A no-donor type dasatinib derivative, and a preparation method and application thereof

Abstract

The application provides a NO donor type dasatinib derivative, a preparation method and application thereof, the derivative is one of compounds 1a-1f and compounds 2a-2f; the derivative has multiple effects of eliminating senescent cells, reducing intraocular pressure and protecting optic nerves, and is a potential new type of therapeutic drug for glaucoma and senescence-related retinopathy.

Description

TECHNICAL FIELD

[0001] The present application relates to a NO donor type dasatinib derivative and its preparation method and application. BACKGROUND

[0002] Glaucoma is a group of diseases characterized by characteristic optic atrophy and visual field defects, and pathologically increased intraocular pressure is the main risk factor. The global prevalence of glaucoma is 3.54%, which is the leading irreversible blinding eye disease in the world. It is estimated that by 2040, 1.12 million people aged 40-80 will suffer from glaucoma [1] . The treatment methods of glaucoma include intraocular pressure reduction therapy and optic nerve protection therapy. The commonly used intraocular pressure reducing drugs include prostaglandin analogues (such as latanoprost eye drops), β-adrenergic receptor blockers (such as timolol eye drops), carbonic anhydrase inhibitors (such as brinzolamide eye drops) and parasympathomimetic drugs (such as pilocarpine eye drops). The above drugs mainly focus on reducing intraocular pressure, but it is not clear whether they can remove aged retinal ganglion cells. There is no report of glaucoma drugs that can reduce intraocular pressure and remove aged cells at the same time.

[0003] Studies have shown that retinal ganglion cell aging is an important pathological mechanism of glaucoma [2] . Senolytics is a class of drugs that can selectively remove senescent cells and delay the progression of age-related diseases. Dasatinib is one of the representative drugs. Animal experiments have shown that intraperitoneal injection of dasatinib can remove retinal senescent cells caused by high intraocular pressure, thereby protecting normal retinal ganglion cells from damage and improving visual function [3] . Studies have shown that nitric oxide (NO) has the effect of reducing intraocular pressure [4][5] . The present application designs and synthesizes a NO donor type dasatinib derivative as a new type of glaucoma drug. This drug has multiple functions of reducing intraocular pressure, removing senescent cells and protecting the optic nerve, and can be used as a therapeutic drug for glaucoma and senile retinal diseases.

[0004] REFERENCES

[0005] [1]. Tham YC, Li X, Wong TY, Quigley HA, Aung T, Cheng CY. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology. 2014 Nov;121(11):2081-90.

[0006] [2]. Zhang Y, Huang S, Xie B, Zhong Y. Aging, Cellular Senescence, and Glaucoma. Aging Dis. 2024 Apr 1;15(2):546-564.

[0007] [3]. Rocha LR, Nguyen Huu VA, Palomino La Torre C, Xu Q, Jabari M, Krawczyk M, Weinreb RN, Skowronska-Krawczyk D. Early removal of senescent cells protects retinal ganglion cells loss in experimental ocular hypertension. Aging Cell. 2020 Feb;19(2):el3089.

[0008] [4]. Toda N, Nakanishi-Toda M. Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy. Prog Retin Eye Res. 2007 May;26(3):205-38.

[0009] [5]. Yang Z, Wu J, Wu K, Luo J, Li C, Zhang J, Zhao M, Mei T, Liu X, Shang B, Zhang Y, Zhao L, Huang Z. Identification of Nitric Oxide-Donating Ripasudil Derivatives with Intraocular Pressure Lowering and Retinal Ganglion Cell Protection Activities. J Med Chem. 2022 Sep 8;65(17):11745-11758. SUMMARY

[0010] The present application aims to overcome the deficiencies of the prior art and provide a NO donor type dasatinib derivative. The NO donor type dasatinib derivative designed and synthesized by the present application is a new type of glaucoma drug, which has multiple effects of eliminating senescent cells, reducing intraocular pressure and protecting the optic nerve.

[0011] To achieve the above-mentioned purpose, the technical scheme adopted is: a NO donor type dasatinib derivative, the derivative is one of compounds 1a-1f, compounds 2a-2f; the structural formula of the compounds 1a-1f, compounds 2a-2f is as follows respectively:

[0012]

[0013] (1a)

[0014]

[0015] (1b)

[0016]

[0017] (1c)

[0018]

[0019] (1d)

[0020]

[0021] (1e)

[0022]

[0023] (1f)

[0024]

[0025] (2a)

[0026]

[0027] (2b)

[0028]

[0029] (2c)

[0030]

[0031] (2d)

[0032]

[0033] (2e)

[0034]

[0035] (2f).

[0036] The present application provides the preparation method of the above-mentioned NO donor type dasatinib derivative, and the preparation methods of the compounds 1a-1f are as follows respectively:

[0037] Dasatinib is reacted with succinic anhydride to obtain an intermediate 2;

[0038] 2-Bromoethanol is reacted with silver nitrate to obtain an intermediate 4a, and the intermediate 4a is reacted with the intermediate 2 by esterification to obtain the compound 1a;

[0039] 3-Bromopropanol is reacted with silver nitrate to obtain an intermediate 4b, and the intermediate 4b is reacted with the intermediate 2 by esterification to obtain the compound 1b;

[0040] 5-Bromopentanol is reacted with silver nitrate to obtain an intermediate 4c, and the intermediate 4c is reacted with the intermediate 2 by esterification to obtain the compound 1d;

[0041] 4-Bromobutyl acetate is reacted with silver nitrate to obtain an intermediate 6, and the intermediate 6 is reacted under alkaline conditions to remove an acetyl group to obtain an intermediate 7, and the intermediate 7 is reacted with the intermediate 2 by esterification to obtain the compound 1c;

[0042] ISMN is reacted with the intermediate 2 by esterification to obtain the compound 1e;

[0043] The intermediate 8 is subjected to a nucleophilic substitution reaction with piperazine under alkaline conditions to obtain an intermediate 9, and the intermediate 9 is further subjected to a nucleophilic substitution reaction with 2-bromoethyl nitrate under alkaline conditions to obtain the compound 1f;

[0044] The structure of the intermediate 2 is as follows:

[0045] ;

[0046] The structure of the intermediate 4a-4c is as follows:

[0047]

[0048] 4a

[0049]

[0050] 4b

[0051]

[0052] 4c;

[0053] The structure of the intermediate 6 is as follows:

[0054] ;

[0055] The structure of the intermediate 7 is as follows:

[0056] ;

[0057] The structure of the ISMN is as follows:

[0058] ;

[0059] The structure of the intermediate 8 is as follows:

[0060] ;

[0061] The structure of the intermediate 9 is as follows:

[0062] ;

[0063] The preparation method of the compounds 2a-2f is as follows respectively:

[0064] The intermediate 11 is obtained by adding glacial acetic acid to the intermediate 10, adding fuming nitric acid after the temperature is raised to 90°C, and further raising the reaction temperature to 120°C;

[0065] The compound 14 and sodium nitrite are reacted under acidic conditions to generate the intermediate 15;

[0066] The intermediate 11 and diethylene glycol are subjected to nucleophilic substitution reaction to obtain the intermediate 12;

[0067] The intermediate 11 and 1,4-butanediol are subjected to nucleophilic substitution reaction to obtain the intermediate 13;

[0068] The intermediate 15 is subjected to a nucleophilic substitution reaction with ethylene glycol to obtain intermediate 16;

[0069] The intermediate 15 is subjected to a nucleophilic substitution reaction with glycollic acid to obtain intermediate 17;

[0070] The intermediate 12 is subjected to an esterification reaction with the intermediate 2 to obtain compound 2a;

[0071] The intermediate 13 is subjected to an esterification reaction with the intermediate 2 to obtain compound 2b;

[0072] The intermediate 16 is subjected to an esterification reaction with the intermediate 2 to obtain compound 2c;

[0073] The intermediate 17 is subjected to an esterification reaction with dasatinib to obtain compound 2d;

[0074] The compound 2a is subjected to a reaction with methanesulfonic acid to form compound 2e;

[0075] The compound 2b is subjected to a reaction with methanesulfonic acid to form compound 2f;

[0076] The intermediate 10 has the following structural formula:

[0077] ;

[0078] The intermediate 11 has the following structural formula:

[0079] ;

[0080] The intermediate 14 has the following structural formula:

[0081] ;

[0082] The intermediate 15 has the following structural formula:

[0083] ;

[0084] The intermediate 12 has the following structural formula:

[0085] ;

[0086] The intermediate 13 has the following structural formula:

[0087] ;

[0088] The intermediate 16 has the following structural formula:

[0089] ;

[0090] The structural formula of the intermediate 17 is as follows:

[0091] .

