Endoperoxide-based anticoagulants and their applications

Abstract

Endoperoxide-based anticoagulants and their applications fall within the biopharmaceutical field. The designed anticoagulants are composed of naphthalene endoperoxide molecules and water-soluble ionic groups (quaternary ammonium salts and sulfonates). The introduction of the ionic groups increases the plasma solubility of the singlet oxygen anticoagulants. The endoperoxide structure acts as a singlet oxygen carrier, allowing thermal cycling to reverse the release of singlet oxygen. Both designed ionic naphthalene endoperoxide compounds can reduce fibrinogen content or affect the conversion of fibrinogen to fibrin, demonstrating significant effects in inhibiting coagulation and enhancing fibrinolysis. Therefore, the two ionic naphthalene singlet oxygen anticoagulants designed have great application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an anticoagulant based on endoperoxide and its application. Background Art

[0002] Traditional antithrombotic drugs have been proven effective in treating thrombotic diseases, but their use is currently limited by various drug and patient factors. Most new antithrombotic drugs today are also limited in their application and promotion due to side effects such as coagulopathy and severe bleeding. There is great interest in developing antithrombotic drugs with high target selectivity, physiological thrombolysis, biocompatibility, and minimal bleeding risk. In the human body, singlet oxygen produced by PMNs has a significant effect on inhibiting blood coagulation and enhancing fibrinolysis. Therefore, the application of singlet oxygen in the study of antithrombotic drugs is of great significance. Summary of the Invention

[0003] The present invention aims to develop an anticoagulant based on endoperoxides. The anticoagulant based on endoperoxides is developed by connecting a naphthalene-based singlet oxygen carrier endoperoxide compound with a water-soluble sulfonate or quaternary ammonium salt group. The endoperoxide molecule, as a singlet oxygen carrier, can release singlet oxygen. The released singlet oxygen has the significant effect of inhibiting blood coagulation and enhancing fibrinolysis. In addition, the introduction of ionic sulfonate and quaternary ammonium salt groups greatly improves the water solubility of the singlet oxygen carrier, thereby having broad application prospects.

[0004] The technical solution of the present invention is an endoperoxide-based anticoagulant, wherein the singlet oxygen anticoagulant has the following structure:

[0005]

[0006] in, Each is independent or

[0007]

[0008] R 1 -R 3 , R 5 -R 7 , R 9 -R 11 each independently represents hydrogen, hydroxy, trimethylsilyl, amino, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkoxy having 1 to 2 carbon atoms, alkylamino having 1 to 6 carbon atoms, alkoxyalkyl having 2 to 6 carbon atoms, alkyl having 1 to 6 carbon atoms, trifluoromethyl, halogen, alkoxycarbonyl having 2 to 6 carbon atoms, aryl having 6 to 20 carbon atoms, heterocycloalkyl having 3 to 10 carbon atoms, or

[0009] Wherein, x is an integer from 1 to 2000;

[0010] R 4 , R 8 , R 12 Each is independent or wherein m is an integer of 0-10, the sum of n1 and n2 is independently an integer of 0-6, the sum of n3 and n4 is independently an integer of 0-8, the sum of n5 and n6 is an integer of 0-7, the sum of n7 and n8 is an integer of 0-10, and the sum of n9 and n1 is independently an integer of 0-10. 10 The sum of is an integer from 0 to 4, and n is an integer from 0 to 6.

[0011] Y + is a monovalent cation, Z - It is a monovalent anion.

[0012] Preferably, R 1 -R 3 , R 5 -R 7 , R 9 -R 11 Each independently represents hydrogen, hydroxyl, trimethylsilyl, amino, alkyl having 1 to 6 carbon atoms, aryl having 6 to 20 carbon atoms, or

[0013] Wherein, x is an integer from 5 to 200;

[0014] R 4 , R 8 , R 12 Each is independent or wherein the sum of n1 and n2 is independently an integer of 0-6, m is an integer of 0-10, and n is an integer of 0-6.

[0015] Y + for Na + , K + or NH4 + ; Z - PF 6- 、BF4 - 、Cl - Br - , I - 、NO3 - 、SO4 2- 、ClO4 - 、CH3COO - 、CH3SO3 - or CF3SO3- .

[0016] The use of the endoperoxide-based anticoagulant as a singlet oxygen carrier to deliver singlet oxygen.

