Benzoyl amidoxime derivative
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-10
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Figure CN121824474A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of medicine, in particular to a benzoyl diamide oxime derivative and a preparation method and application thereof. BACKGROUND
[0002] Nuclear technology has been widely used in all aspects of human life, including scientific research, nuclear power generation, nuclear medical diagnosis and treatment, etc. Nuclear technology has facilitated human life while increasing the risk of exposure to ionizing radiation. Acute radiation syndrome (ARS) is a systemic damage disease caused by whole body or local radiation in a short time, and the clinical manifestations are vomiting, infection, bleeding, etc. Especially under the condition of high-dose whole body irradiation, it may cause severe bacteremia / sepsis, massive loss of body fluids and electrolytes, and the body will suffer devastating blows, with high mortality and high treatment difficulty. According to the size of the irradiation dose and the clinical damage characteristics, it can be divided into three subtypes: hematopoietic acute radiation syndrome, gastrointestinal acute radiation syndrome and neurovascular acute radiation syndrome. At present, the treatment of ARS mainly focuses on hematopoietic acute radiation syndrome, and the main cause of death is the severe damage of acute strong radiation to the hematopoietic system.
[0003] The Toll-like receptor (TLR) family is an important pattern recognition receptor. After the activation of TLRs, a downstream signal cascade reaction can be caused, which activates the MyD88-dependent pathway or the interferon beta TIR domain adapter protein (TRIF)-dependent pathway, and then finally activates transcription factors such as NF-κB, thereby stimulating the secretion of inflammatory cytokines. CBLB502 is a truncated derivative of Salmonella flagellin, and the American Cleveland Company is developing it as an anti-radiation drug. In the mouse models of hematopoietic acute radiation syndrome and gastrointestinal acute radiation syndrome, a single injection of CBLB502 before irradiation can alleviate the radiation damage to the hematopoietic system and gastrointestinal tract of mice, and significantly improve the survival rate of mice. However, protein drugs generally have high production and storage costs, poor injection compliance, and may cause immune reactions, so the development of small molecule TLR agonists for anti-radiation drugs has good prospects. SUMMARY
[0004] The present application aims at the technical defects in the prior art, and provides an O-benzoyl diamide oxime derivative, isomers, prodrugs, pharmaceutically acceptable salts, hydrates or solvates thereof, characterized in that the formula I is as follows,
[0005] Formula I
[0006] wherein R1 is selected from halogen, -CN, hydroxyl, nitro, amino, C1-C6 alkyl, halomethyl, halogenated C1-C4 alkyl, C3-C8 cycloalkyl or heterocycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 alkylthio, halogenated C1-C6 alkoxy, halogenated C1-C6 alkylthio, C2-C4 ester; R2 is selected from halogen, nitro.
[0007] In particular, R1 and R2 are mono- or poly- (2, 3 or 4) substituted on the ring.
[0008] In particular, said halogen is fluorine, chlorine, bromine or iodine.
[0009] Further, said C1-C6 alkylamino is methylamino, ethylamino, propylamino; said C1-C6 alkoxy is methoxy, ethoxy, propoxy; said C1-C6 alkyl is methyl, ethyl, propyl.
[0010] Preferably, R1 is selected from halogen, C1-C3 alkyl.
[0011] Preferably, said O-benzoyl amidoxime derivatives include any one of the following: O-2'-fluoro-5'-nitrobenzoyl-1-(6'-fluoro-2 H -chromen-3-yl)amidoxime O-2'-chloro-5'-nitrobenzoyl-1-(6'-fluoro-2 H -chromen-3-yl)amidoxime O-2'-fluoro-5'-nitrobenzoyl-1-(7'-bromo-2 H -chromen-3-yl)amidoxime O-3'-nitrobenzoyl-1-(7'-bromo-2 H -chromen-3-yl)amidoxime O-2'-chloro-5'-nitrobenzoyl-1-(7'-bromo-2 H -chromen-3-yl)amidoxime O-2'-fluoro-5'-nitrobenzoyl-1-(7'-methyl-2 H -chromen-3-yl)amidoxime O-3'-nitrobenzoyl-1-(7'-methyl-2 H -chromen-3-yl)amidoxime O-2'-amino-5'-fluorobenzoyl-1-(6'-fluoro-2 H -chromen-3-yl)amidoxime The present application also provides a composition comprising the O-benzoyl amidoxime derivative, isomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof according to any one of the preceding claims, and a pharmaceutically acceptable carrier or excipient.
[0012] The present application also provides the use of the O-benzoyl amidoxime derivative, isomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof according to any one of the preceding claims, comprising: for the preparation of a medicament for preventing and / or treating ionizing radiation damage or TLR2 / 1 agonist; optionally, for the preparation of a medicament for preventing and / or treating lethal ionizing radiation damage.
[0013] Further, the present application also provides the use of the O-benzoyl amidoxime derivative, isomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof according to any one of the preceding claims, comprising: for the preparation of a medicament for preventing and / or treating acute radiation sickness, improving survival rate after high-dose radiation exposure, and / or alleviating radiation damage and promoting recovery of the body.