[0092] Preferably, the molar ratio of the dasatinib to succinic anhydride is 4:4.4; the molar ratio of the 2-bromoethanol to silver nitrate is 5:12.5, the molar ratio of the intermediate 4a to the intermediate 2 is 1:1; the molar ratio of the 3-bromopropanol to silver nitrate is 5:12.5, the molar ratio of the intermediate 4b to the intermediate 2 is 1:1; the molar ratio of the 5-bromopentanol to silver nitrate is 5:12.5, the molar ratio of the intermediate 4c to the intermediate 2 is 1:1; the molar ratio of the 4-bromobutyl acetate to silver nitrate is 5:12.5, the molar ratio of the intermediate 7 to the intermediate 2 is 1:1; the molar ratio of the ISMN to the intermediate 2 is 1:1; the molar ratio of the intermediate 8 to piperazine is 1:10; the molar ratio of the intermediate 9 to 2-bromoethyl nitrate is 0.34:1.01;

[0093] The molar ratio of the intermediate 11 to diethylene glycol is 1.36:2.73; the molar ratio of the intermediate 11 to 1,4-butanediol is 1.36:2.73; the molar ratio of the compound 14 to sodium nitrite is 2:7; the molar ratio of the intermediate 15 to ethylene glycol is 2.06:4.12; the molar ratio of the intermediate 15 to glycolic acid is 2.06:4.12; the molar ratio of the intermediate 12 to the intermediate 2 is 1.2:1; the molar ratio of the intermediate 13 to the intermediate 2 is 1.2:1; the molar ratio of the intermediate 16 to the intermediate 2 is 1.2:1; the molar ratio of the intermediate 17 to dasatinib is 1.2:1; the molar ratio of the compound 2a to methanesulfonic acid is 1:6; the molar ratio of the compound 2b to methanesulfonic acid is 1:6.

[0094] The application provides application of the NO donor type dasatinib derivative in preparation of a drug for removing retinal aging cells, reducing intraocular pressure or / and protecting an optic nerve.

[0095] The application provides application of the NO donor type dasatinib derivative in preparation of a drug for treating glaucoma.

[0096] The application provides application of the NO donor type dasatinib derivative in preparation of a drug for treating aging-related retinopathy.

[0097] The application provides application of the NO donor type dasatinib derivative in preparation of a drug for improving visual function.

[0098] Beneficial effects:

[0099] The NO donor type dasatinib derivative prepared by the application has multiple effects of eliminating senescent cells, reducing intraocular pressure and protecting optic nerves, and is a new type of therapeutic drug for glaucoma and senescence-related retinopathy. BRIEF DESCRIPTION OF DRAWINGS

[0100] Figure 1 Figure 1: NO activity and cGMP concentration of different NO donor type dasatinib derivatives in cells; A: NO activity of NO donor type dasatinib derivatives in cells was detected using NO fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The fluorescence intensity of cells was detected by flow cytometry, and the mean fluorescence intensity (MFI) was used as a statistical index. The results showed that, except for 1a compound, the rest of the NO donor type dasatinib derivatives could increase the NO level in cells. B: cGMP is an important downstream molecule of NO for reducing intraocular pressure. The results showed that, except for 2b, the rest of the NO donor type dasatinib derivatives could promote the production of cGMP in cells. Relative MFI: relative mean fluorescence intensity; concentration: concentration; fold of control: fold compared with the control.

[0101] Figure 2: In glaucoma model mice, 2e compound can significantly eliminate retinal senescent cells; Figure A-B: senescence-associated beta-galactosidase (SA-β-Gal) staining of retinal flat mount of normal mice (without eye drop), magnetic bead modeling induced high intraocular pressure mice (Vehicle, dasatinib mesylate and 2e compound eye drop for 10 days), and the number of senescent positive cells was counted. Compared with the Normal group, the number of senescent positive cells per unit area of the retinal flat mount of the Vehicle group mice increased significantly. Compared with the Vehicle group, both dasatinib mesylate and 2e compound eye drops can significantly reduce the number of senescent positive cells per unit area, and there is no statistical difference between the two. Normal: normal group; Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor type dasatinib derivative 2e group; SA-β-Gal positive cells: senescence-associated beta-galactosidase positive cells.

[0102] Figure 3 : In glaucoma model mice, 2e compound can significantly reduce intraocular pressure; 1, 2, 4 and 8 h after single eye drop (Vehicle, dasatinib mesylate and 2e compound) of magnetic bead modeling induced high intraocular pressure mice, the intraocular pressure was measured. Compared with the Vehicle group, there was no statistical difference in intraocular pressure at each time point after eye drop of dasatinib mesylate. Compared with the Vehicle group, 2e compound (10 mM) single eye drop can significantly reduce the intraocular pressure of glaucoma model mice at 2 h and 4 h after eye drop, and the effect of reducing intraocular pressure is most significant at 4 h after single eye drop. ΔIOP: the difference between pre-dropping and post-dropping intraocular pressure; Time after eye drop (hours): the time after eye drop (hours); Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor type dasatinib derivative 2e group.

[0103] Figure 4: In glaucoma model mice, 2e compound can significantly protect retinal ganglion cells; Figure A: retinal flat mount and retinal ganglion cell staining (RBPMS) of Normal mice (without eye drop administration), magnetic bead modeling induced high intraocular pressure mice (Vehicle, dasatinib mesylate and 2e compound eye drop administration for 10 days), and the number of retinal ganglion cells was counted. B: Compared with the Normal group, the number of retinal ganglion cells of the Vehicle group of chronic high intraocular pressure mice induced by magnetic beads decreased significantly. Compared with the Vehicle group, both dasatinib mesylate and 2e compound eye drop administration can protect retinal ganglion cells, and compared with dasatinib mesylate, 2e compound has more significant protective effect on retinal ganglion cells. Normal: normal group; Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor type dasatinib derivative 2e group; RBPMS positive cells: retinal ganglion cell marker RBPMS positive cells.

[0104] Figure 5 : In glaucoma model mice, 2e compound can significantly improve visual function; Figure A: VEP curve of Normal mice (without eye drop administration), magnetic bead modeling induced high intraocular pressure mice (Vehicle, dasatinib mesylate and 2e compound eye drop administration for 10 days). B-C: N1 wave statistical diagram of VEP curve. Compared with the Normal group, the amplitude of N1 wave of the Vehicle group decreased significantly, and the peak time lagged obviously; compared with the Vehicle group, dasatinib mesylate eye drop administration can significantly improve the peak time of N1 wave of chronic high intraocular pressure mice, but there is no statistical difference in amplitude; compared with the Vehicle group, 2e compound eye drop administration can significantly improve the amplitude and peak time of N1 wave of chronic high intraocular pressure mice. Amplitude: amplitude; VEP: visual evoked potential; Scotopic VEP: dark VEP; Time: time; Normal: normal group; Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor type dasatinib derivative 2e group; Peak time: peak time. DETAILED DESCRIPTION

[0105] For the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0106] In our study, by introducing NO donor fragments on the parent nucleus of senolytics-dasatinib, we designed and synthesized two series of NO donor type dasatinib derivatives of nitrate and furazan nitroxide, a total of 12 compounds (1a-1f, 2a-2f), expecting that the candidate molecules enter the eye metabolism to produce NO and dasatinib through local eye drop administration in the eye, respectively, to play a role in reducing intraocular pressure and selective elimination of senescent cells. Our results show that the most active compound 2e performs a dual role in reducing intraocular pressure and eliminating senescent cells. In in vitro experiments, compound 2e exhibits obvious NO release capacity and activity in promoting the production of cyclic guanosine phosphate (3', 5'-cyclophosphate, cGMP) in human trabecular meshwork (HTM) cells. Since 2e is a compound in the form of a methanesulfonate, in subsequent in vivo experiments, we synthesized dasatinib mesylate as a positive drug control by salifying dasatinib with methanesulfonic acid. In in vivo experiments, local eye drop administration of 2e can significantly reduce the intraocular pressure of chronic high intraocular pressure mice induced by magnetic microbeads and eliminate senescent cells in the retinas of the model mice, reduce the death of retinal ganglion cells, and exhibit activity in improving visual function.

