N-methyl nitrosamide compound as well as pharmaceutical composition and application thereof

Abstract

The invention discloses an N-methyl nitrosamide compound as well as a pharmaceutical composition and application thereof. The compound (formula I) is a hypoxia-activated NO donor drug, has the characteristic of effectively releasing NO under a high-selectivity hypoxia condition, so that the activity of myocardial cells is remarkably improved, the in-vivo and in-vitro activity is excellent, and the compound can be used for preventing or treating myocardial hypoxia injury. Meanwhile, the metabolic stability of the compound is improved by an ether bond connection structure, so that the compound has in-vivo metabolic properties beneficial to patent medicines.

Description

Technical Field

[0001] This invention relates to an N-methylnitrosamide compound, its pharmaceutical composition, and its application, and more particularly to an N-methylnitrosamide compound that improves the viability of hypoxic cardiomyocytes, its pharmaceutical composition, and its application. Background Technology

[0002] Coronary artery disease, also known as ischemic heart disease, is caused by organic narrowing or blockage of the coronary arteries, leading to myocardial ischemia and cellular hypoxia, thus causing myocardial damage. Nitric oxide (NO) donor drugs are often used to treat coronary artery disease caused by myocardial ischemia because they can release free NO molecules in the body, thereby promoting vasodilation. However, existing drugs such as nitrates are generally lacking in selectivity, making it difficult to accurately release sufficient NO at hypoxic sites. In addition, their rapid metabolism in the body leads to poor treatment efficacy.

[0003] CN116675620 discloses a class of NO donor compounds that can release NO through nitroreductase catalysis and can also selectively release NO in hypoxic cardiomyocytes. However, the carbonate chain in its structure is not very stable and is easily metabolized. Its drug-like properties need to be improved, which limits the clinical application of this type of compound. Summary of the Invention

[0004] Objectives of the invention: The first objective of this invention is to provide an N-methylnitrosamide compound; the second objective is to provide a pharmaceutical composition constructed from said compound; and the third objective is to provide a pharmaceutical application of said compound and its pharmaceutical composition.

[0005] Technical solution: The N-methylnitrosamide compounds of this invention have the structure of Formula I:

[0006]

[0007] in:

[0008] The definitions of Z1, Z2, X, and Y satisfy any of the following conditions:

[0009] (1) When Z1 is nitro, Z2 is hydrogen, and X and Y are selected from hydrogen, halogen, and C1-C4 alkyl;

[0010] (2) When Z2 is nitro, Z1 is selected from hydrogen, halogen, C1-C4 alkyl, and X and Y are selected from hydrogen, halogen, C1-C4 alkyl.

[0011] Preferably, in the structure:

[0012] When Z1 is nitro, Z2 is hydrogen, and X and Y are both hydrogen or either X or Y is selected from halogens or C1-C4 alkyl groups.

[0013] Preferably, in the structure:

[0014] When Z2 is nitro and Z1 is hydrogen, X and Y are both hydrogen or either X or Y is selected from halogens or C1-C4 alkyl groups.

[0015] When Z2 is nitro and Z1 is selected from halogens or C1-C4 alkyl groups, X and Y are both hydrogen.

[0016] Preferably, in the structure:

[0017] When Z1 is nitro, Z2 is hydrogen, and X and Y are both hydrogen or either X or Y is selected from fluorine, chlorine, methyl, or ethyl.

[0018] When Z2 is nitro and Z1 is hydrogen, X and Y are both hydrogen or either X or Y is selected from chlorine, methyl, or ethyl.

[0019] When Z2 is nitro and Z1 is selected from chlorine, bromine, methyl, or ethyl, X and Y are both hydrogen.

[0020] Preferably, the N-methylnitrosamide compounds of the present invention are selected from any one of the following compounds:

[0021]

[0022] The preparation method of the N-methylnitrosamide compound of the present invention includes the following steps:

[0023]

[0024] (i) 4-hydroxy(N-methyl)aniline hemisulfate reacts with sodium nitrite in the presence of acetic acid to give IM1;

[0025] (ii) Nitrobenzyl alcohol derivative S1, triphenylphosphine (PPh3) and IM1 are dissolved in tetrahydrofuran, and diisopropyl azodicarboxylate (DIAD) is added under nitrogen at 0°C. After the addition is complete, the reaction is moved to room temperature to obtain the N-methylnitrosamide compound I.