[0014] The O-benzoyl amidoxime derivative provided by the present application is a small molecule TLR2 / 1 agonist, which can have an anti-radiation effect by activating the expression of related anti-radiation cytokines in vivo, and has the characteristics of high efficiency, low toxicity and stable quality. Moreover, the excellent efficacy of the compound of the present application in combating lethal radiation damage highlights its outstanding clinical application value and transformation potential. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 The effects of compounds A-2, A-4 and A-7 on the viability of HEK293T and RAW264.7 cells are shown. DETAILED DESCRIPTION
[0016] TERMS
[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.
[0018] Unless otherwise indicated, the present application employs conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Unless specific definitions are provided, the nomenclature employed in connection with, and the procedures involved in, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those with which a person of ordinary skill in the art is acquainted. Generally, the aforementioned techniques and procedures can be performed by conventional methods known to those of skill in the art, and described in various general and more specific references that are cited and discussed throughout the present specification.
[0019] The term "alkyl" refers to an aliphatic hydrocarbon group, which can be straight-chain or branched. Depending on the structure, alkyl can be a monovalent group or a divalent group (i.e., alkylene). In the present application, alkyl is preferably "lower alkyl" having from 1 to 6 carbon atoms, more preferably alkyl having from 1 to 4 carbon atoms. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, and the like. It should be understood that "alkyl" as referred to herein includes all possible configurations and conformations of such alkyl groups, for example, "propyl" as referred to herein includes n-propyl and isopropyl.
[0020] The term "alkylamino" refers to -NH-alkyl, wherein alkyl is as defined herein. Typical alkylamino groups include, but are not limited to, methylamino, ethylamino, propylamino, and the like.
[0021] The term "alkoxy" refers to -O-alkyl, wherein alkyl is as defined herein. Typical alkoxy groups include, but are not limited to, methoxy, ethoxy, propyloxy, and the like.
[0022] The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.
[0023] The term "amino" refers to the -NH2group.
[0024] The term "benzoyl" refers to -CO-phenyl.
[0025] Whole body 8.5 Gy 60 Cobalt-60 gamma rays (dose rate 56.36 cGy / min) irradiation belongs to the extremely high lethal dose of radiation, which is significantly higher than the median lethal dose of mice, and is recognized as a "refractory model" in the field of radiation damage treatment. High-dose radiation can quickly destroy the core functions of the body through a "cascade amplification effect" — irreversible hematopoietic failure caused by the destruction of hematopoietic stem cell pools in a short time, damage to the gastrointestinal mucosal barrier to induce fatal infection and dehydration, while inducing systemic inflammatory response syndrome (SIRS), ultimately leading to multiple organ systemic collapse, and the damage range, severity and repair difficulty are exponentially increased; in contrast, low-dose radiation mostly only causes reversible mild damage to the hematopoietic system and immune system, the damage mechanism is single and does not trigger multiple organ failure, and the corresponding drugs have limited effect on lethal dose radiation.
[0026] Toll-like receptors (TLRs) are a class of proteins that recognize pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), and are involved in the regulation of the innate immune system. TLR is a type I transmembrane protein composed of an extracellular domain, a transmembrane domain and an intracellular domain. After the binding of ligand to Toll-like receptor is triggered, the TLR homodimer or heterodimer activates the intracellular Toll / IL-1R receptor (TIR) domain to cause downstream signal cascade, and then activates the related signal pathway. There are 10 members in the human TLR family, among which TLR1, TLR2, TLR5, TLR10 and TLR6 are located on the cell membrane surface, TLR3, TLR7, TLR8 and TLR9 are located on the membrane of endoplasmic reticulum, endosome and lysosome, and TLR4 is expressed on both the cell membrane surface and the intracellular membrane. In recent years, TLR agonists have become a hot spot in the field of anti-radiation research. Studies have shown that the activation of TLR can enhance the body's response to radiation, alleviate the damage to the hematopoietic system, gastrointestinal tract and immune system caused by radiation by up-regulating the secretion of cytokines such as IL-6, IL-11, IL-12, G-CSF and TNF-α, and thus improve the protection against radiation damage. In addition, the activation of TLRs helps to accelerate the repair of damaged cells, promote tissue regeneration, and improve the recovery ability of the body to radiation damage.
[0027] TLR2 is the member of the TLR family that recognizes the most PAMPs, and can recognize ligands by forming a heterodimer with TLR1 or TLR6, thereby initiating signal transmission. TLR2 mainly recognizes the constituent components of the cell wall of various microorganisms-lipopeptides, and lipopeptides and their derivatives are the earliest discovered TLR2 agonists. The O-benzoyl gem amide oxime derivative represented by formula I provided in the present application has TLR2 / 1 agonist activity, and TLR2 / 6 has the same signal transduction pathway, and has good prospects in the preparation of anti-radiation drugs.
[0028] The present application provides a compound, which is an O-benzoyl gem amide oxime derivative, and the structure is formula I:
[0029] Formula I
[0030] wherein R1 is selected from halogen, -CN, hydroxyl, nitro, amino, C1-C6 alkyl, halomethyl, halogenated C1-C4 alkyl, C3-C8 cycloalkyl or heterocycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 alkylthio, halogenated C1-C6 alkoxy, halogenated C1-C6 alkylthio, C2-C4 ester group; and R2 is selected from halogen, nitro.
