NO donor type dasatinib derivative as well as preparation method and application thereof

Abstract

The invention provides an NO donor type dasatinib derivative as well as a preparation method and application thereof. The derivative is one of a compound 1a-1f and a compound 2a-2f; the derivative has multiple effects of removing senescence cells, reducing intraocular pressure and protecting optic nerves, and is a novel potential treatment medicine for treating glaucoma and senescence-related retinopathy.

Description

Technical Field

[0001] The present invention relates to an NO donor type dasatinib derivative and a preparation method and application thereof. Background Art

[0002] Glaucoma is a group of diseases with characteristic optic atrophy and visual field loss as common features. Pathological increase in intraocular pressure is its main risk factor. The global prevalence of glaucoma is 3.54%, making it the world's leading irreversible blinding eye disease. It is estimated that by 2040, 112 million people aged 40-80 will suffer from glaucoma. [1] . Treatments for glaucoma include intraocular pressure-lowering therapy and optic nerve protective therapy. Currently, commonly used intraocular pressure-lowering drugs include prostaglandin analogs (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 lowering intraocular pressure, but it is not clear whether they can remove senescent retinal ganglion cells. At present, there are no reports of glaucoma drugs that can simultaneously lower intraocular pressure and remove senescent cells.

[0003] Research shows that retinal ganglion cell senescence is an important pathological mechanism of glaucoma [2] Senolytics are 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 invention designs and synthesizes a NO donor type dasatinib derivative as a novel glaucoma drug. The 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 aging-related retinopathy.

[0004] References

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

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

[0007] [3].Rocha LR,Nguyen Huu VA,Palomino La Torre C,Xu Q,Jabari M,KrawczykM,Weinreb RN,Skowronska-Krawczyk D.Early removal of senescent cells protectsretinal ganglion cells loss in experimental ocular hypertension.AgingCell.2020Feb;19(2):e13089.

[0008] [4].Toda N,Nakanishi-Toda M.Nitric oxide:ocular blood flow,glaucoma,and diabetic retinopathy.Prog Retin Eye Res.2007May;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 RipasudilDerivatives with Intraocular Pressure Lowering and Retinal Ganglion CellProtection Activities.J Med Chem.2022Sep8;65(17):11745-11758. Summary of the invention

[0010] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a NO donor dasatinib derivative. The NO donor dasatinib derivative designed and synthesized by the present invention is a new glaucoma drug with multiple functions of removing senescent cells, reducing intraocular pressure and protecting the optic nerve.

[0011] To achieve the above purpose, the technical solution adopted is: a NO donor dasatinib derivative, the derivative is one of compounds 1a-1f and compounds 2a-2f; the structural formulas of compounds 1a-1f and compounds 2a-2f are as follows:

[0012]

[0013]

[0014]

[0015] The present invention provides a method for preparing the above-mentioned NO donor dasatinib derivatives. The preparation methods of the compounds 1a-1f are respectively as follows:

[0016] Dasatinib reacts with succinic anhydride to obtain intermediate 2;

[0017] 2-bromoethanol reacts with silver nitrate to obtain intermediate 4a, and intermediate 4a reacts with intermediate 2 to obtain compound 1a through esterification reaction;

[0018] 3-bromopropanol reacts with silver nitrate to obtain intermediate 4b, and intermediate 4b reacts with intermediate 2 to obtain compound 1b through esterification reaction;

[0019] 5-bromopentanol reacts with silver nitrate to obtain intermediate 4c, and intermediate 4c reacts with intermediate 2 to obtain compound 1c through esterification reaction;

[0020] 4-bromobutyl acetate reacts with silver nitrate to produce intermediate 6, and intermediate 6 is deacetylating under alkaline conditions to produce intermediate 7, and intermediate 7 is reacted with intermediate 2 through esterification to produce compound 1d;

[0021] ISMN reacted with intermediate 2 to give compound 1e;

[0022] Intermediate 8 reacts with piperazine under alkaline conditions to undergo nucleophilic substitution reaction to obtain intermediate 9, and intermediate 9 further reacts with 2-bromoethyl nitrate under alkaline conditions to undergo nucleophilic substitution reaction to obtain compound 1f;

[0023] The structural formula of the intermediate 2 is as follows:

[0024]

[0025] The structural formula of the intermediates 4a-4c is as follows:

[0026]

[0027] The structural formula of the intermediate 6 is as follows:

[0028]

[0029] The structural formula of the intermediate 7 is as follows:

[0030]

[0031] The structural formula of the ISMN is as follows:

[0032]

[0033] The structural formula of the intermediate 8 is as follows:

[0034]

[0035] The structural formula of the intermediate 9 is as follows:

[0036]

[0037] The preparation methods of the compounds 2a-2f are respectively as follows:

[0038] Add glacial acetic acid to the intermediate 10, raise the temperature to 90°C, add fuming nitric acid, and then further raise the reaction temperature to 120°C to obtain the intermediate 11;

[0039] Compound 14 reacts with sodium nitrite under acidic conditions to generate intermediate 15;

[0040] The intermediate 11 undergoes a nucleophilic substitution reaction with diethylene glycol to obtain the intermediate 12;

[0041] The intermediate 11 undergoes a nucleophilic substitution reaction with 1,4-butanediol to obtain the intermediate 13;

[0042] The intermediate 15 undergoes a nucleophilic substitution reaction with ethylene glycol to obtain the intermediate 16;

[0043] The intermediate 15 undergoes a nucleophilic substitution reaction with glycolic acid to obtain the intermediate 17;

[0044] The intermediate 12 undergoes an esterification reaction with the intermediate 2 to obtain compound 2a;

[0045] The intermediate 13 undergoes an esterification reaction with the intermediate 2 to obtain the target 2b;

[0046] The intermediate 16 undergoes an esterification reaction with the intermediate 2 to obtain the target 2c;

[0047] The intermediate 17 undergoes an esterification reaction with dasatinib to obtain compound 2d;

[0048] The compound 2a reacts with methanesulfonic acid to form a compound 2e;

[0049] The compound 2b reacts with methanesulfonic acid to form compound 2f;

[0050] The structural formula of the intermediate 10 is as follows:

[0051]

[0052] The structural formula of the intermediate 11 is as follows:

[0053]

[0054] The structural formula of the intermediate 14 is as follows:

[0055]