[0026] In step (i), the reaction temperature is 0°C and acetic acid is used as the solvent; the definitions of Z1, Z2, X, and Y are as described above.

[0027] The pharmaceutical composition of the present invention comprises the N-methylnitrosamide compound of the present invention and a pharmaceutically acceptable carrier.

[0028] The pharmaceutically acceptable carrier can be an excipient widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to allow the active ingredient to dissolve at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipient can be an inert filler, or it may provide a function such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipient may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0029] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0030] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0031] The N-methylnitrosamide compounds or their pharmaceutical compositions described in this invention are used in the preparation of drugs for the prevention or treatment of myocardial hypoxia-induced damage.

[0032] Preferably, the drug is a hypoxia-activated NO donor drug.

[0033] Preferably, the drug is a drug that improves the viability of myocardial cells under hypoxic conditions.

[0034] Preferably, the drug is a drug for the prevention or treatment of coronary heart disease.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0036] The compound designed in this invention is a hypoxia-activated NO donor drug, exhibiting highly selective and effective NO release under hypoxic conditions, with a release rate exceeding 26%, thereby significantly improving cardiomyocyte viability. It demonstrates excellent in vitro and in vivo activity and can be used to prevent or treat myocardial hypoxia injury. Simultaneously, the ether linkage structure enhances the compound's metabolic stability, giving it favorable in vivo metabolic properties for drug development, and thus broad prospects for clinical application. Attached Figure Description

[0037] Figure 1a The effects of N6 and ISMN on cell viability under normoxic conditions;

[0038] Figure 1b The effects of N6 and ISMN on cell viability under hypoxia;

[0039] Figure 2 The heart-to-body weight ratio in each group within the treatment group;

[0040] Figure 3a The effects of N6 and ISMN on troponin, a marker of myocardial injury, in a mouse model of myocardial hypoxia-injury.

[0041] Figure 3b The effects of N6 and ISMN on creatine kinase, a marker of myocardial injury, in a mouse model of myocardial hypoxia injury under treatment.

[0042] Figure 3c The effects of N6 and ISMN on creatine kinase isoenzymes, a marker of myocardial injury, in a mouse model of myocardial hypoxia injury.

[0043] Figure 4 The heart-to-body weight ratio in each group within the prevention and treatment group;

[0044] Figure 5a The effects of N6 and ISMN on troponin, a marker of myocardial injury, in a mouse model of myocardial hypoxia injury.

[0045] Figure 5b The effects of N6 and ISMN on creatine kinase, a marker of myocardial injury, in a mouse model of myocardial hypoxia injury.

[0046] Figure 5c The effects of N6 and ISMN on creatine kinase isoenzymes, a marker of myocardial injury, in a mouse model of myocardial hypoxia injury.

[0047] Figure 6a The NO release of N6 and ISMN in cardiac tissue in the treatment group and its comparison with the blank group and the model group;

[0048] Figure 6b This study investigated the NO release of N6 and ISMN in cardiac tissue in the prevention and treatment group and compared it with the blank group and the model group. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to the embodiments.

[0050] Example 1: Synthesis of N-methyl-N-nitroso-p-phenol (IM1)

[0051] Under light-protected conditions, 3.4 g (20.0 mmol) of 4-hydroxy(N-methyl)aniline hemisulfate was added to 100 mL of acetic acid and dissolved by sonication. A solution of sodium nitrite (2.8 g, 40.0 mmol) dissolved in 20 mL of water was added dropwise to the acetic acid solution under ice-water bath conditions, and the reaction was continued for 3 h under ice-water bath conditions. 100 mL of water was added to the reaction mixture, and the pH was adjusted to approximately 7 using a saturated sodium bicarbonate aqueous solution. The aqueous solution was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain approximately 2.9 g of a pale yellow solid (IM1), with a yield of 96%.