[0031] The above O-benzoyl amidoxime derivatives also include geometric isomers of the compounds of formula I, pharmaceutically acceptable salts thereof, hydrates or solvates thereof, and pharmaceutical compositions of the compounds with pharmaceutically acceptable carriers or excipients. Among them: isomers or hydrates, such as optical isomers or racemic compounds; pharmaceutically acceptable salts, which can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, bromides, iodides, acetates, propionates, caprylates, acrylates, formates, isobutyrates, heptanoates, decanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, 2-butine-1,4-dioates, 3-cyclohexyl-2,5- dioates, benzoates, chlorobenzoates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, hippurates, β-hydroxybutyrates, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, glutamates, arginates, lysinates, and the like of the compounds of formula I, preferably hydrochlorides and phosphates.
[0032] The O-benzoyl amidoxime derivatives provided by the present application can be administered orally or parenterally. Oral administration can be in the form of tablets, pills, granules, capsules, coated preparations, oral solutions, emulsions, powders; parenteral administration can be in the form of injections, suppositories, or other suitable forms. The excipients used to manufacture the above dosage forms are all common excipients, such as starch, gelatin, acacia, silica, polyethylene glycol; solvents used in liquid dosage forms are water, ethanol, propylene glycol, vegetable oils (such as corn oil, peanut oil, olive oil, etc.), and the like. Other excipients, such as surfactants, lubricants, disintegrants, preservatives, flavoring agents, pigments, and the like, are also common excipients.
[0033] The compounds of formula (I) can be synthesized using standard synthetic techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein. In addition, the solvents, temperatures, and other reaction conditions given herein can be varied in accordance with the skill in the art. As further guidance, the following synthetic methods can also be utilized.
[0034] The reactions can be used in sequence to provide the compounds described herein; or they can be used to synthesize fragments which are subsequently incorporated by the methods described herein and / or methods known in the art.
[0035] Compounds can be synthesized using methods analogous to those described below, by using appropriate alternative starting materials. Starting materials used to synthesize the compounds described herein can be synthesized or can be obtained from commercial sources. The compounds described herein and other related compounds having different substituents can be synthesized using techniques and starting materials known to those skilled in the art. The general methods for preparing the compounds disclosed herein can be derived from reactions known in the art, and the reactions can be modified by reagents and conditions deemed appropriate by those skilled in the art to introduce the various moieties in the molecules provided herein.
[0036] If desired, the reaction products can be isolated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography, and the like. The products can be characterized using conventional methods, including physical constants and spectral data.
[0037] The present application also provides a method for preparing the compound of Formula I, the synthetic procedure for the O-benzoyl diamidoxime derivative is as follows: Step 1: Synthesis of substituted 1-(2 H Synthesis of substituted 3-cyano-2 H Substituted 3-cyano-2-oxindole 4.0 mmol was placed in a 50 mL flask, hydroxylamine hydrochloride 0.500 g (7.2 mmol) was added, ethanol 16 mL was added, triethylamine 1.01 mL (7.2 mmol) was added, and heated to reflux for 2 hours. The solvent was evaporated under reduced pressure, 20 mL of water was added, and insoluble solid appeared. The mixture was filtered, washed with water, and dried under vacuum. The product was used directly for the next step of the synthesis.
[0038]
[0039] Formula 1
[0040] Step 2: Synthesis of O-benzoyl diamidoxime derivative: see Formula 2, substituted benzoic acid compound 2.0 mmol was placed in a 50 mL flask, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) 2.0 mmol was added, and 15 mL of tetrahydrofuran was added. The mixture was stirred at room temperature for 30 minutes. Substituted 1-(2 H Substituted 3-cyano-2-oxindole 1.5 mmol was added, and the reaction was allowed to proceed at room temperature for 6 hours. The reaction mixture was diluted with 20 mL of water, and insoluble solid appeared. The mixture was filtered after being sonicated for 2 minutes, washed with water, and dried under vacuum to obtain the pure product.
[0041]
[0042] Formula 2
[0043] The present application will be further illustrated in connection with the following specific examples, which in no way should be construed as being limiting to the present application.
[0044] The following specific examples illustrate the preparation of the compounds of the present application and their properties. The reagents used in the examples were either commercially available or synthesized according to the literature procedures. The nuclear magnetic resonance spectra were measured on a Bruker 600M superconducting nuclear magnetic resonance spectrometer, and the mass spectra were measured on an Agilent 1260-G6230A mass spectrometer.
[0045] Example 1: Synthesis of O-2'-fluoro-5'-nitrobenzoyl-1-(6'-fluoro-2 H -benzopyran-3-yl) hydrazine (A-1)
[0046] 1-(6'-fluoro-2 H -benzopyran-3-yl) hydrazine:
[0047] 3-cyano-6-fluoro-2 H -benzopyran 0.701 g (4.0 mmol) was placed in a 50 mL flask, hydroxylamine hydrochloride 0.500 g (7.2 mmol) was added, ethanol 16 mL was added, triethylamine 1.01 mL (7.2 mmol) was added, and the mixture was heated to reflux for 2 hours. The solvent was evaporated under reduced pressure, 20 mL of water was added, and insoluble solid appeared. The mixture was filtered, washed with water, and dried under vacuum to obtain a white solid 0.718 g with a yield of 86.2%. The product obtained can be directly used for the synthesis of the next step.
