Iron ion carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an iron ion carrier and a preparation method and application thereof. The iron ion carrier disclosed by the invention has the advantages that the iron ion carrier disclosed by the invention can have relatively good binding activity with iron ions and ferrous ions, and has relatively low cytotoxicity and relatively long in-vivo drug half-life period.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an iron ion carrier and a preparation method and use thereof. Background Art

[0002] Iron is an essential element in living organisms, but since the human body cannot excrete iron at a rate of more than one milligram per day, excess free iron with high oxidative properties can cause oxidative stress, accelerate lipid peroxidation by producing highly active hydroxyl free radicals, damage important organs such as the liver and heart, increase the risk of heart failure, cirrhosis and cancer, arthritis, dyslipidemia, diabetes and gonadal dysfunction, and may be associated with several neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease and β-helical protein-related neurodegeneration, etc.). In addition, patients with refractory anemia such as thalassemia need to be transfused with 1-3 units (1 unit = 200 mg of iron) of red blood cells regularly every 2-4 weeks. When about 50-100 units of red blood cells are transfused, basically irreversible organ damage caused by iron overload and toxicity will occur.

[0003] Chelation therapy has been widely used to improve the condition of patients with iron overload. Chelators can form iron complexes, promote iron excretion, remove plasma non-transferrin-bound iron, remove excess iron from cells, and restore iron levels in the body to safe levels. However, FDA-approved iron chelators, such as deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX), have unfavorable pharmacokinetics, nonspecific tissue distribution, and significant adverse reactions. For example, the half-life of natural DFO is very short (e.g., 5-15 minutes in rodents) and is distributed in non-target tissues, including kidney, muscle, and lung. This in turn can lead to toxic side effects such as endocrine dysfunction, growth retardation, and peripheral neuropathy. And DFP and DFX also show significant toxicity (e.g., gastrointestinal bleeding, granulocytopenia, neutropenia, thrombocytopenia, liver fibrosis, and renal failure), which increases drug-induced mortality. Therefore, a new iron carrier strategy with improved pharmacokinetics and biodistribution, as well as efficacy and safety, is urgently needed.

[0004] Aryloxazoles are a common class of natural siderophore products that play an important role in iron binding and absorption.

[0005] The present disclosure aims to provide a type of iron ion carrier containing an aryl oxazoline structural unit and a preparation method and use thereof. Summary of the invention

[0006] The technical problem to be solved by the present disclosure is to develop a class of iron ion carriers and their preparation methods and uses, in view of the fact that the half-life of the chelating agents currently on the market for improving iron ion overload is very short or they are cytotoxic. The iron ion carriers can have good binding activity with both iron ions and ferrous ions, have lower cytotoxicity, and have a longer in vivo drug half-life.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present disclosure is:

[0008] In one aspect, the present disclosure provides an iron ion carrier and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof, which has a structure shown in the following formula (I):

[0009]

[0010] Q is non-existent or O;

[0011] R1 and R2 are each independently hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 3-6 Cycloalkyl;

[0012] R3 and R4 are each independently N, O, S, -NH2, -NH-, -OH or -SH;

[0013] R5 is C or carbonyl;

[0014] The “…” between R4 and R5 means that R4 and R5 are selected from being connected or not connected, wherein being connected is selected from a single bond or a double bond;

[0015] The “…” between R3 and R5 means that R3 and R5 are selected from being connected or not connected, wherein the connection is selected from a single bond or a double bond;

[0016] When R5 is C, R5 is connected to R3, R4 and the atoms connected thereto to form Wherein, R3 and R4 are each independently N, O or S;

[0017] When R5 is a carbonyl group, R3, R4, and R5 are selected from any of the following:

[0018] (1) R4 and R5 are connected and R3 and R5 are not connected; wherein R4 is -O- or -NH-, and R3 is -NH2, -OH or -SH;

[0019] (2) R3 and R5 are connected and R4 and R5 are not connected; wherein R3 is -O- or -NH-, and R4 is -NH2, -OH or -SH.

[0020] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, when R5 is C, R5 is respectively connected to R3, R4, and the atoms connected thereto to form Wherein, R3 and R4 are each independently N, O or S.

[0021] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof of the present disclosure, when R5 is a carbonyl group, R4 is connected to R5 and R3 is not connected to R5; wherein R4 is -O- or -NH-, and R3 is -NH2, -OH, or -SH.

[0022] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, when R5 is a carbonyl group, R3 is connected to R5 and R4 is not connected to R5; wherein R3 is -O- or -NH-, and R4 is -NH2, -OH, or -SH.

[0023] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, R1 and R2 are each independently hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl. Alkyl is selected from substituted or unsubstituted alkyl; preferably unsubstituted alkyl. Cycloalkyl is selected from substituted or unsubstituted cycloalkyl; preferably unsubstituted cycloalkyl.

[0024] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, R1 and R2 are each independently hydrogen, C 1-5 Alkyl or C 3-5 Cycloalkyl. Alkyl is selected from substituted or unsubstituted alkyl; preferably unsubstituted alkyl. Cycloalkyl is selected from substituted or unsubstituted cycloalkyl; preferably unsubstituted cycloalkyl.

[0025] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, R1 and R2 are each independently hydrogen, C 1-3 Alkyl or C 3-4 Cycloalkyl. Alkyl is selected from substituted or unsubstituted alkyl; preferably unsubstituted alkyl. Cycloalkyl is selected from substituted or unsubstituted cycloalkyl; preferably unsubstituted cycloalkyl.