[0107] Compound 1a of nitrate series The synthetic routes of 1f and dasatinib mesylate are shown in Scheme 1. Briefly, compounds 1a-1e were prepared from 1 by reacting with succinic anhydride to obtain intermediate 2. Compounds 3a-3c, 5 of different carbon chain lengths were reacted with silver nitrate to obtain corresponding 4a-4c and 6, and intermediate 6 was reacted under strong alkaline conditions with LiOH to remove the acetyl group to obtain intermediate 7. Compounds 4a-4c, 7, isosorbide mononitrate (ISMN) were reacted with 2 by esterification to obtain compounds 1a-1e. The synthesis of 1f was carried out by nucleophilic substitution reaction of intermediate 8 with piperazine under alkaline conditions to obtain intermediate 9, and further nucleophilic substitution reaction of intermediate 9 with 2-bromoethyl nitrate under alkaline conditions to obtain compound 1f. In order to improve the water solubility of the compound to prepare eye drops, we designed and synthesized dasatinib mesylate. 1 was used as the raw material, dissolved in dichloromethane, and then methanesulfonic acid was added, and the reaction was carried out at room temperature for 2 h to obtain the target compound dasatinib mesylate.

[0108]

[0109] Scheme 1. Synthetic routes for compounds 1a-1f. Reagents and reaction conditions: a) succinic anhydride, pyridine, DMF ( N , N a) Dimethylformamide, 25℃, 48 h; b) AgNO3, MeCN, reflux, 12 h; c) LiOH, MeOH, 25℃, 4 h; d) EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide), DMAP (4-dimethylaminopyridine), DMF, 25℃, 12 h; e) Piperazine, K2CO3, DMF, 90℃, 12 h; f) 2-bromoethyl nitrate, DMF, 12 h; g) MeSO3H, DCM (dichloromethane), 25℃, 2 h.

[0110] Furazan nitrogen oxide series compound 2a The synthetic route for 2f is shown in Scheme 2. In short, compounds 10 and 14 were reacted with fuming nitric acid and sodium nitrite, respectively, under acidic conditions to generate intermediates 11 and 15. Intermediate 11 underwent nucleophilic substitution reactions with diethylene glycol and 1,4-butanediol, respectively, to give intermediates 12 and 13; intermediate 15 underwent nucleophilic substitution reactions with ethylene glycol and glycolic acid, respectively, to give intermediates 16 and 17. Intermediates 12, 13, and 16 underwent esterification reactions with intermediate 2 to give targets 2a, 2b, and 2c; while intermediate 17 underwent esterification reactions with dasatinib to give compound 2d. To improve the water solubility of the compounds, 2a and 2b were reacted with methanesulfonic acid to form the corresponding target compounds 2e and 2f.

[0111]

[0112] Scheme 2. Synthetic route for compounds 2a-2f. Reagents and reaction conditions: a) Fuming nitric acid, AcOH, 90℃, 3 h; b) Diethylene glycol, 50% (v / v) NaOH aqueous solution, THF (tetrahydrofuran), 4 h; c) 1,4-Butanediol, 50% (v / v) NaOH aqueous solution, THF, 4 h; d) Intermediate 2, TBTU (benzotriazole- N , N , N ', N '-Tetramethylurea hexafluorophosphate borate ester), DMAP (4-dimethylaminopyridine), DMF ( N , Ne) dimethylformamide, 25°C, 12 h; e) NaNO2, 60% H2SO4 aqueous solution, DCM (dichloromethane), 50°C, 0.5 h; f) glycolic acid, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DCM, 25°C, 4 h; g) ethylene glycol, 50% NaOH solution, THF, 4 h; h) dasatinib, TBTU, DMAP, DMF, 25°C, 12 h; i) MeSO3H, DCM, 25°C, 2 h.

[0113] The preparation methods and structural characterization of a total of 12 compounds, 1a-1f and 2a-2f, are as follows:

[0114] 2

[0116] English name:

[0117] 14-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoic acid (2)

[0118] Chinese name:

[0119] 14-(2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutyric acid (2)

[0120] Dasatinib (1948 mg, 4 mmol, 1 equivalent), succinic anhydride (440 mg, 4.4 mmol, 1.1 equivalent), and DMAP (489 mg, 4 mmol, 1 equivalent) were dissolved in 20 mL of DMF and reacted at room temperature for 24 h under TLC monitoring. After the reaction was complete, most of the DMF was removed by rotary evaporation. 50 mL of water was added to the reaction flask, resulting in the precipitation of a large amount of white solid. The mixture was filtered, and the filter cake was washed with methanol (3 × 10 mL) and dried under vacuum to give intermediate 2, 1870 mg. Yield: 81%, white solid. 1 H NMR (300 MHz, DMSO- d 6) δ 11.47 (s, 1H), 9.87 (s, 1H), 8.22 (s, 1H), 7.40 (dd, J= 7.5, 1.5Hz, 1H), 7.29 (dd, J = 7.6, 1.5 Hz, 1H), 7.25 (dd, J = 7.6 Hz, 1H), 6.05 (s, 1H), 4.16 (t, J = 5.7 Hz, 2H), 3.51 (m, 4H), 2.59 (t, J = 5.7 Hz, 2H), 2.44-2.56 (m,8H), 2.41 (s, 3H), 2.24 (s, 3H). ESI-MS (m / z): 588.18 [M + H] +

[0121] The general method for synthesizing intermediates 4a-4c is as follows: Terminal bromoethanol (5 mmol, 1.0 equivalent) / methyl bromocarboxylate (5 mmol, 1.0 equivalent) was dissolved in acetonitrile (40 mL), and silver nitrate (12.5 mmol, 2.5 equivalent) was added. The mixture was heated under reflux for 12 h in the dark. The reaction was monitored by TLC; phosphomolybdic acid was observed as a colorimetric reaction (the nitrate ester compound appeared yellow). Acetonitrile was removed by rotary evaporation. 100 mL of saturated saline and 100 mL of ethyl acetate were added, the mixture was stirred and allowed to stand. The supernatant was transferred to a separatory funnel, extracted, and the organic phases were combined. The mixture was washed three times with 100 mL of saturated saline, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain intermediates 4a-4c.

[0122]

[0123] 4a

[0124] English name: 2-hydroxyethyl nitrate (4a)

[0125] Chinese name: 2-Hydroxyethyl nitrate (4a)

[0126] It is prepared from 2-bromoethanol with a yield of 88% and is a colorless oily substance. 1 H NMR (300 MHz, CDCl3) δ 4.59(t, J = 4.6 Hz, 2H), 3.93 (t, J = 4.6 Hz, 2H), 1.88 (s, 1H).

[0127]

[0128] 4b

[0129] English name: 3-hydroxypropyl nitrate (4b)

[0130] Chinese name: Nitrate-3-hydroxypropyl (4b)

[0131] From 3-bromopropanol as raw material, yield 89%, colorless oil. 1 H NMR (300 MHz, CDCl3) δ 4.61(t, J = 6.3 Hz, 2H), 3.78 (t, J = 6.0 Hz, 2H), 1.98 (p, J = 6.2 Hz, 2H), 1.51 (s,1H).

[0132]

[0133] 4c

[0134] English name: 4-hydroxybutyl nitrate (4c)

[0135] Chinese name: Nitrate-5-hydroxybutyl (4c)

[0136] From 5-bromopentanol as raw material, yield 90%, colorless oil. 1 H NMR (300 MHz, CDCl3) δ 4.50(t, J = 6.6 Hz, 2H), 3.70 (t, J = 6.4 Hz, 2H), 1.85 – 1.77 (m, 2H), 1.69 – 1.61(m, 2H), 1.57 – 1.50 (m, 2H), 1.46 (s, 1H).

