Nitrate donor-albumin coupling medicine as well as preparation method and application thereof

Abstract

The invention relates to the field of medicine, in particular to a nitrite donor-albumin coupling medicine and a preparation method and application thereof. According to the nitrite donor-albumin coupling drug disclosed by the invention, an intelligent drug delivery system with a pH response characteristic is constructed by covalently coupling a nitrite donor compound or a derivative thereof and albumin, so that the bottleneck problems of short half-life period (lt: 1h), non-specific release, poor blood-brain barrier penetrability and the like of a traditional nitrite drug are effectively solved. The coupling medicine can significantly improve cerebral ischemia reperfusion injury through vasodilation regulation and oxidation resistance, and the infarct area is significantly reduced (about 2 times). The breakthrough provides a brand new normal form for developing cerebral apoplexy treatment medicines with targeting accuracy and multi-dimensional treatment, and has important clinical transformation value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and in particular to a nitrite donor-albumin conjugated drug, a preparation method and application thereof. BACKGROUND

[0002] Ischemic stroke is a disease characterized by acute occlusion of cerebral vessels leading to cerebral ischemia and hypoxia, with high morbidity, high disability rate and high mortality. Its prevention and treatment research has become an important topic in the global medical community. NO exhibits dual biological effects in the pathological process of cerebral ischemia: physiological concentration of NO generated by endothelial nitric oxide synthase (eNOS) plays a neuroprotective effect by increasing cerebral blood flow, inhibiting platelet activation, antagonizing oxidative stress, and improving brain microcirculation; while excessive NO produced by neuronal (nNOS) and inducible (iNOS) participates in the process of nerve damage. Pathological mechanism studies show that in the early stage of ischemia, excessive release of excitatory amino acids (glutamate, aspartate) activates the NMDA receptor GluN2B subunit, leading to calcium overload triggering abnormal activation of the nNOS-PSD95 signaling axis, and excessive generation of NO triggers neurotoxicity through free radical effects and interference with the tricarboxylic acid cycle; in the late stage of ischemia, inflammatory factors induce high expression of iNOS, and the NO produced by catalysis combines with superoxide anion to form peroxynitrite anion (ONOO - ), leading to lipid peroxidation, protein nitration and sulfhydryl oxidation, significantly reducing the bioavailability of NO and exacerbating neuronal damage. Studies suggest that endogenous NO generation imbalance during cerebral ischemia-reperfusion can expand the infarct size, but exogenous supplementation of NO donors or regulation of its metabolic pathways may reduce pathological damage to brain tissue by inhibiting oxidative damage and improving microcirculation. This dual effect suggests that spatiotemporal regulation of NO may become a potential target for the treatment of ischemic stroke.

[0003] Nitrite ion (NO2 - ) as a key oxidative metabolite of NO, its therapeutic potential has been reevaluated in recent years. Although sodium nitrite (NaNO2) is questioned for its potential carcinogenicity, pharmacological studies under controlled doses show that in ischemic and hypoxic microenvironments, NO2 - can be efficiently converted to NO through deoxyhemoglobin, xanthine oxidoreductase (XOR), etc., playing a role in vasodilation, antioxidant stress and promoting angiogenesis. For example, experiments on cerebral ischemia models have confirmed that low-dose NaNO2 intravenous infusion can significantly reduce infarct size and reduce brain edema, and its mechanism is closely related to the targeted conversion of NO2 - and sulfhydryl nitrosylation modification in an acidic microenvironment. It is worth noting that NO2 - is rapidly oxidized to nitrate (NO3 -The unique properties of NaNO2, such as avoiding systemic toxicity and selectively releasing active molecules in ischemic areas, make it have unique metabolic advantages. Preclinical studies have further shown that NaNO2 can improve cerebral blood perfusion, inhibit delayed vasospasm, and exhibit neuroprotective effects in primate models. However, its clinical application is limited by three key defects: short half-life (25-30 min), high-dose related hypotension and risk of methemoglobinemia, and long-term use may cause sodium accumulation cardiovascular side effects.

[0004] In view of the inherent defects of inorganic nitrite, researchers have turned to the development of new organic NO2 - donors. By introducing chalcone and other natural product-like structures, the addition / elimination reaction mediated by thiols is used to achieve controllable release of NO2 - , which can significantly reduce the risk of free radical damage. Such compounds not only exhibit excellent infarct area reduction effect in cerebral ischemia models, but also promote endothelial cell proliferation and ischemic penumbra angiogenesis. Notably, by adjusting the rigidity and flexibility of the organic skeleton and the electronegativity of the substituent, the release kinetics of NO2 - can be precisely controlled, providing a new paradigm for addressing the metabolic fluctuations of traditional nitrite. However, existing organic donors still face challenges such as insufficient targeting and poor in vivo stability, and breakthroughs are urgently needed through molecular design and delivery system innovation. SUMMARY

[0005] The present patent addresses the defects of NaNO2 and its use in the treatment of ischemic stroke, such as uncontrolled metabolism, explosive release of NO, and insufficient stability. A novel composite treatment system based on NO2 - donor-recombinant human albumin (rHA) coupling is constructed to achieve synergistic effect. Based on the previous finding that allyl nitro compounds release NO2 - under the action of nucleophiles such as glutathione and cysteine, a class of α-nitromethyl-α, β-unsaturated ketones is designed by replacing the rigid cyclohexenone skeleton with a flexible chain α, β-unsaturated ketone, introducing an aromatic ring system, and adjusting the nucleophilic reactivity at the β position through substituents (electronic and steric control), thereby precisely controlling the release rate of NO2 - . Through in vitro anti-platelet aggregation, nitrite release experiments, and neuron oxygen-glucose deprivation / reoxygenation (OGD / R) model screening, compound 43 with trifluoromethyl substitution is determined as the lead molecule due to its excellent activity and stability. The structural formula of compound 43 is as follows: .

[0006] Based on the above preliminary work, in order to enhance the targeting of ischemic areas, the acid-sensitive prodrug modification of compound 43 is carried out. By constructing a ketal structure with a diol fragment and a ketone carbonyl group, and introducing a fatty long chain linker, compound 40 is obtained. The prodrug is highly stable under physiological pH (7.4) conditions (24 h hydrolysis rate <5%), and is specifically hydrolyzed in the ischemic microenvironment (pH 5.5-5.8), releasing active molecules and triggering the generation of NO2 - . This pH-gated mechanism makes the drug inert in normal tissues, but precisely activated in ischemic areas, improving the selectivity of treatment. The structural formula of compound 40 is as follows: .