[0026] In some preferred embodiments of the present disclosure, according to the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, R1 and R2 are each independently methyl, ethyl, propyl, or hydrogen.

[0027] In some preferred embodiments of the present disclosure, the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof according to the present disclosure has a structure shown in the following formula (I1), formula (I2), formula (I3), formula (I4), or formula (I5):

[0028]

[0029]

[0030] Among them, formula (I1) is recorded as compound SA; formula (I2) is recorded as compound SB; formula (I3) is recorded as compound SC; formula (I4) is recorded as compound SD; formula (I5) is recorded as compound SE.

[0031] In some preferred embodiments of the present disclosure, the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof according to the present disclosure has a structure shown in the following formula (I1):

[0032]

[0033] Herein, formula (I1) is denoted as compound SA.

[0034] In some preferred embodiments of the present disclosure, the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof according to the present disclosure has a structure shown in the following formula (I2):

[0035]

[0036] Wherein, formula (I2) is recorded as compound SB.

[0037] In some preferred embodiments of the present disclosure, the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof according to the present disclosure has a structure shown in the following formula (I3):

[0038]

[0039] Wherein, the formula (I3) is recorded as compound SC.

[0040] In some preferred embodiments of the present disclosure, the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof according to the present disclosure has a structure shown in the following formula (I4):

[0041]

[0042] Wherein, formula (I4) is recorded as compound SD.

[0043] In some preferred embodiments of the present disclosure, the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof according to the present disclosure has a structure shown in the following formula (I5):

[0044]

[0045] Wherein, formula (I5) is recorded as compound SE.

[0046] On the other hand, the present disclosure provides a method for preparing the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor as described above, comprising the following steps:

[0047] (1) synthesizing the iron ion carrier by a fermentation method;

[0048] (2) Using liquid chromatography to separate and obtain an eluent containing the iron ion carrier.

[0049] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, the liquid chromatography conditions are: the eluent is a petroleum ether / ethyl acetate system, which is gradually reduced from a 10:0 petroleum ether / ethyl acetate system (v / v) to a 0:10 petroleum ether / ethyl acetate system (v / v).

[0050] In some preferred embodiments of the present disclosure, the preparation method according to the present disclosure further includes the following steps: subjecting the eluate containing the iron ion carrier to semi-preparative HPLC separation to obtain compound SA, compound SB, compound SC, compound SD, and compound SE.

[0051] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, the semi-preparative HPLC separation conditions are: using an SB-C18 chromatographic column; the UV detection wavelength is set to 232 and 254 nm; the flow rate is 3 mL / min; the mobile phase A is deionized water, and the mobile phase B is chromatographic grade acetonitrile, and the semi-preparative HPLC separation is performed according to 0-18min 20-38% mobile phase B, 18-19min 39-100% mobile phase B, 19-20min 100-20% mobile phase B, and 20-25min 20% mobile phase B.

[0052] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, the SB-C18 chromatographic column is 5 μm, 9.4×250 mm.

[0053] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, compound SA appears at a retention time of 12.8 to 13.2 minutes, compound SB appears at 12.1 to 12.5 minutes, compound SC appears at 8.0 to 8.4 minutes, compound SD appears at 9.4 to 9.8 minutes, and compound SE appears at 6.1 to 6.5 minutes.

[0054] In some preferred embodiments of the present disclosure, according to the preparation method of the present disclosure, compound SA appears at a retention time of 13.0 minutes, compound SB appears at 12.3 minutes, compound SC appears at 8.2 minutes, compound SD appears at 9.6 minutes, and compound SE appears at 6.3 minutes.

[0055] On the other hand, the present disclosure provides the use of the iron ion carrier as described above and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, or the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof obtained by the preparation method as described above, in the preparation of a drug for treating diseases related to iron ion overload.

[0056] In some preferred embodiments of the present disclosure, according to the uses of the present disclosure, the iron ion overload-related diseases include but are not limited to thalassemia or neurodegenerative diseases, etc.

[0057] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the iron ion overload-related disease is selected from thalassemia or neurodegenerative disease.

[0058] In some preferred embodiments of the present disclosure, according to the uses of the present disclosure, the neurodegenerative diseases include but are not limited to Parkinson's disease, Alzheimer's disease or propeller protein-related neurodegenerative diseases, etc.

[0059] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease or propeller protein-related neurodegenerative diseases.

[0060] On the other hand, the present disclosure provides use of the iron ion carrier as described above and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, or the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof obtained by the preparation method as described above, in the preparation of a drug for treating thalassemia.

[0061] On the other hand, the present disclosure provides use of the iron ion carrier as described above and / or its pharmaceutically acceptable salt, solvate, or precursor thereof, or the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof obtained by the preparation method as described above, in the preparation of a drug for treating neurodegenerative diseases.

[0062] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is used for one or more uses selected from the following: 3+ Combine with Fe 2+ Combined with Cu 2+ Combine with Mg 2+ Combined with Mn 2+ Combined with Co 2+ Combined with Ni 2+ Combined with Zn 2+ Binding; blood-brain barrier permeable.

[0063] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is used with Fe 3+ Combine.

[0064] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is used with Fe 2+ Combine.

[0065] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is used with Fe 3+ , Fe 2+ Combine.

[0066] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is used with Cu 2+ Combine.