[0137] 7

[0139] English name: 4-hydroxybutyl nitrate (7)

[0140] Chinese name: Nitrate-4-hydroxybutyl (7)

[0141] Dissolve 4-bromobutyl acetate (5 mmol, 1.0 eq) in acetonitrile (40 mL), add silver nitrate (12.5 mmol, 2.5 eq), avoid light, heat reflux for 12 h. TLC monitor the reaction, molybdenum phosphoric acid coloration (nitrate compounds present yellow), rotary evaporation to remove acetonitrile, add 100 mL saturated brine and 100 mL ethyl acetate, stir and stand, transfer the supernatant to a separatory funnel, extract, combine the organic phase, wash with 100 mL saturated brine for 3 times, dry over anhydrous sodium sulfate, concentrate, column chromatography purification, to obtain intermediate 6. Dissolve intermediate 6 (4 mmol, 1.0 eq) in 10 mL methanol, stir at 0 ℃ for 15 min, add 2M LiOH·H2O aqueous solution (8 mmol, 2.0 eq) dropwise, react at 5 ℃ for 12 h. TLC monitor (PE / EA = 10:1), molybdenum phosphoric acid coloration, after the raw material is completely reacted, rotary evaporation to remove methanol, add 15 mL water, extract with EA (3 × 15 mL), wash with saturated brine (3 × 20 mL), dry over anhydrous sodium sulfate, concentrate to obtain intermediate 7. Yield 86%, brownish yellow oil. 1 H NMR (300 MHz, CDCl3) δ 4.49 (t, J = 6.5 Hz, 2H), 3.69 (t, J =6.2 Hz, 2H),1.91 – 1.77 (m, 2H), 1.71 – 1.62 (m, 2H).

[0142] General synthesis method of compounds 1a-1f: Dissolve 4a-4c, 7 or ISMN (2 mmol, 1 eq), EDCI·HCl (5 mmol, 2.5 eq), DMAP (0.2 mmol, 0.1 eq) and intermediate 2 (2 mmol, 1 eq) in 10 mL DMF, react at room temperature overnight. TLC monitor (EA / MeOH = 40:1), column chromatography purification, to obtain compounds 1a-1f.

[0143]

[0144] 1a

[0145] English name:

[0146] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl (2-(nitrooxy)ethyl) succinate(1a)

[0147] Chinese Name:

[0148] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2- methylpyrimidin-4-yl)piperazin-1-yl)ethyl(2-(nitrooxy)ethyl)succinate (1a)

[0149] Prepared from intermediate 4a and 2 as starting materials in 33% yield as a white solid. 1 H NMR (300 MHz, DMSO- d 6) δ 11.46 (s, 1H), 9.87 (s, 1H), 8.22 (s, 1H), 7.40 (d, J = 5.4 Hz, 1H), 7.32 – 7.28 (m, 1H), 7.26 (s, 1H), 6.06 (s, 1H), 4.74 (t, J = 4.5 Hz, 2H), 4.35 (t, J = 4.5 Hz, 2H), 4.17 (t, J = 5.2 Hz, 2H), 3.57 – 3.46 (m, 4H), 2.66 – 2.54 (m, 6H), 2.50 (m, 4H), 2.41 (s, 3H), 2.24 (s, 3H). 13 C NMR (75 MHz, DMSO-d6) δ 172.30 (x2), 165.65, 163.04, 162.81, 160.40, 157.41, 141.30, 139.30, 133.99, 132.91, 129.51, 128.65, 127.49, 126.17, 83.08, 71.96, 62.05, 60.73, 56.38, 52.85 (x2), 44.02 (x2), 29.03, 28.94, 26.05, 18.78. HRMS (ESI) = [M + H] + = 677.19033.

[0150]

[0151] 1b

[0152] English Name:

[0153] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl (3-(nitrooxy)propyl) succinate(1b)

[0154] Chinese name:

[0155] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl (3-(nitrooxy)propyl) succinate (1b)

[0156] From intermediate 4b and 2 as starting materials, yield 31%, white solid. 1 H NMR (300 MHz, DMSO- d 6) δ 11.51 (s, 1H), 9.90 (s, 1H), 8.23 (s, 1H), 7.41 (dd, J = 7.2, 2.3 Hz, 1H), 7.32– 7.24 (m, 2H), 6.05 (s, 1H), 4.58 (t, J = 6.3 Hz, 2H), 4.17 (t, J = 5.7 Hz, 2H), 4.12 (t, J = 6.2 Hz, 2H), 3.58 – 3.45 (m, 4H), 2.63 – 2.56 (m, 6H), 2.50 (m,4H), 2.41 (s, 3H), 2.25 (s, 3H), 1.99 (q, J = 6.3 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 172.42, 172.36, 165.65, 163.03, 162.81, 160.39, 157.41, 141.30, 139.30,133.99, 132.91, 129.50, 128.65, 127.49, 126.17, 83.09, 71.22, 62.00, 61.01,56.37, 52.83 (×2), 43.99 (×2), 29.08, 26.15, 26.05, 19.04, 18.78. HRMS(ESI) = [M + H] + = 691.20599.

[0157]

[0158] 1c

[0159] English name:

[0160] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl (4-(nitrooxy)butyl) succinate(1c)

[0161] Chinese name:

[0162] 2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl(4-(nitrooxy)butyl)succinate (1c)

[0163] It was prepared from intermediates 7 and 2 as raw materials, with a yield of 33%, and was a white solid. 1 H NMR (300 MHz, DMSO- d 6) δ11.46 (s, 1H), 9.87 (s, 1H), 8.22 (s, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.33 –7.21 (m, 2H), 6.06 (s, 1H), 4.54 (t, J = 6.0 Hz, 2H), 4.17 (t, J = 5.8 Hz, 2H), 4.06 (t, J= 5.8 Hz, 2H), 3.58 – 3.46 (m, 4H), 2.64 – 2.55 (m, 6H), 2.50 (m,4H), 2.41 (s, 3H), 2.24 (s, 3H), 1.77 – 1.62 (m, 4H). 13 C NMR (75 MHz, DMSO-d6)δ 172.42, 172.39, 165.65, 163.03, 162.82, 160.39, 157.41, 141.30, 139.30,133.99, 132.90, 129.51, 128.65, 127.49, 126.17, 83.08, 73.80, 63.89, 62.02,56.39, 52.83 (×2), 44.01 (×2), 29.10, 29.08, 26.05, 24.92, 23.24, 18.78.

[0164]

[0165] 1d

[0166] English name:

[0167] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl (5-(nitrooxy)pentyl) succinate(1d)

[0168] Chinese name:

[0169] 2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl(5-(nitrooxy)pentyl)succinate (1d)

[0170] It is prepared from intermediates 4C and 2 as raw materials, with a yield of 31%, and is a white solid. 1 H NMR (300 MHz, DMSO- d 6) δ11.51 (s, 1H), 9.90 (s, 1H), 8.23 ​​(s, 1H), 7.40 (d, J= 7.2 Hz, 1H), 7.32 –7.24 (m, 2H), 6.05 (s, 1H), 4.50 (t, J = 6.5 Hz, 2H), 4.16 (t, J = 5.8 Hz, 2H),4.02 (t, J = 6.4 Hz, 2H), 3.56 – 3.47 (m, 4H), 2.61 – 2.55 (m, 6H), 2.50 (m,4H), 2.41 (s, 3H), 2.25 (s, 3H), 1.72 – 1.54 (m, 4H), 1.44 – 1.32 (m, 2H). 13 CNMR (75 MHz, DMSO-d6) δ 172.40 (×2), 165.64, 163.02, 162.81, 160.38, 157.41,1.32, 139.29, 133.98, 132.90, 129.50, 128.65, 127.48, 126.17, 83.08, 74.11,64.21, 62.01, 56.40, 52.83 (×2), 44.02 (×2), 29.11, 29.08, 28.08, 26.09,26.05, 22.10, 18.78. HRMS (ESI) = [M + H] + = 719.23729.

