CN112457358A - Compound, nitric oxide donor prodrug compound, preparation method and application thereof - Google Patents

Compound, nitric oxide donor prodrug compound, preparation method and application thereof Download PDF

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CN112457358A
CN112457358A CN202011459460.2A CN202011459460A CN112457358A CN 112457358 A CN112457358 A CN 112457358A CN 202011459460 A CN202011459460 A CN 202011459460A CN 112457358 A CN112457358 A CN 112457358A
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compound
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nitric oxide
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oxide donor
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CN112457358B (en
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杨广宇
何波
汤天辰
徐静思
史君涵
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Shanghai Jiaotong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention relates to the technical field of biological medicines, in particular to a compound, a nitric oxide donor prodrug compound, and a preparation method and application thereof. The compound has a structure shown in a formula I-1, is used for preparing a nitric oxide donor prodrug compound, and has a structure shown in a formula I. The preparation method of the compound comprises the following steps: step c): the compound of formula I-1 is prepared by condensing the compound of formula I-2 with pyrrolidinyl diazodiol sodium salt. TheThe preparation method of the nitric oxide donor prodrug compound comprises the following steps: step d): the compound of formula I is prepared by deacetylating the compound of formula I-1. The nitric oxide donor prodrug compound can be used for controllably releasing nitric oxide under the catalysis of endo-cellulase and can be used for controllably releasing nitric oxide under the natural bio-orthogonal catalysis of endo-cellulase Cel5A-h 38. Therefore, the medicine can be used for preparing the medicine capable of releasing nitric oxide in a controlled manner by utilizing the characteristic of releasing nitric oxide in a controlled manner according to requirements.

Description

Compound, nitric oxide donor prodrug compound, preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a compound, a nitric oxide donor prodrug compound, and a preparation method and application thereof.
Background
Nitric Oxide (NO) is an important gas signaling molecule in cardiovascular, immune and central nervous systems, and plays an important role in the treatment of cancer, inflammation and cardiovascular diseases. However, the physiological activity and efficacy of NO is tissue specific and dose dependent. Therefore, the development of NO delivery systems with targeted controlled release is a hot spot in the research field of NO at present.
In recent years, due to rapid development of pharmaceutical chemistry, NO donor compounds of various structures have been successfully synthesized, including nitrenols, organic nitrates, nitrites thiols, and the like. Among them, azoenolic compounds (Diazeniumdiolate) are the most widely studied and applied NO donors at present, and are generally generated by reacting amine compounds with NO, such as pyrrolidinyl diazodiol sodium salt (NONOate, the same below).
Figure BDA0002830880330000011
However, such compounds have problems of being difficult to store stably, being liable to spontaneous decomposition, being liable to off-target, and the like. Therefore, Wangcong et al designed and synthesized a stable glycosylated NO donor based on galactose units, which can controllably release nitric oxide under the catalysis of microbial galactosidase. However, since endogenous galactosidase is also present in normal tissues and blood of human body, non-specific degradation of part of the prodrug releases NO, limiting its therapeutic effect, and causing side effects such as blood pressure drop and heart rate increase. On the other hand, wild-type β -galactosidase also tends to result in non-specific hydrolysis of endogenous glycosides with unpredictable physiological consequences.
