NO donor compound, preparation method thereof, pharmaceutical composition comprising same, and use thereof
12275686 ยท 2025-04-15
Assignee
Inventors
Cpc classification
A61K31/196
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
International classification
A61K31/196
HUMAN NECESSITIES
Abstract
Disclosed is NO donor compound and preparation method, pharmaceutical composition, and use. The compound has a structure represented by formula The NO donor compound and the pharmaceutical composition thereof, as hypoxia-activated NO donors, may be prepared as drugs for treating myocardial hypoxic damage disease. The prepared drugs can exert efficacy at both the cellular level and the animal level, and the synthesis method of the compound is simple and easy to operate. ##STR00001##
Claims
1. A NO donor compound, wherein a structure of the NO donor compound is represented by a formula I: ##STR00016## wherein: when R1 is nitro group, R2 is selected from the group consisting of hydrogen, halogen, and C.sub.1-C.sub.4 alkyl, R3 is selected from the group consisting of hydrogen, halogen, and C.sub.1-C.sub.4 alkyl; or when R2 is selected from the group consisting of nitro group, R1 is hydrogen, halogen, and C.sub.1-C.sub.4 alkyl, and R3 is selected from the group consisting of hydrogen, halogen, and C.sub.1-C.sub.4 alkyl.
2. The NO donor compound of claim 1, wherein in the formula I, R1 is nitro group, R2 is selected from the group consisting of hydrogen, halogen, and methyl, and R3 is hydrogen.
3. The NO donor compound of claim 1, wherein the NO donor compound is selected from the group consisting of following compounds I1-I4: ##STR00017##
4. A method for preparing the NO donor compound of claim 1, comprising: obtaining the NO donor compound of formula I by acylation reaction between compound 1 and compound 2 as follows: ##STR00018##
5. A pharmaceutical composition, comprising the NO donor compound of claim 1 and a pharmaceutically acceptable carrier.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
DETAILED DESCRIPTION
(5) The present disclosure is further illustrated below in conjunction with examples.
Example 1: Preparation of N-methyl-N-nitroso-p-phenol (Compound 1)
(6) ##STR00005##
(7) 4-Hydroxy (N-methyl) aniline hemisulfate (1.722 g, 10.0 mmol) was added to 50 mL of acetic acid under the dirk condition and dissolved to obtain a mix solution by ultrasound. Sodium nitrite solution, which was obtained by dissolving sodium nitrite (1.380 g, 20.0 mmol) in 10 ml of water, was added dropwise to the mixed solution in an ice-water bath for reaction, and the reaction was continued for 3 h in the ice-water bath after the addition was complete. After the reaction was completed, the reaction solution was transferred to a beaker, 50 mL of water was added into the beaker, the pH of the solution in the beaker was adjusted to 7 with saturated sodium bicarbonate solution, then the solution was extracted with ethyl acetate to obtain organic phases, and the organic phases were combined, dried with anhydrous sodium sulfate, and spin dried to obtain 1.46 g of the product with a yield of 96%.
(8) .sup.1H NMR (600 MHZ, DMSO-d.sub.6): 9.79 (s, 1H), 7.41 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 3.38 (s, 3H) ppm.
Example 2: Preparation of p-nitrobenzyl chloroformate (Compound 2a)
(9) ##STR00006##
(10) P-nitrobenzyl alcohol (0.168 g, 1.1 mmol) and BTC (0.296 g, 1.0 mmol) were dissolved in 20 mL of anhydrous tetrahydrofuran to obtain a mixed solution, N, N-diisopropylethylamine (0.142 g, 1.1 mmol) was added into the mixed solution in the ice-water bath for reaction. The reaction was performed for 30 min under nitrogen protection, and then performed for 5 h at room temperature. After the reaction was completed, the reaction solution was concentrated, and a small amount of anhydrous tetrahydrofuran was used to remove excess phosgene in the reaction solution, and anhydrous dichloromethane was added into the concentrated solution to obtain anhydrous dichloromethane solution for using directly in the next step reaction.