[0052] 1 H NMR (600MHz, DMSO-d6) δ7.76 (d, J = 9.0 Hz, 2H), 7.59 (d, J = 9.0 Hz, 2H), 3.45 (s, 3H).

[0053] Example 2: Synthesis of N-methyl-N-(4-((4-nitrobenzyl)oxy)phenyl)nitrosamide (N1)

[0054] 4-Nitrobenzyl alcohol (510.5 mg, 3.3 mmol), IM1 (507.5 mg, 3.3 mmol), and PPh3 (874.6 mg, 3.3 mmol) were dissolved in anhydrous THF. The solution was cooled in an ice bath, and a DIAD (674.2 mg, 3.3 mmol) / THF solution was slowly added dropwise under nitrogen protection. After the addition was complete, the reaction mixture was allowed to react at room temperature for 5 h. After the reaction was complete, the sample was packed into a silica gel column and eluted with petroleum ether and ethyl acetate in a gradient of 10:1 to 5:1. The eluent was collected, concentrated to dryness under reduced pressure, and the crude product was recrystallized in a 1:2 mixture of ethyl acetate and petroleum ether to give the target compound N1, a white solid, 300.5 mg, in a yield of 31.4%.

[0055] 1H NMR (600MHz, DMSO-d6) δ8.28(d,J=1.8Hz,1H),8.27(d,J=1.9Hz,1H),7.76(s,1H),7.74(s,1H),7.57(d,J= 2.2Hz,1H),7.56(d,J=2.2Hz,1H),7.20(d,J=2.2Hz,1H),7.18(d,J=2.2Hz,1H),5.36(s,2H),3.40(s,3H). 13 C NMR(151MHz,DMSO-d6)δ157.60,147.50,145.23,136.25,129.69,128.68,12 4.10,124.09,121.91,121.90,116.03,116.02,68.80,32.61.HRMS(ESI)m / z calcd for C 14 H 13 N3O4[M+H] + 288.0979, found 288.0982.

[0056] Example 3: Synthesis of N-(4-((2-chloro-4-nitrobenzyl)oxy)phenyl)-N-methylnitrosamide (N2)

[0057] Following the method described in Example 2, 2-chloro-4-nitrobenzyl alcohol was used as a raw material to obtain the target compound N2, a white solid, with a yield of 43.4%.

[0058] 1 H NMR (600MHz, DMSO-d6) δ8.37(d,J=2.3Hz,1H),8.27(dd,J=8.5,2.3Hz,1H),7.90(d,J=8.5Hz,1H),7.59(d,J =2.2Hz,1H),7.58(d,J=2.2Hz,1H),7.23(d,J=2.2Hz,1H),7.22(d,J=2.2Hz,1H),5.36(s,2H),3.41(s,3H). 13 C NMR(151MHz,DMSO-d6)δ157.42,148.13,142.18,136.49,133.29,130.52,12 4.71,122.88,121.95,121.94,116.06,116.04,67.06,32.60.HRMS(ESI)m / z calcd for C 14 H 12 ClN3O4[M+H] +322.0589, found 322.0598.

[0059] Example 4: Synthesis of N-methyl-N-(4-((2-methyl-4-nitrobenzyl)oxy)phenyl)nitrosamide (N3)

[0060] Following the method described in Example 2, using 2-methyl-4-nitrobenzyl alcohol as a raw material, the target compound N3 was obtained as a white solid with a yield of 39.4%.

[0061] 1 H NMR (600MHz, DMSO-d6) δ8.14(d,J=2.0Hz,1H),8.10(dd,J=8.4,2.3Hz,1H),7.73(d,J=8.4Hz,1H),7.58(d,J=2.2H z,1H),7.57(d,J=2.2Hz,1H),7.23(d,J=2.2Hz,1H),7.22(d,J=2.2Hz,1H),5.31(s,2H),3.41(s,3H),2.47(s,3H). 13 C NMR(151MHz,DMSO-d6)δ157.70,147.37,143.29,138.74,136.29,128.73,124.9 3,121.93,121.92,121.37,116.03,116.02,67.61,32.63,18.75.HRMS(ESI)m / z calcd for C 15 H 15 N3O4[M+H] + 302.1135, found 322.1140.