[0048] O-2'-fluoro-5'-nitrobenzoyl-1-(6'-fluoro-2 H -benzopyran-3-yl) hydrazine:
[0049] 2-fluoro-5-nitrobenzoic acid 0.371 g (2.0 mmol) was placed in a 50 mL flask, DMTMM 0.554 g (2.0 mmol) was added, and 15 mL of tetrahydrofuran was added. The mixture was stirred at room temperature for 30 minutes. 1-(6'-fluoro-2 H -benzopyran-3-yl) hydrazine 0.313 g (1.5 mmol) was added, and the mixture was reacted at room temperature for 6 hours. After the reaction was completed, 20 mL of water was added to the reaction mixture, and insoluble yellow solid appeared. After the mixture was ultrasonicated for 2 minutes, it was filtered, and the product was obtained after drying under vacuum with a yield of 90.8%.
[0050] 1 H NMR (600 MHz, DMSO-d6 ) δ 8.77 (dd, J = 6.0, 2.9 Hz, 1H), 8.54 (dt, J = 9.0, 3.6 Hz, 1H), 7.70 (t, J = 9.4 Hz, 1H), 7.31 (s, 1H), 7.11-7.04 (m,2H), 6.91 (dd, J = 9.2, 5.0 Hz, 3H), 4.95 (d, J = 1.0 Hz, 2H). 13 C NMR (151MHz, DMSO-d6) δ 164.06(d, J=267.3Hz), 159.47 (d, J = 3.5 Hz), 156.86 (d, J=237.1Hz), 154.98 (s), 150.22 (s), 143.74 (d, J = 1.5 Hz), 130.01 (d, J = 11.3Hz), 127.50 (s), 124.95 (s), 122.45 (d, J = 8.6 Hz), 119.17 (d, J = 12.9 Hz),118.80 (d, J = 25.1 Hz), 116.93 (d, J = 9.1 Hz), 116.78 (d, J = 24.2 Hz),113.75 (d, J = 24.2 Hz), 63.93 (s). HRMS (ESI-TOF, positive) m / z: calcd forC 17 H 11 F2N3NaO5 [M+Na] + : 398.0559, found 398.0557; calcd for C 34 H 22 F4N6NaO 10 [2M+Na] + : 773.1226; found 773.1234.
[0051] Example 2: Synthesis of O-2'-chloro-5'-nitrobenzoyl-1-(6'-fluoro-2 H -benzopyran-3-yl) hydrazide (A-2)
[0052] A yellow powder, 0.540 g, 91.9% yield was obtained by following the procedure of Example 1, replacing only 2-fluoro-5-nitrobenzoic acid with 2-chloro-5-nitrobenzoic acid.
[0053] 1 H NMR (600 MHz, DMSO- d6 ) δ 8.68 (d, J = 2.8 Hz, 1H), 8.40 (dd, J =8.8, 2.8 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.10-7.03 (m, 2H),6.97-6.85 (m, 3H), 4.94 (d, J = 1.0 Hz, 2H). 13 C NMR (151 MHz, DMSO- d6 ) δ161.16 (s), 156.84 (d, J=236.9Hz), 154.72 (s), 150.21 (s), 146.09 (s), 138.50(s), 132.06 (s), 131.06 (s), 127.19 (s), 126.00 (s), 124.94 (d, J = 4.3 Hz),122.45 (d, J = 9.1 Hz), 116.92 (d, J = 9.1 Hz), 116.76 (d, J = 24.1 Hz),113.73 (d, J = 24.1 Hz), 63.91 (s). HRMS (ESI-TOF, positive) m / z: calcd forC 17 H 11 ClFN3NaO5 [M+Na] + : 414.0263, found 414.0266; calcd for C 34 H 22 Cl2F2N6NaO 10 [2M+Na] + : 805.0635; found 805.0636.
[0054] Example 3: Synthesis of O-2'-fluoro-5'-nitrobenzoyl-1-(7'-bromo-2 H -chromen-3-yl) imidate oxime (A-3)
[0055] 1 -(7'-bromo-2 H Synthesis of O-2'-fluoro-5'-nitrobenzoyl-1 -(7'-bromo-2
[0056] 3-cyano-7-bromo-2 H -benzopyran-3-yl) amidoxime:
[0057] Synthesis of O-2'-fluoro-5'-nitrobenzoyl-1 -(7'-bromo-2 H -benzopyran-3-yl) amidoxime:
[0058] 2-fluoro-5-nitrobenzoic acid 0.371 g (2.0 mmol) was placed in a 50 mL flask, DMTMM 0.554 g (2.0 mmol) was added, 15 mL tetrahydrofuran was added, and the mixture was stirred at room temperature for 30 minutes. 1 -(7'-bromo-2 H -benzopyran-3-yl) amidoxime 0.404 g (1.5 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was completed, 20 mL of water was added to the reaction mixture, and insoluble yellow solids appeared. After ultrasonic treatment for 2 minutes, the mixture was filtered, and the product was obtained by drying under vacuum. The yield was 0.418 g, and the yield was 63.9%.