[0067] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug has blood-brain barrier permeability.

[0068] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug has at least one effect selected from the following:

[0069] It does not cause cytotoxicity to healthy organisms;

[0070] The half-life of the drug in the body is long.

[0071] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug has the following effects: it does not produce cytotoxicity to a healthy body.

[0072] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug has the following effect: the half-life of the drug in vivo is longer.

[0073] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is prepared as a preparation for treating diseases related to iron ion overload; the preparation contains the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor as the sole active ingredient.

[0074] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is prepared as a preparation for treating diseases related to iron ion overload; the preparation comprises the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, precursor thereof, and also comprises a pharmaceutically acceptable carrier.

[0075] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the pharmaceutically acceptable carrier includes one or more of a solvent, a solubilizer, a cosolvent, an emulsifier, a flavoring agent, an olfactory agent, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a pH regulator, a stabilizer, a surfactant, and a preservative. Fillers are also called diluents, such as wheat starch, tapioca starch, corn starch, potato starch, dextrin, microcrystalline cellulose, lactose, etc. Examples of flavoring agents include, but are not limited to, stevioside, glycyrrhizin, mogroside, acesulfame potassium, aspartame, sucralose, isomaltulose, etc. Examples of lubricants include, but are not limited to, magnesium stearate, talc, micropowdered silica gel, magnesium lauryl sulfate, etc.

[0076] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug forms a preparation for treating diseases related to iron ion overload; the preparation comprises the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, precursor thereof, and one or more other active ingredients for treating diseases related to iron ion overload.

[0077] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the preparation is a solid preparation, a semisolid preparation or a liquid preparation.

[0078] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the solid preparation is a tablet, a capsule, a granule or a pill.

[0079] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the semisolid preparation is a gel, a suppository or an ointment.

[0080] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the liquid preparation is an emulsion, a mixture, a suspension or a solution.

[0081] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is prepared into a preparation for treating diseases related to iron ion overload; the content of the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in the unit preparation is 5 to 50 mg, preferably 5 to 30 mg, preferably 10 to 30 mg, preferably 15 to 25 mg, preferably 18 to 22 mg, and more preferably 20 mg.

[0082] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is administered by one or more of oral, intravenous, intraperitoneal, intramuscular, rectal or subcutaneous administration.

[0083] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the drug is administered by one or more of oral, intravenous, and intraperitoneal administration.

[0084] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the medicine is a tablet, a capsule, an injection, a powder, a drop pill, a granule, a syrup, a chewable tablet or a patch.

[0085] In some preferred embodiments of the present disclosure, according to the use of the present disclosure, the medicine is a tablet, a capsule, an injection, a powder, a drop pill, a granule, a syrup or a chewable tablet.

[0086] The present disclosure has the following advantages:

[0087] (1) The present disclosure provides an iron ion carrier that has good binding activity with both iron ions and ferrous ions.

[0088] (2) The present disclosure provides an iron ion carrier with low cytotoxicity, which can reduce toxic side effects and is relatively safe.

[0089] (3) The present disclosure provides an iron ion carrier with a longer drug half-life in vivo.

[0090] (4) The present disclosure provides a method for preparing an iron ion carrier, which can obtain compound SA, compound SB, compound SC, compound SD, and compound SE, which are used for diseases related to iron ion overload.

[0091] (5) The present disclosure provides iron ion carriers, which are expected to provide new strategies for diseases related to iron ion overload. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. The drawings herein are incorporated into the specification and constitute a part of the specification, showing embodiments that conform to the specification, and are used together with the specification to explain the principles of the specification.

[0093] Figure 1 The HPLC detection results of the fermentation product of S. spinosa are shown.

[0094] Figure 2 Gradient elution of unknown chemical components in S. spinosa is shown.

[0095] Figure 3 The structure of compound SA-SE is shown.

[0096] Figure 4 The X-ray single crystal structure of compound SA is shown.

[0097] Figure 5 The 1H NMR spectrum of compound SA in deuterated acetone (acetone-d6) is shown.

[0098] Figure 6 The 13C NMR spectrum of compound SA in deuterated acetone is shown.

[0099] Figure 7 The 1H–1H correlation spectrum of compound SA in deuterated acetone is shown.

[0100] Figure 8 The 1H–13C heteronuclear multiple bond correlation spectrum of compound SA in deuterated acetone is shown.

[0101] Fig. 9 The 1H NMR spectrum of compound SB in deuterated chloroform (CDCl 3 ) is shown.

[0102] Fig.10 The 13C NMR spectrum of compound SB in deuterated chloroform is shown.

[0103] Fig.11 The 1H–1H correlation spectrum of compound SB in deuterated chloroform is shown.

[0104] Fig.12 The 1H–13C heteronuclear multiple bond correlation spectrum of compound SB in deuterated chloroform is shown.

[0105] Fig.13 The 1H NMR spectrum of compound SC in deuterated chloroform is shown.

[0106] Fig.14 The 13C NMR spectrum of compound SC in deuterated chloroform is shown.

[0107] Fig.15 The 1H–1H correlation spectrum of compound SC in deuterated chloroform is shown.

[0108] Fig.16 The 1H–13C heteronuclear multiple bond correlation spectrum of compound SC in deuterated chloroform is shown.

[0109] Fig.17 The 1H NMR spectrum of compound SD in deuterated acetone is shown.

[0110] Fig.18 The 13C NMR spectrum of compound SD in deuterated acetone is shown.

