WO2005118559A1 - A type of formyl peptide receptor-like 1 regulator, preparation method and use thereof - Google Patents

A type of formyl peptide receptor-like 1 regulator, preparation method and use thereof Download PDF

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WO2005118559A1
WO2005118559A1 PCT/CN2005/000736 CN2005000736W WO2005118559A1 WO 2005118559 A1 WO2005118559 A1 WO 2005118559A1 CN 2005000736 W CN2005000736 W CN 2005000736W WO 2005118559 A1 WO2005118559 A1 WO 2005118559A1
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phenyl
dihydro
receptor
butoxy
benzamide
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PCT/CN2005/000736
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French (fr)
Chinese (zh)
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Mingwei Wang
Guangxing Wang
Jianghua Mei
Xiyuan Cheng
Yunxia Xuan
Caihong Zhou
Dequan Ye
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Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/88—Oxygen atoms
    • C07D239/91—Oxygen atoms with aryl or aralkyl radicals attached in position 2 or 3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a class of formyl peptide-like receptor-1 (FPRLl) modulators, and specifically to a class of substituted quinazolinone derivative small molecule organic compounds that can be used as non-peptide FPRLl modulators.
  • FPRL1 modulator As a FPRL1 modulator, it affects the chemotaxis and activation of neutrophils, and plays a pharmacological role in the body's non-specific and specific inflammatory response.
  • the invention also relates to a method for preparing the compound. technical background:
  • Chemotactic agents produced by tissue cells and microorganisms such as N-formyl peptide (fMLF) can cause extravasation, chemotaxis and activation of neutrophils, and exert physiological effects through certain signal transduction pathways, including Inflammatory response, immune regulation, and antiviral infection.
  • these chemokines can enhance the body's non-specific immune response ability, fight against and eliminate pathogenic microorganisms; but their high level of continuous expression causes acute and chronic inflammation, and participates in infectious diseases and allergies with other cytokines The pathological processes of diseases and some immune diseases cause harm to the body. Therefore, as a new target for drug action, chemokines and related receptors provide new research directions for the treatment of many human diseases with high morbidity and mortality.
  • fMLF was the first exogenous Formyl Peptide Receptor (FPR) agonist to be discovered.
  • FPR Formyl Peptide Receptor
  • fMLF can activate the nuclear transcription factor NF- ⁇ , promote the secretion of inflammatory factors such as phagocytes, and start the body's non-specific immune response when it resists the invasion of pathogenic microorganisms.
  • fMLF can also stimulate monocytes to express a series of cytokines such as IL-1, IL-6, and IL-8, etc., through the chain reaction of the latter to transmit information in the immune response, upregulate T cells and antigen delivery It is functional and activates antigen-specific helper T cells 1 and 2 (Thl and Th2), producing immune effects against viral infections.
  • FPRL1 and FPRL2 forml Peptide Receptor Like-2, FPRL2
  • FPRL2 Forml Peptide Receptor Like-2
  • FPR receptor gene knockout mice are more susceptible to bacterial infection than normal mice of the same type.
  • fMLF When fMLF is used to stimulate neutrophils in gene knockout mice in vitro, their chemotaxis function is weakened. After injection of MLF into the body, Its ability to migrate in peripheral blood disappeared (Gao JL, Lee EJ, Murphy PM, J Exp Med 1999, 189 (4): 657-662).
  • FPRLl which is homologous to FPR, is highly expressed on the surface of neutrophils and monocytes, and is specific and non-specific in the body. Plays an important effect in the inflammatory response.
  • FPRL1 belongs to the pertussis toxin-sensitive Gi protein-coupled receptor, and it and FPR are localized on the human chromosome 19ql3.3. FPRL1 and FPR have 69% homology in amino acid sequence. Except for neutrophils and monocytes, FPRL1 is found in liver cells, dendritic cells, stellate cells, microglia, and B lymphocytes. Both cells and T lymphocytes are expressed. It is known that the ligands of FPRL1 have structural diversity. Agonists include the aforementioned N-formylated peptide molecules, Lipoxin A4 (LXA4), which is produced during inflammation and tissue damage, and H. pylori secreted by the stomach. Peptide Hp
  • WKYMVm W-pep
  • W-pep is a hexapeptide identified from a random peptide library.
  • W-pep is a very effective agonist. Further experiments have shown that W-pep works mainly through FPRL1 (Hu, JY et al.
  • FPRL 1 Another peptide MMK-1 identified from a random peptide library is FPRL 1 is a potent and specific chemotactic agonist (Christophe, T. et al., J. Biol. Chem. 2001,276: 21585-21593). FPRL1 serves as a potential drug target for anti-AIDS and amyloidosis A certain experimental basis has been accumulated. At the same time, the screening of immunomodulatory drugs based on FPRL1 receptor agonists and antagonists has also been carried out in several well-known laboratories abroad, and no specific organic small molecule ligand of FPRL1 has been reported to date. content
  • the purpose of the present invention is to design and synthesize a new class of small molecule formyl peptide-like receptor-1 regulators of substituted quinazolinone derivatives;
  • Another object of the present invention is to provide a method for preparing such compounds
  • Yet another object of the present invention is to provide such compounds as modulators of formyl peptide-like receptor-1, and their applications in anti-inflammatory, immunomodulatory and anti-infective drug treatment.
  • the formyl peptide-like receptor-1 modulator according to the present invention has the following general structure I: Of which ⁇ ! And ⁇ are each independently an aromatic group or a substituted aromatic group, wherein the aromatic group refers to a phenyl group, a cyclopentadienyl group, a naphthyl group, a furyl group, a benzofuranyl group, a thiophenol group, a benzothiophenol group, Pyridyl, pyrrolyl, indolyl or quinolinyl; the substituent in the substituted aromatic group is arbitrarily selected from one, two or three of the following groups: alkyl group; hydroxyl group; mercapto group; formamide group Formyloxy; Alkoxy, Alkylamino, Alkylthio, Alkyloxy, Alkylamino, Alkoxycarbonyl or Alkylaminocarbonyl substituted with a halogen atom, a hydroxyl group or an alkoxyl
  • a preferred embodiment of the present invention is a formyl peptide-like receptor- 1 modulator having structural formula I,
  • aryl or cyclofluorenyl For the base, aryl or cyclofluorenyl; X is 0 or 8.
  • Another preferred embodiment of the present invention is a formyl peptide-like receptor-1 modulator having structural formula I, wherein 1 is selected from the following groups:
  • the preferred compound of the invention is 2- (4-methoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (2,4-dimethoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) quinazolin-4-one; 2- (4-di Methylamino-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (2,4-dichloro-phenyl ) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2-4-methyl-phenyl) -2,3-dihydro -3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2-phenyl-2,3-dihydro-3- (4-butoxy-benzamide)- 1H
  • the invention is implemented by the following steps:
  • the solvent used in the cyclization reaction may be N, N-dimethylformamide, N, N-dimethylacetamide, a mixed solvent of N, N-dimethylformamide and glacial acetic acid, ⁇ Mixed solvent of N-dimethylacetamide and glacial acetic acid or mixed solvent of dichloromethane and glacial acetic acid;
  • the preferred catalytic acid is acetic acid or trifluoroacetic acid;
  • reaction temperature is generally 50 ⁇ 100 ° C.
  • Compound II can be prepared by reference to Tetmheron Letters 1997, 38 (49): 8445-8448) by the following method: starting from o-nitrobenzoyl chloride (or o-nitrothiobenzoyl chloride) and 2 equivalents of compound AnNH 2 As a starting material, a coupling reaction occurs in a dichloromethane solution containing triethylamine, and then zinc / glacial acetic acid is used as a reducing agent to prepare an intermediate o-amino compound.
  • compound II can also be prepared in one step by the following simpler method: using isatoic anhydride as the raw material and 1.05 equivalents of compound AnNH 2 in benzene or tetrahydrofuran under reflux.
  • Compound III can be purchased commercially or prepared by oxidation of the corresponding alcohol with Dess-Martin (J Org Chem, 1983, 48: 4155).
  • Compound Ar! NI ⁇ can be purchased commercially or through the corresponding nitro compound Get it.
  • the present invention designs and synthesizes a new class of novel small molecule formyl peptide-like receptor-1 regulators which are substituted for quinazolinone derivatives. These compounds have a certain regulatory effect on the function of FPRL1, and are helpful for intervention by The receptor mediates various pathological processes, such as acute and chronic inflammatory responses, and other cytokines participating in infectious diseases, allergic diseases, and some immune diseases.
  • the compound of the present invention is a specific FPRL1 modulator. It is used in the preparation of anti-inflammatory, anti-infective and immunomodulatory drugs.
  • the compound of the present invention has a relatively simple structure and is easy to prepare. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 is a graphical representation of the effects of different agonists on neutrophil ⁇ -glucosidase release
  • Figure 2 is a graph showing the effect of different agonists on the release of ⁇ -glucosidase from RBL-2H3 cell lines expressing FPRL1, where ⁇ represents W_pep, * represents MMK1, ⁇ represents Cl, and O represents fMLF;
  • Figure 3-1 shows the effect of different agonists on the release of ⁇ -hexosaminidase from RBL-2H3 cell lines expressing FPRL1;
  • Figure 3-2 shows the effects of different agonists on ⁇ -hexose from RBL-2H3 cell lines expressing FPR Effect of aminase release;
  • Figure 4-1 shows the intracellular calcium flow response of FPRL1-expressing RBL-2H3 cells induced by compound C1;
  • Figure 4-2 shows the effect of compound C1 on FBL-expressing RBL-2H3 cells Schematic diagram of intracellular calcium flux response;
  • Figure 5-1 shows the effect of pertussis toxin on the intracellular calcium flux response of RBL-2H3 cells expressing FP
  • Figure 5-2 is a graphical representation of the effect of pertussis toxin on the intracellular calcium flux response of C1-induced RBL-2H3 cells expressing FPRL1;
  • FIG. 6 is a diagram of activation of a cell signal transduction-related kinase Erk caused by compound C1;
  • Fig. 7 is a graph showing the effects of compounds C7 and M8 on the neutrophil degranulation reaction induced by W-pep; Impact icon
  • Figure 9 is a graphical representation of the effect of compound M8 on the intracellular calcium flux response of RBL-2H3 cells expressing FPRL1 caused by W-pep. detailed description
  • Rat basophilic lymphoma cell RBL-2H3 cell line purchased from the American Type Culture Collection;
  • FPR Human formyl peptide receptor
  • FPRL1 formyl peptide-like receptor 1
  • Fluo3 / AM (Molecular Probes, Eugene, OR);
  • SpectroMax 340 plate reader (Molecular Dynamics, Sunnyvale, CA);
  • fMLF N-terminal formylated 3 peptide (N-Fomiyl-Met-Leu-Phe), purchased from (Sigma, St. Louis, MO);
  • W-pep W-peptide (WKYMVm), a synthetic 6 peptide
  • MMK1 synthetic 13 peptide (LESIFRSLLFRVM).
  • RBL-2H3 cells were cultured in DMEM medium containing 10% fetal bovine serum and 2 mM L-glutamine. According to the instructions for use of liposomes (trade name LipofectAmine), FPR or FPRL1 receptor plasmids were transferred into it, and G418 (400 ng / ml) was selected for 4-5 weeks, and cell clones stably expressing the receptor were selected.
  • liposomes trade name LipofectAmine
  • Percoll gradient centrifugation was performed on a peripheral blood reference method from healthy donors (Ulmer, A ⁇ and Flad, HD (1979). J. Immunol. Methods 30, 1-10). The neutrophil isolation efficiency is about 97%, and the survival rate is greater than 98%. Resuspended in serum-free RPMI1640 medium, 2 a cell density of> 106 cells / ml. The number of donor samples required for repeated experiments was not less than 3 cases.
  • the neutrophils isolated according to 2.2 were resuspended in HBSS containing 10 ⁇ M cytochalasin B with a cell density of 6.25 ⁇ 10 6 cells / ml (0.5 ⁇ 10 6 cells / 80 ⁇ 1) and incubated on ice for 15 minutes. , And placed at 37 ° C for 15 minutes. After pre-incubation, 80 ⁇ of cell suspension was mixed with 80 ⁇ of HBSS-HB buffer (containing 20 mM HEPES, (pH 7.4) and 0.2% BSA HBSS) containing different concentrations of compound C1 and each reference, and mixed in After being left at 37 ° C for 10 minutes, it was placed on ice to stop the degranulation reaction.
  • HBSS-HB buffer containing 20 mM HEPES, (pH 7.4) and 0.2% BSA HBSS
  • the supernatant and cells were separated by centrifugation.
  • the supernatant of each test group was collected and the ⁇ -glucosidase release amount was detected according to the method of 2.3.
  • the concentration of compound C1 was set to 0 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M; the concentrations of the reference fMLF and W-pep were set to 0 ⁇ M, 1 ⁇ M, 10nM, 100nM, 100nM; the concentration of the reference MMK1 was set to 0nM , 10nM, 100nM, 300nM, 1000nM.
  • the experimental results are shown in Figure 1.
  • the amount of ⁇ -glucuronidase secreted by the neutrophil after stimulation was dose-dependent, and the effect of compound C1 was similar to that of MMK1, but MMK1 had more Strong affinity.