[0056] The structural formula of the intermediate 15 is as follows:

[0057]

[0058] The structural formula of the intermediate 12 is as follows:

[0059]

[0060] The structural formula of the intermediate 13 is as follows:

[0061]

[0062] The structural formula of the intermediate 16 is as follows:

[0063]

[0064] The structural formula of the intermediate 17 is as follows:

[0065]

[0066] Preferably, the molar ratio of dasatinib to succinic anhydride is 4:4.4; the molar ratio of 2-bromoethanol to silver nitrate is 5:12.5, and the molar ratio of intermediate 4a to intermediate 2 is 1:1; the molar ratio of 3-bromopropanol to silver nitrate is 5:12.5, and the molar ratio of intermediate 4b to intermediate 2 is 1:1; the molar ratio of 5-bromopentanol to silver nitrate is 5:12.5, and the molar ratio of intermediate 4c to intermediate 2 is 1:1; the molar ratio of 4-bromobutyl acetate to silver nitrate is 5:12.5, and the molar ratio of intermediate 7 to intermediate 2 is 1:1; the molar ratio of ISMN to intermediate 2 is 1:1; the molar ratio of intermediate 8 to piperazine is 1:10; the molar ratio of intermediate 9 to 2-bromoethyl nitrate is 0.34:1.01;

[0067] 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.

[0068] The present invention provides the use of the above-mentioned NO donor dasatinib derivative in the preparation of a drug for removing retinal senescent cells, reducing intraocular pressure or / and protecting the optic nerve.

[0069] The present invention provides the use of the above-mentioned NO donor dasatinib derivative in the preparation of a drug for treating glaucoma.

[0070] The present invention provides the use of the above-mentioned NO donor dasatinib derivative in the preparation of a drug for treating age-related retinopathy.

[0071] The present invention provides the use of the above-mentioned NO donor dasatinib derivative in the preparation of a drug for improving visual function.

[0072] Beneficial effects:

[0073] The NO donor type dasatinib derivative prepared by the present invention has the multiple effects of removing senescent cells, reducing intraocular pressure and protecting the optic nerve, and is a potential new therapeutic drug for glaucoma and aging-related retinopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 :NO activity and cGMP concentration of different NO donor dasatinib derivatives in cells; Figure A: NO fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) was used to detect the NO activity of NO donor dasatinib derivatives in cells. The fluorescence intensity of the cells was detected by flow cytometry, and the mean fluorescence intensity (MFI) was used as a statistical indicator and normalized to the control group. The results showed that, except for compound 1a, the other NO donor dasatinib derivatives could increase the NO level in cells. B: cGMP is an important downstream molecule of NO in lowering intraocular pressure. The results showed that, except for 2b, the other NO donor dasatinib derivatives could promote the production of cGMP in cells. Relative MFI: relative mean fluorescence intensity; concentration: concentration; fold of control: compared to the control.

[0075] Figure 2:In glaucoma model mice, 2e compound can significantly eliminate retinal senescent cells; Figure AB: Senescence-associated beta-galactosidase (SA-β-Gal) staining of retinal flat sections of Normal mice (no eye drops) and magnetic bead model-induced high intraocular pressure mice (Vehicle, dasatinib mesylate and 2e compound eye drops for 10 days), and the number of senescence-positive cells was counted. Compared with the Normal group, the number of senescence-positive cells per unit area in the retinal flat sections of the Vehicle group mice increased significantly. Compared with the Vehicle group, eye drops of dasatinib mesylate and 2e compound can significantly reduce the number of senescence-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 dasatinib derivative 2e group; SA-β-Gal positive cells: senescence-associated beta-galactosidase positive cells.

[0076] Figure 3 :In glaucoma model mice, 2e compound can significantly reduce intraocular pressure; IOP was measured 1, 2, 4 and 8h after single eye drop administration (Vehicle, dasatinib mesylate and 2e compound) in magnetic bead model-induced high intraocular pressure mice. Compared with the Vehicle group, there was no statistical difference in intraocular pressure at each time point after eye drop administration of dasatinib mesylate. Compared with the Vehicle group, the intraocular pressure of glaucoma model mice was significantly reduced 2h and 4h after a single eye drop administration of 2e compound (10mM), among which the intraocular pressure-lowering effect was most significant 4h after a single eye drop administration. ΔIOP: intraocular pressure difference before and after eye drop administration; Time after eye drop (hours): time after eye drop administration (hours); Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor dasatinib derivative 2e group.

[0077] Figure 4:In glaucoma model mice, 2e compound can significantly protect retinal ganglion cells; Figure A: Retinal flat mounts and retinal ganglion cell staining (RBPMS) of Normal mice (no eye drops), magnetic bead modeling induced high intraocular pressure mice (Vehicle, dasatinib mesylate and 2e compound eye drops for 10 days), and the number of retinal ganglion cells was counted. B: Compared with the Normal group, the number of retinal ganglion cells in the Vehicle group of mice with chronic high intraocular pressure induced by magnetic beads was significantly decreased. Compared with the Vehicle group, both dasatinib mesylate and 2e compound eye drops can protect retinal ganglion cells, and compared with dasatinib mesylate, 2e compound has a more significant protective effect on retinal ganglion cells. Normal: normal group; Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor dasatinib derivative 2e group; RBPMS positive cells: RBPMS positive cells of retinal ganglion cell marker.

[0078] Figure 5 :In glaucoma model mice, 2e compound can significantly improve visual function; Figure A: VEP curves of Normal mice (no eye drops) and magnetic bead model-induced high intraocular pressure mice (Vehicle, dasatinib mesylate and 2e compound eye drops for 10 days). BC: N1 wave statistics of VEP curves. Compared with the Normal group, the amplitude of the N1 wave in the Vehicle group decreased significantly, and the peak time was significantly delayed; compared with the Vehicle group, dasatinib mesylate eye drops can significantly improve the peak time of the N1 wave in chronic high intraocular pressure mice, but there is no statistical difference in the amplitude between the two; compared with the Vehicle group, 2e compound eye drops can significantly improve the amplitude and peak time of the N1 wave in chronic high intraocular pressure mice. Amplitude: amplitude; VEP: visual evoked potential; Scotopic VEP: scotopic VEP; Time: time; Normal: normal group; Vehicle: solvent control group; Dasatinib Mesylate: dasatinib mesylate group; 2e: NO donor dasatinib derivative 2e group; Peak time: peak time. DETAILED DESCRIPTION