[0111] Fig.19 The 1H–1H correlation spectrum of compound SD in deuterated acetone is shown.

[0112] Fig. 20 The 1H–13C heteronuclear multiple bond correlation spectrum of compound SD in deuterated acetone is shown.

[0113] Fig.21 The 1H NMR spectrum of compound SE in deuterated acetone is shown.

[0114] Fig. 22 The 13C NMR spectrum of compound SE in deuterated acetone is shown.

[0115] Fig.23 The 1H–1H correlation spectrum of compound SE in deuterated acetone is shown.

[0116] Fig.24 The 1H–13C heteronuclear multiple bond correlation spectrum of compound SE in deuterated acetone is shown.

[0117] Fig.25 The color change of compound SA after mixing with different proportions of FeCl3 is shown.

[0118] Fig.26 The UV-visible spectrum detection results of compound SA and FeCl3 mixed in different proportions are shown.

[0119] Fig. 27 The compound SA and Fe 3+ Absorbance results at 500nm and 650nm.

[0120] Fig.28 The compound SA and Fe 3+ Absorbance titration results.

[0121] Fig.29 The compound SA (1) and Fe 2+ Cu 2+ The color of the reaction changes.

[0122] Fig.30 The cytotoxicity of the iron ion carrier of the present invention to HepG2 cells is shown.

[0123] Fig.31 Shown are the effects of iron chelation treatment with different siderophores on healthy HepG2 cells.

[0124] Fig.32 Shown are the effects of iron chelation with different iron carriers on iron-overloaded HepG2 cells.

[0125] Fig.33 Shown is a cellular iron assay of iron-overloaded HepG2 cells after iron chelation treatment.

[0126] Fig.34 The results of EIC-HRMS detection of blood-brain barrier permeability in vivo are shown. DETAILED DESCRIPTION

[0127] Definition and Description

[0128] In order to make it easier to understand the present disclosure, some technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell and tissue culture, microbiology-related terms and laboratory operation steps used herein are terms and routine steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definition and explanation of the relevant terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in the present application are only for describing specific embodiments and are not intended to be limited.

[0129] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0130] As used herein, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or apparatus comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or apparatuses.

[0131] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0132] In the present disclosure, pharmaceutically acceptable salts of ferric ionophores refer to those salts that are non-toxic in dosage administration.

[0133] In the present disclosure, examples of the solvate of the iron ion carrier include, for example, hydrates, alcoholates, and the like.

[0134] In the present disclosure, the precursor of the iron ion carrier refers to a compound that is inactive or has low activity in vitro and releases the iron ion carrier through enzymatic or non-enzymatic conversion in vivo to exert its medicinal effect.

[0135] Saccharopolyspora spinosa, also known as S. spinosa.

[0136] Example

[0137] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

[0138] Example 1

[0139] The preparation method of the iron ion carrier compound of the present invention:

[0140] (1) Fermentation process

[0141] The WHU1107 strain that efficiently produces spinosad was inoculated onto the ABB13 plate medium and cultured at 30°C for 7 days. The culture medium was then fermented according to the fermentation medium described in the literature (Guojun Y, Yuping H, et al., A New Medium for Improving Spinosad Production by Saccharopolyspora spinosa, Jundishapur J. Microbiol., 2016, 9(6): e16765). The culture medium was cultured at 28°C, 250rpm, and 60% humidity for 15 days. The whole bottle of fermentation culture was mixed with anhydrous ethanol at a volume ratio of 1:4, and the supernatant was taken for HPLC analysis after centrifugation. Figure 1 shown.

[0142] HPLC conditions: chromatographic column: Agilent ZORBAX SB-C18 (150×4.6 mm, 3.5 μm), methanol:acetonitrile:water=45:45:10 (containing 0.5% ammonium acetate), isocratic elution for 20 min.

[0143] UV detection wavelength: 250nm

[0144] (2) Identification process

[0145] The WHU1107 strain, which efficiently produces spinosad, was inoculated onto ABB13 plate medium and cultured at 30°C for 7 days. The total extract after fermentation was dissolved in methanol and subjected to column chromatography on a 200-300 mesh silica gel packing. The eluent was a petroleum ether / ethyl acetate system, which was gradually reduced from 10:0 (v / v) to 0:10 (v / v). The iron ion carrier compounds in the effluent began to appear when the petroleum ether:ethyl acetate = 5:5 (v / v). All fractions containing iron ion carrier compounds detected by mass spectrometry were pooled and then chromatographically analyzed using an Angilent ZORBAX SB-C18 column (5μm, 9.4×250mm), with UV detection wavelengths set to 232 and 254nm and a flow rate of 3mL / min. Mobile phase A was deionized water, mobile phase B was chromatographic grade acetonitrile, and semi-preparative HPLC separation was performed according to 0-18 min 20-38% mobile phase B, 18-19 min 39-100% mobile phase B, 19-20 min 100-20% mobile phase B, and 20-25 min 20% mobile phase B. Compound SA (Spinactin A, 1) appeared at a retention time of 13.0 minutes, SB (Spinactin B, 2) appeared at 12.3 minutes, compound SC (Spinactin C, 3) appeared at 8.2 minutes, compound SD (Spinactin D, 4) appeared at 9.6 minutes, and compound SE (Spinactin E, 5) appeared at 6.3 minutes. Figure 2 shown.