[0007] Finally, in order to break through the limitation of small molecule pharmacokinetics, covalent coupling technology is used to covalently modify compound 40 to the Cys 34 site or other surface lysine amino groups of rHA. Through different amino acid covalent warheads, the drug loading of each molecule of albumin is accurately controlled to be 1-8, and rHA-40 conjugates are constructed. This design integrates the multiple advantages of albumin: 1) long circulation half-life; 2) passive targeting of ischemic tissues based on enhanced permeability retention effect (EPR); 3) antioxidant function.

[0008] Molecular design of recombinant human albumin conjugated nitrite donor and mechanism of rHA-40 releasing NO2 - in ischemic environment as Figure 13 shown.

[0009] This innovative system achieves maximum therapeutic effect through multi-level regulation. At the pharmacokinetic level, the albumin carrier prolongs the circulation time and improves the accumulation in ischemic areas; at the pharmacodynamic level, the acid-triggered release mechanism ensures the spatiotemporal generation of NO2 - , and the antioxidant function of albumin realizes multi-target intervention (vasodilation, oxidative stress inhibition, and promotion of angiogenesis). Preclinical studies show that rHA-40 reduces infarct volume by 50% in a rat middle cerebral artery occlusion model, and no significant blood pressure fluctuations or methemoglobinemia are observed, confirming its high efficiency and safety. This "carrier-prodrug-effect molecule" three-in-one design paradigm provides a breakthrough solution for the development of a new generation of stroke treatment drugs. The contents of the present invention are as follows: In the first aspect of the present invention, a nitrite donor-albumin conjugate drug is provided, which comprises the following structure: a) a nitrite donor compound or its derivative, having the following structural formula: ; b) the albumin is selected from at least one of human serum albumin and recombinant human serum albumin.

[0010] Further, in the structure formula of the nitrite donor compound in a), X is at least one selected from carbon atom, nitrogen atom, oxygen atom, and n is 0-3.

[0011] Further, the nitrite donor compound or its derivative is coupled with albumin by chemical bond or non-chemical bond. Preferably, the nitrite donor compound or its derivative is coupled with albumin by chemical bond. More preferably, the nitrite donor compound or its derivative is covalently coupled with albumin by warhead. In an embodiment of the present application, the nitrite donor compound is covalently coupled with the sulfhydryl of the 34th cysteine of albumin by cysteine warhead, or covalently coupled with the amino of the surface lysine of albumin by lysine warhead.

[0012] In the second aspect of the present application, a composition is provided, which comprises the coupled drug and pharmaceutically acceptable additives.

[0013] Further, the additives comprise at least one of lyoprotectants (such as one or more of mannitol, trehalose, sucrose, lactose, glucose, sodium caprylate), preservatives (benzalkonium chloride), antioxidants (sodium ascorbate).

[0014] It should be noted that the present application does not specifically limit the additives in the pharmaceutical field, and any pharmaceutically acceptable additive that can act as a lyoprotectant, preservative, antioxidant is within the protection scope of the present application.

[0015] In the third aspect of the present application, the use of the coupled drug or the composition in the preparation of a drug for treating and / or preventing ischemic cardiovascular and cerebrovascular diseases is provided.

[0016] Further, the use of the coupled drug or the composition in the preparation of a drug for treating and / or preventing ischemic cerebral apoplexy is provided.

[0017] Further, the use of the coupled drug or the composition in the preparation of a drug for treating and / or preventing cerebral ischemia, cerebral apoplexy, myocardial ischemia, myocardial infarction, angina pectoris, arrhythmia, or coronary heart disease and pulmonary arterial hypertension is provided.

[0018] In the fourth aspect of the present application, a method for preparing the coupled drug is provided, which comprises the following steps: S1: mixing albumin with the nitrite donor compound in a molar ratio of 1:1 to 1:10 in a weak alkaline buffer system, preferably, the albumin and the nitrite donor compound are mixed in a molar ratio of 1:1 to 1:8, more preferably, the albumin and the nitrite donor compound are mixed in a molar ratio of 1:1 to 1:4; S2: reacting for 2-24 h at 4-25 ℃ in the dark to generate the conjugated drug, preferably, the reaction time is 8-24 h.

[0019] Further, the pH of the buffer system in step S1 is 7.4-9.0. Preferably, the buffer in the buffer system comprises 1-10 mM EDTA phosphate buffer (PBS).

[0020] Further, the concentration of albumin in step S1 is 200-500 μM, and the concentration of the nitrite donor compound is 500-1000 μM.

[0021] Further, step S2 further comprises a cosolvent, and the concentration of the cosolvent is 1-5% (v / v). Preferably, the cosolvent comprises at least one of DMSO, DMF or PEG-400.

[0022] Further, the method further comprises the step of purifying and drying the obtained conjugated drug.

[0023] In a specific embodiment of the present application, the experimental step of purifying the conjugated drug is purification using an ultrafiltration membrane with a molecular weight cut-off of 10-50 kDa, to remove unbound small molecule compounds.

[0024] Further, the method further comprises the step of ultrafiltration centrifugal concentration of the conjugated drug after ultrafiltration membrane purification, preferably, the specific operation of ultrafiltration concentration is 4 ℃, 10000-15000 rpm, 8-12 min.

[0025] Further, the method further comprises the step of washing the solution of the conjugated drug after ultrafiltration concentration, preferably, washing 3-5 times with PBS with pH 7.0-7.4, and collecting the final conjugated drug solution.

[0026] In a specific embodiment of the present application, the drying step is freeze-drying.

[0027] Further, the step of freeze-drying comprises pre-freezing at -80 ℃ for 24 h, then transferring to a freeze dryer pre-cooled to -50 ℃, and performing a gradient freeze-drying program: primary drying at -35 ℃ for 24 h (vacuum degree <10 Pa), followed by secondary drying at 25 ℃ for 24 h, to finally obtain white loose freeze-dried powder, which is sealed and stored under nitrogen.

[0028] Further, the method further comprises the step of preparing a nitrite donor compound or a derivative thereof.

[0029] It should be noted that the present application does not specifically limit the method for preparing the nitrite donor compound or the derivative thereof, and any method capable of preparing the nitrite donor compound or the derivative thereof is within the scope of the present application.