  • RBL-2H3 cells transfected with FPRL1 receptor were seeded in 48-well plates at a density of 50,000 cells / well. After two days of culture, they were washed twice with HBSS, added to 40 ⁇ HBSS containing 10 ⁇ M cytochalasin B, and incubated on ice for 15 minutes. , And placed at 37 ° C for 15 minutes. After pre-incubation, add 40 ⁇ of HBSS buffer containing different concentrations of compound C1 and each reference, mix, leave at 37 ° C for 10 minutes, and place on ice to stop the degranulation reaction. Centrifuge the supernatant and cells, and take each test group.
  • the supernatant was tested for the amount of ⁇ -glucosidase released according to the method of 2.3.
  • the concentration of compound C1 was set to 0 ⁇ M, 0.01 ⁇ M, 0.03 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M, 100 ⁇ M;
  • the concentrations of the reference MMK1 and W-pep were set to 0 nM, 0.1nM, 0.3nM, 1nM, 3nM, 10nM, 30nM, 100nM, 300nM, 1000nM;
  • the concentration of the reference fMLF is set to 0nM, 1 ⁇ M, 10nM, 100nM, 1000nM, 10000nMo.
  • the experimental group of the RBL-2H3 cell line expressing FPR and the experimental group of the RBL-2H3 cell line expressing FPRL1 were prepared.
  • the experimental cell line was prepared according to the method 2.1 and seeded in a 48-well plate at a density of 50,000 cells / well, and washed with HBSS for two days Two times, add 40 ⁇ L of HBSS containing 10 ⁇ M cytochalasin B, incubate for 15 minutes on ice, and finally incubate at 37 ° C with 1 mM p-nitro-N-acetyl- ⁇ -D-glucosamide for 15 minutes. minute.
  • the dose of compound C1 used to stimulate RBL-2H3 cell line expressing FPR receptor in this experiment was set to 0 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M; the concentrations of the reference fMLF, W-pep, and MMK1 were set to 0 ⁇ M, 10 ⁇ M, 100 ⁇ , ⁇ ;
  • the dose of compound C1 used to stimulate the RBL-2H3 cell line expressing the FPRL1 receptor was set to 0 ⁇ M, 10 ⁇ M, 100 ⁇ M; the concentrations of the reference fMLF and W-pep were set to 0 ⁇ M, 10 ⁇ M, 100 ⁇ M, 100 ⁇ M; control
  • the concentration of product MMK1 was set to 0 nM, 1 nM, 10 nM, 100 nM.
  • the experimental group of RBL-2H3 cell line expressing FPRL1 and the experimental group of RBL-2H3 cell line expressing FPR are assumed. After the cells were seeded in a 24-well plate and cultured for 48 hours, RBL-2H3 cells expressing the two receptors were collected in a trypsin-free digestion solution and incubated with 4 ⁇ M Fluo-3 / AM at 37 ° C for 1 hour. After the cells were washed, Count, 5xl0 5 cells are required for each calcium flux measurement. The PTI spectrofluorimeter was used to detect the fluorescence absorption at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
  • Figure 4-1 and Figure 4-2 are the calcium flux responses induced by compound C1, control W-pep and buffer HBSS on RBL-2H3 cell line expressing FPRL1 and RBL-2H3 cell line expressing FPR, respectively.
  • Figure 5-1 and Figure 5-2 are the effects of pertussis toxin on the calcium flux signal of RBL-2H3 cells induced by the compound to express FPRL1.
  • Each experiment consists of a pertussis toxin-treated group and a non-pertussis toxin-treated group. Pertussis toxin at 100 ng / ml. Cells were briefly rinsed after 18 hours of incubation. The concentration of C1 was set to 100 ⁇ M, and the concentration of W-pep was set to 100 nM.
  • the experimental results show that the positive control W-pep is an FPR and FPRL1 agonist, while the intracellular calcium flow induced by compound C1 is equivalent to W -pep is the same, but it is 1000 times weaker than it.
  • compound C1 is a weak FPR agonist, and only weak calcium flux can be triggered by FPR.
  • the calcium flux response induced by W-pep and compound C1 is mediated by G protein (Gi), which is sensitive to pertussis toxin.
  • RBL-2H3 cells expressing FPRL1 were seeded in a 96-well plate at 20,000 cells / well, cultured overnight, and the calcium flow detection reagent in the FLIPR kit was added, and cultured at 37 ° C for 2 hours.
  • the excitation wavelength is 485. nm
  • emission wavelength is 525 nm.
  • the concentrations of C7 and M8 were set to: 0 ⁇ M, 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ , 33.3 ⁇ M, 100 ⁇ M.
  • the experimental results are shown in Figures 8 and 9, compounds C7 and M8 significantly antagonize the intracellular calcium flow response caused by W-pep, and their antagonism has a certain dose-dependent effect.
  • the RBL-2H3 cells transfected with FPRL1 receptor were stimulated with 100 nMW-pep, 100 nM MKM1 and 100 ⁇ M compound C1, and the cells were collected at various time points (0, 2, 5, 10, 15, 20 minutes), and electrophoresed after lysis.
  • Anti-phosphorylated Erk antibody was used to detect Eri phosphorylation by Western Blot method. Two bands were found: p44 (Erkl) and p42 (Erk2) c.
  • Anti-Erk antibody was used as a control to detect non-phosphorylated Erk in an equal amount of cell lysate.
  • the experimental results are shown in Figure 6.
  • the compounds Cl, W-pep and MMK1 all phosphorylate Erk and reach the peak of the reaction 2-5 minutes after receptor activation. However, the effects of the three ligands are different, and compound C1 is 1000 times weaker than the other two ligands.
  • Compound C1 has the same biological activity as FPRL1 receptor selective agonist MMK1 (short peptide), but the intensity of C1 action is weak. In the calcium flow experiment, the intensity of C1 was 1000 times lower than that of WKYVMm;
  • Compound C1 was confirmed to be a selective agonist of FPRL1 in degranulation experiments and calcium flow experiments;
  • Two compounds, C7 and M8, can antagonize the cell degranulation reaction and intracellular calcium flow caused by WKYVMm, which proves that they are antagonists of the FPRL1 receptor.

Abstract

The present invention relates to a type of novel formyl peptide receptor-like 1 regulators which have the following structure, and their uses in preparing the medicines of antünflammation, antünfection and immunoregulation. The present invention also relates to the preparation method of these compounds.

Description

一类甲酰肽样受体 -1调节剂、 其制备方法和用途 技术领域  A formyl peptide-like receptor-1 modulator, its preparation method and application
本发明涉及一类甲酰肽样受体一 1 (Formyl Peptide Receptor Like-1， FPRLl )调节剂， 具体指一类可作为非肽类 FPRLl调节剂的取代喹唑啉酮衍生物小分子有机化合物， 其作为 FPRL1 调节剂影响嗜中性粒白细胞的趋化和激活， 在机体非特异性和特异性炎症反应中发 挥药理作用。 本发明还涉及该化合物的制备方法。 技术背景：  The present invention relates to a class of formyl peptide-like receptor-1 (FPRLl) modulators, and specifically to a class of substituted quinazolinone derivative small molecule organic compounds that can be used as non-peptide FPRLl modulators. As a FPRL1 modulator, it affects the chemotaxis and activation of neutrophils, and plays a pharmacological role in the body's non-specific and specific inflammatory response. The invention also relates to a method for preparing the compound. technical background:
组织细胞和微生物产生的趋化剂如 N-甲酰肽 (fMLF)能引起嗜中性粒白细胞的外渗、 趋化和激活， 通过一定的信号转导途径发挥生理效应， 包括趋化作用、 炎症反应、 免疫调 节和抗病毒感染等。在感染早期， 这类趋化剂可增强机体非特异性免疫应答能力， 对抗并 清除病原微生物； 但其高水平的持续表达则引起急慢性炎症， 与其他细胞因子共同参与感 染性疾病、 ***反应性疾病和一些免疫性疾病的病理过程， 对机体造成危害。 因此， 趋化 剂及相关受体作为药物作用的新靶点，为许多具有高发病率和死亡率之人类疾病的治疗提 供了新的研究方向。  Chemotactic agents produced by tissue cells and microorganisms such as N-formyl peptide (fMLF) can cause extravasation, chemotaxis and activation of neutrophils, and exert physiological effects through certain signal transduction pathways, including Inflammatory response, immune regulation, and antiviral infection. In the early stage of infection, these chemokines can enhance the body's non-specific immune response ability, fight against and eliminate pathogenic microorganisms; but their high level of continuous expression causes acute and chronic inflammation, and participates in infectious diseases and allergies with other cytokines The pathological processes of diseases and some immune diseases cause harm to the body. Therefore, as a new target for drug action, chemokines and related receptors provide new research directions for the treatment of many human diseases with high morbidity and mortality.
人工合成的 fMLF是第一个被发现的外源性甲酰肽受体（Formyl Peptide Receptor, FPR)激动剂。 研究表明， fMLF 能刺激嗜中性粒白细胞引发一系列生理变化如细胞形状 改变、趋向性移动、粘附至血管壁、噬菌、释放超氧阴离子和脱颗粒反应。另有证据显示， fMLF可激活核转录因子 NF-κΒ, 促使吞噬细胞等分泌炎性因子， 启动机体在抵御病原微 生物入侵时的非特异性免疫应答。在积极对抗感染的同时， 吞噬细胞产生的大量超氧阴离 子和蛋白水解酶会损伤正常组织，如因急性肺损伤而导致的特发性肺纤维化（Mcholls J M, Leo L M, Kam C L， et al., Lancet 2003, 361(9371):1773-1778)。此夕卜， fMLF还能刺激单核细 胞表达一系列细胞因子如 IL-1、 IL-6和 IL-8等， 通过后者的连锁反应在免疫应答中发挥 传递信息、 上调 T细胞和抗原递呈功能， 激活抗原特异性辅助性 T细胞 1和 2 (Thl和 Th2), 产生抗病毒感染的免疫效应。 除 FPR受体外， 迄今还发现了两个人源性的 FPR同 源受体即 FPRL1和 FPRL2 (Formyl Peptide Receptor Like-2, FPRL2),但相关的受体结构、 信号转导通路和病理生理机制研究有待深入开展。  Synthetic fMLF was the first exogenous Formyl Peptide Receptor (FPR) agonist to be discovered. Studies have shown that fMLF can stimulate neutrophils to trigger a series of physiological changes such as changes in cell shape, tropism, adhesion to blood vessel walls, phage, release of superoxide anions, and degranulation reactions. There is also evidence that fMLF can activate the nuclear transcription factor NF-κΒ, promote the secretion of inflammatory factors such as phagocytes, and start the body's non-specific immune response when it resists the invasion of pathogenic microorganisms. While actively fighting infection, a large number of superoxide anions and proteolytic enzymes produced by phagocytic cells can damage normal tissues, such as idiopathic pulmonary fibrosis due to acute lung injury (Mcholls JM, Leo LM, Kam CL, et al ., Lancet 2003, 361 (9371): 1773-1778). In addition, fMLF can also stimulate monocytes to express a series of cytokines such as IL-1, IL-6, and IL-8, etc., through the chain reaction of the latter to transmit information in the immune response, upregulate T cells and antigen delivery It is functional and activates antigen-specific helper T cells 1 and 2 (Thl and Th2), producing immune effects against viral infections. In addition to the FPR receptor, two human-derived FPR homologous receptors, FPRL1 and FPRL2 (Formyl Peptide Receptor Like-2, FPRL2), have been discovered so far, but the related receptor structure, signal transduction pathway, and pathophysiology Research needs to be further developed.
鉴于甲酰肽受体本身可识别菌源性趋化物质， 其在机体对抗感染中的作用不容忽视。 敲除 FPR受体基因的小鼠比同种正常小鼠更易被细菌感染，当用 fMLF在体外刺激基因敲 除小鼠嗜中性粒细胞时， 其趋化功能减弱； 向体内注射 MLF后， 其在外周血中的游走能 力消失（Gao JL, Lee EJ, Murphy PM, J Exp Med 1999, 189(4):657-662)。最新研究成果表明， 与 FPR同源的 FPRLl高表达于嗜中性粒细胞和单核细胞表面， 在机体特异性和非特异性 炎症反应中发挥重要效应。 In view of the fact that the formyl peptide receptor can recognize bacteria-derived chemotactic substances, its role in the body's anti-infection cannot be ignored. FPR receptor gene knockout mice are more susceptible to bacterial infection than normal mice of the same type. When fMLF is used to stimulate neutrophils in gene knockout mice in vitro, their chemotaxis function is weakened. After injection of MLF into the body, Its ability to migrate in peripheral blood disappeared (Gao JL, Lee EJ, Murphy PM, J Exp Med 1999, 189 (4): 657-662). The latest research results show that FPRLl, which is homologous to FPR, is highly expressed on the surface of neutrophils and monocytes, and is specific and non-specific in the body. Plays an important effect in the inflammatory response.