Example 3: Preparation of 4-((methylnitroso) amino) phenyl (4-nitrobenzyl) carbonate (Compound I1)
(11) ##STR00007##
(12) Compound 1 (0.152 g, 1.0 mmol) and N, N-diisopropylethylamine (0.258 g, 2.0 mmol) were dissolved in 20 mL of anhydrous dichloromethane to obtain a mix solution, and the anhydrous dichloromethane solution containing compound 2a obtained in Example 2 was slowly dripped into the mixed solution in the ice-water bath for reaction. After the dripping was completed, the ice-water bath was removed and the reaction was performed for 5 h at room temperature. The reaction solution was washed 3 times with 0.5M dilute hydrochloric acid, water, and saturated saline solution, respectively, to separate the organic phase, the organic phase was dried overnight with anhydrous sodium sulfate, and then concentrated by rotary evaporation to obtain a yellow solid. The yellow solid was recrystallized with a mixed solvent of ethyl acetate and petroleum ether to obtain 285 mg of light-yellow crystal with a yield of 86%.
(13) .sup.1H NMR (600 MHz, DMSO-d.sub.6): 8.28-8.30 (m, 2H), 7.74-7.76 (m, 2H), 7.70-7.72 (m, 2H), 7.45-7.48 (m, 2H), 5.46 (s, 2H), 3.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 153.15, 150.02, 147.87, 143.07, 140.37, 129.32, 124.15, 122.79, 121, 20, 69.03, 32.14 ppm. HRMS (m/z) (ESI): calcd for C.sub.15H.sub.14N.sub.3O.sub.6[M+H].sup.+: 332.08, found: 332.11.
Example 4: Preparation of 4-((methylnitroso) amino) phenyl (2-chloro-4-nitrobenzyl) carbonate (Compound I2)
(14) ##STR00008##
(15) Referring to the method described in Examples 2 and 3, using 2-chloro-4-nitrobenzyl alcohol as raw material, compound I2 was obtained as light-yellow crystal with a yield of 23%.
(16) .sup.1H NMR (600 MHZ, DMSO-d.sub.6): 8.38-8.39 (d, J=2.3 Hz, 1H), 8.28-8.30 (dd, 1H), 7.88-7.90 (d, J=8.5 Hz, 1H), 7.71-7.72 (m, 2H), 7.46-7.48 (m, 2H), 5.49 (s, 2H), 3.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 152.99, 149.95, 148.49, 140.41, 140.18, 133.62, 131.17, 124.78, 122.96, 122.77, 121.25, 66.92, 32.20 ppm. HRMS (m/z) (ESI): calcd for C.sub.15H.sub.13ClN.sub.3O.sub.6Na [M+Na].sup.+: 388.03, found: 388.10.
Example 5: Preparation of 4-((methylnitroso) amino) phenyl (2-methyl-4-nitrobenzyl) carbonate (Compound 13)
(17) ##STR00009##
(18) Referring to the method described in Examples 2 and 3, using 2-methyl-4-nitrobenzyl alcohol as raw material, compound I3 was obtained as light-yellow crystal with a yield of 45%.
(19) .sup.1H NMR (600 MHZ, DMSO-d.sub.6): 8.15-8.16 (d, J=2.2 Hz, 1H), 8.10-8.14 (dd, J=8.4, 2.4 Hz, 1H), 7.71-7.73 (d, J=2.2 Hz, 1H), 7.68-7.70 (m, 2H), 7.45-7.48 (m, 2H), 5.44 (s, 2H), 3.44 (s, 3H), 2.47 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 153.11, 150.01, 147.78, 141.17, 140.37, 139.16, 129.71, 125.06, 122.80, 121.49, 121.21, 67.66, 32.17, 18.77 ppm. HRMS (m/z) (ESI): calcd for C.sub.16H.sub.15N.sub.3O.sub.6[M+H].sup.+: 346.10, found: 346.10.