[0079] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0080] In our study, by introducing NO donor fragments into the nucleus of senolytics, dasatinib, we designed and synthesized two series of NO donor-type dasatinib derivatives, nitrate and furazan N-oxide, totaling 12 compounds (1a-1f, 2a-2f). We expect that the candidate molecules will enter the eye through local eye drops to metabolize and produce NO and dasatinib, which will play the role of lowering intraocular pressure and selectively removing senescent cells to protect the optic nerve. Our results show that the most active compound 2e plays a good role in lowering intraocular pressure and removing senescent cells. In in vitro experiments, compound 2e showed obvious NO release ability and activity in promoting the production of cyclic guanosine monophosphate (3',5'-cyclophosphate, cGMP) in human trabecular meshwork (HTM) cells. Since 2e is a compound in the form of mesylate, in subsequent in vivo experiments, we salified dasatinib with methanesulfonic acid to synthesize dasatinib mesylate as a positive drug control. In in vivo experiments, topical eye drops of 2e were able to significantly reduce intraocular pressure in mice with chronic ocular hypertension induced by magnetic microbeads, eliminate senescent cells in the retina of model mice, reduce the death of retinal ganglion cells, and show activity in improving visual function.

[0081] The synthetic routes of nitrate ester compounds 1a-1f and dasatinib mesylate are shown in Scheme 1. In short, compounds 1a-1e are reacted with succinic anhydride with 1 as raw material to obtain intermediate 2. Compounds 3a-3c and 5 with different carbon chain lengths react with silver nitrate to obtain the corresponding 4a-4c and 6, and intermediate 6 is deacetylating under strong alkaline conditions of LiOH to obtain intermediate 7. Compounds 4a-4c, 7, and isosorbide mononitrate (ISMN) are esterified with 2 to obtain compounds 1a-1e. The synthesis of 1f is through the nucleophilic substitution reaction of intermediate 8 with piperazine under alkaline conditions to obtain intermediate 9, and intermediate 9 is further reacted with 2-bromoethyl nitrate under alkaline conditions to obtain compound 1f. In order to improve the water solubility of the compound for the preparation of eye drops, we designed and synthesized dasatinib mesylate. 1 was used as raw material, dissolved in dichloromethane and then added with methanesulfonic acid. The reaction was carried out at room temperature for 2 h to obtain the target compound dasatinibmesylate.

[0082]

[0083] Scheme 1. Synthesis route of compounds 1a-1f. Reagents and reaction conditions: a) succinic anhydride, pyridine, DMF (N,N-dimethylformamide), 25°C, 48h; b) AgNO 3 , MeCN, reflux, 12h; c) LiOH, MeOH, 25°C, 4h; d) EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide), DMAP (4-dimethylaminopyridine), DMF, 25°C, 12h; e) piperazine, K 2 CO 3 , DMF, 90°C, 12h; f) 2-bromoethyl nitrate, DMF, 12h; g) MeSO 3 H, DCM (dichloromethane), 25° C., 2 h.

[0084] The synthetic route of furazan nitrogen oxide series compounds 2a-2f is shown in Scheme 2. In short, compounds 10 and 14 are used as raw materials, respectively, and reacted with fuming nitric acid and sodium nitrite under acidic conditions to generate intermediates 11 and 15. Intermediate 11 undergoes nucleophilic substitution reaction with diethylene glycol and 1,4-butanediol to obtain intermediates 12 and 13; intermediate 15 undergoes nucleophilic substitution reaction with ethylene glycol and glycolic acid to obtain intermediates 16 and 17. Intermediates 12, 13, and 16 undergo esterification reaction with intermediate 2 to obtain targets 2a, 2b, and 2c; and intermediate 17 undergoes esterification reaction with dasatinib to obtain compound 2d. In order to improve the water solubility of the compound, we reacted 2a and 2b in methanesulfonic acid to form the corresponding target compounds 2e and 2f.

[0085]

[0086] Scheme 2. Synthesis route of compounds 2a-2f. Reagents and reaction conditions: a) fuming nitric acid, AcOH, 90°C, 3h; b) diethylene glycol, 50% aqueous NaOH solution, THF (tetrahydrofuran), 4h; c) 1,4-butanediol, 50% aqueous NaOH solution, THF, 4h; d) intermediate 2, TBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate borate), DMAP (4-dimethylaminopyridine), DMF (N,N-dimethylformamide), 25°C, 12h; e) NaNO 2 , the volume fraction of H is 60% 2 SO 4Aqueous solution, DCM (dichloromethane), 50°C, 0.5h; f) glycolic acid, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DCM, 25°C, 4h; g) ethylene glycol, 50% NaOH solution, THF, 4h; h) dasatinib, TBTU, DMAP, DMF, 25°C, 12h; i) MeSO 3 H, DCM, 25°C, 2h.

[0087] The preparation methods and structural characterizations of a total of 12 compounds 1a-1f, 2a-2f are as follows:

[0088]

[0089] English name:

[0090] 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)

[0091] Chinese name:

[0092] 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)

[0093] 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 DMF and reacted at room temperature for 24 h. TLC was used for monitoring. After the reaction was completed, most of the DMF was evaporated off by rotary evaporation. 50 mL of water was added to the reaction bottle. A large amount of white solid precipitated. The filter cake was filtered and washed with methanol (3×10 mL). It was dried in vacuo to obtain intermediate 2, 1870 mg. The yield was 81%, white solid. 1 H NMR (300 MHz, DMSO-d 6)δ11.47(s,1H),9.87(s,1H),8.22(s,1H),7.40(dd,J=7.5,1.5Hz,1H),7.29(dd,J=7.6,1.5Hz,1H),7.25(dd,J=7.6Hz,1H),6.05(s,1 H),4.16(t,J=5.7Hz,2H),3.51(m,4H),2.59(t,J=5.7Hz,2H),2.44-2.56(m,8H),2.41(s,3H),2.24(s,3H).ESI-MS(m / z):588.18[M+H] +

[0094] General method for the synthesis of intermediates 4a-4c: dissolve terminal bromohydrin (5mmol, 1.0 equivalent) / methyl bromocarboxylate (5mmol, 1.0 equivalent) in acetonitrile (40mL), add silver nitrate (12.5mmol, 2.5 equivalent), protect from light, and heat under reflux for 12h. Monitor the reaction by TLC, color development by phosphomolybdic acid (nitrate compound appears yellow), remove acetonitrile by rotary evaporation, add 100mL saturated brine and 100mL ethyl acetate, stir and stand, transfer the supernatant to a separatory funnel, extract, combine the organic phases, wash 3 times with 100mL saturated brine, dry over anhydrous sodium sulfate, concentrate, and purify by column chromatography to obtain intermediates 4a-4c.