FPRL1属于百日咳毒素敏感的 Gi蛋白偶联受体，它和 FPR均定位在人染色体 19ql3.3。 FPRL1与 FPR在氨基酸序列上有 69%的同源性， 除存在于嗜中性粒细胞禾单核细胞外， 在肝细胞、树突状细胞、星状细胞、 小神经胶质细胞、 B淋巴细胞及 T淋巴细胞等中均有 表达。 已知 FPRL1的配体在结构上具有多样性， 激动剂包括上述 N-甲酰化的肽类分子、 在炎症反应及组织损伤时产生的脂类 Lipoxin A4 (LXA4)、 胃幽门螺杆菌分泌的多肽 Hp FPRL1 belongs to the pertussis toxin-sensitive Gi protein-coupled receptor, and it and FPR are localized on the human chromosome 19ql3.3. FPRL1 and FPR have 69% homology in amino acid sequence. Except for neutrophils and monocytes, FPRL1 is found in liver cells, dendritic cells, stellate cells, microglia, and B lymphocytes. Both cells and T lymphocytes are expressed. It is known that the ligands of FPRL1 have structural diversity. Agonists include the aforementioned N-formylated peptide molecules, Lipoxin A4 (LXA4), which is produced during inflammation and tissue damage, and H. pylori secreted by the stomach. Peptide Hp
(2-20)、细菌的脂多糖（Bae Y S, Park J C, He R, et al" Mol Pharmacol 2003, 64(3):721-730; Betten A, Bylund J, Cristophe T, et al., J Clin Invest 2001, 108(8):1221-1228; Bylund J, Karlsson A, Boulay F, et al., Infect Immun 2002, 70(6):2908-2914)> 炎症急性反应相关蛋白(2-20). Bacterial lipopolysaccharides (Bae YS, Park JC, He R, et al "Mol Pharmacol 2003, 64 (3): 721-730; Betten A, Bylund J, Cristophe T, et al., J Clin Invest 2001, 108 (8): 1221-1228; Bylund J, Karlsson A, Boulay F, et al., Infect Immun 2002, 70 (6): 2908-2914)> Acute response-related proteins
(如淀粉样血清蛋白 Α、 β -淀粉样蛋白 1-42、 HIV-1外壳蛋白片段和病毒蛋白等） (Su S B, Gong W, Gao J L， et al., J Exp Med 1999, 189(2):395-402; Le Y， Gong W, Tiffany H L, et al., J Neurosci 2001, 21(2):RC123; Le Y, Yazawa H, Gong W, et al" J Immunol 2001, 166(3):1448-1451 )等。 研究发现， Cathelicidin C端裂解产物 LL-37具有抗菌作用并可减 弱细菌所产生的内毒素活性， 它经由 FPRL1激活嗜中性粒细胞、 单核细胞和 T细胞发挥 抵抗细菌的功能； 除抗菌外它还能招募噬菌细胞和免疫细胞游走至炎症部位， 增强机体的 免疫应急反应。 WKYMVm (W-pep)是一个从随机肽库中鉴定出来的六肽。 对于 B淋巴 细胞、 单核细胞和外周血中性粒细胞等细胞而言， W-pep是一个非常有效的激动剂。进一 步的实验证明， W-pep 主要通过 FPRL1 发挥作用 （ Hu, J. Y. et al., J Leukoc Biol2001570:155-161 另一个从随机肽库中鉴定出来的多肽 MMK-1，是 FPRL1的有效且 专一性趋化反应激动剂（Christophe, T. et al., J. Biol. Chem. 2001,276: 21585-21593)。 FPRL1 作为抗艾滋病和淀粉样病变的潜在药物治疗靶点已积累了一定的实验基础。 同时， 基于 FPRL1受体激动剂和拮抗剂的免疫调节药物筛选研究也在国外数个著名实验室展开，迄今 尚未报道发现特异性的 FPRL1有机小分子配体。 发明内容 (Such as amyloid serum protein A, β-amyloid 1-42, HIV-1 coat protein fragments and viral proteins, etc.) (Su SB, Gong W, Gao JL, et al., J Exp Med 1999, 189 (2 ): 395-402; Le Y, Gong W, Tiffany HL, et al., J Neurosci 2001, 21 (2): RC123; Le Y, Yazawa H, Gong W, et al "J Immunol 2001, 166 (3) : 1448-1451), etc. The study found that Cathelicidin C-terminal lysate LL-37 has antibacterial effect and can reduce the endotoxin activity produced by bacteria. It activates neutrophils, monocytes and T cells through FPRL1 to exert resistance. Bacterial function; In addition to antibacterial, it can recruit phage cells and immune cells to migrate to the site of inflammation and enhance the body's immune response. WKYMVm (W-pep) is a hexapeptide identified from a random peptide library. For For cells such as B lymphocytes, monocytes, and peripheral blood neutrophils, W-pep is a very effective agonist. Further experiments have shown that W-pep works mainly through FPRL1 (Hu, JY et al. , J Leukoc Biol2001 5 70: 155-161 Another peptide MMK-1 identified from a random peptide library is FPRL 1 is a potent and specific chemotactic agonist (Christophe, T. et al., J. Biol. Chem. 2001,276: 21585-21593). FPRL1 serves as a potential drug target for anti-AIDS and amyloidosis A certain experimental basis has been accumulated. At the same time, the screening of immunomodulatory drugs based on FPRL1 receptor agonists and antagonists has also been carried out in several well-known laboratories abroad, and no specific organic small molecule ligand of FPRL1 has been reported to date. content
本发明的目的在于设计与合成一类新型的取代喹唑啉酮衍生物的小分子甲酰肽样受 体一 1调节剂；  The purpose of the present invention is to design and synthesize a new class of small molecule formyl peptide-like receptor-1 regulators of substituted quinazolinone derivatives;
本发明的另一目的在于提供制备该类化合物的方法；  Another object of the present invention is to provide a method for preparing such compounds;
本发明的再一目的就是提供该类化合物作为甲酰肽样受体一 1调节剂， 在抗炎、 免疫 调节和抗感染药物治疗中的应用。  Yet another object of the present invention is to provide such compounds as modulators of formyl peptide-like receptor-1, and their applications in anti-inflammatory, immunomodulatory and anti-infective drug treatment.
本发明所述的甲酰肽样受体一 1调节剂具有下述通式 I结构：
Figure imgf000003_0001
其中 ^！和^各自独立地为芳香基或取代芳香基， 其中所述的芳香基指苯基、 环戊 二烯基、 萘基、 呋喃基、 苯并呋喃基、 噻酚基、 苯并噻酚基、 吡啶基、 吡咯基、 吲哚基或 喹啉基； 所述的取代芳香基中的取代基任意选自下列基团中的一个、 两个或三个： 烷基； 羟基； 巯基； 甲酰胺基； 甲酰氧基； 被卤素原子、 羟基或烷氧基取代的烷氧基、 烷胺基、 烷巯基、 垸酰氧基、 烷酰胺基、 垸氧羰基或烷胺羰基； 醚； 硫醚； 胺烷基； C2-C6的烯基、 苯基、 苄基、 噻吩基、 C2-C6的烯酰基或苯甲酰基； 被含有包括烷氧基、 烷胺基在内的任 意一个、 两个或者三个基团取代的苯甲酰基、 苄酰基、 噻吩甲酰基或氨酰甲基The formyl peptide-like receptor-1 modulator according to the present invention has the following general structure I:
Figure imgf000003_0001
Of which ^! And ^ are each independently an aromatic group or a substituted aromatic group, wherein the aromatic group refers to a phenyl group, a cyclopentadienyl group, a naphthyl group, a furyl group, a benzofuranyl group, a thiophenol group, a benzothiophenol group, Pyridyl, pyrrolyl, indolyl or quinolinyl; the substituent in the substituted aromatic group is arbitrarily selected from one, two or three of the following groups: alkyl group; hydroxyl group; mercapto group; formamide group Formyloxy; Alkoxy, Alkylamino, Alkylthio, Alkyloxy, Alkylamino, Alkoxycarbonyl or Alkylaminocarbonyl substituted with a halogen atom, a hydroxyl group or an alkoxyl group; Ether; Thioether; Amine alkyl; C 2 -C 6 alkenyl, phenyl, benzyl, thienyl, C 2 -C 6 alkenyl or benzoyl; contained any one including alkoxy, alkylamino , Two or three substituted benzoyl, benzoyl, thienyl or aminoacyl methyl groups
(NH2COCH2); C2-C6的烯基氧羰基； C2-C6的烯基胺羰基； 苯氧羰基； 苯氧胺羰基； 苄 氧羰基； 苄胺羰基； 噻吩氧羰基； 噻吩胺羰基； 氨酰甲基氧羰基； 氨酰甲基胺羰基； 氨酰 甲基酰氧基； 氨酰甲基酰胺基； 烷氧基； 烷胺基； 环烷氧基； 环烷胺基； 胺基及其盐酸盐 或硫酸盐； 酰胺基； 垸氧羰基； 环垸氧羰基； 垸酰氧基； 垸酰胺基； 脲基； 亚脲基； 烷酰 基； 硝基； 羧基； 醛基； 酮； 亚砜； 砜； 磺酸及其盐； 磺酰胺基及 N-取代磺酰胺基； X为 0或8。 (NH 2 COCH 2 ); C 2 -C 6 alkenyloxycarbonyl; C 2 -C 6 alkenylamine carbonyl; phenoxycarbonyl; phenoxyamine carbonyl; benzyloxycarbonyl; benzylamine carbonyl; thienyloxycarbonyl; Thienylaminocarbonyl; aminoacylmethyloxycarbonyl; aminoacylmethylamine carbonyl; aminoacylmethylacyloxy; aminoacylmethylamide; alkoxy; alkylamino; cycloalkoxy; cycloalkylamino Amine and its hydrochloride or sulfate; Amido; Amidocarbonyl; Cycloalkoxycarbonyl; Amidooxy; Amido; Urea; Ureido; Alkanoyl; Nitro; Carboxyl; Aldehyde Ketone; sulfoxide; sulfone; sulfonic acid and its salt; sulfonamide and N-substituted sulfonamide; X is 0 or 8.
本发明的一个优选实施方式是具有结构式 I的甲酰肽样受体一 1调节剂，  A preferred embodiment of the present invention is a formyl peptide-like receptor- 1 modulator having structural formula I,
其中 An与 An为各自独立地选自以下基团：  Where An and An are each independently selected from the following groups:
Figure imgf000004_0001
Figure imgf000004_0001
、 独立选自 H; 垸基； 被卤素原子、 烷氧基或羟基取代的垸基； C2-C6的烯基; 苯基； 苄基； 噻吩基； 氨酰甲基； , Is independently selected from H; embankment group; substituted with a halogen atom, an alkoxy group or a hydroxyl group embankment; C 2 -C 6 alkenyl group; a phenyl group; a benzyl group; thienyl; alanyl methyl;
为院基、 芳基或环垸基； X为 0或8。 For the base, aryl or cyclofluorenyl; X is 0 or 8.
本发明的另一个优选实施方式是具有结构式 I的甲酰肽样受体一 1调节剂， 其中 1选自以下基团： Another preferred embodiment of the present invention is a formyl peptide-like receptor-1 modulator having structural formula I, wherein 1 is selected from the following groups:
Figure imgf000005_0001
Figure imgf000005_0001
X、 X2、 、 R2的定义同前述的定义。 The definitions of X, X 2 , and R 2 are the same as the foregoing definitions.
本发明的优选化合物为 2-(4-甲氧基 -苯基 )-2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(2,4-二甲氧基-苯基) -2,3-二氢 -3-(4-丁氧基-苯甲酰胺) 喹唑啉 -4-酮； 2-(4-二甲 氨基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(2,4-二氯-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-4-甲基-苯基) -2，3-二氢 -3-(4-丁氧 -苯甲酰 胺) -1H-喹唑啉 -4-酮； 2-苯基 -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(4-羟基- 苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) 喹唑啉 -4-酮； 2-(3,4-亚甲二氧基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2- (对甲氧基-苯基) -2,3-二氢 -3-(1-苯基-乙基) -1H- 喹唑啉 _4-酮； 2- (呋喃基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮。  The preferred compound of the invention is 2- (4-methoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (2,4-dimethoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) quinazolin-4-one; 2- (4-di Methylamino-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (2,4-dichloro-phenyl ) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2-4-methyl-phenyl) -2,3-dihydro -3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2-phenyl-2,3-dihydro-3- (4-butoxy-benzamide)- 1H-quinazolin-4-one; 2- (4-hydroxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) quinazolin-4-one; 2 -(3,4-methylenedioxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- ( P-methoxy-phenyl) -2,3-dihydro-3- (1-phenyl-ethyl) -1H-quinazolin-4-one; 2- (furyl) -2,3-di Hydrogen-3- (4-butoxy-benzamide) -1H-quinazolin-4-one.