The problem of donor nonspecific degradation and side effects caused by the donor can be solved by using metabolic enzymes which are deficient endogenously in a human body and a proper prodrug donor compound to construct a natural bio-orthogonal enzyme response NO controlled release system. Cellulose is currently the most abundant renewable resource in nature, and is a sugar chain linked from glucose by β -1, 4-glycosidic linkages. The specific degradation enzyme endocellulase can randomly hydrolyze beta-1, 4-glycosidic bonds in cellulose chains. Such enzymes are widely found in organisms such as bacteria, fungi, actinomycetes, insects, mollusks, nematodes and protozoa in nature. The selectivity of different sources of endo-cellulase on the length of a substrate sugar chain is different, for example, earlier researches of the inventor find that some endo-cellulase exists in ruminant rumen microorganisms, not only can the internal glycosidic bond of macromolecular long-chain cellulose be recognized, but also the endo-cellulase has higher degradation activity on substrates modified by oligosaccharide glycosides such as cellobiose and the like. However, the enzyme and the substrate are just lacking in human endogenous metabolism, and a NO controllable delivery system orthogonal to the natural organism of the human metabolic pathway can be designed by utilizing the combination of the enzyme and the substrate, so that a new thought is provided for the specific targeted research of NO, and a technical support is provided for clinical application.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a compound, a nitric oxide donor prodrug compound, and methods of preparation and use thereof, which are used to solve the problems of the prior art. The invention takes nitrogen monoxide donor pyrrolidinyl diazodiol sodium salt (NONOATE) as a lead compound, and uses cellobiose to carry out structural modification on the compound to obtain a stable glycosylated nitrogen monoxide donor prodrug compound (Cel 2-NONONOATE). The invention also discloses a preparation method of the nitric oxide donor prodrug compound (Cel 2-NONONOate) and application of the compound in specific degradation and release of nitric oxide under the catalysis of endo-cellulase.
To achieve the above and other related objects, the present invention provides, in a first aspect, a compound having a structure represented by formula I-1:
Figure BDA0002830880330000021
in a second aspect, the present invention provides a nitric oxide donor prodrug compound having a structure represented by formula I:
Figure BDA0002830880330000022
in a third aspect, the present invention provides a process for the preparation of the above compound, comprising the steps of:
step c): the compound of formula I-1 is prepared by condensing the compound of formula I-2 with pyrrolidinyl-diazodiol sodium salt, and the reaction equation is as follows:
Figure BDA0002830880330000031
preferably, any one or more of the following features are also included:
c1) in step c), condensing the compound of formula I-2 and pyrrolidinyl diazodiol sodium salt in a dimethylformamide solution;
c2) in the step c), the reaction temperature is 23-25 ℃;
c3) the preparation method further comprises a step b) before the step c): the compound of formula I-2 is prepared by bromination at position 1 from the compound of formula I-3, and the reaction equation is as follows:
Figure BDA0002830880330000032
more preferably, any one or more of the following features are also included:
c11) characteristic c1), the molar ratio of the compound of formula I-2 to the pyrrolidinyl-diazodiol sodium salt is 10: 15, the ratio of the amount of the compound of formula I-2 to dimethylformamide is 10 mmol: 105 mL;
c12) in feature c1), the preparation method further comprises a step of separating and purifying the compound of formula I-1: removing dimethylformamide by rotary evaporation, diluting with dichloromethane, washing with saturated brine, and collecting organic phase with anhydrous Na2SO4Drying, spin-drying, and separating by column chromatography;
b1) in the step b) of the characteristic c3), the compound of the formula I-3 is dissolved in acetic anhydride, and hydrogen bromide/acetic acid solution is added for carrying out 1-bit bromination;
b2) in the step b) of the characteristic c3), the reaction temperature is 23-25 ℃;
b3) in step b) of feature c3), the preparation method further comprises the step of isolating and purifying the compound of formula I-2: adding dichloromethane for dilution, adding saturated sodium bicarbonate solution under ice bath, extracting with separating funnel, back extracting water phase, collecting organic phase, mixing, and adding anhydrous Na2SO4Drying, filtering and spin-drying the solvent;
b4) in step b) of feature c3), the preparation method further comprises, before step b), step a): the compound of formula I-3 is prepared by acetylating and protecting hydroxyl in the compound of formula I-4, and the reaction equation is as follows:
Figure BDA0002830880330000041
even more preferably, any one or more of the following features are also included:
b11) characteristic b1), the amount ratio of the compound of formula I-3, acetic anhydride and hydrogen bromide/acetic acid solution being 10.3 mmol: 7mL of: 17.5 mL;
a1) in the step a) of the characteristic b4), under the protection of inert gas, the compound of the formula I-4 and acetic anhydride realize the acetylation protection of hydroxyl in the compound of the formula I-4 under the pyridine condition to prepare the compound of the formula I-3;
a2) in step a) of feature b4), the compound of formula I-4, acetic anhydride and pyridine are used in a ratio of 14.6 mmol: 18 ml: 30 ml;
a3) in the step a) of the characteristic b4), the reaction temperature is 23-25 ℃;
a4) in the step a) of feature b4), the preparation method further comprises the step of separating and purifying the compound of formula I-3: spin-drying the solvent, diluting with dichloromethane, and sequentially diluting with saturated CuSO4Washed with saturated brine, and the organic phase was washed with anhydrous Na2SO4Drying, filtering and spin-drying the filtrate.