[0095]

[0096]

[0097] English name: 2-hydroxyethyl nitrate (4a)

[0098] Chinese name: 2-Hydroxyethyl nitrate (4a)

[0099] It is prepared from 2-bromoethanol, with a yield of 88%, as a colorless oil. 1 H NMR (300 MHz, CDCl 3 )δ4.59(t,J=4.6Hz,2H),3.93(t,J=4.6Hz,2H),1.88(s,1H).

[0100]

[0101] English name: 3-hydroxypropyl nitrate (4b)

[0102] Chinese name: 3-Hydroxypropyl nitrate (4b)

[0103] It is prepared from 3-bromopropanol, with a yield of 89%, as a colorless oil.1 H NMR (300 MHz, CDCl 3 )δ4.61(t,J=6.3Hz,2H),3.78(t,J=6.0Hz,2H),1.98(p,J=6.2Hz,2H),1.51(s,1H).

[0104]

[0105] English name: 4-hydroxybutyl nitrate (4c)

[0106] Chinese name: 5-Hydroxybutyl nitrate (4c)

[0107] It is prepared from 5-bromopentanol as raw material, with a yield of 90%, as a colorless oil. 1 H NMR (300 MHz, CDCl 3 )δ4.50(t,J=6.6Hz,2H),3.70(t,J=6.4Hz,2H),1.85-1.77(m,2H),1.69-1.61(m,2H),1.57-1.50(m,2H),1.46(s,1H).

[0108]

[0109] English name: 4-hydroxybutyl nitrate(7)

[0110] Chinese name: 4-Hydroxybutyl nitrate (7)

[0111] 4-Bromobutyl acetate (5mmol, 1.0 equivalent) was dissolved in acetonitrile (40mL), and silver nitrate (12.5mmol, 2.5 equivalent) was added. The mixture was protected from light and heated to reflux for 12h. The reaction was monitored by TLC, and phosphomolybdic acid was used for color development (the nitrate compound was yellow). The acetonitrile was removed by rotary evaporation, and 100mL of saturated brine and 100mL 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 and washed three times with 100mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain intermediate 6. Intermediate 6 (4mmol, 1.0 equivalent) was dissolved in 10mL of methanol, stirred at 0°C for 15min, and 2M LiOH·H was added dropwise. 2 O aqueous solution (8mmol, 2.0 equivalents), react at 5°C for 12h. TLC monitoring (PE / EA=10:1), phosphomolybdic acid color development, after the reaction of the raw material is complete, the methanol is removed by rotary evaporation, 15mL of water is added, extracted with EA (3×15mL), washed with saturated brine (3×20mL), dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 7. The yield is 86%, brown oil.1 H NMR (300 MHz, CDCl 3 )δ4.49(t,J=6.5Hz,2H),3.69(t,J=6.2Hz,2H),1.91-1.77(m,2H),1.71-1.62(m,2H).

[0112] General method for the synthesis of compounds 1a-1f: 4a-4c, 7 or ISMN (2 mmol, 1 equivalent), EDCI·HCl (5 mmol, 2.5 equivalent), DMAP (0.2 mmol, 0.1 equivalent) and intermediate 2 (2 mmol, 1 equivalent) were dissolved in 10 mL DMF and reacted at room temperature overnight. TLC monitoring (EA / MeOH=40:1) and column chromatography purification were performed to obtain compounds 1a-1f.

[0113]

[0114] English name:

[0115] 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)

[0116] Chinese name:

[0117] 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)

[0118] Prepared from intermediates 4a and 2, yield 33%, 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=5.4Hz,1H),7.32-7.28(m,1H),7.26(s,1H),6.06(s,1H),4.74(t,J=4.5Hz ,2H),4.35(t,J=4.5Hz,2H),4.17(t,J=5.2Hz,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). 13C NMR(75MHz,DMSO-d6)δ172.30(×2),165.65,163.04,162.81,160.40,157.41,141.30,139.30,133.99,132.91,129.51,128.6 5,127.49,126.17,83.08,71.96,62.05,60.73,56.38,52.85(×2),44.02(×2),29.03,28.94,26.05,18.78.HRMS(ESI)=[M+H] + =677.19033.

[0119]

[0120] English name:

[0121] 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)

[0122] Chinese name:

[0123] 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)

[0124] Prepared from intermediate 4b and 2, yield 31%, white solid. 1 H NMR (300 MHz, DMSO-d 6 )δ11.51(s,1H),9.90(s,1H),8.23(s,1H),7.41(dd,J=7.2,2.3Hz,1H),7.32-7.24(m,2H),6.05(s,1H),4.58(t,J=6.3Hz,2H),4.17(t,J =5.7Hz,2H),4.12(t,J=6.2Hz,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=6.3Hz,2H). 13CNMR(75MHz,DMSO-d6)δ172.42,172.36,165.65,163.03,162.81,160.39,157.41,141.30,139.30,133.99,132.91,129.50,128.65 ,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.

[0125]

[0126] English name:

[0127] 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)

[0128] Chinese name:

[0129] 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)

[0130] Prepared from intermediates 7 and 2, yield 33%, 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=7.3Hz,1H),7.33-7.21(m,2H),6.06(s,1H),4.54(t,J=6.0Hz,2H),4.17(t,J= 5.8Hz,2H),4.06(t,J=5.8Hz,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). 13C NMR(75MHz,DMSO-d6)δ172.42,172.39,165.65,163.03,162.82,160.39,157.41,141.30,139.30,133.99,132.90,129.51,12 8.65,127.49,126.17,83.08,73.80,63.89,62.02,56.39,52.83(×2),44.01(×2),29.10,29.08,26.05,24.92,23.24,18.78.