本发明通过下列步骤实施：  The invention is implemented by the following steps:
Figure imgf000005_0002
Figure imgf000005_0002
II III  II III
其中 An、 Ar2、 X的定义同前面所述。 化合物 II和化合物 III经过环合得到最终产物 化合物 I：化合物 II和 2-3当量的 III在溶剂中酸催化下进行环合反应或在酸催化下用分子 筛脱水进行环合。 反应温度和反应时间根据具体反应物而定， 通常用 TLC来跟踪测定反 应的完成程度，反应完毕后一般采用的后处理方法包括抽滤、浓缩反应液除尽溶剂、萃取、 柱层析分离等。 最终产物化合物 I用 NMR来检测证明。 本发明中， 环合反应所使用的溶 剂可采用 Ν,Ν-二甲基甲酰胺、 Ν,Ν-二甲基乙酰胺、 Ν,Ν-二甲基甲酰胺与冰醋酸的混合溶 剂、 Ν,Ν-二甲基乙酰胺与冰醋酸的混合溶剂或二氯甲烷与冰醋酸的混合溶剂； 优选的催化 用的酸为醋酸或三氟醋酸； 为了使反应快速进行， 优选在反应体系中加入 4Α分子筛； 反 应温度一般为 50~100°C。 The definitions of An, Ar 2 and X are the same as described above. Compound II and compound III are cyclized to obtain the final product compound I: Compound II and 2-3 equivalents of III are cyclized under acid catalysis in a solvent or dehydrated with molecular sieve for cyclization under acid catalysis. The reaction temperature and reaction time depend on the specific reactants. Usually TLC is used to track the reaction. After completion of the reaction, the post-treatment methods generally used include suction filtration, concentration of the reaction solution to remove the solvent, extraction, and column chromatography. The final product Compound I was verified by NMR. In the present invention, the solvent used in the cyclization reaction may be N, N-dimethylformamide, N, N-dimethylacetamide, a mixed solvent of N, N-dimethylformamide and glacial acetic acid, Ν Mixed solvent of N-dimethylacetamide and glacial acetic acid or mixed solvent of dichloromethane and glacial acetic acid; The preferred catalytic acid is acetic acid or trifluoroacetic acid; In order to make the reaction proceed quickly, it is preferable to add 4A molecular sieve; reaction temperature is generally 50 ~ 100 ° C.
化合物 II可参照文献 Tetmheron Letters 1997, 38(49) :8445-8448)通过下列方法制得： 以邻硝基苯甲酰氯（或邻硝基硫代苯甲酰氯）和 2当量化合物 AnNH2为起始原料， 先在 含三乙胺的二氯甲垸溶液中发生偶合反应， 然后以锌 /冰醋酸作为还原试剂制得中间体邻 氨基化合物。
Figure imgf000006_0001
Compound II can be prepared by reference to Tetmheron Letters 1997, 38 (49): 8445-8448) by the following method: starting from o-nitrobenzoyl chloride (or o-nitrothiobenzoyl chloride) and 2 equivalents of compound AnNH 2 As a starting material, a coupling reaction occurs in a dichloromethane solution containing triethylamine, and then zinc / glacial acetic acid is used as a reducing agent to prepare an intermediate o-amino compound.
Figure imgf000006_0001
I  I
当 X=0时， 化合物 II还可通过下列更为简便的方法一步制得： 以靛红酸酐为原料， 与 1.05当量化合物 AnNH2在苯或四氢呋喃中回流得到。 When X = 0, compound II can also be prepared in one step by the following simpler method: using isatoic anhydride as the raw material and 1.05 equivalents of compound AnNH 2 in benzene or tetrahydrofuran under reflux.
Figure imgf000006_0002
Figure imgf000006_0002
化合物 III可以通过商业途径买到或通过相应的醇用 Dess-Martin氧化 （J Org Chem, 1983, 48:4155)来制备， 化合物 Ar!NI^可通过商业途径买到或通过相应的硝基化合物还 原得到。  Compound III can be purchased commercially or prepared by oxidation of the corresponding alcohol with Dess-Martin (J Org Chem, 1983, 48: 4155). Compound Ar! NI ^ can be purchased commercially or through the corresponding nitro compound Get it.
有益效果 Beneficial effect
本发明设计与合成了一类新型的新型的取代喹唑啉酮衍生物的小分子甲酰肽样受体 一 1调节剂，该类化合物对 FPRL1功能具有一定的调节作用，有助于干预由该受体介导的 各种病理过程如急慢性炎症反应、和其他细胞因子共同参与感染性疾病、***反应性疾病 和一些免疫性疾病等， 本发明化合物是一种特异性 FPRL1调节剂， 可以在制备抗炎、 抗 感染及免疫调节药物中得到应用。 本发明化合物结构相对简单， 易于制备。 附图说明  The present invention designs and synthesizes a new class of novel small molecule formyl peptide-like receptor-1 regulators which are substituted for quinazolinone derivatives. These compounds have a certain regulatory effect on the function of FPRL1, and are helpful for intervention by The receptor mediates various pathological processes, such as acute and chronic inflammatory responses, and other cytokines participating in infectious diseases, allergic diseases, and some immune diseases. The compound of the present invention is a specific FPRL1 modulator. It is used in the preparation of anti-inflammatory, anti-infective and immunomodulatory drugs. The compound of the present invention has a relatively simple structure and is easy to prepare. BRIEF DESCRIPTION OF THE DRAWINGS
图 1为不同激动剂对嗜中性粒细胞 β-葡糖苷酶释放的影响图示；  Figure 1 is a graphical representation of the effects of different agonists on neutrophil β-glucosidase release;
图 2为不同激动剂对表达 FPRL1的 RBL-2H3细胞株 β-葡糖苷酶释放的影响图示，其 中躔表示 W_pep, *表示 MMK1,▼表示 Cl， O示表 fMLF; 图 3-1为不同激动剂对表达 FPRL1的 RBL-2H3细胞株 β-己糖胺酶释放的影响图示； 图 3-2为不同激动剂对表达 FPR的 RBL-2H3细胞株 β-己糖胺酶释放的影响图示； 图 4-1 为化合物 C1诱导的表达 FPRL1的 RBL-2H3细胞的胞内钙流反应图示； 图 4-2 为化合物 C1诱导的表达 FPR的 RBL-2H3细胞的胞内钙流反应图示； 图 5-1 为百日咳毒素对 W-pep诱导的表达 FPRL1的 RBL-2H3细胞胞内钙流反应的影 响图示； Figure 2 is a graph showing the effect of different agonists on the release of β-glucosidase from RBL-2H3 cell lines expressing FPRL1, where 躔 represents W_pep, * represents MMK1, ▼ represents Cl, and O represents fMLF; Figure 3-1 shows the effect of different agonists on the release of β-hexosaminidase from RBL-2H3 cell lines expressing FPRL1; Figure 3-2 shows the effects of different agonists on β-hexose from RBL-2H3 cell lines expressing FPR Effect of aminase release; Figure 4-1 shows the intracellular calcium flow response of FPRL1-expressing RBL-2H3 cells induced by compound C1; Figure 4-2 shows the effect of compound C1 on FBL-expressing RBL-2H3 cells Schematic diagram of intracellular calcium flux response; Figure 5-1 shows the effect of pertussis toxin on the intracellular calcium flux response of RBL-2H3 cells expressing FPRL1 induced by W-pep;
图 5-2为百日咳毒素对 C1诱导的表达 FPRL1的 RBL-2H3细胞胞内钙流反应的影响 图示；  Figure 5-2 is a graphical representation of the effect of pertussis toxin on the intracellular calcium flux response of C1-induced RBL-2H3 cells expressing FPRL1;
图 6为化合物 C1引起细胞信号转导相关激酶 Erk的活化图示；  FIG. 6 is a diagram of activation of a cell signal transduction-related kinase Erk caused by compound C1; FIG.
图 7为化合物 C7、 M8对 W-pep引起的嗜中性粒细胞脱颗粒反应的影响图示； 图 8为化合物 C7对 W-pep引起的表达 FPRL1的 RBL-2H3细胞胞内钙流反应的影响 图示；  Fig. 7 is a graph showing the effects of compounds C7 and M8 on the neutrophil degranulation reaction induced by W-pep; Impact icon
图 9为化合物 M8对 W-pep引起的表达 FPRL1的 RBL-2H3细胞胞内钙流反应的影响 图示。 具体实施方式  Figure 9 is a graphical representation of the effect of compound M8 on the intracellular calcium flux response of RBL-2H3 cells expressing FPRL1 caused by W-pep. detailed description
下面结合具体实施实例对本发明作进一步阐述， 但不限制本发明。  The present invention is further described below with reference to specific implementation examples, but the present invention is not limited.
N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1) 的制备  Preparation of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazine (1)
Figure imgf000007_0001
将 6g 4-丁氧基苯甲酰肼溶于 45ml二氯甲烷中，加入 13ml 三乙胺，冰浴下滴加 6. 4g 邻硝基苯甲酰氯的二氯甲垸溶液， 室温搅拌过夜， 过滤， 滤液用饱和碳酸钠和盐水洗涤， 无水硫酸钠干燥。 蒸干溶剂， 剩余物用乙酸乙酯-乙醇重结晶，'得白色晶体 N-(4-丁氧基- 苯甲酰基) -2-硝基 -苯甲酰肼。 将上述所得到的 1 g N-(4-丁氧基-苯甲酰基) -2-硝基-苯甲酰肼溶于 40ml N-(4-丁氧基-苯 甲酰基 )-2-氨基-苯甲酰肼 (1)。 二氯甲烷中，慢慢加入锌粉 1. 05g及冰醋酸 1. 67ml， 搅拌反 应 2小时， 过滤， 滤液用水洗， 无水硫酸钠干燥， 蒸干， 得到白色粉末 N-(4-丁氧基 -苯甲 酰基) -2-氨基-苯甲酰肼 (1)。
Figure imgf000007_0001
6 g of 4-butoxybenzoyl hydrazide was dissolved in 45 ml of dichloromethane, 13 ml of triethylamine was added, and 6.4 g of a solution of o-nitrobenzoyl chloride in dichloromethane was added dropwise under an ice bath, and stirred at room temperature overnight, After filtration, the filtrate was washed with saturated sodium carbonate and brine, and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness, and the residue was recrystallized from ethyl acetate-ethanol to obtain N- (4-butoxy-benzoyl) -2-nitro-benzoylhydrazine as white crystals. 1 g of N- (4-butoxy-benzoyl) -2-nitro-benzoylhydrazine obtained above was dissolved in 40 ml of N- (4-butoxy-benzoyl) -2-amino -Benzoylhydrazine (1). In dichloromethane, 1.05 g of zinc powder and 1.67 ml of glacial acetic acid were slowly added, and the reaction was stirred for 2 hours, filtered, and the filtrate was washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain white powder N- (4-butoxy -Benzoyl) -2-amino-benzoylhydrazine (1).
实施例 2 2-(4-甲氧基 -苯基 )-2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮（化合 物 C1 ) 的制备  Example 2 2- (4-methoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (Compound C1) Preparation
Figure imgf000008_0001
Figure imgf000008_0001
将 0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)溶于 10 ml含 5%冰醋酸的 Ν,Ν- 二甲基乙酰胺中， 加入 0.25 g 4-甲氧基苯甲醛， 在 80°C下加热反应 24小时。反应完毕后， 减压除去反应溶剂， 剩余物用柱层析分离 （硅胶 200-300 目， 用乙酸乙酯-石油醚 （1/3 ) 洗脱）， 得白色固体产物 2-(4-甲氧基 -苯基 )-2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4- 酮 (Cl)。  Dissolve 0.30 g of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazine (1) in 10 ml of N, N-dimethylacetamide containing 5% glacial acetic acid and add 0.25 g of 4-methoxybenzaldehyde was heated at 80 ° C for 24 hours. After completion of the reaction, the reaction solvent was removed under reduced pressure, and the residue was separated by column chromatography (silica gel 200-300 mesh, eluting with ethyl acetate-petroleum ether (1/3)) to obtain 2- (4-form) as a white solid product. Oxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (Cl).
iHNMR (300MHz, DMSO - d6): δ 0.92 (t, 3H)， 1.41 (m, 2H)， 1.67 (m, 2H)， 3.73(s， 3H)， 3.99 (t, 2H), 6.11 (s, 1H)， 6.75 (m， 2H)， 6.91 (m， 4H)， 7.33 - 7.68 (m, 6H )。 iHNMR (300MHz, DMSO-d 6 ): δ 0.92 (t, 3H), 1.41 (m, 2H), 1.67 (m, 2H), 3.73 (s, 3H), 3.99 (t, 2H), 6.11 (s, 1H), 6.75 (m, 2H), 6.91 (m, 4H), 7.33-7.68 (m, 6H).
实施例 3 2-(2,4-二甲氧基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮（化 合物 C2) 的制备  Example 3 2- (2,4-dimethoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one ( Preparation of compound C2)
Figure imgf000008_0002
Figure imgf000008_0002
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.30 g 2,4-二甲氧基苯甲醛溶在 10 ml含 5%冰醋酸的 N，N-二甲基乙酰胺中， 50°C下加热反应 24小时。后处理方法同实施 例 1，得白色固体产物 2-(2,4-二甲氧基-苯基) -2，3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4- 酮 (C2)  0.30 g of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazide (1) and 0.30 g of 2,4-dimethoxybenzaldehyde in 10 ml of 5% glacial acetic acid In N, N-dimethylacetamide, the reaction was heated at 50 ° C for 24 hours. The post-treatment method was the same as in Example 1, and a white solid product 2- (2,4-dimethoxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H was obtained. -Quinazolin-4-one (C2)
1HNMR (300ΜΗζ， DMSO - d6): δ 0.92 (t, 3Η)， 1.42 (m, 2Η)， 1.68 (m, 2H), 3.59 (s, 3H)， 3.72 (s， 3H)， 4.00 (t, 2H), 6.40 (s, 1H )， 6.52 (m, 2H)， 6.73 (m, 2H)， 6.94 - 7.02 (m， 3H)， 7.28 (t, 1H)， 7.48 (m, 1H)， 7.65 (m, 2H)。 1HNMR (300MΗζ, DMSO-d 6 ): δ 0.92 (t, 3Η), 1.42 (m, 2Η), 1.68 (m, 2H), 3.59 (s, 3H), 3.72 (s, 3H), 4.00 (t, 2H), 6.40 (s, 1H), 6.52 (m, 2H), 6.73 (m, 2H), 6.94-7.02 (m, 3H), 7.28 (t , 1H), 7.48 (m, 1H), 7.65 (m, 2H).