[0030] The beneficial effects of the present application include but are not limited to: The nitrite donor-albumin covalent coupling drug disclosed in the present application constructs an intelligent drug delivery system with pH response characteristics by covalent coupling of nitrite prodrugs and albumin, effectively solving the bottleneck problems of short half-life (<1 h), non-specific release and poor blood-brain barrier penetration of traditional nitrite drugs.

[0031] The core technology is to couple ND series nitrite donors and albumin by using amino acid covalent warheads to construct an intelligent delivery system with microenvironment response characteristics: maintaining the prodrug inertness under physiological conditions to reduce systemic toxicity, and specifically activating the nitric oxide signaling pathway in ischemic tissue acid microenvironment. This design realizes the prolongation of drug half-life (about 20 times) and the improvement of bioavailability through the albumin carrier, and at the same time endows the drug with the ability to penetrate the blood-brain barrier.

[0032] The conjugate can significantly improve cerebral ischemia-reperfusion injury through vasodilation regulation and antioxidant, and the infarct area is significantly reduced (about 2 times). This breakthrough provides a new paradigm for developing stroke treatment drugs with both targeted precision and multi-dimensional treatment, and has important clinical translation value. BRIEF DESCRIPTION OF DRAWINGS

[0033] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings: Figure 1 is a schematic diagram of the stability results of 40-Cys, 41-Cys, and compound 43 in physiological saline in test example 1 of the present application.

[0034] Figure 2 is a schematic diagram of the stability results of 40-Cys, 41-Cys, and compound 43 in rat plasma in test example 1 of the present application.

[0035] Figure 3 is a schematic diagram of the original drug release liquid chromatogram of conjugate rHA-40 at 10 min, 30 min, and 3 h in test example 2 of the present application.

[0036] Figure 4 is a release kinetics curve of the original drug compound 43 of the conjugate drug rHA-40 in test example 2 of the present application.

[0037] Figure 5 is a mass spectrum of the original drug release of conjugate drug rHA-40 at 10 min in test example 2 of the present application.

[0038] Figure 6is the mass spectrum of the drug rHA-40 released at 30 min in the test example 2 of the present application.

[0039] Figure 7 is the NO2 released in the cell lysate of the drug rHA-40 in the test example 3 of the present application. - is the result diagram of the release experiment.

[0040] Figure 8 is the result diagram of the in vitro antioxidant experiment of the control drug NBP, compound 40, conjugate rHA-40 and rHA, HSA in the test example 4 of the present application.

[0041] Figure 9 is the result diagram of the neurological deficit scores of tMCAO rats treated with rHA-40 and rHA in the test example 5 of the present application (Note: ****P<0.0001, **P<0.01, *P<0.05, ns P>0.05: compared with the MCAO group; ####P<0.0001, ###P<0.001, #P<0.05: compared with the 640 mg / kg rHA-40 group).

[0042] Figure 10 is the result diagram of the quantitative analysis of the infarct brain area of tMCAO rats treated with rHA-40 and rHA in the test example 5 of the present application (Note: ****P<0.0001, **P<0.01, *P<0.05, ns P>0.05: compared with the MCAO group; ####P<0.0001, ###P<0.001, #P<0.05: compared with the 640 mg / kg rHA-40 group).

[0043] Figure 11 is the brain infarction diagram observed by TTC staining of tMCAO rats treated with rHA-40 and rHA in the test example 5 of the present application.

[0044] Figure 12 is the result diagram of rHA-40 MS in the test example 6 of the present application.

[0045] Figure 13 is the mechanism diagram of the release of NO2 by rHA-40 in the ischemic environment in the present application. - is the mechanism diagram of the release of NO2 by rHA-40 in the ischemic environment in the present application. DETAILED DESCRIPTION

[0046] The present application is further described in detail below with specific examples and drawings, which are only descriptive and not limiting, and cannot limit the protection scope of the present application. The raw materials and equipment used in the specific embodiments of the present application are known products, which are obtained by purchasing commercially available products.

[0047] Example 1 Synthesis of linker compound 13 (1) Synthesis of tert-butyl (8-aminooctyl)carbamate (YY-1)

[0048] Dissolve 1,8-octanediamine (5 g, 34.68 mmol, 1.0 equiv) in anhydrous dichloromethane, after stirring to dissolve, add Et3N (4.0 equiv), cover the constant pressure dropping funnel, dissolve di-tert-butyl dicarbonate (0.6 equiv) in anhydrous dichloromethane, add to the constant pressure dropping funnel, replace nitrogen, add di-tert-butyl dicarbonate drop by drop, stir at room temperature overnight. Monitor the reaction by TLC (DCM:MeOH = 6:1), KMnO4 coloration, after the raw material is completely reacted, filter the reaction mixture, concentrate, purify by column chromatography (DCM:MeOH = 6:1), to obtain intermediate YY-1 (3.5 g). Yield 41%, colorless oil.

[0049] (2) Synthesis of tert-butyl (8-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)octyl)carbamate (YY-2)

[0050] Dissolve intermediate YY-1 (3.5 g, 14.33 mmol, 1.0 equiv) in anhydrous dichloromethane, add Et3N (1.5 equiv), pre-cool at 0 ℃ for 15 min, then add maleic anhydride (1.0 equiv) dissolved in anhydrous dichloromethane drop by drop, replace nitrogen, move to room temperature and stir for 4 h. After the raw material is completely reacted, concentrate, dissolve the concentrate in acetone, add Et3N (1.5 equiv) and acetic anhydride (1.5 equiv), then warm the mixture to 60 ℃, protect with nitrogen, and stir for 20 h. Monitor the reaction by TLC (DCM:MeOH = 8:1), KMnO4 coloration, after the raw material is completely reacted, filter the reaction mixture, concentrate, purify by column chromatography (DCM:MeOH = 8:1), to obtain intermediate YY-2 (1.6 g). Yield 35%, white solid powder.

[0051] (3) Synthesis of 1-(8-aminooctyl)-1H-pyrrole-2,5-dione (YY-3)

[0052] Intermediate YY-2 (1.6 g, 4.97 mmol, 1.0 equiv) was dissolved in ethyl acetate, hydrogen chloride-ethyl acetate solution (2M in Ethyl acetate, 4 equiv) was added, replaced with nitrogen, stirred at room temperature for 6 h. TLC monitoring of the reaction (DCM: MeOH = 10: 1), basic KMNO4 color development, after the raw material reaction was complete, the reaction mixture was filtered, concentrated to obtain intermediate YY-3 (1.3 g). Yield 98%, white solid powder.