[0146] The structures of the compounds were identified by combining HPLC, HPLC-ESI-MS, HR-ESI-MS / MS, 1D NMR, 2D NMR and X-ray single crystal diffraction, and the main product compound SA (Spinactin A, 1) was determined as Figure 4 As shown, as well as analogues compound SB (Spinactin B, 2), compound SC (Spinactin C, 3), compound SD (Spinactin D, 4), compound SE (Spinactin E, 5), among which compound SA, compound SB, compound SC, compound SD, and compound SE are new structures that have not been reported before, as shown Figure 3 The NMR spectrum of compound SA is shown in Figure 5 , Figure 6 , Figure 7 , Figure 8 ; The NMR spectrum of compound SB is shown in Fig. 9 , Fig.10 , Fig.11 , Fig.12 ; The NMR spectrum of compound SC is shown in Fig.13 , Fig.14 , Fig.15 , Fig.16 ; The NMR spectrum of compound SD is shown in Fig.17 , Fig.18 , Fig.19 , Fig. 20 ; The NMR spectrum of compound SE is shown in Fig.21 , Fig. 22 , Fig.23 , Fig.24 .

[0147] The structure of the iron ion carrier compound of the present invention is as follows:

[0148]

[0149] Among them, Q is non-existent or O;

[0150] R1 and R2 are each independently methyl, ethyl, propyl or hydrogen;

[0151] R3 and R4 are each independently N, O, S, -NH2, -NH-, -OH or -SH;

[0152] R5 is C or carbonyl;

[0153] The “…” between R4 and R5 means that R4 and R5 are selected from being connected or not connected, wherein being connected is selected from a single bond or a double bond;

[0154] The “…” between R3 and R5 means that R3 and R5 are selected from being connected or not connected, wherein the connection is selected from a single bond or a double bond;

[0155] When R5 is C, R5 is connected to R3, R4 and the atoms connected thereto to form Wherein, R3 and R4 are each independently N, O or S;

[0156] When R5 is a carbonyl group, R3, R4, and R5 are selected from any of the following:

[0157] (1) R4 and R5 are connected and R3 and R5 are not connected; wherein R4 is -O- or -NH-, and R3 is -NH2, -OH or -SH;

[0158] (2) R3 and R5 are connected and R4 and R5 are not connected; wherein R3 is -O- or -NH-, and R4 is -NH2, -OH or -SH.

[0159] In some preferred embodiments of the present disclosure, the iron ion carrier compound of the present invention has a structure shown in the following formula (I1), formula (I2), formula (I3), formula (I4) or formula (I5):

[0160]

[0161]

[0162] Among them, formula (I1) is compound SA; formula (I2) is compound SB; formula (I3) is compound SC; formula (I4) is compound SD; formula (I5) is compound SE.

[0163] Table 1. NMR results of compound SA and compound SB

[0164]

[0165] Table 2. NMR results of compound SC and compound SD

[0166]

[0167] Table 3. Magnetic results of compound SE

[0168]

[0169]

[0170] Table 4. Single crystal data of compound SA

[0171]

[0172]

[0173] Example 2

[0174] Compound SA and Fe 3+ Chelation

[0175] Since the structure of the iron ion carrier compound of the present invention has similar structural units to the reported iron carrier pyochelin, it is inferred that the iron ion carrier compound of the present invention has the ability to bind iron ions, and the iron ion carrier compound of the present invention is used to bind Fe by spectrophotometry. 3+ ability to be tested.

[0176] Compound SA and Fe 3+ The coordination number

[0177] A 4 mM light yellow solution of the ligand compound SA was reacted with a 400 μM yellow 4 mM FeCl3 solution at different ratios from 10:0 to 0:10. The color change of the reaction was as shown in FIG. Fig.25 As shown, it can be found that when the two are mixed, compound SA can quickly chelate Fe 3+ The corresponding complex is produced, and the whole system obviously changes color. 3+ As the ratio of increases, the color changes from light yellow to blue to red and then to yellow. Fig.25 As shown, the binding of compound SA to Fe 3+ The ability to test the results is as follows Fig.26 As shown, it indicates that the chelating product produced by FeCl3 and the ligand compound SA is complex, and may produce two complexes with different proportions, blue and red respectively.

[0178] Subsequently, we used UV wavelengths of 500nm and 650nm to test the absorbance values ​​A of solutions with different ratios, converted the ligand ratios and plotted the Ax curve to calculate the effect of compound SA on Fe 3+ The chelating ability of Fig. 27 As shown in the figure, the absorbance at 500nm reaches the highest value at 8:2, while the absorbance at 650nm reaches the highest value at 5:5. The coordination numbers of the two points are 4 and 1 respectively, which indicates that Fe 3+ After combining with the ligand compound SA (abbreviated as SpnA), two complexes with different coordination numbers are produced. When the volume fraction of compound SA is high, the 4-coordinated complex Fe(SpnA)4 mainly exists, while when the volume fraction of Fe 3+ When the volume fraction is high, the main component present is 1-coordinated Fe (SpnA).