[0171]

[0172] 1e

[0173] English Name:

[0174] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl((3R,3aR,6S,6aS)-6-(nitrooxy)hexahydrofuro[3,2-b]furan-3-yl) succinate (1e)

[0175] Chinese Name:

[0176] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2- methylpyrimidin-4-yl)piperazin-1-yl)ethyl ((3R,3aR,6S,6aS)-6-(nitrooxy)hexahydrofuro [3,2-b] furan-3-yl)succinate (1e)

[0177] Yield 29%, white solid, from ISMN and intermediate 2. 1 H NMR (300 MHz, DMSO- d 6) δ 11.50 (s, 1H), 9.90 (s, 1H), 8.23 (s, 1H), 7.41 (dd, J = 7.3, 2.3 Hz, 1H), 7.31– 7.25 (m, 2H), 6.06 (s, 1H), 5.51 (td, J = 5.4, 2.1 Hz, 1H), 5.10 (d, J = 3.1Hz, 1H), 4.97 (t, J = 5.3 Hz, 1H), 4.41 (d, J = 5.0 Hz, 1H), 4.17 (t, J = 5.7 Hz,2H), 4.01 – 3.91 (m, 2H), 3.91 – 3.84 (m, 1H), 3.83 – 3.77 (m, 1H), 3.55 – 3.48 (m, 4H), 2.61 – 2.57 (m, 6H), 2.50 (m, 4H), 2.42 (s, 3H), 2.25 (s, 3H). 13 C NMR (75 MHz, DMSO-d6) δ 172.36, 171.78, 165.65, 163.03, 162.82, 160.39, 157.41, 141.30, 139.30, 133.98, 132.90, 129.51, 128.62, 127.48, 126.17, 86.37, 83.08, 82.79, 81.86, 77.39, 73.12, 69.44, 62.07, 56.39, 52.85 (x2), 44.01 (x2), 29.10, 29.06, 26.05, 18.78. HRMS (ESI) = [M + H] + = 761.21146.

[0178]

[0179] 9a

[0180] English name: 2-bromoethyl nitrate (9a)

[0181] Chinese name: 2-bromoethyl nitrate (9a)

[0182] 2-Bromoethanol (3 mmol) was added dropwise to a mixed solution of 70% HNO3 (1.1 mL) and 95% H2SO4 (2.4 mL) under ice bath conditions, and stirred at 0 °C for 1 h. After the reaction was complete, the reaction solution was poured into water (50 mL), extracted with DCM (3 × 50 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give intermediate 9a. The yield was 91%, and it was a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 4.76 (t, J = 6.4 Hz, 2H), 3.56(t, J = 6.4 Hz, 2H).

[0183] 9

[0185] English name:

[0186] N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(piperazin-1-yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide (9)

[0187] Chinese name:

[0188] N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(piperazin-1-yl)pyrimidin-4-yl)amino)thiazolyl-5-carboxamide (9)

[0189] Compound 8 (510 mg, 1.3 mmol, 1 equivalent), piperazine (1120 mg, 13 mmol, 10 equivalent), and DIPEA (453 μL, 2.6 mmol, 2 equivalent) were dissolved in 15 mL of anhydrous 1,4-dioxane and refluxed for 4 h, monitored by TLC. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was filtered. The filter cake was washed with 10 mL of water and 5 mL of methanol, and dried under vacuum to give intermediate 9. The yield was 71%, and the product was a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.25 (s, 1H), 7.40 (dd, J = 7.5, 2.0 Hz, 1H), 7.32 – 7.21 (m, 2H), 6.06 (s, 1H), 3.51 – 3.44 (m, 4H), 2.82 – 2.75 (m, 4H), 2.40 (s, 3H), 2.24 (s, 3H). ESI-MS(m / z): 444.13 [M + H]+

[0190]

[0191] 1f

[0192] English name:

[0193] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl nitrate (1f)

[0194] Chinese name:

[0195] 2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl nitrate (1f)

[0196] Intermediate 9 (150 mg, 0.34 mmol, 1 equivalent), 9a (172 mg, 1.01 mmol, 3 equivalents), and TEA (285 μL, 2.03 mmol, 6 equivalents) were dissolved in 5 mL of anhydrous DMF and reacted at room temperature for 6 h, monitored by TLC (EA / MeOH = 60:1). After the reaction was complete, most of the DMF was removed by rotary evaporation, and the product was purified by column chromatography to give compound 1f 55 mg. Yield: 31%, white solid. 1 H NMR (300 MHz, DMSO- d 6) δ 11.51 (s, 1H), 9.90 (s, 1H), 8.24 (s, 1H), 7.41 (dd, J = 7.2, 2.3 Hz, 1H), 7.30 – 7.23 (m, 2H), 6.07 (s, 1H), 4.68 (t, J=5.3 Hz, 2H), 3.58 – 3.45 (m, 4H), 2.78 – 2.67 (m, 2H), 2.59 – 2.51 (m, 4H), 2.42 (s, 3H), 2.25 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 165.67, 163.01, 162.78,160.39, 157.42, 141.30, 139.29, 133.98, 132.90, 129.50, 128.66, 127.49,126.19, 83.16, 71.13, 54.65, 52.59 (×2), 43.92 (×2), 26.06, 18.78.

[0197] Synthesis of furazolidone nitroxide compounds:

[0198] 10

[0200] English name: 2-(phenylsulfonyl)acetic acid (10)

[0201] Chinese name: 2-(benzenesulfonyl)acetic acid (10)

[0202] Methyl benzenesulfonyl acetate (3 mmol, 1.0 equivalent) was dissolved in 10 mL of methanol and stirred at 0 °C for 15 min. 2 M LiOH·H₂O aqueous solution (9 mmol, 2.0 equivalent) was added dropwise, and the reaction was carried out at 5 °C for 12 h. After the starting material was completely reacted by TLC, the pH was adjusted to 2 with 0.1 M dilute hydrochloric acid. Methanol was removed by rotary evaporation, and 15 mL of water was added. The mixture was extracted with EA (3 × 15 mL), washed with saturated brine (3 × 20 mL), dried over anhydrous sodium sulfate, and concentrated to give intermediate 10. The yield was 89%, and the solid was white. 1 HNMR (300 MHz, CD3OD): δ 7.96 – 7.98 (d, J = 7.6 Hz, 2H), 7.71 – 7.75 (t, J = 7.6Hz, 1H), 7.61 – 7.65 (t, J = 8.0 Hz, 2H), 4.29 (s, 2H).

[0203] 11

[0205] English name: 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11)

[0206] Chinese name: 3,4-bis(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (11)

[0207] 10 mL of glacial acetic acid was added to intermediate 10 (2 mmol, 1.0 equivalent), the temperature was raised to 90 °C, and then 5 mL of fuming nitric acid was added. The reaction temperature was further raised to 120 °C, and the reaction was carried out for 2 h. 50 mL of ice water was poured into the reaction solution, and a white solid precipitated out. After filtration and washing, compound 11 was obtained. The yield was 34%, and the solid was white. 1 H NMR (300 MHz, CDCl3) δ8.25 – 8.09 (m, 4H), 7.86 – 7.73 (m, 2H), 7.72 – 7.60 (m, 4H).

[0208] General method for synthesizing intermediates 12 and 13: Compound 11 (500 mg, 1.36 mmol) was dissolved in THF, the corresponding diol (2.73 mmol) was added, followed by 50% NaOH (2.73 mmol). The reaction was carried out at room temperature for 3–5 h, monitored by TLC (PE / EA = 2:1). After the reaction was completed, the solvent was evaporated and the reaction was carried out by column chromatography (PE / EA = 10:1) to obtain the corresponding compounds 12 and 13.