[0053] (4) Synthesis of 4-((8-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)octyl)amino)-4- oxobutanoic acid (13)

[0054] Intermediate YY-3 (1.3 g, 4.87 mmol, 1.0 equiv) was dissolved in anhydrous DMF, and succinic anhydride (1.5 equiv) and N,N-diisopropylethylamine (2.0 equiv) dissolved in anhydrous DMF were slowly added dropwise, replaced with nitrogen, stirred at room temperature overnight. TLC monitoring of the reaction (DCM: MeOH = 10: 1), basic KMNO4 color development, after the raw material reaction was complete, the reaction mixture was filtered, concentrated, purified by column chromatography (DCM: MeOH = 10: 1) to obtain linker compound 13 (1.31 g). Yield 83%. White solid powder.

[0055] Example 2 Synthesis of linker compound 14 (1) Synthesis of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (L-1)

[0056] 1,8-Diamino-3,6-dioxaoctane (5 g, 33.76 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane, stirred to dissolve, then Et3N (4.0 equiv) was added, a constant pressure dropping funnel was covered, di-tert-butyl dicarbonate (0.6 equiv) was dissolved in anhydrous dichloromethane and added to the constant pressure dropping funnel, replaced with nitrogen, di-tert-butyl dicarbonate was added dropwise, stirred at room temperature overnight. TLC monitoring of the reaction (DCM: MeOH = 6: 1), basic KMNO4 color development, after the raw material reaction was complete, the reaction mixture was filtered, concentrated, purified by column chromatography (DCM: MeOH = 6: 1) to obtain intermediate L-1 (3.6 g). Yield 43%, white solid powder.

[0057] (2) Synthesis of tert-butyl (2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)ethoxy)ethyl)carbamate (L-2)

[0058] Intermediate L-1 (3.6 g, 14.50 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane, Et3N (1.5 equiv) was added, and after being pre-cooled at 0 °C for 15 min, maleic anhydride (1.0 equiv) dissolved in anhydrous dichloromethane was added dropwise, replaced with nitrogen, and stirred at room temperature for 4 h. After the raw material was completely reacted, it was concentrated, dissolved in acetone, and Et3N (1.5 equiv) and acetic anhydride (1.5 equiv) were added, and then the mixture was warmed to 60 °C, protected with nitrogen, and stirred for 20 h. The reaction was monitored by TLC (DCM:MeOH = 8:1), and KMnO4 basic coloration was used. After the raw material was completely reacted, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 8:1) to obtain intermediate L-2 (1.5 g). Yield 32%, milky white oil.

[0059] (3) Synthesis of 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione (L-3)

[0060] Intermediate L-2 (1.5 g, 4.57 mmol, 1.0 equiv) was dissolved in ethyl acetate, and hydrogen chloride-ethyl acetate solution (2M in Ethyl acetate, 4 equiv) was added, replaced with nitrogen, and stirred at room temperature for 6 h. The reaction was monitored by TLC (DCM:MeOH = 10:1), and KMnO4 basic coloration was used. After the raw material was completely reacted, the reaction mixture was filtered and concentrated to obtain intermediate L-3 (1.16 g). Yield 96%, yellow oil.

[0061] (4) Synthesis of 4-((2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)ethoxy)ethyl)amino)-4-oxobutanoic acid (14)

[0062] Intermediate L-3 (1.16 g, 4.39 mmol, 1.0 equiv) was dissolved in anhydrous DMF, and succinic anhydride (1.5 equiv) and N,N-diisopropylethylamine (2.0 equiv) dissolved in anhydrous DMF were slowly added dropwise, replaced with nitrogen, and stirred at room temperature overnight. The reaction was monitored by TLC (DCM : MeOH = 10 : 1), basic KMNO4coloration, and after the starting material was completely reacted, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM : MeOH = 10 : 1) to obtain linker compound 14 (864 mg). Yield 60%. Pale yellow oil.

[0063] Example 3 Synthesis of target compound 40 (1) Synthesis of (E)-2-methyl-3-phenyl-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (34)

[0064] The starting material 31 dibenzylamine (19.6 g, 99 mmol, 5.0 equiv) was dissolved in tert-amyl alcohol, and ammonium persulfate (13.6 g, 59.40 mmol, 3.0 equiv) was added, followed by the starting material 30 1-(3-(trifluoromethyl)phenyl)propan-1-one (4 g, 19.8 mmol, 1.0 equiv) after stirring at room temperature for 15 min, replaced with nitrogen, sealed, and the mixture was warmed to 120 °C and stirred for 24 h. The reaction was monitored by TLC (PE : EA = 20 : 1), and after the starting material was completely reacted, the reaction mixture was filtered with celite, the filter cake was washed with dichloromethane, concentrated, and purified by column chromatography (PE : EA = 20 : 1) to obtain intermediate 34 (3.44 g). Yield 60%. Yellow oil. 1 H NMR (600 MHz, DMSO- d 6) δ 8.08 – 7.90 (m, 3H), 7.78 (t, J = 8.1Hz, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.46 (dd, J = 8.5, 6.8 Hz, 2H), 7.43 – 7.37(m, 1H), 7.16 (d, J = 1.8 Hz, 1H), 2.20 (d, J = 1.4 Hz, 3H)。

[0065] (2) Synthesis of 2-(2,3-dihydroxypropyl)isoindoline-1,3-dione (35)

[0066] The starting material 32 3-chloro-1,2-propanediol (4.0 g, 36.19 mmol, 1.0 equiv) was dissolved in anhydrous DMF, the starting material 33 potassium phthalimide (8.04 g, 43.42 mmol, 1.2 equiv) and a catalytic amount of KI were added, the mixture was warmed to 155 °C, protected by nitrogen, and stirred for 24 h. The reaction was monitored by TLC (DCM : MeOH = 20 : 1), after the starting material was completely reacted, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM : MeOH = 20 : 1) to obtain the intermediate 35 (7.2 g). The yield was 90%. White solid powder. 1 H NMR (600 MHz, DMSO- d 6) δ 7.92 – 7.73 (m, 4H), 4.88(d, J = 5.3 Hz, 1H), 4.63 (t, J = 5.7 Hz, 1H), 3.89 – 3.73 (m, 1H), 3.61 – 3.52(m, 2H), 3.43 – 3.32 (m, 2H).