In a fourth aspect, the present invention provides a method for preparing the above nitric oxide donor prodrug compound, comprising the steps of:
step d): the compound of formula I is prepared by deacetylating a compound of formula I-1 according to the following reaction equation:
Figure BDA0002830880330000042
preferably, any one or more of the following features are also included:
d1) in step d), the compound of formula I-1 is deacetylated in a sodium methoxide and methanol system;
d2) the molar ratio of the compound of formula I-1 to sodium methoxide was 2.67: 0.40, the ratio of the amount of the compound of formula I-1 to methanol was 2.67 mmol: 15 mL;
d3) in the step d), the reaction temperature is 23-25 ℃;
d4) in step d), the preparation method further comprises the steps of separating and purifying the compound shown in the formula I: after the reaction is finished, adding methanol for dilution, then adding activated strong-acid cation exchange resin, adjusting the pH value to 7-8, filtering, and spin-drying to obtain yellow oily liquid; the yellow oily liquid was dissolved in distilled water and lyophilized.
In a fifth aspect, the present invention provides the use of a compound as described above for the preparation of a nitric oxide donor prodrug compound having the structure shown in formula I:
Figure BDA0002830880330000051
the sixth aspect of the present invention provides the use of the aforementioned pro-drug of nitric oxide donor compound for the controlled release of nitric oxide under the catalysis of an endocellulase.
Preferably, the nitric oxide donor prodrug compound is applied to controllable release of nitric oxide under natural bioorthogonal catalysis of endo-cellulase Cel5A-h 38.
The invention relates to a method for screening endo-cellulase orthogonal to human metabolic organisms by utilizing endo-cellulase reported in literature [ Biotechnol.Biofuels.2019,12(1) ] to react on different types of glycosidic bonds under physiological conditions (pH 7.4 and 37 ℃). Specifically, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-mannopyranoside, p-nitrophenyl-alpha-L-fucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside containing a human body common endogenous glycosidic bond are dissolved in PBS buffer solution with pH of 7.4. Four kinds of pure enzyme solutions of the endo-cellulase, namely Cel5A-h23, Cel5A-h28, Cel5A-h38 and Cel5A-h49, which are derived from literature reports are respectively added into a substrate solution, reaction is carried out at 37 ℃, and whether the endo-cellulase has nonspecific degradation effect on common endogenous glycosidic bonds of a human body is determined.
The invention also utilizes the method of catalyzing Cel 2-NONONOate prodrug donor glycosidic bond degradation by using endocellulase screened in the literature [ Biotechnol.Biofuels.2019,12(1) ] to release NO. Specifically, the synthesized compound shown in the formula (I) is dissolved in PBS buffer solution with pH 7.4, purified endo-cellulase with different doses is added, the reaction is carried out at 37 ℃, and the cumulative release amount of nitric oxide in the solution after the reaction is detected by a Greiss method.
The application of the nitric oxide donor prodrug compound in preparing a nitric oxide controllable-release medicine.
The nitric oxide donor prodrug compound is applied to the preparation of medicines for treating diabetes lower limb ischemia, skin injury and myocardial infarction diseases.
Compared with the prior art, the invention has the outstanding advantages that the NO donor compound (the pyrrolidinyl diazodiol sodium salt) is combined with cellobiose by adopting a chemical bonding method, the stability of the NO donor is improved, and the cellobiose bond in the NO donor compound can be specifically degraded by using the endocellulase deficient in human endogenous metabolism to release NO. On one hand, non-specific degradation of a prodrug donor is avoided, and the targeting of NO delivery is improved. Meanwhile, the selected endo-cellulase substrate has specificity and has no influence on endogenous glycosidic bonds of a human body. Thus constructing a biomedical delivery system which is orthogonal to the natural organism of human metabolism and has the function of controllable release of NO.