[0059] 1 H NMR (600 MHz, DMSO- d6 ) δ 8.76 (dd, J = 6.0, 2.9 Hz, 1 H), 8.54 (dt, J = 9.0, 3.4 Hz, 1 H), 7.70 (t, J = 9.4 Hz, 1 H), 7.33 (s, 1 H), 7.19-7.13 (m, 2H), 7.12 (s, 1 H), 6.89 (s, 2H), 4.99 (s, 2H). 13 C NMR (151 MHz, DMSO- d6) δ 164.04 (d, J = 266.9 Hz), 159.46 (d, J = 3.4 Hz), 154.95 (s), 154.84 (s), 143.74 (d, J = 2.8 Hz), 130.00 (d, J = 11.4 Hz), 129.36 (s), 127.49 (s), 124.78 (s), 124.76 (s), 123.70 (s), 122.62 (s), 120.51 (s), 119.17 (d, J = 12.9 Hz), 118.80 (d, J = 24.8 Hz), 118.60 (s), 64.10 (s). HRMS (ESI-TOF, positive) m / z: calcd for C 17 H 11 BrFN3NaO5 [M+Na] + : 457.9758, found 457.9751; calcd forC 34 H 22 79 Br 81 BrF2N6NaO 10 [2M+Na] + : 894.9604; found 894.9607.
[0060] Example 4: Synthesis of O-3'-nitrobenzoyl-1-(7'-bromo-2 H -benzopyran-3-yl) imidate oxime (A-4)
[0061] Following the procedure of Example 3, only replacing 2-fluoro-5-nitrobenzoic acid with 3-nitrobenzoic acid, afforded yellow powder 0.519 g in 82.7% yield.
[0062] 1 H NMR (600 MHz, DMSO- d6 ) δ 8.83-8.78 (m, 1H), 8.65-8.58 (m, 1H),8.50 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.34 (s, 1H),7.20-7.14 (m, 2H), 7.12 (s, 1H), 6.99 (s, 2H), 5.00 (d,J = 1.1 Hz, 2H). 13 CNMR (151 MHz, DMSO- d6 ) δ 161.70 (s), 154.83 (s), 154.37 (s), 147.96 (s), 135.73 (s), 130.68 (s), 130.42 (s), 129.31 (s), 127.63 (s), 124.74 (s), 124.55 (s), 124.07(s), 123.99(s), 122.55 (s), 120.58 (s), 118.59 (s), 64.17(s). HRMS (ESI-TOF, positive) m / z: calcd for C 17 H 12 BrN3NaO5 [M+Na] + : 439.9853,found 439.9859; calcd for C 34 H 24 79 Br 81 BrN6NaO 10 [2M+Na] + : 858.9793; found858.9801.
[0063] Example 5: Synthesis of O-2'-chloro-5'-nitrobenzoyl-1-(7'-bromo-2 H -benzopyran-3-yl) imidate oxime (A-5)
[0064] Following the procedure of Example 3, only replacing 2-fluoro-5-nitrobenzoic acid with 2-chloro-5-nitrobenzoic acid, afforded yellow powder 0.563 g, 82.9% yield.
[0065] 1 H NMR (600 MHz, DMSO- d6 ) δ 8.67 (d, J = 2.8 Hz, 1H), 8.40 (dd, J =8.8, 2.8 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.33 (s, 1H), 7.15 (ddd, J= 20.6, 10.3, 1.6 Hz, 3H), 6.91 (s, 2H), 4.97 (d, J = 1.1 Hz, 2H). 13 C NMR (151MHz, DMSO- d6 ) δ 161.16 (s), 154.83 (s), 154.68 (s), 146.09 (s), 138.50 (s),132.06 (s), 131.07 (s), 129.35 (s), 127.19 (s), 125.98 (s), 124.75 (s),123.71 (s), 122.61 (s), 120.52 (s), 118.59 (s), 64.08 (s). HRMS (ESI-TOF,positive) m / z: calcd for C 17 H 11 81 BrClN3NaO5 [M+Na] + : 475.9443, found 475.9451;calcd for C 34 H 22 79 Br 81 BrCl2N6NaO 10 [2M+Na] + : 926.9013; found 926.9018.
[0066] Example 6: Synthesis of O-2'-fluoro-5'-nitrobenzoyl-1-(7'-methyl-2 H -benzopyran-3-yl) hydrazine (A-6)
[0067] 1-(7'-methyl-2 H -benzopyran-3-yl) hydrazine
[0068] 3-nitro-7-methyl-2 H -benzopyran 0.685 g (4.0 mmol) was placed in a 50 mL flask, hydroxylamine hydrochloride 0.500 g (7.2 mmol) was added, ethanol 16 mL was added, triethylamine 1.01 mL (7.2 mmol) was added, and heated to reflux for 2 hours. The solvent was evaporated under reduced pressure, 20 mL of water was added, and insoluble solid appeared, which was filtered, washed with water, and dried under vacuum to obtain white solid 0.597 g with a yield of 73.2%. The product obtained can be directly used for the synthesis of the next step.