[0131]

[0132]

[0133] English name:

[0134] 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)

[0135] Chinese name:

[0136] 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)

[0137] Prepared from intermediate 4c and 2, yield 31%, 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=7.2Hz,1H),7.32-7.24(m,2H),6.05(s,1H),4.50(t,J=6.5Hz,2H),4.16(t,J=5.8Hz,2H ),4.02(t,J=6.4Hz,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). 13CNMR(75MHz,DMSO-d6)δ172.40(×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.

[0138]

[0139] English name:

[0140] 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)

[0141] Chinese name:

[0142] 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)

[0143] Prepared from ISMN and intermediate 2, yield 29%, white solid. 1 H NMR (300 MHz, DMSO-d 6)δ11.50(s,1H),9.90(s,1H),8.23(s,1H),7.41(dd,J=7.3,2.3Hz,1H),7.31-7.25(m,2H) ,6.06(s,1H),5.51(td,J=5.4,2.1Hz,1H),5.10(d,J=3.1Hz,1H),4.97(t,J=5.3Hz,1H),4 .41(d,J=5.0Hz,1H),4.17(t,J=5.7Hz,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(75MHz,DMSO-d6)δ172.36,171.78,165.65,163.03,162.82,160.39,157.41,141.30,139.30,133.98,132.90,129.51,128.62,127.48,1 26.17,86.37,83.08,82.79,81.86,77.39,73.12,69.44,62.07,56.39 ,52.85(×2),44.01(×2),29.10,29.06,26.05,18.78.HRMS(ESI)=[M+H] + =761.21146.

[0144]

[0145] English name: 2-bromoethyl nitrate (9a)

[0146] Chinese name: 2-bromoethyl nitrate (9a)

[0147] 2-Bromoethanol (3 mmol) was added dropwise to 70% HNO under ice bath conditions. 3 (1.1 mL) and 95% H 2 SO 4 (2.4 mL) of the mixed solution, stirred at 0 ° C for 1 h. After the reaction was completed, 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 obtain intermediate 9a. The yield was 91%, colorless oil. 1 H NMR (300 MHz, CDCl 3)δ4.76(t,J=6.4Hz,2H),3.56(t,J=6.4Hz,2H).

[0148]

[0149] English name:

[0150] N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(piperazin-1-yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide(9)

[0151] Chinese name:

[0152] N-(2-Chloro-6-methylphenyl)-2-((2-methyl-6-(piperazin-1-yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide (9)

[0153] Compound 8 (510 mg, 1.3 mmol, 1 equivalent), piperazine (1120 mg, 13 mmol, 10 equivalents) and DIPEA (453 μL, 2.6 mmol, 2 equivalents) were dissolved in 15 mL of anhydrous 1,4-dioxane, refluxed for 4 h, and monitored by TLC. After the reaction was completed, 20 mL of water was added to the reaction solution, filtered, and the filter cake was washed with 10 mL of water and 5 mL of methanol, and vacuum dried to obtain intermediate 9. The yield was 71%, white solid. 1 H NMR(300MHz,DMSO-d6)δ9.92(s,1H),8.25(s,1H),7.40(dd,J=7.5,2.0Hz,1H),7.32-7.21(m,2H),6.0 6(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]+

[0154]

[0155] English name:

[0156] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl nitrate(1f)

[0157] Chinese name:

[0158] 2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidin-4-yl)piperazin-1-yl)ethyl nitrate (1f)

[0159] Intermediate 9 (150 mg, 0.34 mmol, 1 equivalent), 9a (172 mg, 1.01 mmol, 3 equivalents), TEA (285 μL, 2.03 mmol, 6 equivalents) were dissolved in 5 mL of anhydrous DMF, reacted at room temperature for 6 h, and monitored by TLC (EA / MeOH=60:1). After the reaction was completed, most of the DMF was removed by rotary evaporation, and purified by column chromatography to obtain 55 mg of compound 1f. The yield was 31%, and it was a white solid. 1 H NMR (300 MHz, DMSO-d 6 )δ11.51(s,1H),9.90(s,1H),8.24(s,1H),7.41(dd,J=7.2,2.3Hz,1H),7.30-7.23(m,2H),6.07(s,1H),4 .68(t,J=5.3Hz,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 )δ165.67,163.01,162.78,160.39,157.42,141.30,139.29,133.98,132.90,129.50 ,128.66,127.49,126.19,83.16,71.13,54.65,52.59(×2),43.92(×2),26.06,18.78.

[0160] Synthesis of Furazan Nitrogen Oxides:

[0161]

[0162] English name: 2-(phenylsulfonyl)acetic acid (10)

[0163] Chinese name: 2-(phenylsulfonyl)acetic acid (10)

[0164] Methyl benzenesulfonyl acetate (3 mmol, 1.0 equivalent) was dissolved in 10 mL of methanol, stirred at 0 °C for 15 min, and 2 M LiOH·H was added dropwise. 2O aqueous solution (9 mmol, 2.0 equivalents), react at 5 ° C for 12 hours. After the reaction of the raw material is completed, the pH is adjusted to 2 with 0.1 M dilute hydrochloric acid, and the methanol is removed by rotary evaporation. 15 mL of water is added, extracted with EA (3×15 mL), washed with saturated brine (3×20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 10. The yield is 89%, and the product is a white solid. 1 H NMR (300MHz, CD 3 OD): δ7.96-7.98(d,J=7.6Hz,2H),7.71-7.75(t,J=7.6Hz,1H),7.61-7.65(t,J=8.0Hz,2H),4.29(s,2H).

[0165]

[0166] English name: 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide(11)

[0167] Chinese name: 3,4-Bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11)

[0168] 10 mL of glacial acetic acid was added to the intermediate 10 (2 mmol, 1.0 equivalent), the temperature was raised to 90°C, 5 mL of fuming nitric acid was added, and the reaction temperature was further raised to 120°C. After reacting for 2 h, 50 mL of ice water was poured into the reaction solution, and a white solid precipitated. Compound 11 was obtained by filtration and washing. The yield was 34%, white solid. 1 H NMR (300 MHz, CDCl 3 )δ8.25-8.09(m,4H),7.86-7.73(m,2H),7.72-7.60(m,4H).

[0169] General method for the synthesis of intermediates 12 and 13: Compound 11 (500 mg, 1.36 mmol) was dissolved in THF, the corresponding diol (2.73 mmol) was added, and then 50% NaOH (2.73 mmol) was added. The reaction was carried out at room temperature for 3-5 h and monitored by TLC (PE / EA=2:1). After the reaction was completed, the solvent was dried and column chromatography (PE / EA=10:1) was performed to obtain the corresponding compounds 12 and 13.