[0062] Example 5: Synthesis of N-methyl-N-(4-((2-nitrobenzyloxy)phenyl)nitrosamide (N4)

[0063] Following the method described in Example 2, using 2-nitrobenzyl alcohol as a raw material, the target compound N4 was obtained as a white solid with a yield of 41.5%.

[0064] 1 H NMR (600MHz, DMSO-d6) δ8.15(s,1H),8.14(s,2H),7.82-7.80(m,1H),7.64(dt,J=5.9,3.5Hz,1H),7.57(d,J =2.2Hz,1H),7.56(d,J=2.2Hz,1H),7.18(d,J=2.2Hz,1H),7.17(d,J=2.2Hz,1H),5.53(s,2H),3.41(s,3H).13 C NMR (151MHz, DMSO-d6) δ157.56,147.97,136.34,134.47×2,132.71,129.71,125.33,121.95,121.94,115.99,115.98,67.17,32.62.HRMS (ESI) m / z calcd for C 14 H 13 N3O4[M+H] + 288.0979, found 288.0980.

[0065] Example 6: Synthesis of N-(4-((2-chloro-6-nitrobenzyloxy)phenyl)-N-methylnitrosamide (N5)

[0066] Following the method described in Example 2, using 2-chloro-6-nitrobenzyl alcohol as a starting material, the target compound N5 was obtained as a white solid with a yield of 44.1%.

[0067] 1 H NMR (600MHz, DMSO-d6) δ7.99(dd,J=8.1,1.1Hz,1H),7.95(dd,J=8.2,1.0Hz,1H),7.71(t,J=8.2Hz,1H),7.59( d,J=2.2Hz,1H),7.57(d,J=2.3Hz,1H),7.15(d,J=2.2Hz,1H),7.14(d,J=2.2Hz,1H),5.39(s,2H),3.42(s,3H). 13 CNMR(151MHz,DMSO-d6)δ157.66,151.61,136.57,135.83,134.47,131.84,1 27.93,123.95,121.96,121.95,115.74,115.73,64.03,32.63.HRMS(ESI)m / z calcd for C 14 H 12 ClN3O4[M+H] + 322.0589, found 322.0594.

[0068] Example 7: Synthesis of N-(4-((3-fluoro-4-nitrobenzyloxy)phenyl)-N-methylnitrosamide (N6)

[0069] Following the method described in Example 2, using 3-fluoro-4-nitrobenzyl alcohol as a raw material, the target compound N6 was obtained as a white solid with a yield of 36.1%.

[0070] 1 H NMR (600MHz, DMSO-d6) δ8.21(t,J=8.2Hz,1H),7.69(d,J=11.9Hz,1H),7.58(d,J=2.1Hz,1H),7.57(d,J=2 .2Hz,1H),7.53(d,J=8.5Hz,1H),7.20(d,J=2.1Hz,1H),7.19(d,J=2.1Hz,1H),5.34(s,2H),3.40(s,3H). 13 C NMR(151MHz,DMSO-d6)δ157.40,156.00,154.27,147.21(d,J=8.3Hz),136.34,127 .04,123.98,121.93,121.94,117.19,117.05,116.05,68.21,32.63.HRMS(ESI)m / z calcd for C 14 H 12 FN3O4[M+H] + 306.0885, found 306.0885.