[0179] Measuring the siderophore complexation reaction stability constant:

[0180] Since the absorbance of the solution is proportional to the concentration of the complex, under the conditions of two different total molar numbers, the two points with the same absorbance value A in the curve should have the same concentration of the complex. Therefore, if we change the total molar number of the reaction system and test the absorbance value again, we can combine the two measurement results to list the equation to obtain the concentration of compound SA and Fe 3+ The concentration of the chelate formed is used to further calculate the equilibrium constant K. Since in the previous experiment, compound SA and Fe 3+ The reaction product at 400 nm has a faster reaction efficiency and a stronger absorbance value, so we further adjusted the total molar number of the reaction system to 200 μM and measured the reaction between compound SA and Fe 3+ After incubation, the absorbance at 500 nm changes with the molar ratio as shown in Fig.28 shown.

[0181] Therefore, when the absorbance value A is constant, we can combine the two reactions to list the following equation to obtain the reaction ratio of compound SA and Fe when the total molar number is 200 μM. 3+ The concentration X of the Fe(SpnA)4 product generated by the reaction is 0.01799. Based on this, the reaction constant K is further calculated to be 1.946×105. When K>105, the chemical reaction can be considered complete. Therefore, at this time, it can be considered that the compound SA and Fe 3+ The combination can be completely reacted into the product Fe(SpnA)4.

[0182]

[0183] Example 3

[0184] Chelation of compound SA with other metals

[0185] Therefore, in addition to testing the compound SA and Fe 3+ In addition to the chelating ability of compound SA to Fe 2+ ,Mg 2+ , Mn 2+ ,Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ The binding capacity of other metals with important biological activities was tested by mixing equal amounts of compound SA with the corresponding metal ions and incubating them at 30°C for 10 h. 2+ Cu 2+ After mixing, the entire reaction system shows obvious color changes visible to the naked eye, such as Fig.29 As shown, SA(1) indicated that the chelation reaction had successfully occurred.

[0186] The properties of copper ligands and the planar square geometry exhibited by copper complexes are very likely to cause DNA-specific damage very similar to platinum compounds. Copper carriers such as tetrathiomolybdate and clioquinol have been used for cancer therapy and have demonstrated anticancer activity in vitro and in preclinical models. The synergistic effect of disulfiram and copper gluconate is currently being studied in a phase II clinical trial for recurrent glioblastoma. In addition to Fe 3+ In addition, compound SA has 2+ , Fe 2+ Its chelating ability of other metals indicates that in addition to its iron carrier function, it has the potential to be further developed as a carrier of other metals.

[0187] Example 4

[0188] Cytotoxicity of the iron ion carrier compounds of the present invention

[0189] Since the FDA-approved iron carrier drugs commonly used in clinical practice, deferiprone (DFP) and deferoxamine (DFO) have high cytotoxicity, in order to explore the potential of the iron ion carrier compounds of the present invention for subsequent activity research, the CCK-8 method was first used to test the cytotoxicity of compound SA, compound SB, compound SC, compound SD, and compound SE to healthy HepG2 cells. The results are as follows Fig.30 , indicating that within the test concentration of 200 μM, compound SA, compound SB, compound SC, compound SD, and compound SE did not produce obvious cytotoxicity to healthy HepG2 cells, indicating that IC50>>200 μM, which is better than the IC50 of DFP reported in the literature (100 μM).

[0190] Example 5

[0191] Therapeutic activity of the iron ion carrier compounds of the present invention on iron-overloaded cells

[0192] After constructing an iron overload cell model by treating with ferric ammonium citrate (FAC), we further studied the effects of compound SA and iron carriers commonly used in the clinical treatment of iron overload diseases, deferiprone (DFP) and deferoxamine (DFO), on the activity of iron overloaded HepG2 cells. The 6-well culture plate inoculated with iron overloaded HepG2 cells was washed with PBS, and the medium was replaced with 30 μM of the iron carrier compounds to be tested (DFP, DFO, compound SA, compound SB, compound SC, compound SD, compound SE). After culturing for 2 days, CCK-8 detection was performed, and the following test results were obtained. Fig.31 and Fig.32 As shown:

[0193] (1) For healthy HepG2 cells, the cell activity after treatment with 15 μM iron ion carrier compounds of the present invention (Compound SA, Compound SB, Compound SC, Compound SD, Compound SE) and solvent DMSO was not much different, which is consistent with the conclusion that the iron ion carrier compounds of the present invention have low cytotoxicity.

[0194] (2) For healthy HepG2 cells, after treatment with 15 μM deferiprone DFP and deferoxamine DFO, cell activity decreased, with DFP reducing cell activity by 11.34% (p<0.05) and DFO reducing it by 30.79% (p<0.0001) compared with the untreated control. This may be due to the greater cytotoxicity of DFP and DFO to HepG2 cells.

[0195] (3) After healthy HepG2 cells were treated with FAC for iron, the cell activity decreased by 14.47% compared with the untreated group (p<0.05), indicating that iron overload caused by excessive iron affects cell activity.

[0196] (4) For iron-overloaded HepG2 cells, the cell activity of the five iron ion carrier compound treatment groups of the present invention (compound SA, compound SB, compound SC, compound SD, compound SE) increased compared with the DMSO treatment group and recovered to the level of untreated healthy HepG2 cells, indicating that compound SA, compound SB, compound SC, compound SD, and compound SE all have a certain therapeutic effect on iron-overloaded HepG2 cells.

[0197] (5) For iron-overloaded HepG2 cells, there was no significant difference in cell activity between the iron carriers deferoxamine DFO and deferiprone DFP treatment groups and the solvent DMSO control group, which may be due to the high cytotoxicity at the 15 μM treatment concentration.