实施例 4 2-(4-二甲氨基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 （化 合物 C3) 的制备  Example 4 2- (4-dimethylamino-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (compound C3) Preparation
Figure imgf000009_0001
Figure imgf000009_0001
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.27 g 4-二甲氨基苯甲醛在 10 ml 含 5%冰醋酸的 N，N-二甲基乙酰胺中 60°C下加热反应 24小时。 后处理方法同实施例 1， 得浅黄色固体产物 2-(4-二甲氨基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 (C3) 0.30 g of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazide (1) and 0.27 g of 4-dimethylaminobenzaldehyde in 10 ml of N, N- The reaction was heated at 60 ° C in dimethylacetamide for 24 hours. The post-treatment method was the same as in Example 1, and a pale yellow solid product 2- (4-dimethylamino-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quine was obtained. Oxazolin-4-one (C3)
1HNMR (300MHz， DMSO - d6): δ 0.92 (t, 3H)， 1.42 (m, 2H)， 1.68 (m, 2H)， 2.84 (s, 6H)， 4.00 (t, 2H), 6.04 (s, 1H)， 6.68-6.79 (m, 4H)， 6.92 (m, 2H)， 7.17-7.30 (m， 4H ), 7.61 (m， 2H)。 1HNMR (300MHz, DMSO-d 6 ): δ 0.92 (t, 3H), 1.42 (m, 2H), 1.68 (m, 2H), 2.84 (s, 6H), 4.00 (t, 2H), 6.04 (s, 1H), 6.68-6.79 (m, 4H), 6.92 (m, 2H), 7.17-7.30 (m, 4H), 7.61 (m, 2H).
实施例 5 2-(2,4-二氯-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 （化合 物 C4) 的制备  Example 5 2- (2,4-dichloro-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (Compound C4 ) Preparation
Figure imgf000009_0002
Figure imgf000009_0002
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.32 g 2,4-二氯苯甲醛在 10 ml含 5%冰醋酸的 Ν,Ν-二甲基乙酰胺中 100Ό下加热反应 24小时。 后处理方法同实施例 1， 得 白色固体产物 2-(2,4-二氯-苯基) -2,3-二氢 -3-(4-丁氧基-苯甲酰胺 )-1Η-喹唑啉 -4-酮 (C4)。  0.30 g of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazine (1) and 0.32 g of 2,4-dichlorobenzaldehyde in 10 ml of 5% glacial acetic acid N, N -Heat reaction at 100 ° F in dimethylacetamide for 24 hours. The post-treatment method was the same as that in Example 1. The white solid product 2- (2,4-dichloro-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quine was obtained. Oxazolin-4-one (C4).
1HNMR (300MHz， DMSO - d6): δ 0.92 (t, 3Η)， 1.42 (m, 2Η ), 1.69 (m, 2H )， 4.00 (t， 2H)， 6.58 (s, 1H)， 6.78 (m, 1H)， 6.99 (m, 2H), 7.35 (m, 1H)， 7.53-7.80 (m, 7H)， 7.93 (m, 1H)。  1HNMR (300MHz, DMSO-d6): δ 0.92 (t, 3Η), 1.42 (m, 2Η), 1.69 (m, 2H), 4.00 (t, 2H), 6.58 (s, 1H), 6.78 (m, 1H ), 6.99 (m, 2H), 7.35 (m, 1H), 7.53-7.80 (m, 7H), 7.93 (m, 1H).
. 实施例 6 2-4-甲基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮（化合物 C5) 的制备 Example 6 2-4-methyl-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (compound C5) Preparation
Figure imgf000010_0001
Figure imgf000010_0001
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.22 g 4-甲基苯甲醛在 10 ml含 5%冰醋酸的 Ν,Ν-二甲基乙酰胺中 80°C下加热反应 24小时。后处理方法同实施例 1，得白 色固体产物 2-4-甲基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 (C5)。  0.30 g N- (4-butoxy-benzoyl) -2-amino-benzoyl hydrazide (1) and 0.22 g 4-methylbenzaldehyde in 10 ml N, N-di The reaction was heated at 80 ° C in methylacetamide for 24 hours. The post-treatment method was the same as in Example 1, and a white solid product of 2-methyl-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazoline- 4-keto (C5).
1HNMR (300MHz， DMSO - d6)： δ 0.91 (t， 3H)， 1.40 (m, 2H)， 1.68 (m， 2H)， 2.31 (m， 3H)， 3.99 (t， 2H)， 6.11 (s, 1H )， 6.78 (m, 2H), 6.94 (m， 2H), 7.18 - 7.35 (m， 6H)， 7.58 (m， 2H) o 1HNMR (300MHz, DMSO-d 6 ): δ 0.91 (t, 3H), 1.40 (m, 2H), 1.68 (m, 2H), 2.31 (m, 3H), 3.99 (t, 2H), 6.11 (s, 1H), 6.78 (m, 2H), 6.94 (m, 2H), 7.18-7.35 (m, 6H), 7.58 (m, 2H) o
实施例 7 2-苯基 -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 （化合物 C6) 的 制备  Example 7 Preparation of 2-phenyl-2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (Compound C6)
Figure imgf000010_0002
Figure imgf000010_0002
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.19 g苯甲醛在 10 ml含 5%冰醋 酸的 Ν,Ν-二甲基乙酰胺中 50°C下加热反应 24小时。 后处理方法同实施例 1， 得白色固体 产物 2-苯基 -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 (C6)。  0.30 g of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazine (1) and 0.19 g of benzaldehyde in 10 ml of N, N-dimethylacetamide with 5% glacial acetic acid The reaction was heated at 50 ° C for 24 hours. The post-treatment method was the same as in Example 1. The white solid product 2-phenyl-2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (C6) was obtained. .
1HNMR (300MHz， DMSO - d6): δ 0.92 (t, 3Η ), 1.40 (m, 2H)， 1.67 (m， 2H )， 3.99 (t， 2H), 6.15 (s, 1H)， 6.82-7.00 (m, 4H)， 7.46 (m, 4H), 7.58 - 7.69 (m, 5H)。 1HNMR (300MHz, DMSO-d 6 ): δ 0.92 (t, 3Η), 1.40 (m, 2H), 1.67 (m, 2H), 3.99 (t, 2H), 6.15 (s, 1H), 6.82-7.00 ( m, 4H), 7.46 (m, 4H), 7.58-7.69 (m, 5H).
实施例 8 2-(4-羟基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 （化合物 Example 8 2- (4-hydroxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one (compound
C7) 的制备 C7) Preparation
Figure imgf000011_0001
Figure imgf000011_0001
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.22 g 4-羟基苯甲醛在 10 ml含 5%冰醋酸的 Ν,Ν-二甲基乙酰胺中 70Ό下加热反应 24小时。后处理方法同实施例 1，得浅 黄色固体产物 2-(4-羟基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 (C7)。 0.30 g N- (4-butoxy-benzoyl) -2-amino-benzoyl hydrazide (1) and 0.22 g 4-hydroxybenzaldehyde in 10 ml N, N-dimethylformaldehyde with 5% glacial acetic acid The reaction was heated at 70 ° F for 24 hours in acetylacetamide. The post-treatment method was the same as that in Example 1, and a pale yellow solid product 2- (4-hydroxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazoline was obtained. 4-one (C7).
1HNMR (300MHz, DMSO - d6): δ 0.92 (t, 3Η), 1.39 (m, 2H), 1.66 (m， 2H )， 3.99 (t， 2H)， 6.04 (s, 1H )， 6.73-6.91 (m, 6H)， 7.22 (m， 1H), 7.30 (m, 2H)， 7.58 (m， 2H)， 7.67 (m， 1H)。 1HNMR (300MHz, DMSO-d 6 ): δ 0.92 (t, 3Η), 1.39 (m, 2H), 1.66 (m, 2H), 3.99 (t, 2H), 6.04 (s, 1H), 6.73-6.91 ( m, 6H), 7.22 (m, 1H), 7.30 (m, 2H), 7.58 (m, 2H), 7.67 (m, 1H).
实施例 9 2-(3，4-亚甲二氧基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 Example 9 2- (3,4-Methylenedioxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one
(化合物 C8) 的制备 Preparation of (Compound C8)
Figure imgf000011_0002
Figure imgf000011_0002
0.30 g N-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.28 g胡椒醛在 10 ml含 5%冰醋 酸的 Ν,Ν-二甲基乙酰胺中 100°C下加热反应 24小时。 后处理方法同实施例 1， 得白色固 体产物 2-(3,4-亚甲二氧基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮 (C8)。  0.30 g of N- (4-butoxy-benzoyl) -2-amino-benzoylhydrazide (1) and 0.28 g of piperonal in 10 ml of N, N-dimethylacetamide with 5% glacial acetic acid The reaction was heated at 100 ° C for 24 hours. The post-treatment method was the same as in Example 1, and the white solid product 2- (3,4-methylenedioxy-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide)- 1H-quinazolin-4-one (C8).
1HNMR (300MHz, DMSO - d6): δ 0.92 (t, 3Η), 1.44 (m， 2H), 1.68(m, 2H )， 4.01 (t， 2H), 6.00 (s， 2H), 6.07 (s, 1H ), 6.75-6.91 (m, 6H)， 7.03 (m, 1H)， 7.27 (m, 2H), 7.68 (m, 2H) c 1HNMR (300MHz, DMSO-d 6 ): δ 0.92 (t, 3Η), 1.44 (m, 2H), 1.68 (m, 2H), 4.01 (t, 2H), 6.00 (s, 2H), 6.07 (s, 1H), 6.75-6.91 (m, 6H), 7.03 (m, 1H), 7.27 (m, 2H), 7.68 (m, 2H) c
实施例 10 2- (对甲氧基-苯基) -2,3-二氢 -3-(l-苯基-乙基) -1H-喹唑啉 -4-酮 （化合物 M8) 的制备 Example 10 Preparation of 2- (p-methoxy-phenyl) -2,3-dihydro-3- (l-phenyl-ethyl) -1H-quinazolin-4-one (compound M8)
Figure imgf000012_0001
Figure imgf000012_0001
0.35 gN-(l-苯基-乙基) -2-氨基-苯甲酰胺（2)和 0.30 g对甲氧基苯甲醛在 10 ml含 5% 冰醋酸的 Ν,Ν-二甲基乙酰胺中 100°C下加热反应 24小时。 后处理方法同实施例 1， 得白 色固体产物 2- (对甲氧基-苯基) -2,3-二氢 -3-(1-苯基-乙基) -1H-喹唑啉 -4-酮（Μ8)。 0.35 g N- (l-phenyl-ethyl) -2-amino-benzamide (2) and 0.30 g p-methoxybenzaldehyde in 10 ml N, N-dimethylacetamide with 5% glacial acetic acid The reaction was heated at 100 ° C for 24 hours. The post-treatment method was the same as that in Example 1. The white solid product 2- (p-methoxy-phenyl) -2,3-dihydro-3- (1-phenyl-ethyl) -1H-quinazoline-4 was obtained. -Ketone (M8).
!HNMR (300MHz, DMSO - d6): δ 1.19(d, 3H), 3.63(m, IH), 3.70(s， 3H)， 6.17(s, 1H)， 6.78 (d, 2H)， 7.08(d, 2H ), 7.12(m, 5H), 7.24(m, 4H)。 ! HNMR (300MHz, DMSO-d 6 ): δ 1.19 (d, 3H), 3.63 (m, IH), 3.70 (s, 3H), 6.17 (s, 1H), 6.78 (d, 2H), 7.08 (d , 2H), 7.12 (m, 5H), 7.24 (m, 4H).