[0071] Example 8: Evaluation of the effects of compounds on NO release from H9c2 cardiomyocytes under normoxic and hypoxic conditions using the Griess method

[0072] 1. Experimental Methods

[0073] After resuscitating H9c2 cardiomyocytes, they were cultured in DMEM medium and placed in a CO2 incubator to promote cell adhesion and growth. Once the cells reached a suitable density, they were seeded into multi-well plates and cultured in a CO2 incubator (37℃, 5% CO2, saturated humidity) for 24 h until the cells were fully adhered. The supernatant was then discarded, and 2 mL of fresh DMEM medium was added, along with a specific concentration (10 μM) of the compound and the control drug isosorbide mononitrate (ISMN). The cells were cultured for 4 h in either a normoxic incubator (37℃, 21% O2) or a hypoxic incubator (37℃, 1% O2). The supernatant was then discarded, and the cells were collected into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded. The cells were resuspended in 1 mL of PBS, centrifuged again, and the PBS was discarded. Add 100 μL of cell lysis buffer (for NO assay) to a centrifuge tube, incubate on ice for 30 seconds, then centrifuge at 13300 rpm for 5 min at 4 °C and collect the supernatant. Transfer 50 μL of the supernatant to a 96-well plate, with three replicates per sample. Add 50 μL of Griess reagent I and 50 μL of Griess reagent II sequentially. Measure the OD value of each well using a full-wavelength microplate reader at 540 nm. The NO concentration is calculated using a standard curve.

[0074] 2. Experimental Results

[0075] Table 1. NO release from H9c2 cardiomyocytes after 4 hours of compound treatment under normoxic and hypoxic conditions.

[0076] compound NO release rate under hypoxia (%) Ascorbic NO release (%) N1 13.83 4.44 N2 18.07 2.41 N3 15.12 5.54 N4 8.12 3.46 N5 20.83 7.02 N6 26.54 3.89 ISMN 21.20 22.67

[0077] Note: NO release = measured NO concentration / calculated NO concentration. Data is expressed as the average SD of three independent experiments.

[0078] As shown in Table 1, treatment of cardiomyocytes with different compounds under normoxic conditions resulted in relatively low NO release. However, under hypoxic conditions, treatment of cardiomyocytes with compounds N1–N6 effectively induced NO release, with release rates ranging from 8.12% to 26.54%, demonstrating hypoxia-activated NO donors. The control compound isosorbide mononitrate (ISMN) released a certain amount of NO after treatment of cardiomyocytes under both normoxic and hypoxic conditions; its NO release rate under normoxic conditions was 22.67%, while under hypoxia it was 21.20%, which are similar, indicating that ISMN is not hypoxia-selective in NO release.

[0079] Example 9: Effects of compound N6 on the viability of H9c2 cardiomyocytes under normoxic and hypoxic conditions

[0080] 1. Experimental Methods

[0081] Cells were washed twice with 5 mL PBS, and H9c2 monolayer cultured cardiomyocytes were digested with 1 mL trypsin. A single-cell suspension was prepared using serum-containing culture medium, and 5000–10000 cells were seeded per well in a 96-well plate. Each well contained 100 μL of culture medium, and the edge wells were filled with sterile purified water. The plate was transferred to a CO2 incubator and cultured at 37°C, 5% CO2, and saturated humidity for 24 h until cell adhesion. The 100 μL of culture medium in each well was aspirated, and 50 μL of fresh culture medium was added. Different concentrations of compound N6 and the control drug isosorbide mononitrate were administered, 50 μL per well. Three parallel groups and one normoxic control group (concentration gradients of 10, 1, 0.1, and 0.01 μM) were established. The normoxic culture group was incubated in a normal incubator at 37°C with 5% CO2 for 4 hours; the hypoxic group was incubated in a hypoxic incubator (1% O2 concentration) at 37°C for 4 hours. After the culture was completed, 10 μL of CCK8 reagent was added to each well, and the cells were cultured in a normoxic incubator for another 4 hours. The culture was then terminated, and the absorbance of each well was measured at 450 nm using a microplate reader to calculate the cell viability of cardiomyocytes at each concentration.