[0170]

[0171] English name:

[0172] 4-(2-(2-hydroxyethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide(12)

[0173] Chinese name:

[0174] 4-(2-(2-Hydroxyethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12)

[0175] Prepared from intermediate 11 and diethylene glycol, yield 30%, white solid. 1 H NMR (300 MHz, CDCl 3 )δ8.19-8.15(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-).

[0176]

[0177] English name:

[0178] 4-(4-hydroxybutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide(13)

[0179] Chinese name:

[0180] 4-(4-Hydroxybutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13)

[0181] It is prepared from intermediate 11 and 1,4-butanediol, with a yield of 45%, as a white solid. 1 H NMR (300 MHz, CDCl 3 )δ8.19-8.15(m,2H),7.77-7.83(m,1H),7.62-7.70(m,2H),4.39-4.50(t,J =6Hz,2H),3.74-3.78(t,J=6Hz,2H),2.11-2.15(m,2H),1.97-2.02(m,2H).

[0182]

[0183] English name: 3-methyl-4-nitro-1,2,5-oxadiazole 2-oxide(15)

[0184] Chinese name: 3-Methyl-4-nitro-1,2,5-oxadiazole 2-oxide (15)

[0185] Towards 60% H 2 SO 4 To a mixed solution of 1.72 g (6 mL) and compound 14 (1.72 g, 20 mmol, 1 equivalent) in DCE (20 mL) was added NaNO in batches. 2 (4.83 g, 70 mmol, 3.5 equivalents), react at 50 ° C for 1 h, and monitor by TLC. After the reaction is completed, the organic phase is separated and 3% Na 2 CO 3 The organic phase was washed with aqueous solution until it was completely colorless, washed with saturated brine (3×20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain 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 obtain intermediate 15. 1 H NMR (300 MHz, CDCl 3 )δ2.51(s,3H).

[0186] General method for the synthesis of compounds 16 and 17: Dissolve compound 15 (300 mg, 2.06 mmol) in 10 mL THF, add the corresponding diol (4.12 mmol), then add 50% NaOH (4.12 mmol), react at room temperature for 3-5 h, monitor the reaction by TLC (PE / EA=2:1), spin dry the solvent after completion, and obtain the corresponding compound by column chromatography (PE / EA=2:1).

[0187]

[0188] English name: 4-(2-hydroxyethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide(16)

[0189] Chinese name: 4-(2-hydroxyethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide (16)

[0190] Prepared from intermediate 15 and ethylene glycol, yield 22%, white solid. 1 H NMR (500MHz, DMSO-d6) δ4.99 (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).

[0191]

[0192] English name: 4-(carboxymethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide(17)

[0193] Chinese name: 4-(Carboxymethyloxy)-3-methyl-1,2,5-oxadiazole 2-oxide (17)

[0194] Prepared from intermediate 15 and glycolic acid, yield 31%, white solid. 1 H NMR(500MHz,DMSO-d6)δ4.84(s,1H),2.01(s,3H).

[0195] General method for the synthesis of target compounds 2a-2d: Compound 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 DMF and reacted at room temperature for 12 h. TLC monitoring (EA / MeOH=40:1) and column chromatography purification were performed to obtain compounds 2a-2d.

[0196]

[0197] English name:

[0198] 4-(2-(2-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino))-2-methylpy rimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide(2a)

[0199] Chinese name:

[0200] 4-(2-(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)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (2a)

[0201] Prepared from intermediates 12 and 2, yield 59%, white solid. 1 H NMR (500 MHz, DMSO-d6 ) 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 (×2), 129.50, 128.80 (×2), 128.04, 127.49, 126.17, 110.97, 83.08, 71.28, 68.85, 68.21, 63.89, 60.23, 56.38, 52.83 (×2), 44.01 (×2), 29.06, 28.99, 26.05, 18.78. HRMS (ESI) = [M + H] + = 900.22065.

[0202]

[0203] English Name:

[0204] 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)

[0205] Chinese Name:

[0206] 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)

[0207] Prepared from intermediates 13 and 2, 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=8.5,1.3Hz,2H),7.92-7.87(m,1H ),7.77-7.72(m,2H),7.39(dd,J=7.7,1.8Hz,1H),7.30-7.23(m,2H),6.05(s,1H),4.41( t,J=6.2Hz,2H),4.15(t,J=5.8Hz,2H),4.09(t,J=6.4Hz,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). 13 CNMR(75MHz,DMSO-d6)δ172.42,172.40,165.64,163.03,162.80,160.40,159.30, 157.40,141.30,139.30,137.65,136.61,133.98,132.91,130.50(×2),129.50,12 8.80(×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.

[0208]

[0209] English name:

[0210] 4-(2-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyri midin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide(2c)

[0211] Chinese name:

[0212] 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)

[0213] Prepared from intermediates 16 and 2, yield 59%, 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=7.8,1.8Hz,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=5.8Hz,2H),3.53-3.49(m,4H),2.61-2.57(m,6H),2.50(m,4H),2.40(s,3H),2.24(s,3H),2.04(s,3H).HRMS(ESI)=[M+H] + =752.19783.

[0214]

[0215] English name:

[0216] 4-(2-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyrimidi n-4-yl)piperazin-1-yl)ethoxy)-2-oxoethoxy)-3-methyl-1,2,5-oxadiazole 2-oxide(2d)

[0217] Chinese name:

[0218] 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)

[0219] Prepared from intermediates 17 and 1, yield 59%, 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=7.3Hz,1H),7.30-7.23(m,2H),6.05(s,1H),5.11(s,2H),4.30(t,J=5 .6Hz,2H),3.54-3.46(m,4H),2.63(t,J=5.4Hz,2H),2.50(m,4H),2.41(s,3H),2.24(s,3H),2.10(s,3H).HRMS(ESI)=[M+H] + =666.16195.