[0067] (3) Synthesis of E)-2-((2-(1-phenylprop-1-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-1,3-dioxolan-4-yl)methyl)isoindoline-1,3-dione (36) Figure 3 )

[0068] The intermediate 34 (2.0 g, 6.89 mmol, 1.0 equiv) was dissolved in toluene, the intermediate 35 (4.6 g, 20.67 mmol, 3.0 equiv) and p-toluenesulfonic acid (119 mg, 0.69 mmol, 0.1 equiv) were added, the mixture was warmed to 120 °C, protected by nitrogen, and stirred for 72 h. The reaction was monitored by TLC (PE : EA = 5 : 1), the reaction mixture was filtered, concentrated, and purified by column chromatography (PE : EA = 5 : 1) to obtain the intermediate 36 (1.3 g). The yield was 37%. Yellow oil. 1 H NMR(600 MHz, DMSO- d6) δ 7.87 - 7.83 (m, 1H), 7.82 - 7.75 (m, 4H), 7.74 - 7.66 (m, 2H), 7.64 - 7.60 (m, 2H), 7.52 - 7.44 (m, 1H), 7.38 - 7.27 (m, 3H), 6.88 - 6.77 (m, 1H), 4.52 - 4.41 (m, 1H), 4.24 - 4.20 (m, 1H), 4.00 - 3.96 (m, 1H), 3.94 - 3.84 (m, 1H), 3.80 - 3.74 (m, 1H), 1.64 (dd, J = 5.4, 1.4 Hz, 3H). J

[0069] (4) Synthesis of (Z)-2-((2-(3-bromo-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methyl)isoindoline-l,3-dione (37)

[0070] Intermediate 36 (1.3 g, 2.64 mmol, 1.0 equiv) was dissolved in dry carbon tetrachloride, N-bromosuccinimide (563 mg, 3.16 mmol, 1.2 equiv) and azobisisobutyronitrile (4.33 mg, 0.026 mmol, 0.01 equiv) were added, the mixture was warmed to 85 °C, stirred for 6 h under nitrogen protection. TLC monitoring of the reaction (PE : EA = 5: 1), after the raw material reacted completely, the reaction mixture was filtered, concentrated, the concentrate was dissolved with dichloromethane, extracted with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, the organic phase was concentrated and purified by column chromatography (PE : EA = 5 : 1) to obtain intermediate 37 (858 mg). Yield 57%. Yellow oil.

[0071] (5) Synthesis of (E)-2-((2-(3-nitro-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methyl)isoindoline-l,3-dione (38)

[0072] ​Intermediate 37 (858 mg, 1.50 mmol, 1.0 equiv) was dissolved in dry diethyl ether, silver nitrite (693 mg, 4.51 mmol, 3.0 equiv) was added, nitrogen was purged, and the reaction was stirred at room temperature for 24 h. The reaction was monitored by TLC (PE : EA = 5 : 1), and after the starting material was consumed, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE : EA = 5 : 1) to give intermediate 38 (234 mg). Yield 29%. Yellow oil.

[0073] (6) Synthesis of (E)-(2-(3-nitro-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methanamine (39)

[0074] Intermediate 38 (234 mg, 0.43 mmol, 1.0 equiv) was dissolved in dry ethanol, 80% hydrazine hydrate (109 mg, 2.17 mmol, 4.0 equiv) was added, the mixture was warmed to 80 °C, and the reaction was stirred under nitrogen for 2 h. The reaction was monitored by TLC (DCM : MeOH = 20 : 1), and after the starting material was consumed, the reaction mixture was filtered, concentrated, and the concentrate was dissolved in dichloromethane, extracted with saturated brine (3 x 20 mL), dried over anhydrous sodium sulfate, and concentrated, and purified by column chromatography (DCM : MeOH = 20 : 1) to give intermediate 39 (92 mg). Yield 52%. Yellow oil. 1 H NMR (600 MHz, DMSO- d 6) δ 7.85 - 7.63 (m, 4H), 7.47 - 7.25 (m, 5H), 6.92 (s, 1H), 5.39 - 5.05 (m, 2H), 4.24 - 3.79 (m, 3H), 2.80 - 2.54 (m, 2H), 2.11 - 1.88 (m, 2H).

[0075] (7) Synthesis of (E)-(2-(3-nitro-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methanamine (39) N 1 -((2-(3-nitro-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methyl)succinamide (40) H -((2-(3-nitro-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methyl)succinamide (40) N 4 -((2-(3-nitro-l-phenylprop-l-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-l,3-dioxolan-4-yl)methyl)succinamide (40)

[0076] Intermediate 39 (22 mg, 0.054 mmol, 1.0 equiv) and linker compound 13 (21 mg, 0.065 mmol, 1.2 equiv) were dissolved in dry dichloromethane, O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were added, the solution was stirred until clear, intermediate 39 (1.0 equiv) was added, the nitrogen was replaced and the reaction was stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM : MeOH = 20 : 1) and when the starting material was consumed, the reaction mixture was filtered, concentrated and purified by column chromatography (DCM : MeOH = 20 : 1) to give the target compound 40 (14 mg). Yield 35%. Yellowish oil. 1 H NMR (600 MHz, DMSO- d 6) δ 8.14 - 7.93 (m, 1H), 7.91 - 7.54 (m, 6H), 7.52 - 7.09 (m, 6H), 6.99 (d, J = 1.3 Hz, 2H), 5.75 (s, 1H), 5.12 (d, J = 1.7 Hz, 1H), 3.41 - 3.35 (m, 2H), 3.30 (s, 2H), 3.24 - 3.01 (m, 2H), 2.98 (q, J = 6.5 Hz, 2H), 2.71 - 2.67 (m, 8H), 2.34 - 2.20 (m, 4H), 2.02 - 1.96 (m, 2H), 1.46 (t, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, DMSO- d6) δ 174.72, 172.31, 171.53, 171.43, 171.41, 141.33, 138.70, 137.19, 134.90, 134.66, 131.00, 130.83, 130.60, 130.37, 130.10, 130.04, 129.74, 129.63, 129.52, 129.50, 129.43, 129.36, 129.22, 129.19, 128.93, 128.81, 128.56, 128.45, 126.40, 126.09, 126.07, 124.57, 122.83, 122.80, 109.43, 76.55, 76.24, 73.21, 68.44, 68.34, 41.02, 40.75, 38.91, 38.71, 37.51, 35.60, 31.75, 31.61, 31.33, 31.30, 31.26, 31.19, 30.86, 30.31, 29.76, 29.55, 29.49, 29.46, 29.44, 29.33, 29.29, 29.22, 29.20, 29.15, 29.05, 28.90, 28.36, 27.03, 26.76, 26.54, 25.58, 22.55, 14.38.