[0209] 12

[0211] English name:

[0212] 4-(2-(2-hydroxyethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12)

[0213] Chinese name:

[0214] 4-(2-(2-hydroxyethoxy)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (12)

[0215] It was prepared from intermediate 11 and diethylene glycol, with a yield of 30%, and was a white solid. 1H NMR (300 MHz, CDCl3) δ 8.19-8.15 (m, 2H, ArH), 7.77-7.83 (m, 1H, ArH), 7.62-7.70 (m, 2H, ArH), 4.59-4.62 (m, 4H, -OCH2-), 4.02-4.05 (m, 4H, -OCH2-).

[0216] 13

[0218] English name:

[0219] 4-(4-hydroxybutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13)

[0220] Chinese name:

[0221] 4-(4-hydroxybutoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (13)

[0222] It was prepared from intermediate 11 and 1,4-butanediol as raw materials, with a yield of 45%, and was a white solid. 1 H NMR (300 MHz, CDCl3) δ 8.19-8.15 (m, 2H), 7.77 – 7.83 (m, 1H), 7.62 – 7.70 (m, 2H), 4.39 –4.50 (t, J = 6 Hz, 2H), 3.74 – 3.78 (t, J = 6 Hz, 2H), 2.11 – 2.15 (m, 2H), 1.97 – 2.02 (m, 2H).

[0223] 15

[0225] English name: 3-methyl-4-nitro-1,2,5-oxadiazole 2-oxide (15)

[0226] Chinese name: 3-methyl-4-nitro-1,2,5-oxadiazole 2-oxide (15)

[0227] NaNO2 (4.83 g, 70 mmol, 3.5 equivalents) was added fractionally to a mixed solution of 60% H2SO4 (6 mL) and compound 14 (1.72 g, 20 mmol, 1 equivalent) in DCE (20 mL). The reaction was carried out at 50 °C for 1 h, monitored by TLC. After the reaction was complete, the organic phase was separated and washed with 3% Na2CO3 aqueous solution until it was completely colorless. The mixture was washed with saturated brine (3 × 20 mL), dried over anhydrous sodium sulfate, and concentrated to give a mixture of 3- and 4-nitroso isomers. The resulting mixture was dissolved in 3 mL of toluene, refluxed for 3 h, and rotary evaporated to give intermediate 15. 1 H NMR (300 MHz, CDCl3) δ 2.51 (s, 3H).

[0228] The general method for synthesizing compounds 16 and 17: Compound 15 (300 mg, 2.06 mmol) was dissolved in 10 mL of THF, the corresponding diol (4.12 mmol) was added, followed by 50% NaOH (4.12 mmol). The reaction was carried out at room temperature for 3-5 h. The reaction was detected by TLC (PE / EA = 2:1). After the reaction was completed, the solvent was evaporated and the corresponding compounds were obtained by column chromatography (PE / EA = 2:1).

[0229] 16

[0231] English name: 4-(2-hydroxyethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (16)

[0232] Chinese name: 4-(2-hydroxyethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (16)

[0233] It was prepared from intermediate 15 and ethylene glycol as raw materials, with a yield of 22%, and was a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 4.99 (t, J = 5.6 Hz, 1H), 4.37 – 4.33 (m, 2H), 3.75 (td, J = 5.5, 4.3 Hz, 2H), 2.05 (s, 3H).

[0234] 17

[0236] English name: 4-(carboxymethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (17)

[0237] Chinese name: 4-(carboxymethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (17)

[0238] It was prepared from intermediate 15 and glycolic acid, with a yield of 31%, and was a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 4.84 (s, 1H), 2.01 (s, 3H).

[0239] The general method for synthesizing target compounds 2a-2d was as follows: Compounds 12, 13, 16, or 17 (1.2 mmol, 1.2 equivalents), TBTU (1.2 mmol, 1.2 equivalents), DMAP (2 mmol, 2 equivalents), and dasatinib or intermediate 2 (1 mmol, 1 equivalent) were dissolved in 10 mL of DMF and reacted at room temperature for 12 h. The reaction was monitored by TLC (EA / MeOH = 40:1), and purified by column chromatography to obtain compounds 2a-2d.

[0240]

[0241] 2a

[0242] English name:

[0243] 4-(2-(2-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino))-2-methylpyrimidi n-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (2a)

[0244] Chinese name:

[0245] 4-(2-(2-((4-(2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutyryl)oxy)ethoxy)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (2a)

[0246] Prepared from intermediate 12 and 2 as starting materials in 59% yield as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 9.87 (s, 1H), 8.23 (s, 1H), 8.05 - 8.02 (m, 2H), 7.91 (dt, J = 7.4 Hz, 1H), 7.76 (dt, J = 7.8 Hz, 2H), 7.41 (d, J = 7.6 Hz, 1H), 7.32 - 7.25 (m, 2H), 6.07 (s, 1H), 4.54 (t, J = 4.4 Hz, 2H), 4.20 - 4.15 (m, 4H), 3.83 (t, J = 4.3 Hz, 2H), 3.72 (t, J = 4.8 Hz, 2H), 3.54 - 3.50 (m, 4H), 2.61 - 2.58 (m, 6H), 2.50 (m, 4H), 2.42 (s, 3H), 2.26 (s, 3H). 13 C NMR (75 MHz, DMSO-d6) δ 172.45, 172.35, 165.64, 163.03, 162.80, 160.39, 159.36, 157.40, 141.30, 139.30, 137.70, 136.61, 133.98, 132.90, 130.47 (x2), 129.50, 128.80 (x2), 128.04, 127.49, 126.17, 110.97, 83.08, 71.28, 68.85, 68.21, 63.89, 60.23, 56.38, 52.83 (x2), 44.01 (x2), 29.06, 28.99, 26.05, 18.78. HRMS (ESI) = [M + H] + = 900.22065.

[0247]

[0248] 2b

[0249] English Name:

[0250] 4-(4-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (2b)

[0251] Chinese Name:

[0252] 4-(4-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-)yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (2b)

[0253] From intermediate 13 and 2 as starting material, yield 59%, white solid. 1 H NMR (500 MHz, DMSO- d 6) δ11.44 (s, 1H), 9.86 (s, 1H), 8.21 (s, 1H), 8.01 (dd, J = 8.5, 1.3 Hz, 2H), 7.92– 7.87 (m, 1H), 7.77 – 7.72 (m, 2H), 7.39 (dd, J = 7.7, 1.8 Hz, 1H), 7.30 –7.23 (m, 2H), 6.05 (s, 1H), 4.41 (t, J = 6.2 Hz, 2H), 4.15 (t, J = 5.8 Hz, 2H),4.09 (t, J = 6.4 Hz, 2H), 3.52 – 3.47 (m, 4H), 2.60 – 2.57 (m, 6H), 2.50 (m,4H), 2.40 (s, 3H), 2.24 (s, 3H), 1.84 – 1.78 (m, 2H), 1.71 – 1.65 (m, 2H). 13CNMR (75 MHz, DMSO-d6) δ 172.42, 172.40, 165.64, 163.03, 162.80, 160.40,159.30, 157.40, 141.30, 139.30, 137.65, 136.61, 133.98, 132.91, 130.50 (×2),129.50, 128.80 (×2), 128.65, 127.48, 126.18, 110.93, 83.08, 71.48, 64.00,62.01, 56.39, 52.83 (×2), 44.00 (×2), 29.11, 29.08, 26.04, 25.06, 24.92,18.78. HRMS (ESI) = [M + H] + = 884.22422.

[0254]

[0255] 2c

[0256] English name:

[0257] 4-(2-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (2c)

[0258] Chinese name:

[0259] 4-(2-((4-(2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-)yl)piperazin-1-yl)ethoxy)-4-oxobutyryl)oxy)ethoxy)3-methyl-1,2,5-oxadiazole 2-oxide (2c)

[0260] It was prepared from intermediates 16 and 2 as raw materials, with a yield of 59%, and was a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ11.42 (s, 1H), 9.85 (s, 1H), 8.21 (s, 1H), 7.40 (dd, J= 7.8, 1.8 Hz, 1H), 7.30– 7.25 (m, 2H), 6.05 (s, 1H), 4.57 – 4.54 (m, 2H), 4.43 – 4.41 (m, 2H), 4.15(t, J HRMS (ESI) = [M + H] + = 752.19783.