[0220] General method for the synthesis of compounds 2e and 2f: Compounds 2a and 2b (0.5 mmol, 1.0 equivalent) were suspended in 10 mL of ether, and methanesulfonic acid (3 mmol, 6.0 equivalent) was added. The reaction was allowed to react at room temperature for 3 h, and monitored by TLC (EA / MeOH=40:1). After the reaction of the raw materials was complete, the solvent was removed by rotary evaporation. 2 mL of isopropanol was added, and the mixture was heated until it was just clear. Then, 10 mL of ether was slowly added dropwise to the reaction solution, and a large amount of white solid was gradually precipitated. The mixture was centrifuged, and the supernatant was removed by suction. The solid was dissolved in 2 mL of isopropanol again. The operation was repeated 3 times, and the white solid obtained was dried under vacuum to obtain compounds 2e and 2f.

[0221]

[0222] English name:

[0223] 4-(2-(2-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino))-2-methylpy rimidin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide tri mesylate(2e)

[0224] Chinese name:

[0225] 4-(2-(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)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide trimesylate (2e)

[0226] Prepared from compound 2a, yield 65%, white solid. 1 H NMR (500 MHz, DMSO-d 6 )δ9.92(s,1H),8.26(s,1H),8.04(d,J=7.7Hz,2H),7.92(t,J=7.4Hz,1H),7.77(t,J=7.8Hz,2H),7 .42(d,J=8.7Hz,1H),7.32-7.27(m,2H),6.21(s,1H),4.55(t,J=4.3Hz,2H),4.41(t,J=5.1Hz,4H), 4.20(t,J=4.7Hz,2H),3.83(t,J=4.1Hz,2H),3.73(t,J=4.7Hz,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).

[0227]

[0228] English name:

[0229] 4-(4-((4-(2-(4-(6-((5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)amino)-2-methylpyri midin-4-yl)piperazin-1-yl)ethoxy)-4-oxobutanoyl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole2-oxide tri mesylate(2f)

[0230] Chinese name:

[0231] 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 trimesylate (2f)

[0232] Prepared from compound 2b, yield 71%, white solid. 1 H NMR (500 MHz, DMSO-d 6 )δ11.64(s,1H),9.89(s,1H),8.24(s,1H),8.01(d,J=7.4Hz,2H),7.90(t,J=7.5Hz,1H),7.75(t ,J=7.8Hz,2H),7.40(d,J=7.6Hz,1H),7.31-7.24(m,2H),6.18(s,1H),4.43(t,J=5.7Hz,2H),4.1 1(t,J=6.4Hz,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).

[0233] Since the target compound enters the anterior chamber after eye drops and regulates TM cells by releasing NO, we chose HTM cells as a cell model to test the NO release of the compound in HTM cells. According to the design concept of the compound, after entering the TM cells, the compound can release NO through enzymatic or non-enzymatic metabolism. We used the NO fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FMDA) to detect the release of NO. Each target compound was incubated with HTM cells at a concentration of 5μM at 37°C for 12h, and then the DAF-FM DA fluorescent probe was added and incubated for 30min. The fluorescence intensity of the cells was detected by flow cytometry, and the mean fluorescence intensity (MFI) was used as a statistical indicator and normalized to the control group. The results showed that most of the target compounds were able to increase the NO level in HTM cells ( Figure 1 A), among which compounds 1f, 2a and 2e had the most significant effects, which was consistent with our original design idea.

[0234] cGMP is an important downstream molecule for NO to exert its physiological activity, and the activity of NO in reducing intraocular pressure also requires the participation of cGMP. Therefore, we explored whether compound treatment would promote cGMP production in HTM cells. After incubating HTM cells with each target compound at a concentration of 5 μM for 24 hours, the cell supernatant was collected and the cGMP concentration in the cell supernatant was determined by enzyme-linked immunosorbent assay. The results showed that most of the target compounds could promote cGMP production in HTM cells and increase the concentration of cGMP. Among them, compound 2e had the most obvious activity in increasing cGMP levels ( Figure 1 B). Considering the significant activity of compound 2e in releasing NO and upregulating cGMP levels, we selected compound 2e for subsequent in vivo pharmacodynamic experiments.

[0235] In order to evaluate whether our target compound can also play the role of clearing senescent cells in vivo, we stained the retina of mice with chronic ocular hypertension induced by magnetic microbeads with senescence-associated beta-galactosidase (SA-β-gal) and counted the number of senescence-positive cells per unit area. Starting from the second day after the mouse model was established, eye drops were administered three times a day. After 10 days of continuous eye drops, the mouse retinas were collected and retinal flat slides were prepared, followed by SA-β-gal staining and counting the number of senescence-positive cells per unit area. ( Figure 2A, B). The results showed that the number of senescence-positive cells per unit area of ​​retinal flat mounts in the vehicle control group was significantly increased compared with the normal group (***p<0.001), and after eye drops of dasatinib mesylate or 2e, the number of senescence-positive cells per unit area of ​​both groups was significantly decreased compared with the vehicle group (***p<0.001). Similar to the results of retinal sections, there was no significant difference between the Dasatinib Mesylate group and the 2e group ( Figure 2 B). The above results show that both 2e and dasatinib mesylate can play a significant role in clearing senescent cells in vivo, and there is no significant difference in the effects of the two. The above data show that the number of senescent cells in mice with chronic ocular hypertension induced by magnetic microbeads increased significantly, and the administration of dasatinib mesylate and 2e by eye drops can significantly reduce the number of senescent cells per unit area.

[0236] Next, we investigated the effects of target compound 2e and dasatinib mesylate on intraocular pressure in a mouse model of chronic ocular hypertension induced by magnetic microbeads. We selected time points of 1, 2, 4, and 8 h after eye drop administration for intraocular pressure measurement and statistics ( Figure 3 The experimental results showed that the administration of 2e (10mM) to the model mice by eye drops could significantly reduce the intraocular pressure of the mice at 2h and 4h after administration, and the effect of reducing intraocular pressure in the 2e group mice was the most obvious at 4h after eye drops (3.6mmHg, **p<0.01, Figure 3 ), and then at 8h after administration, there was no significant difference in intraocular pressure level compared with the Vehicle group. However, no significant reduction in intraocular pressure was detected in the Vehicle group and the Dasatinib Mesylate group at each time point after administration. The above results show that the NO donor dasatinib derivative 2e exhibits a significant effect of lowering intraocular pressure compared with the parent compound.