Drawings
FIG. 1 shows the degradation activity of four candidate endocellulases on different types of glycosidic bonds;
FIG. 2 is a graph showing the cumulative amount of NO released by an endocellulase to controllably catalyze the degradation of Cel 2-NONONAte prodrug compound.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
Example (b):
1. synthesis of Azenol-type Donor Compound (Cel 2-NONONAte) with Cellobioside modification
(1) Cellobiose (5.00g,14.6mmol) was weighed into a 100mL three-necked flask, anhydrous pyridine (30mL) was added under nitrogen, acetic anhydride (18mL) was added dropwise under ice bath using a constant pressure dropping funnel, and after completion of the addition, the ice bath was removed and stirred at room temperature for 12 hours. The reaction disappeared. The solvent was oil-pumped dry, diluted with 100mL DCM dichloromethane, and successively saturated CuSO4(100 mL. times.2), and washed with saturated saline (100 mL). Anhydrous for organic phaseNa2SO4Drying, filtering and spin-drying the filtrate. 7.0g of a white solid powder of wholly acetylated cellobiose was obtained at a yield of 70%.
(2) Fully acetylated cellobiose (7.0g,10.3mmol) was weighed out and dissolved in acetic anhydride (7 mL). A33% HBr/AcOH solution (17.5mL) was slowly added dropwise with stirring in an ice bath, and the ice bath was removed and stirred at room temperature for 1 hour. Adding DCM (100mL) for dilution, slowly adding into saturated sodium bicarbonate solution (1.8L) dropwise under ice bath, monitoring with pH paper, extracting with separating funnel, back-extracting the water phase once, collecting the organic phases, combining, and adding anhydrous Na2SO4Drying, filtering and spin-drying the solvent to obtain 7.0g of white solid powder of the 2,3,6,2 ', 3', 4 ', 6' -hepta-O-acetyl-beta-D-cellobiose bromide, with the yield of 97%. This intermediate was used in the next reaction without additional purification.1H NMR(400MHz,Chloroform-d)δ6.53(d,J＝4.1Hz,1H),5.54(apparent t,J＝9.7Hz,1H),5.20-5.03(m,2H),4.94(dd,J＝9.2,7.9Hz,1H),4.77(dd,J＝10.0,4.0Hz,1H),4.59-4.48(m,2H),4.37(dd,J＝12.5,4.5Hz,1H),4.27-4.14(m,2H),4.06(dd,J＝12.5,2.3Hz,1H),3.84(apparent t,J＝9.7Hz,1H),3.68(ddd,J＝9.8,4.5,2.3Hz,1H),2.14(s,3H),2.09(s,3H),2.09(s,3H),2.05(s,3H),2.05(s,3H),2.01(s,3H),1.99(s,3H)
(3) NONONAte (2.3g,15mmol) was weighed into a 250mL three-necked flask, and magneton, freshly activated molecular sieve (2g) was placed, purged three times with an oil pump, protected with nitrogen, and charged with 49mL anhydrous DMF. Peracetylcellulose disaccharide bromoglycoside (7.0g,10mmol) was weighed into another single-neck flask, poured into 56mL of DMF under nitrogen protection, and dissolved with stirring. The DMF solution of 2,3,6,2 ', 3', 4 ', 6' -hepta-O-acetyl-beta-D-cellobiose bromide was slowly added dropwise to a three-necked flask containing pyrrolidinyl-diazodiol sodium salt NONONOATE under ice-bath, and the ice-bath was removed and stirred at room temperature for 4 hours. The oil pump was forced to rotary evaporate most of the DMF solvent, dilute with DCM (100mL), wash with saturated brine (100 mL. times.2), and dry Na was added to the organic phase2SO4Drying and spin-drying to obtain brown oily liquid. And (4) performing column chromatography separation, wherein an eluent is PE, acetone is 5:1, and 2.6g of yellow solid is obtained, and the yield is 36%.1H NMR(400MHz,Chloroform-d)δ5.27-5.19(m,2H),5.14(dd,J＝8.6,4.8Hz,2H),5.07(t,J＝9.5Hz,1H),4.93(t,J＝8.5Hz,1H),4.51(d,J＝8.2Hz,2H),4.39(dd,J＝12.5,4.2Hz,1H),4.10(dd,J＝12.0,4.7Hz,1H),4.05(d,J＝12.5Hz,1H),3.85(d,J＝8.7Hz,1H),3.69(dd,J＝20.8,9.1Hz,2H),3.62-3.53(m,4H),2.12(s,3H),2.09(s,3H),2.03(s,3H),2.06(s,3H),2.01(s,3H),1.98(s,3H),1.95(s,3H)；13C NMR(101MHz,Chloroform-d)δ170.49,170.30,170.19,169.77,169.30,169.30,169.04,100.80,100.17,75.92,73.28,72.91,72.59,71.97,71.57,69.54,67.77,61.80,61.53,50.62,23.01,20.84,20.64,20.64,20.52,20.52,20.52,20.52；LC-MS(m/z):[M+Na]+＝773.