[0017] The endoperoxide-based anticoagulant is used for preparing drugs for anticoagulation and enhancing fibrinolysis.

[0018] The present invention has the beneficial effects of introducing naphthalene endoperoxide molecules and ionic sulfonates or quaternary ammonium salts into a singlet oxygen transport system in an endoperoxide-based anticoagulant. The water solubility of the endoperoxide molecules is improved by the sulfonate group and the quaternary ammonium salt group, so that the endoperoxide molecules can be dissolved in plasma. Singlet oxygen is released at human body temperature to regulate the body's coagulation and fibrinolysis mechanisms. The release rate of singlet oxygen can be regulated by designing different endoperoxide structures. The endoperoxide system introduced with the ionic sulfonate group and the quaternary ammonium salt group exhibits good water solubility and also shows significant activity in in vitro anticoagulation tests. The ionic naphthalene singlet oxygen carriers involved in the present invention have good biocompatibility and therapeutic activity and have broad application prospects.

[0019] A singlet oxygen anticoagulant containing a sulfonate group is obtained by reflux hydrolysis of a pentafluorophenol sulfonate compound (Compound A) in a 5% w / w NaOH ethanol solution. A singlet oxygen anticoagulant containing a quaternary ammonium group is obtained by reacting an amino compound (Compound B) with methyl iodide and then with sodium hexafluorophosphate. The corresponding pentafluorophenol sulfonate compounds and amino compounds can be obtained using conventional chemical synthesis methods. The specific conversion process is as follows:

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Graph showing the change in singlet oxygen release intensity of endoperoxy compound 10 over time.

[0022] Figure 2 Graph showing the change in singlet oxygen release intensity of endoperoxy compound 11 over time.

[0023] Figure 3 This is a graph showing the change in singlet oxygen release intensity over time in the blank control group.

[0024] Figure 4 This is an analysis of the singlet oxygen release of endoperoxygen compounds 10, 11 and the control group.

[0025] Figure 5 is the endoperoxide 10 in CD3OD at 37℃ 1 Time evolution of H NMR spectra.

[0026] Figure 6is the endoperoxide 11 in CD3OD at 37℃ 1 Time evolution of H NMR spectra.

[0027] Figure 7 This is a graph showing the half-life calculation of endoperoxide 10.

[0028] Figure 8 This is a graph showing the half-life calculation of endoperoxide 11.

[0029] Figure 9 This is the in vitro anticoagulation data of endoperoxygen compound 10 and compound 8 as a control.

[0030] Figure 10 This is the in vitro anticoagulation data of endoperoxy compound 11 and compound 9 as a control. DETAILED DESCRIPTION

[0031] The present invention is illustrated but not limited by the following examples in which all parts and percentages are by weight unless otherwise indicated.

[0032] The specific embodiments of the present invention are described in detail below in conjunction with the technical solutions:

[0033] Example 1

[0034]

[0035] Step a: Preparation of compound 2

[0036] A mixture of methoxy(cyclooctadiene)iridium dimer (331 mg, 0.5 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (268 mg, 1.0 mmol), bis-pinacolatodiborane (2.79 g, 11.0 mmol) and 1,4-dimethylnaphthalene (1.54 mL, 10.0 mmol) was dissolved in cyclohexane (38 mL) and heated at 60 ° C. for about 20 hours. After the reaction, the reaction mixture was cooled to room temperature, and the organic phase was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: n-hexane), and the eluent was removed by rotary evaporation to obtain 2.62 g of white crystals. 1 H NMR(400MHz,Chloroform-d)δ8.48–8.39(m,1H),7.86(dd,J=8.4,0.7Hz,1H),7.80 (dd,J=8.4,1.2Hz,1H),7.15–7.02(m,2H),2.61(s,3H),2.53(s,3H),1.27(s,12H).

[0037] Step b: Preparation of compound 3

[0038] Compound 2 (500 mg, 1.77 mmol) and sodium periodate (1.136 g, 5.31 mmol) were stirred in 12 mL THF: H2O (4: 1, v / v) for 30 minutes, at which time an aqueous HCl solution (1 N, 1240 μL) was added to the suspension. The reaction mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction mixture was diluted with water and extracted with EtOAc. The combined organic phases were washed with water and then washed with brine. The organic phase was dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane to methanol volume ratio 98: 2) to obtain 326 mg of a white solid compound. 1 HNMR (400MHz, DMSO-d6) δ8.60(t,J=1.0Hz,1H),8.26(s,2H),8.03–7.95(m,2H),7.29(pd,J=6.3,5.6,1.7Hz,2H),2.71(s,3H),2.66(s,3H).