[0198] After that, the intracellular iron content was further measured using the o-phenanthroline method. In a 6-well cell culture plate, iron-overloaded cells were treated with 15 μM iron carrier for 2 days, then digested with trypsin, washed twice with 1× PBS solution, and collected cell samples. After quick freezing in liquid nitrogen, they were placed in a -80°C refrigerator for later use. Before the test, we lysed the cell samples by ice bath ultrasonication, and used the hydroxylamine hydrochloride in the kit to remove Fe 3+ Reduction to Fe 2+ , Fe 2+ Under weak acid conditions, it forms an orange-red complex with o-phenanthroline, and the corresponding ultraviolet absorption is detected at 510nm for quantitative analysis, and the following test results are obtained: Fig.33 As shown:

[0199] (1) The intracellular iron content of iron-overloaded cells (FAC) treated with FAC was significantly increased by 54.9% compared with normal cells (Control) (p<0.01).

[0200] (2) Treatment with different iron carriers can reduce the iron ion level in iron-overloaded cells to normal cell (Control) levels.

[0201] (3) Deferiprone (DFP) and compound SA reduced the iron ion concentration in iron-overloaded cells the most. DFP reduced the iron ion concentration in iron-overloaded HepG2 cells by 53.2% (p<0.01), and compound SA reduced it by 45.4% (p<0.01).

[0202] (4) Treatment with deferoxamine (DFO) and compounds SB, SC, SD, and SE can also reduce the intracellular iron concentration of iron-overloaded HepG2 cells to normal cell levels.

[0203] Example 6

[0204] Blood-brain barrier permeability of compound SA

[0205] The brain is also an important organ susceptible to iron overload, and elevated iron ion concentrations can be detected in the brain tissue of many patients with neurodegenerative diseases. However, the means of treating brain iron deposition in clinical studies are still limited, partly because most existing iron chelating drugs cannot cross the blood-brain barrier (Abbruzzese et al., 2011). Therefore, blood-brain barrier permeability is also an important indicator for evaluating the effect of iron carriers.

[0206] The blood-brain barrier permeability of compound SA and the iron carriers deferiprone (DFP) and deferoxamine (DFO) commonly used in clinical practice was evaluated through in vivo experiments. The test was divided into compound SA, DFP, DFO and negative control PBS groups. We administered 5 mg / kg of the drug to seven-week-old C57BL / 6J mice by intraperitoneal injection. 60 minutes after administration, 100 mg / kg of sodium amobarbital was injected intraperitoneally for anesthesia. After blood was collected, sterile 1× PBS buffer was used for cardiac perfusion until the mouse liver and kidneys turned white to remove blood from the brain tissue as much as possible.

[0207] After the perfusion, the mice were confirmed dead by cervical dislocation and the brain was sampled. At this time, there should be no visible blood in the meningeal vessels. The blood was also centrifuged at 4°C, 1000g for 5 minutes to collect plasma. The brain tissue and plasma samples were quickly frozen with liquid nitrogen and then stored in a -80°C refrigerator.

[0208] Before the test, the centrifuge tube containing the brain tissue was thawed on ice, and 200 μL of pre-cooled 1× PBS buffer was added, and then the mouse brain tissue was homogenized in an ice bath using an ultrasonic disruptor. Then, 100 μL of tissue homogenate and thawed serum samples were taken out with a cut gun tip, and 3 volumes of chromatographic grade methanol containing 0.1% formic acid were added to each of them. After vortex mixing for 10 minutes, centrifuge at 13200 rpm for 10 minutes to fully precipitate the protein. The supernatant was transferred to a new 1.5 mL centrifuge tube, placed in a freeze dryer for overnight drying, fully dissolved with 50 μL of chromatographic grade methanol, and centrifuged at 13200 rpm for 10 minutes before taking the supernatant for EIC-HRMS detection. The corresponding standards of compounds SA, DFP and DFO were subjected to the same operation.

[0209] The blood-brain barrier permeability EIC-HRMS test results of each iron carrier-treated sample are as follows Fig.34 As shown, we can find that:

[0210] (1) The molecular ions corresponding to the three iron carriers could not be detected in the PBS control.

[0211] (2) The molecular ion peaks corresponding to deferiprone DFP, deferoxamine DFO and compound SA with the same retention time as the standard can be detected in serum.

[0212] (3) Only the molecular ion peaks of compounds SA and DFP were detected in brain tissue samples, indicating that they can pass through the blood-brain barrier.

[0213] (4) After the same treatment, the response intensity of DFP in serum and brain tissue homogenate samples was the same, while the response intensity of compound SA in brain tissue was significantly stronger than that in plasma, indicating that compound SA has stronger blood-brain barrier permeability than DFP.

[0214] (5) After the same administration time, the remaining response intensity of compound SA in plasma and brain tissue samples was significantly greater than that of DFP and DFO samples. It is speculated that this may be caused by its slower metabolism in the body. The drug metabolism study of the iron ion carrier compound of the present invention can be carried out in the future.

[0215] Therefore, we can conclude that the relationship between the blood-brain barrier permeability of the three iron carriers tested is compound SA>DFP>DFO, and compound SA may have a better effect in treating local brain tissue iron deposition caused by neurodegenerative diseases.