[0069] O-2'-fluoro-5'-nitrobenzoyl-1-(7'-methyl-2 H Synthesis of O-2'-fluoro-5'-nitrobenzoyl-1-(7'-methyl-2
[0070] 2-Fluoro-5-nitrobenzoic acid 0.371 g (2.0 mmol) was placed in a 50 mL flask, DMTMM 0.554 g (2.0 mmol) was added, 15 mL tetrahydrofuran was added, and it was stirred at room temperature for 30 minutes. 1-(7'-methyl-2 H O-2'-fluoro-5'-nitrobenzoyl-1-(7'-methyl-2
[0071] 1 H NMR (600 MHz, DMSO- d6 ) δ 8.76 (dd, J = 6.0, 2.9 Hz, 1H), 8.54 (dt, J = 9.0, 3.6 Hz, 1H), 7.69 (t, J = 9.4 Hz, 1H), 7.32 (s, 1H), 7.08 (d, J =7.6 Hz, 1H), 6.88 – 6.76 (m, 3H), 6.71 (s, 1H), 4.92 (d, J = 0.9 Hz, 2H),2.27 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 164.04 (d, J = 267.3 Hz), 159.53 (d, J = 3.5 Hz), 155.20 (s), 154.06 (s), 143.74 (d, J = 2.3 Hz), 140.91 (s), 129.95 (d, J = 11.3 Hz), 127.76 (s), 127.47 (s), 125.72 (s), 122.53 (s), 121.99 (s), 119.25 (d, J = 13.0 Hz), 118.79 (d, J = 25.1 Hz), 118.69 (s), 116.13 (s), 63.72 (s), 21.15 (s). HRMS (ESI-TOF, positive) m / z: calcd for C 18 H 14 FN3NaO5 [M+Na] + : 394.0810, found 394.0808; calcd for C 36 H 28 F2N6NaO 10 [2M+Na] + : 765.1727;found 765.1728.
[0072] Example 7: Synthesis of O-3'-nitrobenzoyl-l-(7'-methyl-2 H -benzopyran-3-yl) hydrazidoxime (A-7)
[0073] Following the procedure of Example 6, only replacing 2-fluoro-5-nitrobenzoic acid with 3-nitrobenzoic acid, afforded yellow powder 0.426 g in 80.4% yield.
[0074] 1 H NMR (600 MHz, DMSO- d6 ) δ 8.82-8.79 (m, 1H), 8.61 (d, J = 7.8 Hz,1H), 8.50 (dd, J = 8.2, 1.4 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.33 (s, 1H),7.09 (d, J = 7.6 Hz, 1H), 6.93 (s, 2H), 6.80 (d, J= 7.6 Hz, 1H), 6.71 (s,1H), 4.94 (s, 2H), 2.27 (s, 3H). 13 C NMR (151 MHz, DMSO- d6 ) δ 161.75 (s),154.63 (s), 154.04 (s), 147.95 (s), 140.81 (s), 135.71 (s), 130.77 (s),130.40 (s), 127.70 (s), 127.58 (s), 125.48 (s), 124.05 (s), 122.51 (s),122.28 (s), 118.75 (s), 116.13 (s), 63.79 (s), 21.14 (s). HRMS (ESI-TOF,positive) m / z: calcd for C 18 H 15 N3NaO5 [M+Na] + : 376.0904, found 376.0909; calcdfor C 36 H 30 N6NaO 10 [2M+Na] + : 729.1916; found 729.1926.
[0075] Example 8: Synthesis of O-2'-amino-5'-fluorobenzoyl-1-(6'-fluoro-2 H -benzopyran-3-yl) imidoxyl (A-8)
[0076] Following the procedure of Example 1, only replacing 2-fluoro-5-nitrobenzoic acid with 2-amino-5-fluorobenzoic acid, afforded yellow powder 0.405 g, 78.2% yield.
[0077] 1 H NMR (600 MHz, DMSO- d6 ) δ 7.89 (dd, J = 10.2, 3.1 Hz, 1H), 7.29 (s,1H), 7.22-7.16 (m, 1H), 7.09-7.02 (m, 2H), 6.90 (dd, J = 8.2, 4.6 Hz, 1H),6.80 (dd, J= 9.3, 5.0 Hz, 3H), 6.60 (s, 2H), 4.95 (d, J = 0.9 Hz, 2H). 13 CNMR (151 MHz, DMSO- d6 ) δ 164.14 (d, J = 2.7 Hz), 156.86 (d, J=237.1Hz),153.15 (s), 152.49 (d, J = 1.7 Hz), 150.17 (d, J = 1.4 Hz), 148.54 (s), 125.62(s), 124.18 (d, J = 1.7 Hz), 122.65 (d, J = 9.1 Hz), 122.18 (d, J = 23.4 Hz),117.75 (d, J = 7.1 Hz), 116.87 (d, J = 8.2 Hz), 116.53 (d, J = 22.7 Hz),115.60 (d, J = 23.3 Hz), 113.61 (d, J = 24.2 Hz), 107.29 (d, J = 6.9 Hz),64.01 (s). HRMS (ESI-TOF, positive) m / z: calcd for C 17 H 13 F2N3NaO3 [M+Na] + :368.0817, found 368.0819; calcd for C 34 H 26 F4N6NaO6 [2M+Na] + : 713.1742; found713.1752.
[0078] The structural formula of the compounds of Examples 1-8 is shown in Table 1.