[0039] Step c: Preparation of compound 4

[0040] A mixture of compound 3 (326 mg, 1.63 mmol) and bismuth (III) nitrate pentahydrate (1.58 g, 3.26 mmol) was dissolved in toluene (38 mL) and heated at 80°C for 4 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and then filtered through filter paper. The residue was washed with ethyl acetate and then with dichloromethane. The organic phase was concentrated in vacuo, and the crude product was purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate volume ratio 99:1) to obtain 180 mg of the compound as a yellow solid. 1 HNMR (400MHz, Chloroform-d) δ8.90(d,J=2.3Hz,1H),8.20(dd,J=9.3,2.3Hz,1H),8.05(d,J=9.2Hz,1H),7.36–7.26(m,2H),2.68(s,3H),2.63(s,3H).

[0041] Step d: Preparation of compound 5

[0042] A mixture of compound 4 (302 mg, 1.50 mmol) and stannous chloride dihydrate (1.69 g, 7.50 mmol) was dissolved in anhydrous ethanol (3.5 mL) and heated at 70 ° C under an argon atmosphere for 30 min. After the reaction, the reaction solution was cooled and then poured into ice water. The pH was adjusted to slightly alkaline (pH 8) by adding 5% sodium hydroxide aqueous solution. The resulting alkaline mixture was stirred for one hour. The aqueous mixture was extracted three times with ethyl acetate, and the organic phase was thoroughly washed with brine and dried over sodium sulfate. After evaporation of the solvent, 230 mg of white slightly red needle-shaped crystals were obtained. 1 HNMR(400MHz,Chloroform-d)δ7.76(d,J=8.9Hz,1H),7.09–7.01(m,2H),6.90(ddd,J=6.7,5.9,1.7Hz,2H),2.52(s,3H),2.48(s,3H). 13 C NMR(101MHz,Chloroform-d)δ143.70,134.15,132.26,130.19,126.99,126.74,126.12,123.08,117.25,106.33,19.48,19.32.HRMS(ESI,m / z):[M+H]+calcd for target,172.1121; fond 172.1124.

[0043] Step e: Preparation of Compound 6

[0044] In a 50 mL round-bottom flask, compound 5 (103 mg, 0.60 mmol) was dissolved in a mixture of anhydrous ethanol (3.0 mL) and aqueous HBF4 (48%, 0.15 mL). t-BuONO (0.16 mL) was then added dropwise at 0°C. After stirring at room temperature for one hour, diethyl ether (10 mL) was added to precipitate the diazo compound. The precipitated dark green solid was filtered and washed with diethyl ether (3 x 10 mL) to yield 130 mg of the product. 1 HNMR(400MHz,DMSO-d6)δ9.72(d,J=2.1Hz,1H),8.52(d,J=9.3Hz,1H),8.49–8.4 2(m,1H),7.80(d,J=7.3Hz,1H),7.68(d,J=7.3Hz,1H),2.73(s,3H),2.71(s,3H).

[0045] Step f: Preparation of Compound 7

[0046] Compound 6 (405 mg, 1.5 mmol), 1,4-diazabicyclo[2.2.2]octane bis(sulfur dioxide) adduct (480 mg, 2.0 mmol), cuprous iodide (38 mg, 0.2 mmol), and pentafluorophenol (185 mg, 1.0 mmol) were dissolved in acetonitrile (10 mL), and the mixture was stirred at 85°C for 12 hours. After the reaction, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (25 mL), filtered through a silica plug, and washed with ethyl acetate (40 mL). The filtrate was then dried over anhydrous magnesium sulfate and concentrated under vacuum. The crude product was purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate volume ratio 98:2) to obtain compound 7 (140 mg). 1 HNMR(400MHz,Chloroform-d)δ8.69(d,J=1.9Hz,1H),8.22(d,J=9.0Hz,1H),7.98 (dd,J=9.0,2.0Hz,1H),7.46(d,J=7.1Hz,1H),7.40(d,J=7.2Hz,1H),2.72(s,6H). 19 F NMR

[0047] (377MHz, Chloroform-d)δ-150.35,-150.40,-155.40,-155.46,-155.52,-161.03,-161.08(d,J=4.4Hz),-161.14.