实施例 11 2- (呋喃基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -IH-喹唑啉 -4-酮（化合物 W1 ) 的制备  Example 11 Preparation of 2- (furyl) -2,3-dihydro-3- (4-butoxy-benzamide) -IH-quinazolin-4-one (compound W1)
Figure imgf000012_0002
Figure imgf000012_0002
0.10 gN-(4-丁氧基 -苯甲酰基) -2-氨基-苯甲酰肼 (1)和 0.096g康醛在 10 ml含 2ml冰醋 酸和 2ml N，N-二甲基乙酰胺二氯甲烷中 60°C下加热反应 2小时。后处理方法同实施例 1， 得白色固体产物 2- (呋喃基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) 喹唑啉 -4-酮 （Wl )。 iHNMR (300MHz, CDC13): δ 0.97(t, 3H), 1.46(m, 2H), 1.79(m, 2H)， 3.96(t, 2H), 6.27(m, IH), 6.3 l(s, IH), 6.39(m, IH), 6.70(d, IH), 6.79(d, 2H)， 6.84(t, 1H)， 7.34(m, 2H), 7.69(d, 2H), 7.91(d, IH). 0.10 g of N- (4-butoxy-benzoyl) -2-amino-benzoyl hydrazide (1) and 0.096 g of Conaldehyde in 10 ml containing 2 ml of glacial acetic acid and 2 ml of N, N-dimethylacetamide di The reaction was heated at 60 ° C for 2 hours in methyl chloride. The post-treatment method was the same as in Example 1 to obtain 2- (furanyl) -2,3-dihydro-3- (4-butoxy-benzamide) quinazolin-4-one (W1) as a white solid product. iHNMR (300MHz, CDC1 3 ): δ 0.97 (t, 3H), 1.46 (m, 2H), 1.79 (m, 2H), 3.96 (t, 2H), 6.27 (m, IH), 6.3 l (s, IH ), 6.39 (m, IH), 6.70 (d, IH), 6.79 (d, 2H), 6.84 (t, 1H), 7.34 (m, 2H), 7.69 (d, 2H), 7.91 (d, IH) .
实施例 12 生物活性测试  Example 12 Biological activity test
1.实验材料与仪器  Experimental materials and instruments
大鼠嗜碱性淋巴瘤细胞 RBL-2H3细胞株，购自美国模式菌种收集中心 (American Type Culture Collection);  Rat basophilic lymphoma cell RBL-2H3 cell line, purchased from the American Type Culture Collection;
人源甲酰肽受体（FPR)、 甲酰肽样受体 1 (FPRL1 )真核表达质粒（伊利诺大学芝加 哥分校）；  Human formyl peptide receptor (FPR), formyl peptide-like receptor 1 (FPRL1) eukaryotic expression plasmid (University of Illinois at Chicago)
胎牛血清、 DMEM培养基（GIBCO/BRL); 细胞松弛素 B 、对硝基 -N-乙酰基 -β-D-葡糖酰胺、 4-甲基伞形基 -β-D-葡糖苷酸水合物 (Sigma, St. Louis, MO); Fetal bovine serum, DMEM medium (GIBCO / BRL); Cytochalasin B, p-nitro-N-acetyl-β-D-glucamide, 4-methylumbellyl-β-D-glucuronide hydrate (Sigma, St. Louis, MO);
抗磷酸化 Erk抗体和抗 Erk抗体（Cell Signaling Technologies);  Anti-phosphorylated Erk antibody and anti-Erk antibody (Cell Signaling Technologies);
Fluo3/AM (Molecular Probes, Eugene, OR);  Fluo3 / AM (Molecular Probes, Eugene, OR);
百日咳毒素 (List Laboratories, Campbell, CA);  Pertussis toxin (List Laboratories, Campbell, CA);
G418 (Invitrogen, Carlsbad, CA);  G418 (Invitrogen, Carlsbad, CA);
月旨质体 LipofectAmine (Invitrogen, Carlsbad, CA);  LipofectAmine (Invitrogen, Carlsbad, CA);
FLIPR钙流检测试剂盒（Molecular Devices, San Diego, CA)  FLIPR Calcium Flow Detection Kit (Molecular Devices, San Diego, CA)
Forma二氧化碳培养箱（Forma, USA);  Forma carbon dioxide incubator (Forma, USA);
PTI荧光检测仪 (Photon Technology International, Lawrenceville, NJ);  PTI fluorescence detector (Photon Technology International, Lawrenceville, NJ);
SpectroMax 340读板机 (Molecular Dynamics, Sunnyvale, CA);  SpectroMax 340 plate reader (Molecular Dynamics, Sunnyvale, CA);
FlexStation 384荣光读板机 (Molecular Devices, San Diego, CA)。  FlexStation 384 Honor Plate Reader (Molecular Devices, San Diego, CA).
fMLF: N端甲酰化 3肽（N-Fomiyl-Met-Leu-Phe )， 购自（Sigma, St. Louis, MO)；  fMLF: N-terminal formylated 3 peptide (N-Fomiyl-Met-Leu-Phe), purchased from (Sigma, St. Louis, MO);
W-pep: W-肽 （WKYMVm), 人工合成的 6肽；  W-pep: W-peptide (WKYMVm), a synthetic 6 peptide;
MMK1 : 人工合成的 13肽（LESIFRSLLFRVM)。  MMK1: synthetic 13 peptide (LESIFRSLLFRVM).
2. 实验方法及结果  2. Experimental methods and results
2.1 表达 FPR或 FPRL1受体的 RBL-2H3细胞株培养  2.1 RBL-2H3 cell line culture expressing FPR or FPRL1 receptor
RBL-2H3细胞培养在含 10%胎牛血清和 2 mM L型谷氨酰胺的 DMEM培养基中。按 脂质体 （商品名 LipofectAmine) 的使用说明， 将 FPR或 FPRL1受体质粒转入其中， 以 G418 (400 ng/ml)选择 4-5周， 挑选稳定表达受体的细胞克隆。  RBL-2H3 cells were cultured in DMEM medium containing 10% fetal bovine serum and 2 mM L-glutamine. According to the instructions for use of liposomes (trade name LipofectAmine), FPR or FPRL1 receptor plasmids were transferred into it, and G418 (400 ng / ml) was selected for 4-5 weeks, and cell clones stably expressing the receptor were selected.
2.2分离嗜中性粒细胞  2.2 Isolation of neutrophils
采取健康供者的外周血参照文献方法（Ulmer, A丄 and Flad, H.D. (1979). J. Immunol. Methods 30, 1-10)进行 Percoll梯度离心。 嗜中性粒细胞的分离效率约为 97% , 存活率大 于 98%。 将其重悬在无血清的 RPMI1640培养基中， 细胞密度为 2> 106个 /ml。 重复实验 所需血细胞的供者样本数不少于 3例。 Percoll gradient centrifugation was performed on a peripheral blood reference method from healthy donors (Ulmer, A 丄 and Flad, HD (1979). J. Immunol. Methods 30, 1-10). The neutrophil isolation efficiency is about 97%, and the survival rate is greater than 98%. Resuspended in serum-free RPMI1640 medium, 2 a cell density of> 106 cells / ml. The number of donor samples required for repeated experiments was not less than 3 cases.
2.3 β-葡糖苷酶释放量的检测方法  2.3 Detection method of β-glucosidase release
将 20 μΐ样品和 20 μΐ 10 mM 4-甲基伞形基 -β-D-葡糖苷酸水合物在含 0.1% Triton X-100的 0.1 M醋酸钠溶液（pH 4.0) 中于 37°C下孵育 15分钟。 加入 300 μΐ含有 50 mM 甘氨酸和 5 mM EDTA的终止液 (pH 10.4)终止反应。立即用 PTI分光荧光计在 365 nm激发 波长和 460 nm发射波长下检测荧光反应。细胞内总的 β-葡糖苷酶量从含 0.1% Triton X- 100 细胞裂解液中测得。 结果以上清中 β-葡糖苷酶含量占细胞中总葡糖苷酶含量的百分比表 示。  20 μΐ sample and 20 μΐ 10 mM 4-methylumbellyl-β-D-glucuronide hydrate in a 0.1 M sodium acetate solution (pH 4.0) containing 0.1% Triton X-100 at 37 ° C Incubate for 15 minutes. The reaction was stopped by adding 300 μΐ of a stop solution (pH 10.4) containing 50 mM glycine and 5 mM EDTA. Immediately detect the fluorescence reaction with a PTI spectrofluorometer at an excitation wavelength of 365 nm and an emission wavelength of 460 nm. The total amount of β-glucosidase in cells was measured from 0.1% Triton X-100 cell lysate. Results The percentage of β-glucosidase content in the above supernatant to the total glucosidase content in the cells is shown.
2.4 β-己糖苷酶释放量的检测方法  2.4 Detection method of β-hexosidase release
将 20 μΐ样品和 10 μΐ 1 mM的对硝基 -N-乙酰 -β-D-葡糖酰胺于 37°C孵育 1小时后，加 After incubating 20 μΐ samples and 10 μΐ 1 mM p-nitro-N-acetyl-β-D-glucamide at 37 ° C for 1 hour, add
】2 入 200 μΐ θ.1 Μ Na2C03和 0.1 Μ NaHC03 ( H 10) 终止反应， 用 SpectroMax 340读扳机 测定 405 nm处的吸光值。 细胞内总的 β-己糖苷酶量从 0.1% Triton X-100细胞裂解液中测 得。 结果以上清中 β-己糖苷酶含量占细胞中总 β-己糖苷酶含量的百分比表示。 】2 The reaction was stopped by adding 200 μΐ θ. 1 M Na 2 C0 3 and 0.1 M NaHC0 3 (H 10), and the absorbance at 405 nm was measured with a SpectroMax 340 reading trigger. The total amount of β-hexosidase in the cells was measured from 0.1% Triton X-100 cell lysate. Results The percentage of β-hexosidase in the above supernatant to the total β-hexosidase content in cells was expressed.
2.5 细胞脱颗粒反应实验  2.5 Cell degranulation reaction experiment
2.5.1 化合物 C1对嗜中性粒细胞脱颗粒反应的影响  2.5.1 Effect of compound C1 on neutrophil degranulation response
将按照 2.2方法分离得到的嗜中性粒细胞重悬在含有 10 μΜ细胞松弛素 Β的 HBSS 中， 细胞密度为 6.25χ106个 /ml (0.5xl06个 / 80 μ1)， 冰上孵育 15分钟， 再置于 37°C放置 15分钟。预孵化后，取 80 μΐ细胞悬液分别与 80 μΐ含不同浓度的化合物 C1及各对照品的 HBSS-HB缓冲液（含 20 mM HEPES, (pH 7.4)和 0.2% BSA的 HBSS)混合， 于 37°C放 置 10分钟后置于冰上终止脱颗粒反应， 离心分离上清和细胞， 取各测试组上清， 按照 2.3 的方法检测其中 β-葡糖苷酶的释放量。本实验中化合物 C1的浓度设置为 0μΜ、0. 1μΜ、 1μΜ、 10μΜ、 ΙΟΟμΜ; 对照品 fMLF及 W-pep的浓度设置为 0ηΜ、 1ηΜ、 10nM、 100nM、 ΙΟΟΟηΜ; 对照品 MMK1的浓度设置为 0nM、 10nM、 100nM、 300nM、 1000nM。 实验结果如图 1所示， 被剌激后的嗜中性粒细胞 β-葡糖苷酸酶的分泌量与给药浓度呈剂量依赖性关系，其中化合 物 C1的效应与 MMK1相近， 但 MMK1具有更强的亲和力。 The neutrophils isolated according to 2.2 were resuspended in HBSS containing 10 μM cytochalasin B with a cell density of 6.25 × 10 6 cells / ml (0.5 × 10 6 cells / 80 μ1) and incubated on ice for 15 minutes. , And placed at 37 ° C for 15 minutes. After pre-incubation, 80 μΐ of cell suspension was mixed with 80 μΐ of HBSS-HB buffer (containing 20 mM HEPES, (pH 7.4) and 0.2% BSA HBSS) containing different concentrations of compound C1 and each reference, and mixed in After being left at 37 ° C for 10 minutes, it was placed on ice to stop the degranulation reaction. The supernatant and cells were separated by centrifugation. The supernatant of each test group was collected and the β-glucosidase release amount was detected according to the method of 2.3. In this experiment, the concentration of compound C1 was set to 0 μM, 0.1 μM, 1 μM, 10 μM, 100 μM; the concentrations of the reference fMLF and W-pep were set to 0ηM, 1ηM, 10nM, 100nM, 100nM; the concentration of the reference MMK1 was set to 0nM , 10nM, 100nM, 300nM, 1000nM. The experimental results are shown in Figure 1. The amount of β-glucuronidase secreted by the neutrophil after stimulation was dose-dependent, and the effect of compound C1 was similar to that of MMK1, but MMK1 had more Strong affinity.