[0077] Example 4 Synthesis of target compound 41 (1) Steps (1) - (6) in Example 3 were repeated to obtain the intermediate 39.

[0078] (2) (E)- N 1 - (2-(2-(2,5-dioxo-2,5-dihydro-1 H - pyrrol-1-yl)ethoxy)ethoxy)ethyl)- N 4 Synthesis of ((2-(3-nitro-1-phenylprop-1-en-2-yl)-2-(3-(trifluoromethyl)phenyl)-1,3-dioxolan-4-yl)methyl)succinamide (41)

[0079] Intermediate 39 (32 mg, 0.078 mmol, 1.0 equiv) and linker compound 14 (31 mg, 0.094 mmol, 1.2 equiv) were dissolved in dry dichloromethane, O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were added, the solution was stirred until clear, intermediate 39 (1.0 equiv) was added, the nitrogen was replaced and the reaction was stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM : MeOH = 20 : 1) and when the starting material was consumed, the reaction mixture was filtered, concentrated and purified by column chromatography (DCM : MeOH = 20 : 1) to give the target compound 41 (24 mg). Yield 42%. Pale yellow oil. 1 H NMR (600 MHz, DMSO- d 6) δ 7.85 - 7.73 (m, 4H), 7.72 - 7.62 (m, 2H), 7.46 - 7.30 (m, 5H), 7.14 - 7.03 (m, 2H), 7.01 (s, 2H), 5.36 - 5.18 (m, 1H), 5.12 (s, 1H), 3.56 (t, J = 5.7 Hz, 2H), 3.51 (t, J = 5.6 Hz, 2H), 3.49 - 3.46 (m, 2H), 3.45 - 3.41 (m, 2H), 3.35 - 3.32 (m, 2H), 3.19 - 3.10 (m, 4H), 2.26 (d, J = 10.8 Hz, 4H), 2.19 (d, J = 18.4 Hz, 2H). 13 C NMR (151 MHz, DMSO- d6) δ 172.41, 172.28, 171.77, 171.36, 141.44, 141.34, 138.72, 137.21, 135.01, 134.66, 134.58, 131.40, 131.01, 130.84, 130.38, 130.18, 130.11, 130.09, 130.05, 129.85, 129.64, 129.50, 129.46, 129.23, 129.19, 128.93, 128.82, 128.78, 128.57, 128.30, 128.08, 127.47, 127.26, 126.41, 126.17, 126.10, 126.08, 122.83, 122.81, 109.44, 76.56, 76.24, 75.88, 74.82, 73.21, 72.97, 69.93, 69.80, 69.60, 68.45, 68.33, 67.40, 63.95, 41.25, 41.03, 40.97, 40.76, 38.98, 37.27, 35.60, 31.75, 31.16, 31.12, 31.08, 31.05, 31.04, 31.01, 30.65, 30.31, 29.55, 29.49, 29.44, 29.29, 29.19, 29.15, 29.05, 27.03, 25.58, 22.55, 21.40, 19.79, 19.07, 14.39, 13.99, 12.44.

[0080] Example 5 Coupling and optimization of compound 40 / 41 with recombinant human albumin a) Compound 40 / 41 was dissolved in PBS buffer (pH 7.4, containing 1 mM EDTA) containing 5% DMSO (v / v) to prepare a 300 mM stock solution, which was sterilized by 0.22 μm filter and ready for use; b) In a temperature-controlled stirring reactor, rHA solution (concentration 30 mg / mL, PBS buffer pH 7.2) was mixed with compound stock solution at a molar ratio of 3:1, using a gradient feeding strategy (added in 3 times with 2 hours interval), incubated at 4 ℃ with constant speed of 200 r / min for 8-12 h to generate covalent conjugate; c) Purification by ultrafiltration membrane with molecular weight cut-off 10-50 kDa, remove unbound small molecule compounds, concentrate the protein solution in ultrafiltration centrifuge tube (4 ℃, 12000 rpm, 10 min), wash 4 times with PBS (pH 7.2), collect the final coupling solution; d) The concentrated solution was divided into 2R borosilicate glass vials (2 mL / vial), pre-frozen at -80 ℃ for 24 h, then transferred to a pre-cooled freeze dryer at -50 ℃, and a gradient freeze-drying program was performed: primary drying at -35 ℃ for 24 h (vacuum degree <10 Pa), followed by secondary drying at 25 ℃ for 24 h, finally obtaining white loose lyophilized powder, which was sealed with nitrogen and stored.

[0081] Synthesis of positive drug compound 43 (1) Synthesis of intermediate (Z)-2-(bromomethyl)-3-phenyl-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one

[0082] Intermediate 34 (1 g, 3.44 mmol, 1.0 equiv) was dissolved in anhydrous carbon tetrachloride, N-bromosuccinimide (736 mg, 4.13 mmol, 1.2 equiv) and azobisisobutyronitrile (5.66 mg, 0.034 mmol, 0.01 equiv) were added, the mixture was warmed to 85 ℃, protected by nitrogen, and stirred for 24 h. TLC monitoring of the reaction (PE : EA = 20 : 1), after the raw material was completely reacted, the reaction mixture was filtered, concentrated, the concentrate was dissolved in dichloromethane, extracted with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, the organic phase was concentrated and purified by column chromatography (PE : EA = 20 : 1) to obtain intermediate 42 (928 mg). Yield 73%. Yellow oil.

[0083] (2) Synthesis of positive drug 43 (E)-2-(nitromethyl)-3-phenyl-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one

[0084] Intermediate 42 (928 mg, 2.51 mmol, 1.0 equiv) was dissolved in anhydrous ether, silver nitrite (1.2 g, 7.54 mmol, 3.0 equiv) was added, and the reaction was stirred at room temperature for 24 h under nitrogen. The reaction was monitored by TLC (PE:EA = 10:1), and after the starting material was completely consumed, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE:EA = 10:1) to give the target compound 43 (320 mg). Yield 38%. Yellow oil. 1 H NMR (600 MHz, DMSO- d 6) δ 8.11 (d, J =7.8 Hz, 1H), 8.05 (d, J = 7.4 Hz, 2H), 7.84 (t, J = 7.9 Hz, 1H), 7.71 (s, 1H),7.50 (s, 5H), 5.75 (s, 2H). 13 C NMR (151 MHz, DMSO- d 6) δ 195.38, 149.13,138.25, 133.89, 133.60, 130.82, 130.52, 130.12, 130.01, 129.91, 129.83,129.47, 129.28, 126.07, 126.04, 126.02, 125.99, 72.80, 40.94.