[0079] Table 1 Structural formula of the compounds of Examples 1-8
[0080] Experiment 1: In vitro TLR2 / 1 agonistic activity screening
[0081] The TLR2 / 1 agonistic activity of the compound was evaluated by using a HEK293T cell dual luciferase reporter gene model expressing hTLR2 / 1 constructed by transient co-transfection. The DNA plasmids of hTLR2 and hTLR1 were introduced into HEK293T cells by using transient co-transfection technology, so that they could temporarily express hTLR2 / 1. At the same time, the firefly luciferase (FLuc) gene plasmid for detecting the agonistic level of NF-κB and the sea cucumber luciferase (RLuc) gene plasmid as an internal reference were also introduced into HEK293T. The catalytic luminescence intensity of the two fluorescent enzymes was detected by using a dual luciferase reporter gene detection kit at different wavelengths, and the RLU (fold) ratio was used as an evaluation index of agonistic activity.
[0082] The compounds in Table 1 were prepared into 10 mM stock solutions with DMSO, and diluted with DMEM (H) medium to 10 μM to obtain the medium solution of the compounds to be screened. The logarithmic growth phase HEK293T cells were centrifuged at 900 r / min for 5 min, the cells were resuspended with DMEM (H) complete medium, and the cell density was adjusted to 5×10 5 / mL. 5 million cells were inoculated in each well of a 96-well culture plate. 0.7 mL of Opti-MEM medium (purchased from Thermo Fisher Scientific) was added to a sterile 1.5 mL centrifuge tube (centrifuge tube A), 30.03 μL of ExFect transfection reagent (purchased from Nanjing Nvwaizan Biotechnology Co., Ltd.) was added, and the mixture was gently mixed with a pipette. 0.7 mL of Opti-MEM medium was added to another sterile 1.5 mL centrifuge tube (centrifuge tube B), and 7000 ng of pcDNA3.1(+)-hTLR2, 7000 ng of pcDNA3.1(+)-hTLR1, 875 ng of pNFκB-luc, and 140 ng of pRL-SV40-C plasmid (all purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) were sequentially added, and the mixture was gently mixed with a pipette. The ExFect-Opti-MEM in the centrifuge tube A was added dropwise to the plasmid solution in the centrifuge tube B, gently mixed, and incubated at room temperature for 20 minutes to obtain a transfection working solution. When the cells grew to 70-80% of the well plate bottom area, the transfection working solution was added dropwise to the 96-well plate at 20 μL per well, gently mixed, and then incubated in a 37°C, 5% CO2 incubator. After 24 hours, the medium in each well was replaced with the medium solution of the compound to be screened at a final concentration of 10 μM, and three replicate wells were set up for each compound. Blank controls and positive controls (Pam3CSK4) were also set up. After another 24 hours of incubation, the fluorescence was detected according to the instructions of the Biyun Tian dual luciferase reporter gene detection kit (RG088S), and the RLU (fold) ratio of the catalytic luminescence intensity of the firefly luciferase and the sea cucumber luciferase was used as an evaluation index of agonistic activity. The experimental results are shown in Table 2.
[0083] Table 2. Results of in vitro TLR2 / 1 agonist activity screening of compounds
[0084] Experimental results showed that, compared with the blank group, all compounds exhibited significant TLR2 / 1 agonist activity at a concentration of 10 μM.
[0085] Experiment 2: Cytotoxicity Experiment
[0086] Cytotoxicity experiments were conducted using A-2, A-4, and A-7, which exhibit strong in vitro TLR2 / 1 agonist activity. HEK293T and RAW264.7 cells in logarithmic growth phase were adjusted to a density of 5 × 10⁻⁶ cells / year. 5 50,000 cells were seeded per well in 96-well plates at a concentration of / mL. After 24 hours, the culture medium in each well was replaced with a concentration gradient of compounds A-2, A-4, and A-7 (100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.12μM, 1.56μM, 0.78μM, 0.39μM), 100μL per well, with three replicates for each concentration. A negative control and a blank control were also established; the negative control was treated with 1‰ DMSO (v / v), and the blank control was treated with cell-free medium. After another 24 hours of culture, 10μL of CCK-8 reagent was added to each well, and the OD value at 450nm was measured using a microplate reader after 2 hours.
[0087] Substitute the values into the following formula to calculate cell viability: Cell viability (%) = [(OD with added drugs - OD blank) / (OD negative - OD blank)] × 100% OD dosing: absorbance of cells, CCK-8 solution and drug solution.
[0088] OD blank: Absorbance of cells containing culture medium and CCK-8 solution.
[0089] OD negative: Absorbance of cells and CCK-8 solution but no drug solution.
[0090] Depend on Figure 1 It can be seen that compounds A-2, A-4, and A-7 at different concentrations showed no significant toxicity to HEK293T and RAW264.7 cells, indicating that compounds A-2, A-4, and A-7 have good safety for cells.