2.5.2化合物 C1对转染 FPRL1受体的 RBL-2H3细胞株脱颗粒反应的影响  2.5.2 Effect of compound C1 on degranulation reaction of RBL-2H3 cell line transfected with FPRL1 receptor
转染 FPRL1受体的 RBL-2H3细胞以 50000个 /孔的密度接种于 48孔板， 培养两天后 用 HBSS洗两遍， 加入 40μ 含有 10 μΜ细胞松弛素 Β的 HBSS中， 冰上孵育 15分钟， 再置于 37°C放置 15分钟。 预孵化后， 加入 40 μΐ含不同浓度的化合物 C1及各对照品的 HBSS缓冲液混合， 于 37°C放置 10分钟后置于冰上终止脱颗粒反应， 离心分离上清和细 胞， 取各测试组上清， 按照 2.3的方法检测其中 β-葡糖苷酶的释放量。本实验中化合物 C1 的浓度设置为 0μΜ、 0. 01μΜ、 0. 03μΜ、 0. 1μΜ、 0. 3μΜ、 1μΜ、 3μΜ、 10μΜ、 30μΜ、 ΙΟΟμΜ; 对照品 MMK1及 W-pep的浓度设置为 0nM、 0. 1ηΜ、 0. 3ηΜ、 1ηΜ、 3nM、 10nM、 30nM、 100nM、 300nM、 1000nM; 对照品 fMLF的浓度设置为 0nM、 1ηΜ、 10nM、 100nM、 1000nM、 10000nMo 实验结果如图 2所示， fMLF作为 FPR受体的激动剂， 不诱导经由 FPRL1介导的脱颗粒 作用， W-pep是最强效的 FPRL1受体激动剂，化合物 C1 和 MMK1的效应检测结果相似， 但 MMK1在强度上胜过化合物 Cl。 经计算， 相应的 EC5Q值分别为： 化合物 Cl = 1.88 x 1(Γ6Μ; MMKl =7.17 x 10-8M; W-pep = 2.29 x 10'8M。 RBL-2H3 cells transfected with FPRL1 receptor were seeded in 48-well plates at a density of 50,000 cells / well. After two days of culture, they were washed twice with HBSS, added to 40 μ HBSS containing 10 μM cytochalasin B, and incubated on ice for 15 minutes. , And placed at 37 ° C for 15 minutes. After pre-incubation, add 40 μΐ of HBSS buffer containing different concentrations of compound C1 and each reference, mix, leave at 37 ° C for 10 minutes, and place on ice to stop the degranulation reaction. Centrifuge the supernatant and cells, and take each test group. The supernatant was tested for the amount of β-glucosidase released according to the method of 2.3. In this experiment, the concentration of compound C1 was set to 0 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM; the concentrations of the reference MMK1 and W-pep were set to 0 nM, 0.1nM, 0.3nM, 1nM, 3nM, 10nM, 30nM, 100nM, 300nM, 1000nM; The concentration of the reference fMLF is set to 0nM, 1ηM, 10nM, 100nM, 1000nM, 10000nMo. The experimental results are shown in Figure 2. FPR receptor agonist does not induce degranulation via FPRL1. W-pep is the most potent FPRL1 receptor agonist. The effects of compounds C1 and MMK1 are similar, but MMK1 outperforms the compound in strength. Cl. After calculation, the corresponding EC 5Q values are: Compound Cl = 1.88 x 1 (Γ 6 Μ; MMKl = 7.17 x 10- 8 M; W-pep = 2.29 x 10 ' 8 M.
2.5.3化合物 CI选择性介导 RBL-2H3细胞株脱颗粒反应实验  2.5.3 Compound CI selectively mediates degranulation reaction of RBL-2H3 cell line
设表达 FPR的 RBL-2H3细胞株实验组和表达 FPRL1的 RBL-2H3细胞株实验组，实 验细胞株按照 2.1方法制备并以 50000个 /孔的密度接种于 48孔板， 培养两天后用 HBSS 洗两遍， 加入 40μί含有 10 μΜ细胞松弛素 Β的 HBSS中， 冰上共孵育 15分钟， 最后在 37°C下和 1 mM对硝基 -N-乙酰基 -β-D-葡糖酰胺孵育 15分钟。 冲洗细胞后， 用不同剂量 的化合物 C1及各对照品的 HBSS-HB缓冲液于 37°C剌激 10分钟后， 将培养板置于冰上 终止脱颗粒反应。 取各测试组上清， 按照 2.4的方法检测其中 β-己糖胺 |的释放量。 本实 验中用于刺激表达 FPR受体的 RBL-2H3细胞株的化合物 C1的剂量设' 为 0μΜ、1μΜ、10μΜ、 ΙΟΟμΜ; 对照品 fMLF、 W-pep及 MMKl的浓度设置为 ΟηΜ、 10ηΜ、 ΙΟΟηΜ, ΙΟΟΟηΜ; 用 于剌激表达 FPRL1受体的 RBL-2H3细胞株的化合物 C1的剂量设置为 ΟμΜ、 10μΜ、 ΙΟΟμΜ; 对照品 fMLF及 W-pep的浓度设置为 0ηΜ、 10ηΜ、 100ηΜ、 ΙΟΟΟηΜ; 对照品 MMKl的浓度 设置为 0nM、 1ηΜ、 10nM、 100nM。 实验结果如图 3-1及图 3-2所示， MMKl和化合物 C1 选择性地激活表达 FPRL1受体细胞 β-己糖胺酶分泌途径， 但对表达 FPR细胞 β-己糖胺酶 的分泌则无影响。 比较发现， fMLF选择性地作用于 FPR而非 FPRL1 ; W-pep对 FPR和 FPRL1均有激动效应， 在表达上述两种受体的细胞上均引发脱颗粒作用。 The experimental group of the RBL-2H3 cell line expressing FPR and the experimental group of the RBL-2H3 cell line expressing FPRL1 were prepared. The experimental cell line was prepared according to the method 2.1 and seeded in a 48-well plate at a density of 50,000 cells / well, and washed with HBSS for two days Two times, add 40 μL of HBSS containing 10 μM cytochalasin B, incubate for 15 minutes on ice, and finally incubate at 37 ° C with 1 mM p-nitro-N-acetyl-β-D-glucosamide for 15 minutes. minute. After washing the cells, stimulate the cells with different doses of compound C1 and HBSS-HB buffer at 37 ° C for 10 minutes, and then place the culture plate on ice. Stop the degranulation reaction. The supernatant of each test group was taken, and the β-hexosamine | release amount was detected according to the method of 2.4. The dose of compound C1 used to stimulate RBL-2H3 cell line expressing FPR receptor in this experiment was set to 0 μM, 1 μM, 10 μM, 100 μM; the concentrations of the reference fMLF, W-pep, and MMK1 were set to 0 ηM, 10 ηM, 100 ηΜ , ΙΟΟΟηΜ; The dose of compound C1 used to stimulate the RBL-2H3 cell line expressing the FPRL1 receptor was set to 0 μM, 10 μM, 100 μM; the concentrations of the reference fMLF and W-pep were set to 0ηM, 10ηM, 100ηM, 100ηM; control The concentration of product MMK1 was set to 0 nM, 1 nM, 10 nM, 100 nM. The experimental results are shown in Figure 3-1 and Figure 3-2. MMK1 and compound C1 selectively activate the β-hexosaminidase secretion pathway of cells expressing FPRL1 receptors, but secrete β-hexosaminidase secretion of FPR cells. No effect. The comparison found that fMLF selectively acts on FPR rather than FPRL1; W-pep has an agonistic effect on both FPR and FPRL1, and triggers degranulation on cells expressing both receptors.
2.5.4化合物 C7和化合物 M8对 W-pep引起的嗜中性粒细胞脱颗粒反应的影响 实验方法同 2.5.1中所述， 预孵化后的 80 μΐ细胞悬液分别与 80 μΐ含 100 nM W-pep, 100 μΜ C7和 M8的 HBSS-HB缓冲液混合。 实验结果如图 7所示， 100 μ Μ的化合物 C7 和 Μ8均可抑制 100 nM W-pep刺激人嗜中性粒细胞对 β-葡糖苷酶的分泌，其中化合物 Μ8 的效应稍强于 C7。  2.5.4 Effect of compound C7 and compound M8 on neutrophil degranulation reaction induced by W-pep The experimental method is the same as described in 2.5.1. The pre-incubated 80 μΐ cell suspension and 80 μΐ 100 nM W-pep, 100 μM C7 and M8 HBSS-HB buffer were mixed. The experimental results are shown in Figure 7. Both compounds C7 and M8 at 100 μM could inhibit 100 nM W-pep from stimulating human neutrophils to secrete β-glucosidase. Among them, the effect of compound M8 was slightly stronger than that of C7.
2.6钙流实验  2.6 Calcium Flow Experiment
2.6.1化合物 C1引发 RBL-2H3细胞内钙流效应实验  2.6.1 Effect of compound C1 on calcium flux in RBL-2H3 cells
设表达 FPRL1的 RBL-2H3细胞株实验组和表达 FPR的 RBL-2H3细胞株实验组。 细 胞接种于 24孔板培养 48小时后， 用无胰酶消化液分别收集表达两种受体的 RBL-2H3细 胞，与 4 μΜ Fluo-3/AM在 37°C下孵育 1小时，细胞冲洗后，计数，每次钙流测定需 5xl05 个细胞。使用 PTI分光荧光计检测激发波长为 488 nm和发射波长为 525 nm下荧光吸收值。 图 4-1、图 4-2分别为化合物 C1、对照品 W-pep及缓冲液 HBSS在表达 FPRL1的 RBL-2H3 细胞株和表达 FPR的 RBL-2H3细胞株上诱导的钙流反应。 图 5-1、 图 5- 2为百日咳毒素 对化合物诱导表达 FPRL1的 RBL-2H3细胞钙流信号的影响，每个实验设百日咳毒素处理 组和非百日咳毒素处理组， 其中对百日咳毒素处理组给予 100 ng/ml的百日咳毒素， 孵育 18小时后将细胞简单冲洗。 C1浓度均设定为 100 μΜ， W-pep的浓度设置为 100 nM. 实验 结果表明， 阳性对照品 W-pep是 FPR和 FPRL1激动剂， 而化合物 C1引发的细胞内钙流 在效能上与 W-pep—致， 但在强度上比其弱 1000倍。 对比发现， 化合物 C1是 FPR弱激 动剂， 通过 FPR仅能触发微弱钙流。 此外， W-pep和化合物 C1诱导的钙流反应是通过对 百日咳毒素敏感的 G蛋白 （Gi)所介导的 The experimental group of RBL-2H3 cell line expressing FPRL1 and the experimental group of RBL-2H3 cell line expressing FPR are assumed. After the cells were seeded in a 24-well plate and cultured for 48 hours, RBL-2H3 cells expressing the two receptors were collected in a trypsin-free digestion solution and incubated with 4 μM Fluo-3 / AM at 37 ° C for 1 hour. After the cells were washed, Count, 5xl0 5 cells are required for each calcium flux measurement. The PTI spectrofluorimeter was used to detect the fluorescence absorption at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Figure 4-1 and Figure 4-2 are the calcium flux responses induced by compound C1, control W-pep and buffer HBSS on RBL-2H3 cell line expressing FPRL1 and RBL-2H3 cell line expressing FPR, respectively. Figure 5-1 and Figure 5-2 are the effects of pertussis toxin on the calcium flux signal of RBL-2H3 cells induced by the compound to express FPRL1. Each experiment consists of a pertussis toxin-treated group and a non-pertussis toxin-treated group. Pertussis toxin at 100 ng / ml. Cells were briefly rinsed after 18 hours of incubation. The concentration of C1 was set to 100 μM, and the concentration of W-pep was set to 100 nM. The experimental results show that the positive control W-pep is an FPR and FPRL1 agonist, while the intracellular calcium flow induced by compound C1 is equivalent to W -pep is the same, but it is 1000 times weaker than it. By comparison, compound C1 is a weak FPR agonist, and only weak calcium flux can be triggered by FPR. In addition, the calcium flux response induced by W-pep and compound C1 is mediated by G protein (Gi), which is sensitive to pertussis toxin.
2.6.2化合物 C7和化合物 M8对 W-pep诱导表达 FPRL1的 RBL-2H3细胞内钙流的影 响  2.6.2 Effect of compound C7 and compound M8 on W-pep-induced calcium flux in RBL-2H3 cells expressing FPRL1
表达 FPRL1的 RBL-2H3细胞以 20000个 /孔接种于 96孔板， 培养过夜， 加入 FLIPR 试剂盒中的钙流检测试剂， 37°C培养 2小时。 加入不同浓度的 C7或 M8， 37°C孵育 30 分钟， 加入 6 nM的 W-pep, 应用 FlexStation 384荧光读板机进行检测， 激发波长为 485 nm， 发射波长为 525 nm。 C7和 M8的浓度设置为： 0μΜ、 0. 001μΜ、 0. 01μΜ、 0. 1μΜ、 1μΜ、 10μ 、 33. 3μΜ、 100μΜ。 实验结果如图 8和图 9所示， 化合物 C7和 Μ8显著拮抗 W-pep 引起的胞内钙流反应， 且其拮抗作用具有一定的剂量依赖效应。 RBL-2H3 cells expressing FPRL1 were seeded in a 96-well plate at 20,000 cells / well, cultured overnight, and the calcium flow detection reagent in the FLIPR kit was added, and cultured at 37 ° C for 2 hours. Add different concentrations of C7 or M8, incubate at 37 ° C for 30 minutes, add 6 nM W-pep, and use FlexStation 384 fluorescence plate reader for detection. The excitation wavelength is 485. nm, emission wavelength is 525 nm. The concentrations of C7 and M8 were set to: 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μ, 33.3 μM, 100 μM. The experimental results are shown in Figures 8 and 9, compounds C7 and M8 significantly antagonize the intracellular calcium flow response caused by W-pep, and their antagonism has a certain dose-dependent effect.