[0085] The following demonstrates the beneficial effects of the present application compared to the positive drug (43), butylphthalide (NBP) and human serum albumin through specific test examples.

[0086] Test Example 1 Stability Study of Compounds To simulate the actual effect of albumin conjugates, the covalent coupling products of small molecules and N-acetyl-L-cysteine (40-Cys, 41-Cys) were used as research objects. First, the stability of 40-Cys, 41-Cys and the original drug compound 43 in physiological saline was tested. The samples were dissolved in physiological saline containing 10% DMSO (20 μM), incubated at 37 °C, and sampled periodically to detect the change in compound peak area by high performance liquid chromatography (HPLC). As shown in Figure 1 Figure 1, 40-Cys and 41-Cys remained stable in physiological saline within 48 h, and the stability of 40-Cys was better than that of 41-Cys. The original drug compound 43 had partially degraded after 1 h.

[0087]

[0088] Further test the 40-Cys, 41-Cys and the original drug compound 43 in rat plasma stability, the sample dissolved in 10% DMSO in rat plasma (20 μM) after incubation at 37 ℃, regularly sampling and by high performance liquid chromatography (HPLC) detection of compound peak area change. Results can be seen, the original drug 43 in plasma rapidly decomposed, 2 h completely degraded. Ketal structure modified 40-Cys and 41-Cys significantly improved the plasma stability, of which compound 40-Cys stability performance is better, 24 h only degraded 15.22% ( Figure 2 ). The results of the study showed that the application of ethylene glycol ketal protecting group can effectively enhance the stability of the compound in different media, this structure modification can prevent the compound from being released too early before reaching the treatment area, so as to maintain the therapeutic effect.

[0089] Test example 2 in vitro metabolic study of compound Based on the stability advantage obtained earlier, the experiment further explores the possibility of whether the conjugate rHA-40 can remove the protecting group and convert into the original drug compound 43 under acidic environment. The experiment uses PBS buffer solution with pH 5.8 to simulate the acidic microenvironment of ischemic and hypoxic tissues, and then a certain concentration of conjugate rHA-40 is dissolved in the buffer system and incubated at physiological temperature (37.4℃). At each time point, the degradation of the conjugate is dynamically monitored by HPLC. The results are shown in Figures 3-6 HPLC combined with release kinetics curve analysis found that the conjugate rHA-40 rapidly degraded in the acidic buffer system, and the original drug compound 43 was not significantly generated after 4 h. The mass spectrum result of 30 min showed that the released compound was the original drug compound 43 (the 10 min sample did not show a characteristic peak because the release amount was lower than the detection limit of the instrument), indicating that the conjugate can be activated under specific acidic conditions to release the original drug.

[0090] Test example 3 nitrite ion release study of compound Based on the acidic release characteristics of the conjugate, the experiment evaluated the NO2 - release level of the conjugate rHA-40 by Griess reagent method. The experiment used oxygen-glucose deprivation / reoxygenation (OGD / R) treated cell lysate to construct an in vitro injury model, and 100 μM rHA-40 solution and PBS control were added to the reaction system, respectively, and incubated at 37 ℃ constant temperature shaking bed (200 rpm) for 1 h and 12 h. After mixing the sample, 40 μL of the reaction solution was added to a 96-well enzyme-labeled plate (n=3), and an equal volume of Griess reagent was added. The absorbance was measured at 540 nm wavelength using an enzyme-labeled instrument to detect the NO2- The results are shown in Figure 6. The rHA-40 group showed different degrees of NO2 Figure 7 release at 1 h and 12 h. The amount of NO2 - generated at 1 h was significantly increased by 1.7 times compared with the control group, and the sustained release trend was maintained at 12 h, indicating that the conjugate can specifically and slowly release NO2 - in a simulated ischemic microenvironment, which can be further reduced to NO and exert its pharmacological effects. -

[0091] In vitro antioxidant study of the compound of Test Example 4 The total antioxidant capacity assay kit was used to evaluate the antioxidant activity of the positive control drug NBP, the compound 40, the conjugate rHA-40, and rHA and HSA in vitro. The single concentration (50 mg / mL) comparison showed that rHA had significantly higher antioxidant capacity than human serum albumin (p<0.001) due to its high free thiol content. The albumin-small molecule conjugation strategy significantly improved the antioxidant capacity of the conjugate rHA-40 compared with the small molecule drugs NBP and 40 (as shown in Figure 7). Figure 8

[0092] In vivo anti-cerebral ischemia activity study of the compound of Test Example 5 To systematically evaluate the in vivo anti-cerebral ischemia activity of the conjugate rHA-40, the rat transient middle cerebral artery occlusion (tMCAO) model was used to simulate the pathological state of cerebral ischemia. The low, medium, and high dose groups (40, 160, and 640 mg / kg) and the rHA control group (40 and 640 mg / kg) were set up, and the same volume of solvent was given to the sham operation group and the model group. After ischemia for 2 h, the wire plug was removed for reperfusion injury and drug intervention was given at the same time. The neurological function recovery and cerebral infarction volume changes were evaluated by neurological function score (Longa method) and TTC staining at 24 h after the operation.

[0093] 1. Establishment of rat tMCAO model and drug administration 1.1 Experimental animals: 42 SD rats (180-200 g, male) were purchased from Zhejiang Vantong Lihua Experimental Animal Technology Co., Ltd. 1.2. Experimental method ​​SD rats were acclimated for 3 days (temperature 25 °C, humidity 40%), and the model group and drug group rats were fasted for 12 h before surgery, with free access to water. After isoflurane anesthesia, the rats were placed in a supine position. After skinning and disinfecting the neck, the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed, and the CCA and ECA were ligated. Then, a thread plug was inserted into the ICA through the ECA until it reached the origin of the middle cerebral artery, and the plug was left in place for 2 h. Then, the thread plug was removed from the ICA to perform reperfusion, and the drugs for each drug group were injected into the tail vein simultaneously, as shown in Table 1. The sham operation group mice were only freed from the blood vessels, without embolizing the middle cerebral artery.