Example 6: Preparation of 4-((methylnitroso) amino) phenyl (2-nitrobenzyl) carbonate (Compound I4)
(20) ##STR00010##
(21) Referring to the method described in Examples 2 and 3, 2-nitrobenzyl alcohol was used as a raw material, compound I4 was obtained as light-yellow crystal with a yield of 73%.
(22) .sup.1H NMR (600 MHZ, DMSO-d.sub.6): 8.17-8.19 (dd, 1H), 7.85-7.88 (m, 1H), 7.79-7.80 (m, 1H), 7.70-7.73 (m, 2H), 7.68-7.69 (m, 1H), 7.45-7.47 (m, 2H), 5.65 (s, 2H), 3.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 153.04, 149.98, 147.82, 140.40, 134.85, 130.81, 130.25, 130.06, 125.49, 122.77, 121.24, 67.09, 32.16 ppm. HRMS (m/z) (ESI): calcd for C.sub.15H.sub.14N.sub.3O.sub.6[M+H].sup.+: 332.09, found: 332.02.
Example 7: Preparation of 4-((methylnitroso) amino) phenyl (2-nitro-4-chlorobenzyl) carbonate (Compound I5)
(23) ##STR00011##
(24) Referring to the method described in Examples 2 and 3, using 2-nitro-4-chlorobenzyl alcohol as raw material, compound I5 was obtained as light-yellow crystal with a yield of 46%.
(25) .sup.1H NMR (600 MHZ, DMSO-d.sub.6): 8.26-8.27 (d, J=2.2 Hz, 1H), 7.94-7.96 (dd, 1H), 7.81-7.83 (d, J=8.4 Hz, 1H), 7.70-7.72 (m, 2H), 7.45-7.46 (m, 2H), 5.62 (s, 2H), 3.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 152.94, 149.96, 148.47, 140.42, 134.51, 134.21, 131.84, 129.81, 125.32, 122.75, 121.24, 66.53, 32.15 ppm. HRMS (m/z) (ESI): calcd for C.sub.15H.sub.13ClN.sub.3O.sub.6Na [M+Na].sup.+: 388.03, found: 388.00.
Example 8: Preparation of 4-((methylnitroso) amino) phenyl (2-nitro-4-bromobenzyl) carbonate (Compound I6)
(26) ##STR00012##
(27) Referring to the method described in Examples 2 and 3, using 2-nitro-4-bromobenzyl alcohol as raw material, compound I6 was obtained as light-yellow crystal with a yield of 33%.
(28) .sup.1H NMR (600 MHZ, DMSO-d.sub.6): 8.36-8.37 (d, J=2.1 Hz, 1H), 8.07-8.09 (dd, 1H), 7.73-7.75 (d, J=8.3 Hz, 1H), 7.70-7.72 (m, 2H), 7.44-7.46 (m, 2H), 5.60 (s, 2H), 3.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d 6): 8152.94, 149.96, 148.49, 140.42, 137.45, 131.94, 130.19, 128.03, 122.75, 122.16, 121.24, 66.58, 32.15 ppm. HRMS (m/z) (ESI): calcd for C.sub.15H.sub.13BrN.sub.3O.sub.6[M+H].sup.+: 431.98, found: 431.90.
Example 9: Preparation of 4-((methylnitroso) amino) phenyl (2-nitro-4-methylbenzyl) carbonate (Compound 17)
(29) ##STR00013##
(30) Referring to the method described in Examples 2 and 3, using 2-nitro-4-methylbenzyl alcohol as raw material, compound I7 was obtained as light-yellow crystal with a yield of 51%.