[0048] Step g: Preparation of compound 8

[0049] Pentafluorophenylsulfonate was dissolved in 5% w / w sodium hydroxide solution in ethanol and heated under reflux for 30 minutes. After the reaction, 5% aqueous HCl was added to adjust the pH to between 4 and 6. The aqueous reaction mixture was extracted three times with ethyl acetate. The organic phase was then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and diethyl ether was added to precipitate 1,4-dimethylnaphthalenesulfonate. The pure product was obtained after filtration and washing with diethyl ether. 1 HNMR (400MHz, Methanol-d4) δ8.55(d,J=1.8Hz,1H),8.08(d,J=8.8Hz,1H),7.93(dd,J=8.8,1.9Hz,1H),7.28(s,2H),2.68(s,3H),2.65(s,3H). 13C NMR(101MHz,Methanol-d4)δ141.28,133.22,133.12,132.11,131.75,127.43,126.84,124.67,122.16,121.99,17.95,17.89.HRMS(ESI,m / z):[M-Na]-calcd for target,235.0434; fond235.0435.

[0050] Step h: Preparation of Compound 9

[0051] To a mixture of compound 5 (86 mg, 0.5 mmol) and potassium carbonate (345 mg, 2.5 mmol) in acetonitrile (5 mL), iodomethane (355 mg, 2.5 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was then filtered, and the filtrate was concentrated in vacuo. The residue was washed several times with ether. The iodine salt was dispersed in 3 mL of acetone, and a saturated NH4PF6 aqueous solution was then added until a clear solution was obtained. After the solution was further stirred for 4 hours, the solvent was evaporated under reduced pressure, and a white solid precipitated out, filtered, washed with water, and then dried to obtain 116 mg of compound as a white solid. 1 HNMR (400MHz, DMSO-d6) δ8.35(d,J=2.8Hz,1H),8.27(d,J=9.4Hz,1H),8.15(dd,J=9.4,2.8Hz,1H),7.43(s,2H),3.74(s,9H),2.72(s,3H),2.67(s,3H). 13 C NMR(101MHz,DMSO-d6)δ144.53,133.82,132.66,132.27,132.02,128.83,128.71,127.84,117.86,116.53,56.88,19.34,19.25.HRMS(ESI,m / z):M+calcd for target,214.1590; fond 214.1594.

[0052] Step i: Preparation of compound 10

[0053] Compound 8 (20.3 mg, 0.07 mmol) was dissolved in deuterated methanol. Microparticles of methylene blue were added to give the solution a distinct blue color. The reaction mixture was cooled to 0°C in an ice bath. Red light (18 W, 630 nm) was irradiated while oxygen was passed through. After 6 hours of reaction, the methylene blue was removed by passing through 200-mesh activated carbon, and the solvent was evaporated under reduced pressure to yield compound 10. 1H NMR (400MHz, Methanol-d4) δ7.79(d,J=1.6Hz,1H),7.76(dd,J=7.8,1.7Hz,1H),7.41(d,J=7.7Hz,1H),6.76–6.68(m,2H),1.84(s,3H),1.83(s,3H). 13 C NMR(101MHz,Methanol-d4)δ143.51,143.27,141.47,139.10,138.83,124.07,119.61,117.46,78.49,78.39,14.89,14.82.HRMS(ESI,m / z):[M-Na]-calcd for target,267.0332; fond 267.0326.

[0054] Step j: Preparation of compound 11

[0055] Compound 9 (21.6 mg, 0.06 mmol) was dissolved in deuterated methanol. Microparticles of methylene blue were added to give the solution a distinct blue color. The reaction mixture was cooled to 0°C in an ice bath. Red light (18 W, 630 nm) was irradiated while oxygen was passed through. After 6 hours of reaction, the methylene blue was removed by passing through 200-mesh activated carbon, and the solvent was evaporated under reduced pressure to yield compound 11. 1 H NMR (400MHz, Methanol-d4) δ7.86 (d, J=2.7Hz, 1H), 7.81 (dd, J=8.4, 2.8Hz, 1H), 7. 59(d,J=8.4Hz,1H),6.76(d,J=2.0Hz,2H),3.68(s,9H),1.91(s,3H),1.86(s,3H). 13 C NMR(101MHz,DMSO-d6)δ146.25,143.49,143.23,139.74,139.48,121.84,119.17,113.51,78.86,78.34,57.04,16.30,16.13.HRMS(ESI,m / z):M+calcd for target,246.1488; fond 246.1493.