[0082] 2. Experimental Results

[0083] Under normoxic conditions (Figure 1a), compound N6 slightly improved cell viability at 0.1 μM and 1 μM, but had some effect on cell viability at low concentrations of 0.01 μM and high concentrations of 10 μM, exhibiting low toxicity. Under hypoxic conditions (Figure 1b), compound N6 significantly restored the viability of hypoxic cells within the concentration range of 0.01–1 μM, with the improvement increasing with increasing concentration. However, at a high concentration of 10 μM, it actually reduced cell viability, indicating that high concentrations of NO have a damaging effect on cells. The control drug isosorbide mononitrate showed no improvement in cell viability under normoxic conditions, only exhibiting a certain ability to improve cell viability under hypoxic conditions, but still inferior to compound N6. Based on the results in Table 1, the representative hypoxia-activated NO donor compound N6 designed in this invention can effectively release NO under hypoxic conditions and improve cardiomyocyte viability within a certain concentration range, with activity superior to the existing drug isosorbide mononitrate.

[0084] Example 10: Therapeutic effect of compound N6 on myocardial hypoxia injury in mice

[0085] 1. Experimental Materials

[0086] (1) Compound N6, ISMN, isoproterenol, cardiac troponin kit, creatine kinase kit and creatine kinase isoenzyme kit.

[0087] (2) C57 mice, 8-10 weeks old, male.

[0088] 2. Experimental Methods

[0089] (1) Establishment of hypoxia model: Mice were subcutaneously injected with isoproterenol 10 mg / kg once a day for 3 consecutive days. After the model was established, the mice were administered N6 at 8 mg / kg and 16 mg / kg and ISMN at 5 mg / kg (equimolar dose with 8 mg / kg N6) via intraperitoneal injection, once a day for 3 consecutive days. After administration, the mice were sacrificed, and the heart tissue was collected and the size and weight of the heart were measured and recorded.

[0090] (2) Collect cardiac tissue homogenate by centrifugation at 12000 rpm for 15 min, and use enzyme-linked immunosorbent assay (ELISA) to determine the levels of cardiac troponin, creatine kinase and creatine kinase isoenzyme.

[0091] 3. Experimental Results

[0092] The heart weight ratio of the mice was calculated, and the results are as follows: Figure 2 As shown, the heart weight ratio in the model group mice increased from 0.49% in the control group to 0.60%, indicating collagen fiber proliferation and cardiac enlargement under hypoxic conditions. The mean heart weight ratio in the ISMN group was 0.55%, lower than that in the model group. The mean heart weight ratios in the N6 treatment groups were 0.54% (low dose) and 0.51% (high dose), respectively. The high dose of N6 almost restored heart weight to normal levels. Overall, N6 effectively reduced the increase in heart weight, with the high dose being more effective than the low dose, showing a dose-dependent effect.

[0093] As shown in Figure 3, compared with the model group, the levels of troponin, creatine kinase, and creatine kinase isoenzymes were significantly reduced in the treatment group, with N6 showing better efficacy than ISMN. This indicates that N6 can be used to treat hypoxic myocardial injury, and its efficacy is superior to the existing drug ISMN.

[0094] Example 11: The preventive and therapeutic effects of compound N6 on myocardial hypoxia injury in mice.

[0095] 1. Experimental Materials

[0096] (1) Compound N6, ISMN, isoproterenol, cardiac troponin kit, creatine kinase kit and creatine kinase isoenzyme kit.

[0097] (2) C57 mice, 8-10 weeks old, male.

[0098] 2. Experimental Methods

[0099] (1) Establishment of hypoxia model: Mice were subcutaneously injected with isoproterenol 10 mg / kg once a day for 3 consecutive days. At the same time as establishing the model, the mice were also given drugs, namely 8 mg / kg N6 and 5 mg / kg ISMN (equimolar dose with 8 mg / kg N6), by intraperitoneal injection and gavage, once a day for 3 consecutive days. After the drug administration was completed, the mice were sacrificed, the heart tissue was collected, and the size and weight of the heart were measured and recorded.

[0100] (2) Collect cardiac tissue homogenate by centrifugation at 12000 rpm for 15 min, and use enzyme-linked immunosorbent assay (ELISA) to determine the levels of cardiac troponin, creatine kinase and creatine kinase isoenzyme.