(31) .sup.1H NMR (600 MHz, DMSO-d.sub.6): 8.00-8.01 (m, 1H), 7.70-7.72 (m, 2H), 7.66-7.67 (d, J=0.9 Hz, 2H), 7.43-7.46 (m, 2H), 5.60 (s, 2H), 3.44 (s, 3H), 2.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 153.04, 149.99, 147.77, 140.57, 140.38, 135.29, 130.22, 127.75, 125.56, 122.74, 121.20, 67.02, 32.12, 20.74 ppm. HRMS (m/z) (ESI): calcd for C.sub.16H.sub.16N.sub.3O.sub.6[M+H].sup.+: 346.10, found: 345.90.
Example 10: Preparation of 4-((methylnitroso) amino) phenyl (2-nitro-5-chlorobenzyl) carbonate (Compound 18)
(32) ##STR00014##
(33) Referring to the method described in Examples 2 and 3, using 2-nitro-5-chlorobenzyl alcohol as raw material, compound I8 was obtained as light-yellow crystal with a yield of 37%.
(34) .sup.1H NMR (600 MHz, DMSO-d.sub.6): 8.21-8.23 (d, J=8.8 Hz, 1H), 7.83-7.84 (d, J=2.3 Hz, 1H), 7.76-7.79 (dd, 1H), 7.71-7.73 (m, 2H), 7.46-7.48 (m, 2H), 5.65 (s, 2H), 3.44 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 152.82, 149.98, 146.36, 140.43, 139.48, 133.27, 130.05, 129.67, 127.63, 122.76, 121.23, 66.56, 32.14 ppm. HRMS (m/z) (ESI): calcd for C.sub.15H.sub.13ClN.sub.3O.sub.6Na [M+Na].sup.+: 388.03, found: 388.00.
Example 11: Preparation of 4-((methylnitroso) amino) phenyl (2-nitro-5-methylbenzyl) carbonate (Compound 19)
(35) ##STR00015##
(36) Referring to the method described in Examples 2 and 3, using 2-nitro-5-methylbenzyl alcohol as raw material, compound I9 was obtained as light-yellow crystal with a yield of 48%.
(37) .sup.1H NMR (600 MHz, DMSO-d.sub.6): 8.09-8.11 (d, J=8.4 Hz, 1H), 7.72-7.73 (d, J=2.2 Hz, 1H), 7.71-7.72 (d, J=2.3 Hz, 1H), 7.57-7.58 (m, 1H), 7.48-7.49 (m, 1H), 7.46-7.47 (d, J=2.3 Hz, 1H), 7.45-7.46 (d, J=2.2 Hz, 1H), 5.63 (s, 2H), 3.44 (s, 3H), 2.47 (s, 3H) ppm. .sup.13C NMR (150 MHz, DMSO-d.sub.6): 153.00, 150.01, 145.93, 145.49, 140.40, 130.95, 130.47, 130.26, 125.70, 122.78, 121.24, 67.21, 32.15, 21.53 ppm. HRMS (m/z) (ESI): calcd for C.sub.16H.sub.15N.sub.3O.sub.6Na [M+Na].sup.+: 368.09, found: 368.10.
Example 12: Detection of NO Release Level of the Compound after Interacting with H9c2 Cells Under Normoxia or Hypoxia Condition Using Griess Method
(38) H9c2 cells were revived, cultured with DMEM medium, and placed in a carbon dioxide incubator to allow the cells to adhere to the wall for growth. After the cells grew to reach an appropriate density, cells were inoculated in a multi-well plate and placed in the carbon dioxide incubator for culture for 24 h at 37 C., 5% CO.sub.2, and saturated humidity until the cells adhered to the wall, the supernatant was discarded, and 2 mL of fresh DMEM medium was added. Then, a certain concentration (5, 10, 15 M) of compounds and the reference drug of isosorbide dinitrate were added. After culturing in a normoxic incubator (37 C., 21% O.sub.2) or an anoxic incubator (37 C., 1% O.sub.2) for 4 h, the supernatant was removed, and the cells in the multi-well plate were collected in 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 1 mL of PBS was added into the centrifuge tubes to resuspend the cells, and the cells were centrifuged again, the PBS was discarded. 100 L of cell lysis buffer (special for NO measurement) was added to the centrifuge tubes, placed on ice for half a minute, and then centrifuged for 5 min at 4 C. and 13300 rpm, and the supernatant was collected. 50 L of the supernatant was taken in a 96-well plate, three parallel groups were set for each sample, and 50 L of Griess reagent I and 50 L of Griess reagent II were added in sequence. The OD value of each well was measured at 540 nm with a full-wavelength microplate reader, and the NO concentration was calculated using the standard curve, as shown in Tables 1 and 2.