[0261]

[0262] 2d

[0263] English name:

[0264] 4-(2-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-2-oxoethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (2d)

[0265] Chinese name:

[0266] 4-(2-(2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-2-oxoethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (2d)

[0267] It was prepared from intermediates 17 and 1 as raw materials, with a yield of 59%, and was a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ11.45 (s, 1H), 9.85 (s, 1H), 8.22 (s, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.30 –7.23 (m, 2H), 6.05 (s, 1H), 5.11 (s, 2H), 4.30 (t, J= 5.6 Hz, 2H), 3.54 – 3.46(m, 4H), 2.63 (t, J = 5.4 Hz, 2H), 2.50 (m, 4H), 2.41 (s, 3H), 2.24 (s, 3H), 2.10 (s, 3H). HRMS (ESI) = [M + H] + = 666.16195.

[0268] The general method for synthesizing compounds 2e and 2f is as follows: Compounds 2a and 2b (0.5 mmol, 1.0 equivalent) were suspended in 10 mL of diethyl ether, and methanesulfonic acid (3 mmol, 6.0 equivalent) was added. The reaction was carried out at room temperature for 3 h, monitored by TLC (EA / MeOH = 40:1). After the starting materials reacted completely, the solvent was removed by rotary evaporation. 2 mL of isopropanol was added, and the mixture was heated until just clear. Then, 10 mL of diethyl ether was slowly added dropwise to the reaction solution, and a large amount of white solid gradually precipitated. The mixture was centrifuged, the supernatant was removed, and the solid was dissolved again in 2 mL of isopropanol. This process was repeated 3 times. The white solid obtained by vacuum drying was the product of compounds 2e and 2f.

[0269]

[0270] 2e

[0271] English name:

[0272] 4-(2-(2-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino))-2-methylpyrimidi n-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide tri mesylate (2e)

[0273] Chinese name:

[0274] 4-(2-(2-((4-(2-(4-(6-((5-(((2-chloro-6-methylphenyl)carbamoyl)thiazolyl-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutyryl)oxy)ethoxy)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide trimethylsulfonate (2e)

[0275] Prepared from compound 2a as starting material in 65% yield as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 9.92(s, 1H), 8.26 (s, 1H), 8.04 (d, J = 7.7 Hz, 2H), 7.92 (t, J = 7.4 Hz, 1H),7.77 (t, J = 7.8 Hz, 2H), 7.42 (d, J = 8.7 Hz, 1H), 7.32 – 7.27 (m, 2H), 6.21(s, 1H), 4.55 (t, J = 4.3 Hz, 2H), 4.41 (t, J = 5.1 Hz, 4H), 4.20 (t, J = 4.7Hz, 2H), 3.83 (t, J = 4.1 Hz, 2H), 3.73 (t, J = 4.7 Hz, 2H), 3.67 – 3.61 (m,2H), 3.53 – 3.48 (m, 2H), 3.35 – 3.26 (m, 2H), 3.19 (s, 2H), 2.68 – 2.63 (m,4H), 2.47 (s, 3H), 2.39 (s, 9H), 2.26 (s, 3H).

[0276]

[0277] 2f

[0278] English Name:

[0279] 4-(4-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide tri mesylate (2f)

[0280] Chinese Name:

[0281] 4-(4-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2- methylpyrimidin-4-yl) piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)butyloxy)-3- (phenylsulfonyl)-1,2,5-oxadiazole 2-oxide trimethylsulfonate (2f)

[0282] Prepared from compound 2b in 71% yield as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 11.64 (s, 1H), 9.89 (s, 1H), 8.24 (s, 1H), 8.01 (d, J = 7.4 Hz, 2H), 7.90 (t, J = 7.5 Hz, 1H), 7.75 (t, J = 7.8 Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.31 - 7.24 (m, 2H), 6.18 (s, 1H), 4.43 (t, J = 5.7 Hz, 2H), 4.11 (t, J = 6.4 Hz, 2H), 3.67 - 3.56 (m, 2H), 3.52 - 3.46 (m, 2H), 3.33 - 3.24 (m, 2H), 3.21 - 3.11 (m, 2H), 2.69 - 2.62 (m, 4H), 2.45 (s, 3H), 2.35 (s, 9H), 2.24 (s, 3H), 1.85 - 1.80 (m, 2H), 1.73 - 1.67 (m, 2H).

[0283] Since the target compounds enter the anterior chamber after eye drop administration and modulate TM cells by releasing NO, we selected HTM cells as a cell model to test the NO release of the compounds in HTM cells. According to the design idea of the compounds, the compounds can be metabolized to release NO through enzymatic or non-enzymatic pathways after entering TM cells. We used the NO fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) to detect the release of NO. Each target compound was incubated with HTM cells at 37 °C for 12 h at a concentration of 5 μM, and then DAF-FM DA fluorescent probe was added for incubation for 30 min. The fluorescence intensity of the cells was detected by flow cytometry, and the mean fluorescence intensity (MFI) was used as a statistical indicator. The control group was used as a standard for normalization. The results showed that most of the target compounds could increase the NO level in HTM cells (A), among which compounds 1f, 2a and 2e had the most significant effect, which was consistent with our initial design idea. Figure 1

[0284] cGMP is an important downstream molecule for the physiological activity of NO, and the activity of NO in reducing intraocular pressure also requires the participation of cGMP. Therefore, we explored whether the treatment of compounds would promote the production of cGMP in HTM cells. After each target compound was incubated with HTM cells at a concentration of 5 μM for 24 h, the cell supernatant was collected, and the cGMP concentration in the cell supernatant was determined using enzyme-linked immunosorbent assay. The results showed that most of the target compounds could promote the production of cGMP in HTM cells, and increase the concentration of cGMP. Among them, compound 2e had the most obvious activity in up-regulating the level of cGMP (Fig. 1B). Considering the significant activity of compound 2e in releasing NO and up-regulating the level of cGMP, we selected compound 2e for subsequent in vivo pharmacodynamic experiments.

[0285] ​To evaluate whether our target compounds can also exert the activity of eliminating senescent cells in vivo, we performed senescence-associated beta-galactosidase (SA-β-gal) staining on retinal flatmounts of the chronic ocular hypertensive mice induced by magnetic microbeads, and counted the number of senescent positive cells per unit area. From the second day after the mice were modeled, eye drops were administered three times a day, and after 10 days of continuous eye drop administration, the mice were taken for retinal sampling and retinal flatmounts were prepared, followed by SA-β-gal staining and counting the number of senescent positive cells per unit area. (Fig. 2A, B). The results showed that the number of senescent positive cells per unit area of the retinal flatmounts of the vehicle control (Vehicle) group was significantly increased compared with the normal (Normal) group mice (*** p <0.001), and after eye drop administration of dasatinib mesylate or 2e, the number of senescent positive cells per unit area was significantly decreased compared with the Vehicle group (*** p <0.001). Similar to the results of the retinal sections, there was no significant difference between the Dasatinib Mesylate group and the 2e group (Fig. 2B). The above results showed that both 2e and dasatinib mesylate can exert a significant effect of eliminating senescent cells in vivo, and there is no significant difference between the two. The above data showed that the senescent cells of the chronic ocular hypertensive mice induced by magnetic microbeads were significantly increased, and both eye drop administration of dasatinib mesylate and 2e can significantly reduce the number of senescent cells per unit area.

[0286] Next, we explored the effects of target compound 2e and dasatinib mesylate on intraocular pressure of the chronic ocular hypertensive mouse model induced by magnetic microbeads. We selected the time points of 1, 2, 4, and 8 h after eye drop administration for intraocular pressure measurement and statistics (Fig. 3). The experimental results showed that eye drop administration of 2e (10 mM) to the model mice can significantly reduce the intraocular pressure of the mice at 2 h and 4 h after administration, and at 4 h after eye drop administration, the 2e group of mice showed the most obvious effect of reducing intraocular pressure (3.6 mmHg, ** p <0.01, Fig. 3), and then at 8 h after administration, there was no significant difference in intraocular pressure compared with the Vehicle group. However, no significant reduction in intraocular pressure was detected at each time point after administration in the Vehicle group and the Dasatinib Mesylate group. The above results showed that the NO donor type dasatinib derivative 2e exhibited a significant effect of reducing intraocular pressure compared with the parent compound.