(4) 2,3,6,2 ', 3', 4 ', 6' -hepta-O-acetyl-. beta. -D-cellobioside-NOnoate (2.00g,2.67mmol) was weighed into a 50mL single-necked flask, and anhydrous methanol (15mL) was poured and dissolved with stirring. Na (9.0mg,0.40mmol) was weighed into a 5mL round bottom flask, connected to a tee, and purged with nitrogen balloon three times, and 1mL of anhydrous methanol was poured. The freshly prepared sodium methoxide solution was slowly poured into the above 50mL single-necked flask with stirring in an ice bath, and the ice bath was removed and the reaction was allowed to proceed at room temperature for 1h with stirring. Adding 30mL of methanol for dilution, adding activated strong-acid cation exchange resin, adjusting the pH value to 7-8, filtering, and spin-drying to obtain yellow oily liquid, dissolving the yellow oily liquid in 2mL of distilled water, and freeze-drying to obtain 1.1g of Cel-NONONONOATE white solid with the yield of 91%.1H NMR(400MHz,D2O)δ5.20(d,J＝8.2Hz,1H),4.51(d,J＝7.9Hz,1H),3.98-3.93(m,2H),3.84(dd,J＝12.4,4.1Hz,1H),3.87-3.80(m,1H),3.75-3.70(m,4H),3.64-3.54(m,5H),3.52-3.44(m,2H),3.40(apparent,J＝9.1Hz,1H),3.30(apparent,J＝8.6Hz,1H),2.01-1.91(m,4H)；13C NMR(101MHz,D2O)δ102.51,102.32,77.70,75.97,75.46,75.31,74.07,73.12,70.86,69.44,60.56,59.58,50.90,22.51；LC-MS(m/z):[M+Na]+＝478.65.
2. Screening of human endogenous glycosidic bond bioorthogonal endo-cellulase
(1) 46.34mg of p-nitrophenyl-beta-D-cellobioside (pNPCel2) was weighed out and dissolved in PBS (pH 7.4) buffer, and the volume was adjusted to 100mL to prepare a substrate solution with a final concentration of 1 mmol/L. Preheating 450 μ l of the above substrate solution in 37 deg.C water bath for 5min, adding 50 μ l of the above substrate solutionCel5A-h23, Cel5A-h28, Cel5A-h38 and Cel5A-h49 pure enzymes react for 10min at 37 ℃, and 1mL of 1mol/L Na is immediately added2CO3The solution stops the reaction. And (3) determining the degradation conditions of the four kinds of endo-cellulases on the specific fiber glycosidic bond. The results show (fig. 1): the degradation activity of the cellobioside bonds of the three enzymes, namely Cel5A-h23, Cel5A-h28, Cel5A-h38 and Cel5A-h49, under physiological conditions is 0.011, 0.010, 11.02 and 0.192U/mg respectively.