(39) TABLE-US-00001 TABLE 1 NO release amount of H9c2 cells treated with compounds for 4 h under the normoxia condition Concentration (M) NO release amount (%) * Compound 5 10 15 I1 0.24 0.84 0.16 I2 1.58 3.34 0.93 I3 0.43 0.92 0.32 I4 1.02 2.35 0.98 I5 .sup.a 0.15 I6 0.17 I7 0.12 I8 0.27 I9 0.23 Isosorbide 36.30 41.63 36.89 dinitrate .sup.b * denotes that NO release amount = measured NO concentration/calculated NO concentration, data are expressed as the mean SD of three independent experiments: *p < 0.05, **p < 0.01; .sup.a denotes that the NO release amount is not measured, and .sup.b denotes calculation based on of the NO release amount.
(40) TABLE-US-00002 TABLE 2 NO release amount of H9c2 cells treated with compounds for 4 h under the hypoxia condition Concentration (M) NO release (%) * Compound 5 10 15 I1 25.24 24.38 20.15 I2 34.75 30.96 27.37 I3 40.58 45.42 43.21 I4 20.78 20.62 18.33 I5 .sup.a 0.96 I6 0.94 I7 1.83 I8 0.93 I9 0.95 Isosorbide 22.73 27.41 24.90 dinitrate .sup.b * denotes that NO release amount = measured NO concentration/calculated NO concentration, data are expressed as the mean SD of three independent experiments: *p < 0.05, **p < 0.01; .sup.a denotes that the NO release amount is not measured, .sup.b denotes calculation based on of the NO release amount.
(41) As shown in Tables 1 and 2, the NO release amount of cells after being treated with different concentrations of compounds under the normoxia condition are very small, which are all lower than the maximum release amount of compound I2 (<3.34%, 10 M). But under the hypoxia condition, except for compounds I5-I9, after the cells being treated with different concentrations of compounds I1-I4, the compounds I1-I4 can effectively cause release of NO in the cells, with the NO release amount ranging from 20.15% to 45.45%, indicating hypoxia-activated NO donors. The reference drug of isosorbide dinitrate (ISDN) releases a certain amount of NO in the cells after treating cells under the normoxia or hypoxia condition. The isosorbide dinitrate causes a large NO release amount (up to 41.63%, (10 M)) in the cells under the normoxia condition. The isosorbide dinitrate also cause a NO release amount in the cells under the hypoxia condition, but the NO release amount is less than that under the normoxia condition, and lower than the NO release amount of compounds I2 and I3 under the hypoxia condition.