[0056] Example 2

[0057] In vitro singlet oxygen release experiment of endoperoxides. Using SOSG as a singlet oxygen capture reagent, endoperoxides (0.5 mM) and SOSG (1.25 μM) were mixed and the release of singlet oxygen was detected using an excitation light of 504 nm. Figure 1 、 23 and 4 respectively show the singlet oxygen release of endoperoxy compounds 10, 11 and the blank control group. The horizontal axis is the wavelength and the vertical axis is the change in the singlet oxygen release intensity. Figure 4 This is an analysis diagram of singlet oxygen release. The results show that endoperoxides can release singlet oxygen rapidly.

[0058] Example 3

[0059] Experiment on the rate of ring reduction reaction of endoperoxides at 37℃. Figure 5 、 6 As shown, the conversion of endoperoxy compounds 10 and 11 to compounds 8 and 9 is respectively 1 H NMR changes, using deuterated methanol as the solvent, were used to calculate the half-life based on the time-dependent changes in the endoperoxy compound content. Based on the appearance / disappearance of the normalized integrated values ​​of selected peaks, compound 10 was observed to have a half-life of 2.0 hours at 37°C, and compound 11 had a half-life of 3.3 hours at 37°C. The rate constant and half-life were calculated based on the first-order reaction rate equation. The equation is as follows:

[0060] ln[A]=ln[A0]-kt

[0061] t 1 / 2 =0.693 / k

[0062] In the equation, A is the percentage of endoperoxides in the total substance in the system at time t, A0 is the initial content of endoperoxides, k is the reaction rate constant, and t 1 / 2 is the half-life. The equation obtained based on the test results is as follows Figure 7 、 8 shown.

[0063] Example 4

[0064] The in vitro anticoagulation test of endoperoxygen compound 10 and endoperoxygen compound 11 was carried out at 37°C using sterile anticoagulated sheep blood (sodium citrate: blood, 1 / 9, v / v). In order to accurately separate the effects of singlet oxygen on the coagulation parameters tested, compound 8 and compound 9 were used as controls for endoperoxygen compound 10 and endoperoxygen compound 11, respectively, and the compounds were incubated with sheep plasma for 30 minutes. Then, the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were tested using a blood coagulation analyzer (PUN-2048B) using the corresponding assay kits. Each test was repeated three times (n=3), and the results are shown as mean ± standard deviation (SD). The coagulation test results are shown in Figure 2. Figure 9 、 10As shown, the abscissa represents the final concentration of the tested compounds (0-10 mM), and the ordinate represents the test results of the corresponding coagulation parameters. The test results show that both endoperoxides exhibited concentration-dependent prolongations of TT and FIB times and decreased FIB concentrations compared to their control compounds. This indicates that the two designed endoperoxides reduce normal fibrinogen content or affect the conversion of fibrinogen to fibrin, demonstrating significant effects in inhibiting coagulation and enhancing fibrinolysis. Therefore, the two singlet oxygen anticoagulants we designed have great potential for application.

Claims

1. An anticoagulant based on endoperoxides, characterized in that The anticoagulant has the following structure: Y + for Na + , K + or NH4 + ; Z - PF6 - 、BF4 - 、Cl - Br - , I - 、NO3 - 、ClO4 - 、CH3COO - 、CH3SO3 - or CF3SO3 - .

2. The endoperoxide-based anticoagulant according to claim 1, characterized in that The anticoagulant has the following structure:

3. Use of the endoperoxide-based anticoagulant according to any one of claims 1 to 2 in the preparation of a drug for delivering singlet oxygen as a singlet oxygen carrier.

4. Use of the endoperoxide-based anticoagulant according to any one of claims 1 to 2 in the preparation of a medicament for enhancing coagulation inhibition and activating fibrinolytic activity.

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