[0101] 3. Experimental Results

[0102] The heart weight ratio of the mice was calculated, and the results are as follows: Figure 4 As shown in the figure, the heart weight ratio increased from 0.51% in the control group to 0.68% in the model group, indicating collagen fiber proliferation and cardiac enlargement under hypoxic conditions. The mean heart weight ratio in the ISMN intraperitoneal injection group was 0.61%, while that in the N6 intraperitoneal injection group was 0.53%, almost close to the normal heart weight ratio. However, the heart weight ratio in the gavage administration group was higher than that in the intraperitoneal injection group, indicating that intraperitoneal administration was superior to gavage administration in terms of therapeutic effect. It is noteworthy that both N6 and ISMN can slow the increase in heart weight, but N6 was more effective than ISMN in both intraperitoneal injection and gavage administration.

[0103] As shown in Figure 5, compared with the model group, the levels of troponin, creatine kinase, and creatine kinase isoenzyme in the drug-treated group were significantly reduced. Among them, N6 was more effective than ISMN in both intraperitoneal injection and gavage administration, indicating that N6 can prevent and treat hypoxic myocardial injury and is more effective than the existing drug ISMN, especially when administered via intraperitoneal injection.

[0104] Example 12: Effect of compound N6 on cardiac NO release in mice with myocardial hypoxia injury

[0105] 1. Experimental Materials

[0106] NO detection kit (Shanghai Beyotimes Biotechnology Co., Ltd.), control group, mouse myocardial hypoxia injury model group and drug-treated group mouse heart tissue homogenate.

[0107] 2. Experimental Methods

[0108] (1) Collect heart tissue homogenate by centrifugation at 12000 rpm for 15 min;

[0109] (2) Prepare the working solution of the NO detection kit, add heart tissue homogenate for incubation, and use an ELISA reader to detect and analyze the NO level of each group.

[0110] 3. Experimental Results

[0111] As shown in Figure 6, compared to the model group, the treatment group ( Figure 6a ) and prevention and control group ( Figure 6b NO levels were significantly elevated in the study, with N6 showing a greater effect than ISMN.

[0112] Example 13: Evaluation of the in vivo pharmacokinetic properties of compound N6

[0113] 1. Experimental Materials

[0114] (1) Compound N6.

[0115] (2) Blab / c mice, 6-8 weeks old, female, 3 mice in total.

[0116] 2. Experimental Methods

[0117] (1) Dissolve N6 in 10% DMSO / 20% Solutol HS15 / 70% (10% HP-β-CD) and administer at a dose of 8 mg / kg via intraperitoneal injection.

[0118] (2) Blood samples were drawn at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 7 h and 24 h after drug administration. All blood samples were transferred to plastic microcentrifuge tubes containing anticoagulant and centrifuged at 4000 g and 4 °C for 5 min. The supernatant was transferred to microcentrifuge tubes without anticoagulant and the plasma was stored in a refrigerator at -75±15 °C. The drug concentration in the plasma was tested by LC-MS, the data were processed, a standard curve was plotted, and the relevant pharmacokinetic parameters were calculated.

[0119] 3. Experimental Results

[0120] Table 2. Pharmacokinetic parameters of compound N6 in mice.

[0121] Parameters CompoundN6 Dose (mg / kg, ip) 8 <![CDATA[T 1 / 2 (h)]]> 2.39 <![CDATA[T max (h)]]> 2.08 <![CDATA[C max (ng / mL)]]> 450 Cl (mL / min / kg) 6.45 <![CDATA[AUC 0-last (ng.h / mL)]]> 626 <![CDATA[AUC 0-inf (ng.h / mL)]]> 640

[0122] As shown in Table 2, compound N6 exhibited low clearance and moderate half-life after intraperitoneal injection of 8 mg / kg, indicating good pharmacokinetic properties.

[0123] Example 14: In vivo pharmacokinetic study of compound I3

[0124] 1. Experimental Materials

[0125] (1) Compound I3 (Compound I3 disclosed in CN116675620).