[0094] Table 1. Drug administration table for each group

[0095] 1.3. Evaluation index (1) Scoring method for mouse neurological behavior defects After the rats woke up, the Zea Longa 5-point scoring standard was used for scoring: 0 points represent no neurological defect symptoms; 1 point represents an inability to fully extend the contralateral forelimb; 2 points represent walking while turning to the paretic side; 3 points represent walking while falling to the paretic side; 4 points represent an inability to walk independently, with impaired consciousness; and 5 points represent death. A cumulative score of 1 point or higher is considered a successful model; any score of 5 points, subarachnoid hemorrhage upon dissection, or no neurological defect symptoms (0 points) are considered a failed model.

[0096] (2) TTC staining method for detecting mouse brain infarct area After the mice were sacrificed by decapitation under anesthesia, the intact brain tissue was removed, frozen at -20 °C for 30 min, and coronally sectioned at a thickness of about 2 mm. The sections were immersed in 2% 2,3,5 triphenyltetrazolium chloride (TTC) dye solution, and photographed after 20 min of 37 °C dark staining. Image J software was used for analysis and calculation of the percentage of brain infarct volume. J

[0097] 1.4 Statistics All data were analyzed using GraphPad Prism 8 software. For comparisons between multiple groups, two-way ANOVA analysis was used, combined with Tukey's test for pairwise comparisons between groups to determine significant differences between different groups. This statistical method is suitable for handling complex data involving multiple independent variables and can provide more accurate difference analysis results.

[0098] 2. Results Pharmacodynamic experiments confirmed that rHA-NO2 - ​The donor conjugate rHA-40 exhibits significant multidimensional efficacy in the treatment of ischemic stroke. Through the evaluation of the tMCAO rat model, the rHA-40 high-dose group (10 mg / kg) reduced the brain infarction volume ratio from 38.22% to 18.69% (p<0.0001, TTC staining quantification as shown in the accompanying Figure 11 , and showed a clear dose-effect relationship (infarction volume of low / middle / high dose groups was 29.15%, 23.74%, and 18.69%, respectively). The neurological behavior score (mNSS) further verified its functional protection effect, and the high-dose group (640 mg / kg) significantly reduced the neurological deficit score by 77.27% (from 3.67 to 0.83) compared with the model group (p<0.0001), which was significantly better than the pure recombinant human albumin control group (p<0.001), indicating that the conjugation strategy significantly improved the anti-cerebral ischemia activity through structural synergistic effect (as Figures 9-10 ).

[0099] Studies have shown that the optimized design of rHA-40 achieves double synergies: (1) Albumin conjugation improves the stability of the prodrug by 3.2 times (the plasma half-life is extended from 1 h to 72 h), and triggers specific hydrolysis of the ketal bond through the acidic microenvironment (pH 6.2-6.5) in the ischemic area, with a local NO2 - release amount 5.7 times higher than that in normal tissues (LC-MS / MS detection); (2) The albumin carrier itself removes hydroxyl radicals (·OH clearance rate of 89%) and inhibits lipid peroxidation (MDA level reduction of 71%), and cooperates with released NO to regulate the eNOS / AMPK pathway, enhancing microvascular perfusion (brain blood flow increased by 65% by laser Doppler) and reducing half-dark zone neuronal apoptosis (TUNEL+ cells reduced by 58%). This targeted delivery and multi-mechanism synergistic strategy provides a molecular basis for the excellent efficacy of rHA-40.

[0100] Test Example 6 Screening of conjugation process To optimize the conjugation process of the target compound 40 and recombinant human albumin, the present patent focuses on three core factors: the conjugation ratio of small molecules to rHA, reaction temperature, and time, and designs 12 different conjugation conditions. High-resolution mass spectrometer (MS, Thermo Scientific Q Exactive) combined with ultra-high performance liquid chromatograph (UPLC, Thermo Scientific Vanquish Flex) is used for systematic characterization of the product. Through systematic evaluation, condition 12 exhibits the optimal conjugation effect (small molecule 40: rHA is 3.0: 1, temperature is 4 ℃, and reaction time is 8 h), mass spectrometry analysis shows that the proportion of recombinant human albumin monomer modification product under this condition reaches 100% (as shown in the accompanying Figure 12 ). Figure 13It is known that compound 40 and rHA are covalently coupled. The multi-molecular modification and the residue of unreacted prototype are completely avoided. Different coupling conditions and the proportion of different components in the obtained coupled drugs are shown in Table 2. In the subsequent process verification, this condition shows good reproducibility, and the yield of single-molecule modification is always maintained above 90%.

[0101] Table 2 Coupling conditions and coupling results of compound 40 and rHA

[0102] The above merely illustrates the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but determined by the claims of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A nitrite donor-albumin conjugate drug, characterized by: The conjugated drug comprises the following structure: a) a nitrite donor compound or a derivative thereof, wherein the nitrite donor compound has the following structure: ； b) the albumin, wherein the albumin comprises at least one of human serum albumin, recombinant albumin, bovine serum albumin, ovalbumin and mouse serum albumin.

2. The conjugated pharmaceutical according to claim 1, characterized in that, In the structure of the nitrite donor compound in a), X is at least one of a carbon atom, a nitrogen atom and an oxygen atom, and n is 0-3.

3. The conjugated drug according to claim 1, wherein The nitrite donor compound or the derivative thereof is connected to the albumin by a chemical bond or a non-chemical bond.

4. The conjugated drug according to claim 1, wherein The nitrite donor compound or the derivative thereof is covalently coupled to the albumin by a warhead.

5. A composition characterized in that, The composition comprises the conjugated drug according to any one of claims 1-4 and an additive acceptable in the medical field.

6. Use of the conjugated drug according to any one of claims 1-4 or the composition according to claim 5 in the preparation of a drug for treating and / or preventing ischemic cardiovascular and cerebrovascular diseases.

7. A process for the preparation of the conjugated pharmaceutical according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1: mixing the albumin according to b) of claim 1 and the nitrite donor compound according to a) in a molar ratio of 1:1 to 1:10 in a weak alkaline buffer system; S2: reacting in the dark at 4-25 ℃ for 2-24 h to generate the conjugated drug.

8. The method of claim 7, wherein, The method further comprises the steps of purifying and drying the obtained conjugated drug.

9. The method of claim 7, wherein, The pH of the buffer system in step S1 is 7.4-9.

0.

10. The method of claim 7, wherein, In step S2, a cosolvent is further included, and the concentration of the cosolvent is 1-5% (v / v).

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