the logarithmic growth phase were seeded in 6-well plates (1×10 5 cells / well). After the cells adhered, the corresponding drug-containing medium was added. The cells were collected, washed with PBS, and stained with FITC-Annexin-V and PI. Flow cytometry (BECKMAN COULTER CytoFLEX) was used to detect cell apoptosis.

[0217] Previous studies have shown that both FAK and NO can induce apoptosis in cancer cells by regulating the expression of apoptosis-related signals and proteins (such as p53). Considering the inconsistent anti-tumor activity and kinase inhibitory activity of hybrids and TAE226 against cancer cells, we further investigated the induction of apoptosis of I6 in MDA-MB-231 cells to better clarify the mechanism of these compounds in inhibiting cell proliferation.

[0218] As Figure 9 shown, compound I6 significantly increased the degree of apoptosis in MDA-MB-231 cells in a dose-dependent manner. At concentrations of 0.1, 0.2, and 0.4 μM, the apoptosis rates were 18.58%, 25.98%, and 71.02% respectively, and the ability of I6 to induce apoptosis was significantly stronger than that of the control TAE226. At the same time, I3, I5, and I 12 also had significantly stronger ability to induce apoptosis than TAE226 at the same concentration. The effects of I1, I2, I4, I 13 , I 14 , I 15 , I 16 , I 17 , I 20 were slightly worse than that of I6.

[0219] Example 13

[0220] Compound I6 of the present invention inhibits the metastasis of triple-negative breast cancer in vivo

[0221] A lung metastasis tumor model was established by the method of tail vein inoculation of MDA-MB-231 cells. Mice were randomly divided into 4 groups (n = 4 / group). Compounds I6 (15 and 30 mg / kg) and TAE226 (30 mg / kg) were dissolved in a suspension of 0.5% CMC-Na and administered daily (po) starting from the successful establishment of the model for a total of 6 weeks. Body weight was recorded every 3 days. Lung tissues were taken for HE staining and photographed.

[0222] The complex physiological events involved in tumor metastasis are the main reasons leading to the failure of anti-cancer treatment. Therefore, we evaluated the in vivo anti-tumor activity of compound I6 in a tail vein experimental MDA-MB-231 metastatic tumor animal model. Mice were randomly divided into treatment groups (n = 3) 24 h after inoculation and were treated with I6 (15 mg / kg, 30 mg / kg) or the positive control TAE226 (30 mg / kg) daily for 6 weeks. The results showed that compound I6 did not cause a significant decrease in body weight during the treatment period and the body weight of mice was higher than that of the positive drug group ( Figure 10 C). The lung is one of the most common metastatic sites of breast cancer. The total number of surface metastases in the lung was used for preliminary quantitative analysis. As Figure 10As shown in Figure B, Ⅰ6 has an obvious inhibitory effect on tumor metastasis, and is superior to the positive control TAE226 at the same dose. To more intuitively measure the tumor burden, lung organs were collected for H&E staining analysis ( Figure 10 D). It is worth noting that compound Ⅰ6 inhibited the size and number of liver metastases. Histological analysis of tumor tissues showed that partial necrosis of tumor cells occurred in each drug administration group, with karyopyknosis and karyolysis, which was most obvious in the Ⅰ6 (30 mg / kg) group. At the same time, Ⅰ3, Ⅰ5 and Ⅰ 12 also had an obvious inhibitory effect on tumor metastasis that was stronger than that of TAE226 at the same concentration. Ⅰ1, Ⅰ2, Ⅰ4, Ⅰ 13 , Ⅰ 14 , Ⅰ 15 , Ⅰ 16 , Ⅰ 17 , Ⅰ 20 had slightly worse effects than Ⅰ6.

[0223] A series of NO-donating FAK kinase inhibitors I1-I 20 were designed and synthesized in the present invention. All of them have good in vitro activity against triple-negative breast cancer. Among them, the in vitro anti-proliferative activities of I3, I5, I6 and I 12 are superior to the positive drug TAE226. Especially for compound I6, its IC 50 value against MDA-MB-231 cells is 8 times that of TAE226. Further studying the drug action mechanism, compound I6 inhibited the invasion and metastasis ability of MDA-MB-231 cells concentration-dependently by inhibiting FAK phosphorylation and enhancing p53 expression to disrupt focal adhesion, and also promoted tumor cell apoptosis concentration-dependently. During the exploration of the synergistic effect of NO, it was found that the anti-proliferative activity of the compound was proportional to the intracellular NO release amount. The in vivo experimental results in mice showed that compound I6 could effectively inhibit the metastasis of triple-negative breast cancer on the basis of ensuring safety.

[0224] The above are only the preferred embodiments of the present invention. It should be pointed out that: for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A 2,4-dianilinopyrimidine derivative, or a pharmaceutically acceptable salt thereof, characterized in that, The 2,4-dianilinopyrimidine derivative is shown as general formula I: Wherein: L is selected from: C3-C 12 linear alkanes, substituted C3-C 12 linear alkanes, C2-C4 alkynes, substituted C2-C4 alkynes, C2-C4 alkenes, substituted C2-C4 alkenes, substituted C3-C6 cycloalkanes; benzene ring, substituted benzene ring; The substituted C3-C 12 The number of substituents in the linear alkane, substituted C2-C4 alkyne, substituted C2-C4 alkene, substituted C3-C6 cycloalkane, and substituted benzene ring is 1-3, and the substituent is a C1-C4 alkane.

2. The 2,4-dianilinopyrimidine derivative according to claim 1, characterized in that, The number of substituents in the substituted C3-C6 cycloalkane and the substituted benzene ring is 1 or 2, and the substitution positions of the two substituents in the substituted C3-C6 cycloalkane and the substituted benzene ring are in the meta or para positions.

3. The 2,4-dianilinopyrimidine derivative according to claim 1, characterized in that, L is selected from:

4. The 2,4-dianilinopyrimidine derivative according to claim 1, characterized in that, selected from the following compounds:

5. A method for preparing the 2,4-dianilinopyrimidine derivative according to claim 1, characterized in that, The synthetic route of the 2,4-dianilinopyrimidine derivative shown as general formula I is as follows: The preparation method includes the following steps: A. Compound II and compound III are reacted with an acid at a certain temperature in a solvent state to obtain compound IV; B. Compound IV and compound V are reacted with a conditional reagent in a solvent state to obtain compound I; The conditional reagent is one or both of carbodiimide hydrochloride and 4-dimethylaminopyridine.

6. The method for preparing the 2,4-dianilinopyrimidine derivative according to claim 5, characterized in that, In step A, the solvent is one or both of isopropanol and ethanol; the acid is hydrochloric acid; the reaction temperature is 90-100 °C; In step B, the solvent is one or both of anhydrous N,N-dimethylformamide and N,N-dimethylacetamide; the reaction temperature is room temperature.

7. A pharmaceutical composition comprising a therapeutically effective amount of the 2,4-dianilinopyrimidine derivative according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

8. Use of the 2,4-dianilinopyrimidine derivative according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for preventing and / or treating triple-negative breast cancer.

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