(2) 30.125mg of p-nitrophenyl-beta-D-glucopyranoside (pNPGlu), 28.525mg of p-nitrophenyl-beta-D-xylopyranoside (pNPXyl), 30.125mg of p-nitrophenyl-beta-D-mannopyranoside (pNPMan), 28.53mg of p-nitrophenyl-alpha-L-fucopyranoside (pNPFuc), 30.125mg of p-nitrophenyl-beta-D-galactopyranoside (pNPGal) and 31.523mg of p-nitrophenyl-beta-D-glucopyranoside (pNPGlc) are respectively weighed and dissolved in PBS (pH 7.4) buffer solution, and the solution is subjected to constant volume to 100mL to prepare a substrate solution with the final concentration of 1 mmol/L. Respectively taking 450 mu L of the substrate solution, preheating the substrate solution in a water bath at 37 ℃ for 5min, adding 50 mu L of Cel5A-h23, Cel5A-h28, Cel5A-h38 and Cel5A-h49 pure enzyme, reacting for 100min at 37 ℃, and immediately adding 1mL of 1mol/L Na2CO3The solution stops the reaction. Determining the degradation conditions of the enzymes Cel5A-h23, Cel5A-h28, Cel5A-h38 and Cel5A-h49 on human endogenous glycosidic bonds. The results show (fig. 1): the three enzymes Cel5A-h23, Cel5A-h28 and Cel5A-h49 have certain degradation activity on galactose bonds endogenously existing in a human body under physiological conditions, and the endo-cellulase Cel5A-h38 has no degradation activity on common human endogenous glycoside bonds under physiological conditions.
3. Endocellulase catalyzed glycosylation NO donor prodrug Cel 2-NONONOATE controllable release of NO
4.554mg of Cel 2-NONONOate were weighed out and dissolved in PBS (pH 7.4) buffer to a volume of 10mL to prepare a prodrug solution at a final concentration of 1 mmol/L. 1.25mL of the prodrug solution is added with pure enzymes Cel5A-h23, Cel5A-h28, Cel5A-h38 and Cel5A-h49 with different final concentrations respectively, and reacted at 37 ℃. At the same time, a blank control without enzyme was set. Then, samples were taken for 2min, 5min, 10min, 15min, 20min, 25min, 30min, 40min, 50min, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 9h, 12h, 18h, 24h, 48h, 72h and 96h, respectively, and the cumulative amount of NO released from the reaction solution was measured by the Griess method. The results show (fig. 2): the synthesized glycosylated NO donor prodrug Cel 2-NONONAte shown in the formula (I) is stable under physiological conditions, can be specifically degraded and release NO under the catalysis of endo cellulase, and can control the release rate and the cumulative release amount of NO by adjusting the addition amount of the enzyme.
And (4) conclusion: according to the experimental results, the synthetic cellobioside-modified NO donor prodrug Cel 2-NONONAte has higher stability, and a controllable NO release system is constructed by utilizing the specific catalytic activity of the endo cellulase. Particularly, the method solves the problem of non-specific degradation of a glycosylated NO donor delivery system based on the natural bioorthogonality of an endo-cellulase Cel5A-h38 delivery system and a human endogenous metabolic pathway, has a larger clinical application prospect, and can be used for treating lower limb ischemia, skin injury, myocardial infarction diseases and the like.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (13)

1. A compound having a structure according to formula I-1:
Figure FDA0002830880320000011
2. a nitric oxide donor prodrug compound having the structure of formula I:
Figure FDA0002830880320000012
3. a process for the preparation of a compound according to claim 1, comprising the steps of:
step c): the compound of formula I-1 is prepared by condensing the compound of formula I-2 with pyrrolidinyl-diazodiol sodium salt, and the reaction equation is as follows:
Figure FDA0002830880320000013
4. a method of preparing a compound as claimed in claim 3, further comprising any one or more of the following features:
c1) in step c), condensing the compound of formula I-2 and pyrrolidinyl diazodiol sodium salt in a dimethylformamide solution;
c2) in the step c), the reaction temperature is 23-25 ℃;
c3) the preparation method further comprises a step b) before the step c): the compound of formula I-2 is prepared by bromination at position 1 from the compound of formula I-3, and the reaction equation is as follows:
Figure FDA0002830880320000014
5. the method of preparing a compound of claim 4, further comprising any one or more of the following features:
c11) characteristic c1), the molar ratio of the compound of formula I-2 to the pyrrolidinyl-diazodiol sodium salt is 10: 15, the ratio of the amount of the compound of formula I-2 to dimethylformamide is 10 mmol: 105 mL;