[0091] Experiment 3: Survival Experiment of Irradiated Mice After 30 Days
[0092] The compounds A-2, A-4, A-7 in Table 1 were selected to carry out the irradiated mouse 30-day survival experiment. Adult male C57 / BL mice were bred by Beijing Sbiopharm Co., Ltd., and the body weight of the mice was 20-22 g. The experimental animal license number was SCXK (Jing) 2019-0010. The mice were bred in a SPF laboratory, 10 mice per cage, fed with specially formulated feed for mice, free water, and the animal laboratory temperature was maintained at 25°C, the relative humidity was 40%-70%, and the daily light was 12 hours. The solvent was 20% hydroxypropyl-β-cyclodextrin (HPCD) containing normal saline, and the compounds A-2, A-4, A-7 were prepared into 8 mg / mL suspensions with 20% HPCD normal saline, respectively.
[0093] The experimental setup included a radiation control group (given 20% HPCD-containing normal saline) and an experimental group (80 mg / kg). Both the radiation control group and the experimental group were administered intraperitoneally, with each administration being 0.2 mL per mouse. The irradiation dose rate was 56.36 cGy / min. 60 The mice were irradiated with Co γ rays, and the irradiation dose rate was 56.36 cGy / min. The survival of the mice was observed for 30 days, with the irradiation day being 0 days. The survival of the mice from 0 days to 30 days was observed, and the body weight of the mice was measured at 1, 4, 7, 10, 14, 18, 22, and 30 days after irradiation. The changes in the body weight of the mice were recorded for 30 days.
[0094] The survival and body weight changes after irradiation are shown in Tables 3-4. The results showed that after being given a whole-body irradiation of 8.5 Gy, all mice in the radiation control group died within 14 days; the survival rates of the experimental groups A-7 and A-2 were 100% and 70%, respectively. Statistical analysis of the survival curves of the mice was performed using GraphPad Prism 9.5.1 software, and the Log-Rank test was used for comparison between groups. The survival rates of the A-7 and A-2 groups were significantly higher than those of the control group (p<0.0001, <0.001, and <0.0001, respectively), and the survival rate of the A-7 group was as high as 100% under high-dose irradiation. As shown in Table 4, the body weight of the mice in the radiation control group continued to decrease after irradiation; the body weight of the mice in the A-2 and A-7 groups began to recover after 14 days, especially the mice in the A-7 group, which had basically recovered to the pre-irradiation level after 18 days of irradiation.
[0095] Table 3. Survival experiment of mice
[0096] Table 4. Changes in the body weight of irradiated mice for 30 days
[0097] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.
Claims
1. An O-benzoyl benzoyl oxime derivative, its isomer, its prodrug, its pharmaceutically acceptable salt, its hydrate, or its solvent compound, characterized in that, Equation I is as follows. Formula I Wherein, R1 is selected from halogen, -CN, hydroxyl, nitro, amino, C1-C6 alkyl, halomethyl, haloC1-C4 alkyl, C3-C8 cycloalkyl or heterocycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 alkylthio, haloC1-C6 alkoxy, haloC1-C6 alkylthio, C2-C4 ester; R2 is selected from halogen and nitro.
2. The O-benzoyl genamidooxime derivative according to claim 1, characterized in that, R1 and R2 are mono- or poly-substituted on the ring.
3. The O-benzoyl amide oxime derivative according to claim 1 or 2, characterized in that, The halogen is fluorine, chlorine, bromine or iodine.
4. The O-benzoyl genamidooxime derivative according to claim 3, characterized in that, The C1-C6 alkylamino group is selected from methylamino, ethylamino, and propylamino; the C1-C6 alkoxy group is selected from methoxy, ethoxy, and propoxy; and the C1-C6 alkyl group is selected from methyl, ethyl, and propyl.
5. The O-benzoyl genamidooxime derivative according to claim 3, characterized in that, R1 is selected from halogens and C1-C3 alkyl groups.
6. The O-benzoyl genamidooxime derivative according to claim 1, characterized in that, Includes any of the following: O-2'-Fluoro-5'-Nitrobenzoyl-1-(6'-Fluoro-2 H -benzopyran-3-yl)gamidoxime O-2'-Chloro-5'-Nitrobenzoyl-1-(6'-Fluoro-2') H -benzopyran-3-yl)gamidoxime O-2'-Fluoro-5'-Nitrobenzoyl-1-(7'-Bromo-2') H -benzopyran-3-yl)gamidoxime O-3'-Nitrobenzoyl-1-(7'-Bromo-2- H -benzopyran-3-yl)gamidoxime O-2'-Chloro-5'-Nitrobenzoyl-1-(7'-Bromo-2'- H -benzopyran-3-yl)gamidoxime O-2'-Fluoro-5'-Nitrobenzoyl-1-(7'-Methyl-2 H -benzopyran-3-yl)gamidoxime O-3'-Nitrobenzoyl-1-(7'-methyl-2 H -benzopyran-3-yl)gamidoxime O-2'-Amino-5'-Fluorobenzoyl-1-(6'-Fluoro-2'- H -benzopyran-3-yl)gammoxime.
7. A composition comprising the O-benzoyl benzoylamidooxime derivative, its isomer, its prodrug, its pharmaceutically acceptable salt, its hydrate or its solvent compound, as well as a pharmaceutically acceptable carrier or excipient, according to any one of claims 1-6.
8. Use of any of the O-benzoyl benzoylamidoxame derivatives, isomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvent compounds thereof according to claims 1-6, characterized in that, Used to prepare drugs for the prevention and / or treatment of ionizing radiation damage.