[0287] Glaucoma-induced damage and death of retinal ganglion cells is a significant cause of vision loss in patients. Therefore, we used retinal ganglion cell-specific antibody RBPMS for immunofluorescence staining to evaluate whether 2e could exert a protective effect on retinal ganglion cells (Figure 4A). As shown in Figure 4B, compared with the Normal group, the number of retinal ganglion cells in mice with chronic ocular hypertension induced by magnetic microbeads was significantly reduced (**** p <0.0001), while both dasatinib mesylate and 2e, administered as eye drops, have a protective effect on retinal ganglion cells, and 2e has a more significant protective effect on retinal ganglion cells than dasatinib mesylate (*). p <0.05). The above results indicate that, thanks to the synergistic effect of 2e in clearing senescent cells and reducing intraocular pressure, 2e can exert a significant protective effect on retinal ganglion cells in mice with chronic intraocular hypertension induced by magnetic microbeads, and its activity is superior to that of the parent compound.

[0288] In glaucoma patients and multiple mouse models of glaucoma, visual function is significantly impaired, specifically manifested as abnormalities in visual electrophysiology, namely, a decrease in N1 wave amplitude and a delay in peak time in visual evoked potentials (VEPs). Therefore, we aim to test whether 2e can exert a protective effect on visual function in mice by clearing senescent cells and reducing intraocular pressure through VEP detection. Representative VEP curves for each group of mice are shown in the figure. Figure 5 A) Compared to the Normal group, mice with chronic ocular hypertension induced by magnetic microbeads showed a significant decrease in the amplitude of the N1 wave and a marked lag in peak duration. These data indicate that chronic ocular hypertension induced by magnetic microbeads significantly impairs visual function in mice. Intraocular administration of 2e significantly increased the N1 wave amplitude in the model mice. While dasatinib mesylate administration showed a trend towards increasing N1 wave amplitude, it was not significantly different from the Vehicle group (Figure 5B). Furthermore, both 2e and dasatinib mesylate significantly improved the lag in N1 wave peak duration, but there was no significant difference between the two (Figure 5C). Therefore, intraocular administration of the NO donor dasatinib derivative 2e can significantly improve visual function.

[0289] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A NO donor type dasatinib derivative, characterized in that, The derivative is one of compounds 1b-1f, compound 2a, 2c-2f; the structural formula of the compound 1b-1f, compound 2a, 2c-2f is as follows respectively: （1b） （1c） （1d） （1e） （1f） （2a） （2c） （2d） （2e） （2f）。 2. A process for the preparation of a NO-donor type dasatinib derivative according to claim 1, characterized by, The preparation method of the compound 1b-1f is as follows respectively: Dasatinib reacts with succinic anhydride to obtain intermediate 2; 3-bromopropanol reacts with silver nitrate to obtain intermediate 4b, and the intermediate 4b reacts with the intermediate 2 through esterification to obtain the compound 1b; 5-bromopentanol reacts with silver nitrate to obtain intermediate 4c, and the intermediate 4c reacts with the intermediate 2 through esterification to obtain the compound 1d; 4-bromobutyl acetate reacts with silver nitrate to obtain intermediate 6, and the intermediate 6 is reacted under alkaline conditions to remove acetyl to obtain intermediate 7, and the intermediate 7 reacts with the intermediate 2 through esterification to obtain the compound 1c; ISMN reacts with the intermediate 2 through esterification to obtain the compound 1e; The intermediate 8 reacts with piperazine under alkaline conditions to occur nucleophilic substitution to obtain intermediate 9, and the intermediate 9 further reacts with 2-bromoethyl nitrate under alkaline conditions to occur nucleophilic substitution to obtain the compound 1f; The structural formula of the intermediate 2 is as follows: ； The structural formula of the intermediate 4b-4c is as follows: 4b 4c； The structural formula of the intermediate 6 is as follows: ； The structural formula of the intermediate 7 is as follows: ； The structural formula of the ISMN is as follows: ； The structural formula of the intermediate 8 is as follows: ； The structural formula of the intermediate 9 is as follows: ； The preparation method of the compound 2a, 2c-2f is as follows respectively: The intermediate 10 is added with glacial acetic acid, the temperature is raised to 90 DEG C, fuming nitric acid is added, and then the reaction temperature is further raised to 120 DEG C, and the reaction is carried out to obtain the intermediate 11; The compound 14 and sodium nitrite are reacted under acidic conditions to obtain the intermediate 15; The intermediate 11 occurs nucleophilic substitution with diethylene glycol to obtain the intermediate 12; The intermediate 11 occurs nucleophilic substitution with 1, 4-butanediol to obtain the intermediate 13; The intermediate 15 occurs nucleophilic substitution with ethylene glycol to obtain the intermediate 16; The intermediate 15 occurs nucleophilic substitution with glycollic acid to obtain the intermediate 17; The intermediate 12 occurs esterification with the intermediate 2 to obtain the compound 2a; The intermediate 13 occurs esterification with the intermediate 2 to obtain the compound 2b; The intermediate 16 occurs esterification with the intermediate 2 to obtain the compound 2c; The intermediate 17 occurs esterification with dasatinib to obtain the compound 2d; The compound 2a is reacted with methanesulfonic acid to form the compound 2e; The compound 2b is reacted with methanesulfonic acid to form the compound 2f; The structural formula of the intermediate 10 is as follows: ； The structural formula of the intermediate 11 is as follows: ； The structural formula of the intermediate 14 is as follows: ； The structural formula of the intermediate 15 is as follows: ； The structural formula of the intermediate 12 is as follows: ； The structural formula of the intermediate 13 is as follows: ； The structural formula of the intermediate 16 is as follows: ； The structural formula of the intermediate 17 is as follows: ； The structural formula of the compound 2b is as follows: 。 3. The method for preparing the NO donor-type dasatinib derivative according to claim 2, characterized in that, the molar ratio of the dasatinib to succinic anhydride is 4:4.4; the molar ratio of the 3-bromopropanol to silver nitrate is 5:12.5, the molar ratio of the intermediate 4b to the intermediate 2 is 1:1; the molar ratio of the 5-bromopentanol to silver nitrate is 5:12.5, the molar ratio of the intermediate 4c to the intermediate 2 is 1:1; the molar ratio of the 4-bromobutyl acetate to silver nitrate is 5:12.5, the molar ratio of the intermediate 7 to the intermediate 2 is 1:1; the molar ratio of the ISMN to the intermediate 2 is 1:1; the molar ratio of the intermediate 8 to piperazine is 1:10; the molar ratio of the intermediate 9 to 2-bromoethyl nitrate is 0.34:1.01; the molar ratio of the intermediate 11 to diethylene glycol is 1.36:2.73; the molar ratio of the intermediate 11 to 1,4-butanediol is 1.36:2.73; the molar ratio of the compound 14 to sodium nitrite is 2:7; the molar ratio of the intermediate 15 to ethylene glycol is 2.06:4.12; the molar ratio of the intermediate 15 to glycolic acid is 2.06:4.12; the molar ratio of the intermediate 12 to the intermediate 2 is 1.2:1; the molar ratio of the intermediate 13 to the intermediate 2 is 1.2:1; the molar ratio of the intermediate 16 to the intermediate 2 is 1.2:1; the molar ratio of the intermediate 17 to dasatinib is 1.2:1; the molar ratio of the compound 2a to methanesulfonic acid is 1:6; the molar ratio of the compound 2b to methanesulfonic acid is 1:

6.

4. The use of the NO-donor dasatinib derivative of claim 1 in the preparation of a medicament for removing retinal senescent cells, reducing intraocular pressure or / and protecting optic nerve.

5. The use of the NO-donor dasatinib derivative of claim 1 in the preparation of a medicament for treating glaucoma.

6. The use of the NO-donor dasatinib derivative of claim 1 in the preparation of a medicament for treating senescence-associated retinopathy.

7. The use of the NO-donor dasatinib derivative of claim 1 in the preparation of a medicament for improving visual function.

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