c12) in feature c1), the preparation method further comprises a step of separating and purifying the compound of formula I-1: removing dimethylformamide by rotary evaporation, and adding dichloromethaneDiluting with alkane, washing with saturated brine, and adding anhydrous Na to organic phase2SO4Drying, spin-drying, and separating by column chromatography;
b1) in the step b) of the characteristic c3), the compound of the formula I-3 is dissolved in acetic anhydride, and hydrogen bromide/acetic acid solution is added for carrying out 1-bit bromination;
b2) in the step b) of the characteristic c3), the reaction temperature is 23-25 ℃;
b3) in step b) of feature c3), the preparation method further comprises the step of isolating and purifying the compound of formula I-2: adding dichloromethane for dilution, adding saturated sodium bicarbonate solution under ice bath, extracting with separating funnel, back extracting water phase, collecting organic phase, mixing, and adding anhydrous Na2SO4Drying, filtering and spin-drying the solvent;
b4) in step b) of feature c3), the preparation method further comprises, before step b), step a): the compound of formula I-3 is prepared by acetylating and protecting hydroxyl in the compound of formula I-4, and the reaction equation is as follows:
Figure FDA0002830880320000021
6. the method of preparing a compound of claim 5, further comprising any one or more of the following features:
b11) characteristic b1), the amount ratio of the compound of formula I-3, acetic anhydride and hydrogen bromide/acetic acid solution being 10.3 mmol: 7mL of: 17.5 mL;
a1) in the step a) of the characteristic b4), under the protection of inert gas, the compound of the formula I-4 and acetic anhydride realize the acetylation protection of hydroxyl in the compound of the formula I-4 under the pyridine condition to prepare the compound of the formula I-3;
a2) in step a) of feature b4), the compound of formula I-4, acetic anhydride and pyridine are used in a ratio of 14.6 mmol: 18 ml: 30 ml;
a3) in the step a) of the characteristic b4), the reaction temperature is 23-25 ℃;
a4) in step a) of feature b4), the preparation method further comprises formulaI-3 separation and purification steps of the compound: spin-drying the solvent, diluting with dichloromethane, and sequentially diluting with saturated CuSO4Washed with saturated brine, and the organic phase was washed with anhydrous Na2SO4Drying, filtering and spin-drying the filtrate.
7. The method of making a nitric oxide donor prodrug compound of claim 2, comprising the steps of:
step d): the compound of formula I is prepared by deacetylating a compound of formula I-1 according to the following reaction equation:
Figure FDA0002830880320000031
8. the method of making a nitric oxide donor prodrug compound of claim 7, further comprising any one or more of the following features:
d1) in step d), the compound of formula I-1 is deacetylated in a sodium methoxide and methanol system;
d2) the molar ratio of the compound of formula I-1 to sodium methoxide was 2.67: 0.40, the ratio of the amount of the compound of formula I-1 to methanol was 2.67 mmol: 15 mL;
d3) in the step d), the reaction temperature is 23-25 ℃;
d4) in step d), the preparation method further comprises the steps of separating and purifying the compound shown in the formula I: after the reaction is finished, adding methanol for dilution, then adding activated strong-acid cation exchange resin, adjusting the pH value to 7-8, filtering, and spin-drying to obtain yellow oily liquid; the yellow oily liquid was dissolved in distilled water and lyophilized.
9. The use of a compound as claimed in claim 1 for the preparation of a nitric oxide donor prodrug compound having the structure shown in formula I:
Figure FDA0002830880320000032
10. use of the nitric oxide donor prodrug compound of claim 2 for the controlled release of nitric oxide catalysed by an endocellulase.
11. The use of the nitric oxide donor prodrug compound of claim 10 for the controlled release of nitric oxide under natural bioorthogonal catalysis by endocellulase Cel5A-h 38.
12. Use of the nitric oxide donor prodrug compound of claim 2 for the preparation of a controlled release nitric oxide drug.
13. The use of the nitric oxide donor prodrug compound of claim 2 in the manufacture of a medicament for the treatment of diabetic lower limb ischemia, skin injury and myocardial infarction.
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