[0237] The damage and death of retinal ganglion cells caused by glaucoma is an important cause of visual loss in patients. Therefore, we used the retinal ganglion cell-specific antibody RBPMS for immunofluorescence staining to evaluate whether 2e can play a protective role on retinal ganglion cells ( Figure 4 A). Figure 4As shown in B, compared with the Normal group, the number of retinal ganglion cells in mice with chronic ocular hypertension induced by magnetic microbeads decreased significantly (****p<0.0001), and eye drops of dasatinib mesylate and 2e can both have a protective effect on retinal ganglion cells, and compared with dasatinib mesylate, 2e has a more significant protective effect on retinal ganglion cells (*p<0.05). The above results show that thanks to the synergistic effect of 2e in clearing senescent cells and reducing intraocular pressure, 2e can play a significant protective role on retinal ganglion cells in mice with chronic ocular hypertension induced by magnetic microbeads, and its activity is better than that of the parent compound.

[0238] In glaucoma patients and multiple glaucoma mouse models, visual function is significantly impaired, which can be manifested as abnormal visual electrophysiology, that is, in the visual evoked potential (VEP), the amplitude of the N1 wave is reduced and the peak time is delayed. Therefore, we hope to test whether 2e can protect the visual function of mice by removing senescent cells and reducing the intraocular pressure of mice through the detection of VEP. The representative VEP curves of each group of mice are shown in the figure ( Figure 5 A), the amplitude of N1 wave in mice with chronic ocular hypertension induced by magnetic microbeads was significantly decreased compared with the Normal group, and the peak time was significantly delayed. These data indicate that chronic ocular hypertension induced by magnetic microbeads has caused significant damage to the visual function of mice. The administration of 2e by eye drops can significantly increase the amplitude of N1 wave in model mice. Although the administration of dasatinib mesylate has a certain trend of increasing the amplitude of N1 wave in mice, there is no significant difference compared with the Vehicle group ( Figure 5 B). In addition, both 2e and dasatinib mesylate significantly improved the lag in the peak time of the N1 wave, but there was no significant difference between the two ( Figure 5 C). Therefore, eye administration of the NO-donating dasatinib derivative 2e significantly improved visual function.

[0239] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A NO donor dasatinib derivative, characterized in that: The derivative is one of compounds 1a-1f and compounds 2a-2f; the structural formulas of compounds 1a-1f and compounds 2a-2f are as follows:

2. A method for preparing the NO donor dasatinib derivative according to claim 1, characterized in that: The preparation methods of the compounds 1a-1f are respectively as follows: Dasatinib reacts with succinic anhydride to obtain intermediate 2; 2-bromoethanol reacts with silver nitrate to obtain intermediate 4a, and intermediate 4a reacts with intermediate 2 to obtain compound 1a through esterification reaction; 3-bromopropanol reacts with silver nitrate to obtain intermediate 4b, and intermediate 4b reacts with intermediate 2 to obtain compound 1b through esterification reaction; 5-bromopentanol reacts with silver nitrate to obtain intermediate 4c, and intermediate 4c reacts with intermediate 2 to obtain compound 1c through esterification reaction; 4-bromobutyl acetate reacts with silver nitrate to produce intermediate 6, and intermediate 6 is deacetylating under alkaline conditions to produce intermediate 7, and intermediate 7 is reacted with intermediate 2 through esterification to produce compound 1d; ISMN reacted with intermediate 2 to give compound 1e; Intermediate 8 reacts with piperazine under alkaline conditions to undergo nucleophilic substitution reaction to obtain intermediate 9, and intermediate 9 further reacts with 2-bromoethyl nitrate under alkaline conditions to undergo nucleophilic substitution reaction to obtain compound 1f; The structural formula of the intermediate 2 is as follows: The structural formula of the intermediates 4a-4c is as follows: 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 methods of the compounds 2a-2f are respectively as follows: Add glacial acetic acid to the intermediate 10, raise the temperature to 90°C, add fuming nitric acid, and then further raise the reaction temperature to 120°C to obtain the intermediate 11; Compound 14 reacts with sodium nitrite under acidic conditions to generate intermediate 15; The intermediate 11 undergoes a nucleophilic substitution reaction with diethylene glycol to obtain the intermediate 12; The intermediate 11 undergoes a nucleophilic substitution reaction with 1,4-butanediol to obtain the intermediate 13; The intermediate 15 undergoes a nucleophilic substitution reaction with ethylene glycol to obtain the intermediate 16; The intermediate 15 undergoes a nucleophilic substitution reaction with glycolic acid to obtain the intermediate 17; The intermediate 12 undergoes an esterification reaction with the intermediate 2 to obtain compound 2a; The intermediate 13 undergoes an esterification reaction with the intermediate 2 to obtain the target 2b; The intermediate 16 undergoes an esterification reaction with the intermediate 2 to obtain the target 2c; The intermediate 17 undergoes an esterification reaction with dasatinib to obtain compound 2d; The compound 2a reacts with methanesulfonic acid to form a compound 2e; The compound 2b reacts with methanesulfonic acid to form 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:

3. The method for preparing the NO donor dasatinib derivative according to claim 2, characterized in that: The molar ratio of dasatinib to succinic anhydride is 4:4.4; the molar ratio of 2-bromoethanol to silver nitrate is 5:12.5, and the molar ratio of intermediate 4a to intermediate 2 is 1:1; the molar ratio of 3-bromopropanol to silver nitrate is 5:12.5, and the molar ratio of intermediate 4b to intermediate 2 is 1:1; the molar ratio of 5-bromopentanol to silver nitrate is 5:12.5, and the molar ratio of intermediate 4c to intermediate 2 is 1:1; the molar ratio of 4-bromobutyl acetate to silver nitrate is 5:12.5, and the molar ratio of intermediate 7 to intermediate 2 is 1:1; the molar ratio of ISMN to intermediate 2 is 1:1; the molar ratio of intermediate 8 to piperazine is 1:10; the molar ratio of 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. Use of the NO donor dasatinib derivative according to claim 1 in the preparation of a drug for removing retinal senescent cells, reducing intraocular pressure or / and protecting the optic nerve.

5. Use of the NO donor dasatinib derivative according to claim 1 in the preparation of a drug for treating glaucoma.

6. Use of the NO donor dasatinib derivative according to claim 1 in the preparation of a medicament for treating age-related retinopathy.

7. Use of the NO donor dasatinib derivative according to claim 1 in the preparation of a drug for improving visual function.

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