[0126] (2) SD rats, male, 170-183g; 3 rats in the intravenous injection group (A1-A3) and 3 rats in the gavage group (B1-B3), for a total of 6 rats.

[0127] 2. Experimental Methods

[0128] (1) Dissolve I3 in 10% DMSO / 50% PEG400 / 40% H2O, and administer 1 mg / kg via intravenous injection; administer 5 mg / kg via gavage.

[0129] (2) Blood samples were drawn at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 7 h and 24 h after drug administration. All blood samples were transferred to plastic microcentrifuge tubes containing anticoagulant and centrifuged at 4000 g and 4 °C for 5 min. The supernatant was transferred to microcentrifuge tubes without anticoagulant and the plasma was stored in a refrigerator at -75±15 °C. The drug concentration in the plasma was tested by LC-MS, the data were processed, a standard curve was plotted, and the relevant pharmacokinetic parameters were calculated.

[0130] 3. Experimental Results

[0131] Table 3. Individual and mean plasma concentrations of compound I3 (dose 1 mg / kg) at different time points after intravenous administration to rats.

[0132]

[0133]

[0134] Note: BLOQ represents below the check threshold, and NA represents no useful value.

[0135] Table 4. Individual and mean plasma drug concentrations of compound I3 (dose 5 mg / kg) at different time points after gavage administration to rats.

[0136]

[0137] Note: BLOQ represents below the check threshold, and NA represents no useful value.

[0138] As shown in Tables 3 and 4, no metabolic data related to the parent compound I3 could be detected in rat blood, whether administered intravenously or by gavage, indicating that I3 is rapidly metabolized in vivo.

Claims

1. An N-methylnitrosamide compound, characterized in that, It has the structure of Formula I: in: The definitions of Z1, Z2, X, and Y satisfy any of the following conditions: (1) When Z1 is nitro, Z2 is hydrogen, and X and Y are selected from hydrogen, halogen, and C1-C4 alkyl; (2) When Z2 is nitro, Z1 is selected from hydrogen, halogen, C1-C4 alkyl, and X and Y are selected from hydrogen, halogen, C1-C4 alkyl.

2. The N-methylnitrosamide compound according to claim 1, characterized in that, In the structure: When Z1 is nitro, Z2 is hydrogen, and X and Y are both hydrogen or either X or Y is selected from halogens or C1-C4 alkyl groups.

3. The N-methylnitrosamide compound according to claim 1, characterized in that, In the structure: When Z2 is nitro and Z1 is hydrogen, X and Y are both hydrogen or either X or Y is selected from halogens or C1-C4 alkyl groups. When Z2 is nitro and Z1 is selected from halogens or C1-C4 alkyl groups, X and Y are both hydrogen.

4. The N-methylnitrosamide compound according to claim 1, characterized in that, In the structure: When Z1 is nitro, Z2 is hydrogen, and X and Y are both hydrogen or either X or Y is selected from fluorine, chlorine, methyl, or ethyl. When Z2 is nitro and Z1 is hydrogen, X and Y are both hydrogen or either X or Y is selected from chlorine, methyl, or ethyl. When Z2 is nitro and Z1 is selected from chlorine, bromine, methyl, or ethyl, X and Y are both hydrogen.

5. The N-methylnitrosamide compound according to claim 1, characterized in that, Selected from any one of the following compounds:

6. A pharmaceutical composition, characterized in that, It comprises the N-methylnitrosamide compound of claim 1 and a pharmaceutically acceptable carrier.

7. The use of an N-methylnitrosamide compound of claim 1 or a pharmaceutical composition of claim 6 in the preparation of a medicament for the prevention or treatment of myocardial hypoxia injury.

8. The application according to claim 7, characterized in that, The drug in question is a hypoxia-activated NO donor drug.

9. The application according to claim 7, characterized in that, The drug described is a drug that improves the viability of myocardial cells under hypoxic conditions.

10. The application according to claim 7, characterized in that, The aforementioned medication is for the prevention or treatment of coronary heart disease.

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