2.7化合物 C1引起细胞信号转导相关激酶 Erk的活化  2.7 Compound C1 Activates Cell Signaling-Related Kinase Erk
分别用 100nMW-pep、 ΙΟΟηΜΜΜΚΙ和 ΙΟΟμΜ化合物 C1 刺激转染 FPRL1 受体的 RBL-2H3细胞， 分别在各个作用时间点 (0、 2、 5、 10、 15、 20分钟)收集细胞， 裂解后进 行电泳并用抗磷酸化 Erk抗体以 Western Blot方法检测 Eri 磷酸化， 发现两个条带： p44 (Erkl )和 p42 (Erk2) c 抗 Erk抗体作为对照检测等量细胞裂解液中非磷酸化 Erk。 实验 结果如图 6所示， 化合物 Cl、 W-pep和 MMK1均可在受体活化后 2— 5分钟使 Erk发生 磷酸化并达到反应的高峰。 但三种配体的效应有强度差异， 化合物 C1较其他两个配体弱 1000倍。  The RBL-2H3 cells transfected with FPRL1 receptor were stimulated with 100 nMW-pep, 100 nM MKM1 and 100 μM compound C1, and the cells were collected at various time points (0, 2, 5, 10, 15, 20 minutes), and electrophoresed after lysis. Anti-phosphorylated Erk antibody was used to detect Eri phosphorylation by Western Blot method. Two bands were found: p44 (Erkl) and p42 (Erk2) c. Anti-Erk antibody was used as a control to detect non-phosphorylated Erk in an equal amount of cell lysate. The experimental results are shown in Figure 6. The compounds Cl, W-pep and MMK1 all phosphorylate Erk and reach the peak of the reaction 2-5 minutes after receptor activation. However, the effects of the three ligands are different, and compound C1 is 1000 times weaker than the other two ligands.
3. 实验结论  3. Experimental conclusions
( 1) 化合物 C1与 FPRL1受体选择性激动剂 MMK1 (短肽） 具有相同的生物活性， 但 C1作用的强度较弱。 在钙流实验中 C1的激动强度比 WKYVMm降低 1000倍；  (1) Compound C1 has the same biological activity as FPRL1 receptor selective agonist MMK1 (short peptide), but the intensity of C1 action is weak. In the calcium flow experiment, the intensity of C1 was 1000 times lower than that of WKYVMm;
(2) 脱颗粒实验和钙流实验中证实化合物 C1是 FPRL1的受体选择性激动剂； (2) Compound C1 was confirmed to be a selective agonist of FPRL1 in degranulation experiments and calcium flow experiments;
(3) 化合物 C1引发的细胞内钙流经由对百日咳毒素敏感的 Gi蛋白介导，说明 FPRL1 受体与百日咳毒素敏感的 Gi蛋白相偶联。 (3) Intracellular calcium flow induced by compound C1 is mediated by Gi protein that is sensitive to pertussis toxin, indicating that the FPRL1 receptor is coupled to Gi protein that is sensitive to pertussis toxin.
(4) 化合物 C1激活 FPRL1受体后能触发信号转导途径中 Erk磷酸化反应。  (4) The activation of FPRL1 receptor by compound C1 can trigger Erk phosphorylation in signal transduction pathway.
(5) C7和 M8两个化合物可以拮抗 WKYVMm引起的细胞脱颗粒反应和细胞内钙 流， 证实其为 FPRL1受体的拮抗剂。  (5) Two compounds, C7 and M8, can antagonize the cell degranulation reaction and intracellular calcium flow caused by WKYVMm, which proves that they are antagonists of the FPRL1 receptor.

Claims

权利要求 Rights request
1、 一类具有下述通式结构的甲酰肽样受体一 1调节剂:  1. A class of formyl peptide-like receptors with the following general structure: 1 modulators:
Figure imgf000017_0001
其中 1和 2各自独立地为芳香基或取代芳香基， 其中所述的芳香基指苯基、 环戊 二烯基、 萘基、 呋喃基、 苯并呋喃基、 噻酚基、 苯并噻酚基、 吡啶基、 吡咯基、 吲哚基或 喹啉基； 所述的取代芳香基中的取代基任意选自下列基团中的一个、 两个或三个： 垸基； 羟基； 巯基； 甲酰胺基； 甲酰氧基； 被卤素原子、 羟基或烷氧基取代的烷氧基、 垸胺基、 烷巯基、 烷酰氧基、 烷酰胺基、 烷氧羰基或烷胺羰基； 醚； 硫醚； 胺垸基； C2-C6的烯基、 苯基、 苄基、 噻吩基、 c2-c6的烯酰基或苯甲酰基； 被含有包括烷氧基、 烷胺基在内的任 意一个、 两个或者三个基团取代的苯甲酰基、 苄酰基、 噻吩甲酰基或氨酰甲基
Figure imgf000017_0001
Wherein 1 and 2 are each independently an aromatic group or a substituted aromatic group, wherein the aromatic group refers to a phenyl group, a cyclopentadienyl group, a naphthyl group, a furyl group, a benzofuranyl group, a thiophenol group, and a benzothiophene Group, pyridyl, pyrrolyl, indolyl or quinolinyl; the substituent in the substituted aromatic group is arbitrarily selected from one, two or three of the following groups: amidino; hydroxyl; thiol; Amido; formyloxy; alkoxy, amido, alkylthio, alkanoyloxy, alkamido, alkoxycarbonyl or alkaminocarbonyl substituted by halogen atom, hydroxyl or alkoxy; ether; sulfur Ethers; Amino groups; C 2 -C 6 alkenyl, phenyl, benzyl, thienyl, c 2 -c 6 alkenyl or benzoyl groups; containing alkoxy, alkylamino groups Any one, two or three groups substituted with benzoyl, benzoyl, thienyl or aminoacylmethyl
(NH2COCH2); C2-C6的烯基氧羰基； C2-C6的烯基胺羰基； 苯氧羰基； 苯氧胺羰基； 苄 氧羰基； 苄胺羰基； 噻吩氧羰基； 噻吩胺羰基； 氨酰甲基氧羰基； 氨酰甲基胺羰基； 氨酰 甲基酰氧基； 氨酰甲基酰胺基； 烷氧基； 垸胺基； 环烷氧基； 环垸胺基； 胺基及其盐酸盐 或硫酸盐； 酰胺基； 垸氧羰基； 环烷氧羰基； 垸酰氧基； 烷酰胺基； 脲基； 亚脲基； 烷酰 基； 硝基； 羧基； 醛基； 酮； 亚砜； 砜； 磺酸及其盐； 磺酰胺基及 N-取代磺酰胺基； X为 0或8。 (NH 2 COCH 2 ); C 2 -C 6 alkenyloxycarbonyl; C 2 -C 6 alkenylamine carbonyl; phenoxycarbonyl; phenoxyamine carbonyl; benzyloxycarbonyl; benzylamine carbonyl; thienyloxycarbonyl; Thienylamine carbonyl; aminoacyloxycarbonyl; aminoacylmethylamine carbonyl; aminoacylmethylacyloxy; aminoacylmethylamide; alkoxy; amido; cycloalkoxy; cycloamido Amine and its hydrochloride or sulfate; Amido; Amidocarbonyl; Cycloalkoxycarbonyl; Amidooxy; Alkamido; Urea; Ureido; Alkanoyl; Nitro; Carboxyl; Aldehyde Ketone; sulfoxide; sulfone; sulfonic acid and its salt; sulfonamide and N-substituted sulfonamide; X is 0 or 8.
2、 根据权利要求 1所述的甲酰肽样受体一 1调节剂， 其特征在于其中 Art与 A 为各 自独立地选自以下基团：  2. The formyl peptide-like receptor-1 modulator according to claim 1, wherein Art and A are each independently selected from the following groups:
Figure imgf000017_0002
Figure imgf000018_0001
Figure imgf000017_0002
Figure imgf000018_0001
Ri R2独立选自 H; 烷基； 被卤素原子、 烷氧基或羟基取代的烷基； ¾- 的烯基； 苯基； 苄基； 噻吩基； 氨酰甲基； Ri R 2 is independently selected from H ; alkyl ; alkyl substituted with halogen atom, alkoxy or hydroxy; alkenyl of ¾-; phenyl; benzyl; thienyl; aminoacylmethyl;
为烷基、 芳基或环垸基。  Is alkyl, aryl or cyclofluorenyl.
3、根据权利要求 1所述的甲酰肽样受体一 1调节剂，其特征在于其中 选自以下基 团：  3. The formyl peptide-like receptor-1 modulator according to claim 1, characterized in that it is selected from the group consisting of:
Figure imgf000018_0002
Ar2为 ， 其中 n为 0或 1
Figure imgf000018_0002
Ar 2 is, where n is 0 or 1
4、 根据权利要求 1所述的甲酰肽样受体一 1调节剂， 其特征在于包括以下化合物- 2-(4-甲氧基-苯基) -2,3-二氢 -3-(4-丁氧基-苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(2,4-二甲氧基-苯 基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(4-二甲氨基-苯基) -2,3-二氢 -3-(4- 丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(2,4-二氯-苯基) -2，3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H- 喹唑啉 _4-酮； 2-4-甲基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑啉 -4-酮； 2-苯基 -2,3- 二氢 -3-(4-丁氧基-苯甲酰胺) -1H-喹唑啉 -4-酮； 2-(4-羟基-苯基) -2,3-二氢 -3-(4-丁氧基-苯甲 酰胺) -1H-喹唑啉 -4-酮； 2-(3,4-亚甲二氧基-苯基) -2,3-二氢 -3-(4-丁氧基 -苯甲酰胺) -1H-喹唑 啉 _4_酮； 2- (对甲氧基-苯基) -2,3-二氢 -3-(l-苯基-乙基) -1H-喹唑啉 -4-酮； 2- (呋喃基) -2,3-二 氢 -3-(4-丁氧基 -苯甲酰胺) 喹唑啉 -4-酮。 4. The formyl peptide-like receptor-1 modulator according to claim 1, comprising the following compound: 2- (4-methoxy-phenyl) -2,3-dihydro-3- ( 4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (2,4-dimethoxy-phenyl) -2,3-dihydro-3- (4-butane Oxy-benzamide) -1H-quinazolin-4-one; 2- (4-dimethylamino-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide ) -1H-quinazolin-4-one; 2- (2,4-dichloro-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quine Oxazoline_4-one; 2-4-methyl-phenyl) -2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2 -Phenyl-2,3-dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (4-hydroxy-phenyl) -2,3- Dihydro-3- (4-butoxy-benzamide) -1H-quinazolin-4-one; 2- (3,4-methylenedioxy-phenyl) -2,3-dihydro -3- (4-butoxy-benzamide) -1H-quinazole _4_one; 2- (p-methoxy-phenyl) -2,3-dihydro-3- (l-phenyl-ethyl) -1H-quinazolin-4-one; 2- ( Furyl) -2,3-dihydro-3- (4-butoxy-benzamide) quinazolin-4-one.
5、 权利要求 1 所述的甲酰肽样受体一 1 调节剂的制备方法， 其特征在于由化合物
Figure imgf000019_0001
和化合物 Ar2CHO在溶剂中酸催化下进行环合反应或在酸催化下用分子筛 脱水进行环合制得。 5. The method for preparing a formyl peptide-like receptor-1 modulator according to claim 1, characterized in that it comprises a compound
Figure imgf000019_0001
It is prepared by carrying out cyclization reaction with compound Ar 2 CHO under acid catalysis in a solvent or dehydration with molecular sieve under acid catalysis.
6、 根据权利要求 5所述的甲酰肽样受体一 1调节剂的制备方法， 其特征在于环合反 应所用的溶剂为 Ν,Ν-二甲基甲酰胺、 Ν,Ν-二甲基乙酰胺、 Ν,Ν-二甲基甲酰胺与冰醋酸的 混合溶剂、 Ν,Ν-二甲基乙酰胺与冰醋酸的混合溶剂或二氯甲烷与冰醋酸的混合溶剂。  6. The method for preparing a formyl peptide-like receptor-1 modulator according to claim 5, characterized in that the solvent used in the cyclization reaction is N, N-dimethylformamide, Ν, N-dimethyl Acetamide, a mixed solvent of N, N-dimethylformamide and glacial acetic acid, a mixed solvent of N, N-dimethylacetamide and glacial acetic acid, or a mixed solvent of methylene chloride and glacial acetic acid.
7、 根据权利要求 5所述的甲酰肽样受体一 1调节剂的制备方法， 其特征在于催化用 酸为醋酸或三氟醋酸。  7. The method for preparing a formyl peptide-like receptor-1 modulator according to claim 5, wherein the catalyzing acid is acetic acid or trifluoroacetic acid.
8、 根据权利要求 5所述的甲酰肽样受体一 1调节剂的制备方法， 其特征在于所述分 子筛为 4Α分子筛。 '  8. The method for preparing a formyl peptide-like receptor-1 modulator according to claim 5, wherein the molecular sieve is a 4A molecular sieve. '
9、 根据权利要求 5所述的甲酰肽样受体一 1调节剂的制备方法， 其特征在于反应温 度为 50~100°C。  9. The method for preparing a formyl peptide-like receptor-1 modulator according to claim 5, characterized in that the reaction temperature is 50 to 100 ° C.
10、 权利要求 1所述的甲酰肽样受体一 1调节剂在制备抗炎、 抗感染及免疫调节药物 中的应用。  10. The use of a formyl peptide-like receptor-1 modulator according to claim 1 in the preparation of anti-inflammatory, anti-infective and immunomodulatory drugs.
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