[0216] The present disclosure proposes a new type of iron ion carrier containing an aryl oxazoline structural unit, which has good binding activity with both iron and ferrous ions. Cell experiments have shown that compound SA can restore the activity of iron-overloaded cells at therapeutic concentrations, and can significantly reduce the total iron concentration in iron-overloaded cells to normal levels, with no significant difference in effect from DFP, and compound SA has lower cytotoxicity (IC50>>200μM). In addition, in addition to iron-overloaded cells, compound SA can significantly reduce the total iron and ferrous ion levels in neurodegenerative β-helical protein-related neurodegenerative cells with iron accumulation. Animal experiments have shown that compound SA is less toxic in mice (LD50>>100mg / kg), and has better blood-brain barrier permeability than DFP and DFO, and a longer drug half-life in vivo.

[0217] The foregoing description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present disclosure and various different selections and changes. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. An iron ion carrier and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof, which has a structure represented by the following formula (I): In the formula, Q is non-existent or O; R1 and R2 are each independently hydrogen, substituted or unsubstituted C 1-6 Alkyl, or substituted or unsubstituted C 3-6 Cycloalkyl; R3 and R4 are each independently N, O, S, -NH2, -NH-, -OH or -SH; R5 is C or carbonyl; The "..." between R4 and R5 means that R4 and R5 are selected to be connected or not connected, wherein, The connection is selected from a single bond or a double bond; "..." between R3 and R5 means that R3 and R5 are selected from being connected or not connected, wherein the connection is selected from a single bond or a double bond; When R5 is C, R5 is connected to R3, R4 and the atoms connected thereto to form Wherein, R3 and R4 are each independently N, O or S; When R5 is a carbonyl group, R3, R4, and R5 are selected from any of the following: (1) R4 and R5 are connected and R3 and R5 are not connected; wherein R4 is -O- or -NH-, and R3 is -NH2, -OH or -SH; (2) R3 and R5 are connected and R4 and R5 are not connected; wherein R3 is -O- or -NH-, and R4 is -NH2, -OH or -SH.

2. The iron ion carrier and / or its pharmaceutically acceptable salt, solvate or precursor thereof according to claim 1, characterized in that: Preferably, R1 and R2 are each independently hydrogen, C 1-3 Alkyl and C 3-4 Cycloalkyl; Preferably, R1 and R2 are each independently methyl, ethyl, propyl or hydrogen; Preferably, the iron ion carrier has a structure shown in the following formula (I1), formula (I2), formula (I3), formula (I4) or formula (I5): Among them, formula (I1) is recorded as compound SA; formula (I2) is recorded as compound SB; formula (I3) is recorded as compound SC; formula (I4) is recorded as compound SD; formula (I5) is recorded as compound SE.

3. The method for preparing the iron ion carrier and / or its pharmaceutically acceptable salt, solvate or precursor thereof according to claim 1 or 2, characterized in that: The following steps are involved: (1) synthesizing the iron ion carrier by a fermentation method; (2) separating and obtaining an eluent containing the iron ion carrier by a liquid chromatography method; Preferably, the liquid chromatography conditions are as follows: the eluent is a petroleum ether / ethyl acetate system, which is gradually reduced from a 10:0 petroleum ether / ethyl acetate system (v / v) to a 0:10 petroleum ether / ethyl acetate system (v / v); Preferably, the method further comprises the following steps: subjecting the eluate containing the iron ion carrier to semi-preparative HPLC separation to obtain compound SA, compound SB, compound SC, compound SD and compound SE.

4. Use of the iron ion carrier according to claim 1 or 2 and / or its pharmaceutically acceptable salt, solvate or precursor thereof, or the iron ion carrier obtained by the preparation method according to claim 3 and / or its pharmaceutically acceptable salt, solvate or precursor thereof in the preparation of a drug for treating diseases related to iron ion overload.

5. The use according to claim 4, characterized in that The iron overload-related disease is selected from thalassemia or neurodegenerative disease; Preferably, the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease or a propeller protein-associated neurodegenerative disease.

6. The use according to claim 4 or 5, characterized in that The drug is used for one or more purposes selected from the following: 3+ Combine with Fe 2+ Combined with Cu 2+ Combine with Mg 2+ Combined with Mn 2+ Combined with Co 2+ Combined with Ni 2+ Combined with Zn 2+ Binding; has blood-brain barrier permeability; Preferably, the drug has at least one effect selected from the following: Not cytotoxic to healthy organisms; or, The half-life of the drug in the body is long.

7. The use according to any one of claims 4 to 6, characterized in that The medicine is prepared into a preparation for treating diseases related to iron ion overload; the preparation uses the iron ion carrier and / or its pharmaceutically acceptable salt, solvate or precursor as the sole active ingredient.

8. The use according to any one of claims 4 to 6, characterized in that The drug is prepared into a preparation for treating diseases related to iron ion overload; the preparation comprises the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, precursor thereof, and also comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes one or more of a solvent, a solubilizer, a cosolvent, an emulsifier, a flavoring agent, an olfactory agent, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a pH regulator, a stabilizer, a surfactant, and a preservative.

9. The use according to any one of claims 4 to 6, characterized in that The drug is prepared as a preparation for treating diseases related to iron ion overload; the preparation comprises the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, precursor thereof, and one or more other active ingredients for treating diseases related to iron ion overload.

10. The use according to any one of claims 4 to 9, characterized in that The drug forms a preparation for treating diseases related to iron ion overload; the content of the iron ion carrier and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in a unit preparation is 5 to 50 mg, preferably 5 to 30 mg, preferably 10 to 30 mg, and more preferably 20 mg.

Images