Example 13: Improvement Effect of the Compound on the Vitality of H9c2 Cells Under the Normoxia or Hypoxia Condition
(42) The cells were washed twice with 5 mL PBS, 1 mL trypsin was added to digest the monolayer cultured H9c2 cells, a single cell suspension was prepared with serum-containing culture medium, and 5000-10000 cells per well were inoculated in a 96-well culture plate. The volume of culture medium per well is 100 L, and the edge wells are filled with sterilized pure water. The culture plate was moved into a carbon dioxide incubator for culture for 24 h at 37 C., 5% CO.sub.2, and saturated humidity until the cells adhere to the wall. 100 L culture medium in the well was aspirated, 50 L fresh culture medium was added into the well, and different concentrations of compounds and reference drug isosorbide dinitrate were given respectively to the well. 50 L drug was added to each well, 3 parallel groups and 1 normoxic group without drug addition as control were set, and concentration gradient of the drug is 10, 1, 0.1, 0.01, 0.001 M. The normoxic group was incubated in a normal incubator at 5% CO.sub.2 and 37 C. for 4 h; and the hypoxic group was incubated in a hypoxic incubator (concentration of 021%) at 37 C. for 4 h. After the culture was completed, 10 L CCK8 reagent was added to each well and the normoxic group and the hypoxic group continued to be cultured in a normoxic incubator for 4 h, and the culture was terminated. The absorbance of each well was measured at 450 nm with a microplate reader to calculate the cell survival rate of cells at different concentrations, as shown in
(43) Under the normoxic condition (as shown in
Example 14: Detection of NO Release Level of Compound I1 in the Heart of Mouse Hypoxia Model Using NO Detection Kit
(44) (a) Drugs, Reagents, and Experimental Animals
(45) NO detection kit (Biyuntian Biotechnology Co., Ltd., Shanghai), heart tissue homogenate of hypoxia modeling and drug-given mouse, and blank control group mouse, and 6-8-week-old male Kunming mouse.
(46) (b) Experimental Principle
(47) NO has chemical activity and is rapidly converted into NO.sub.3.sup. and NO.sub.2.sup. in the body, and NO.sub.2.sup. is further converted into NO.sub.3.sup.. NO.sup.3 is reduced to NO.sub.2.sup. using Griess reagent, NO.sub.2.sup. has characteristic absorption at 540 nm, the absorbance is detected at 540 nm by a microplate reader, and the NO concentration is calculated using a standard curve.
(48) (c) Experimental Operation
(49) (1) Hypoxia model (85 mg/kg isoproterenol), the drug-given group was intraperitoneally injected with 20 mg/kg compound I1 before hypoxia modeling; (2) Cardiac tissue homogenate protein was obtained by centrifugation at 12,000 rpm for 15 min; (3) The working solution in the NO detection kit was prepared, the working solution was added to each group of proteins, after incubation, and each group of proteins was detected with an microplate reader to analyze the NO content in each group, as shown in
(50) As shown in
Example 15: Study on Regulatory Effect of Compound I1 on the Expression Level of Myocardial Injury Marker Proteins in Myocardial Hypoxia Mouse Using Western Blot
(51) (a) Drugs, Reagents, and Experimental Animals
(52) Western Blot gel preparation kit (Biyuntian Biotechnology Co., Ltd., Shanghai), BCA detection kit for protein quantification (Biyuntian Biotechnology Co., Ltd., Shanghai), ionization buffer, wet transfer solution, primary antibody (anti-TSC2-P, Abgent, 1:1000, A-AP3825a; anti-mTORC1, Abcam, 1:1000, ab120224; anti-GAPDH, Bioworld Technology, 1:500, AP0063), secondary antibody (Goat Anti-Rabbit IgG (H+L) HRP, 1:3000, Bioworld Technology, BS13278), 6-8-week-old male Kunming mouse.
(53) (b) Experimental Principle
(54) The changes of protein expression levels are analyzed based on Western Blot bands.
(55) (c) Experimental Operation
(56) (1) Mouse heart tissue protein was extracted and centrifuged at 12,000 rpm for 15 min to collect the protein, the protein was quantified using a BCA kit, and added to boiling water in loading buffer and cook for 5 min for later use; (2) SDS-PAGE gel was prepared and the sample was loaded to separate protein bands; (3) After transferring the protein bands to the PVDF membrane, the membrane was blocked with milk to incubate primary and secondary antibodies in sequence. The protein bands were visualized using a chemiluminescence developer to analyze the changes of protein expression in each group, as shown in
(57) As shown in