WO2019071395A1 - 一种抗白色念珠菌的二芳基硫族化合物及其制备和应用 - Google Patents

一种抗白色念珠菌的二芳基硫族化合物及其制备和应用 Download PDF

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WO2019071395A1
WO2019071395A1 PCT/CN2017/105400 CN2017105400W WO2019071395A1 WO 2019071395 A1 WO2019071395 A1 WO 2019071395A1 CN 2017105400 W CN2017105400 W CN 2017105400W WO 2019071395 A1 WO2019071395 A1 WO 2019071395A1
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candida albicans
compound
diaryl
chalcogenide
diarylchalcogenide
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PCT/CN2017/105400
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English (en)
French (fr)
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邓音乐
张珉
赵朔
谭振达
孙秀云
蒙丽丽
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华南农业大学
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Priority to PCT/CN2017/105400 priority Critical patent/WO2019071395A1/zh
Priority to CN201780025371.7A priority patent/CN109843289B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • 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
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles

Definitions

  • the invention belongs to the field of biomedical technology. More specifically, it relates to a diaryl chalcogenide against Candida albicans and its preparation and use.
  • Candida albicans is a fungal disease widely spread in humans. It is an important conditional pathogenic fungus that usually causes acute, subacute or chronic infections. It is also one of the most important pathogens for hospital-acquired infections. . On healthy human mucosal surfaces, such as the oral cavity and intestinal tract, Candida albicans usually does not cause disease, but in patients with impaired or suppressed immune system, such as chemotherapy patients, organ transplant patients or AIDS patients, it can cause serious system. Sexual infections have a mortality rate of up to 40%.
  • Fluconazole drugs are widely used. Fluconazole plays a bacteriostatic role by inhibiting fungal replication, but with the abuse of antibiotics, the more resistant the drug The more serious it is.
  • the technical problem to be solved by the invention is to overcome the defects and deficiencies of the existing anti-Candida albicans drugs, and to design a compound against Candida albicans by using a novel antibacterial strategy, starting from the yeast-hyphae biphasic characteristic of Candida albicans, targeted It has designed a new type of compound with high efficiency, low toxicity and low resistance, which has important scientific significance and application prospects.
  • Another object of the present invention is to provide a process for the preparation of the diarylchalcogenide.
  • a further object of the invention is to provide the use of the diarylchalcogenide.
  • the diaryl chalcogenide is prepared by hydrogenating a nitrogen-containing heterocyclic compound and a diaryl sulfur/
  • the selenoether compound is mixed, dimethyl sulfoxide is added as a solvent and iodine as a promoter, and after stirring, the reaction mixture is cooled to room temperature, and the reaction liquid is diluted with ethyl acetate, then washed and dried, and the solvent is evaporated under reduced pressure to give a crude product. Purification by column chromatography gave the diarylchalcogenide.
  • the hydrogenated nitrogen-containing heterocyclic compound, the diarylsulfide/selenoether compound and the iodine element have a molar ratio of 0.5 to 1.5:0.5 to 1.5:1 to 2.
  • the hydrogenated nitrogen-containing heterocyclic compound, the diarylsulfide/selenide compound and the iodine element have a molar ratio of 1:1:1.5.
  • the hydrogenated nitrogen-containing heterocyclic compound is indoline.
  • the diarylsulfide/selenoether compound is diphenyl sulfide or diphenyl selenide.
  • the solvent dimethyl sulfoxide and the iodine element are used in an amount of 1 to 2 mL: 1 to 2 mmol.
  • the solvent dimethyl sulfoxide and iodine are used in an amount of 2 mL: 1.5 mmol.
  • the stirring reaction is carried out by stirring the reaction at 100 ° C for 4 to 20 hours.
  • the washing is washed several times with water.
  • Yeast-hyphae biphasic properties are characteristic of Candida albicans.
  • the free yeast state is not toxic to the host during infection, mainly exerts the action of adhering to the receptor tissue, and then undergoes a transition from a yeast state to a mycelial state to promote invasion, and then enters the invagination with the mycelial state as a pathogen. Organize to further play a toxic role.
  • the morphological transformation of yeast-hyphae is an important process for Candida albicans to exert virulence.
  • the present invention utilizes a novel bacteriostatic strategy to design a compound against Candida albicans, and starts from the yeast-hyphae biphasic characteristic of Candida albicans, and specifically designs a novel compound which is highly efficient, low in toxicity, and resistant to drug resistance.
  • Candida albicans was used as the test object to investigate the effects of the diaryl chalcogenide designed and synthesized by the present invention on the adhesion, hyphal formation rate, cytotoxicity and survival rate of Candida albicans.
  • the purpose is to further influence the toxic effects of Candida albicans in mammals by detecting the interference of compounds on the virulence factors of Candida albicans.
  • the results showed that the compound had a good inhibitory effect on the adhesion of Candida albicans, mycelial morphology transformation and pathogenicity.
  • the compound itself is less toxic and does not affect the growth of Candida albicans and human cells.
  • the anti-Candida albicans refers to inhibiting the adhesion of Candida albicans, the transformation of mycelium morphology, Pathogenicity and / or virulence effects on cells.
  • a drug against Candida albicans containing the diarylchalcogenide or a drug for preventing an infectious disease caused by Candida albicans should also be within the scope of the present invention.
  • the invention provides a novel diaryl chalcogen compound which has good inhibitory effect on the adhesion of Candida albicans, mycelium morphology transformation and pathogenicity. Moreover, the compound itself is less toxic and does not affect the growth of human cells, and has a good application prospect in the development of novel antifungal drugs, especially for the anti-candida infection drug.
  • the present invention also found that the diaryl chalcogen compound only has a certain inhibitory effect on the growth of Candida albicans, indicating that the effect of the compound on the Candida albicans strain does not depend mainly on killing bacteria, but on inhibiting bacteria. Adhesion, hyphal morphological transformation and pathogenicity are therefore less susceptible to drug resistance.
  • Figure 1 is a diagram showing the synthesis process of a diarylchalcogenide.
  • Figure 2 is a nuclear magnetic resonance spectrum of Compound No. 3.
  • Figure 3 is a carbon spectrum of Compound No. 3.
  • Figure 4 is a nuclear magnetic hydrogen spectrum of Compound No. 8.
  • FIG. 5 is a carbon spectrum of Compound No. 8.
  • Figure 6 is a graph showing the effect of a diaryl chalcogen compound on the adhesion of Candida albicans; wherein, Figure (A) shows the effect of 13 compounds at a final concentration of 100 ⁇ M on the adhesion of Candida albicans cells to polystyrene.
  • Figure (B) is a graph showing the inhibition rates of four compounds of 1, 2, 3, and 8 at different concentrations of 6.25 ⁇ M to 100 ⁇ M; DMSO as a control; the data shows the average of 8 biological replicates. The error bar reflects the standard deviation.
  • Figure 7 is a graph showing the effect of diarylchalcogenide on the formation of Candida albicans hyphae; wherein, Figure (A) is the formation of 13 synthetic diaryl chalcogenides at a final concentration of 100 ⁇ M against Candida albicans hyphae Figure (B) shows the inhibition effect of compounds No. 1, 2, 3, and 8 at different concentrations of 6.25 ⁇ M to 100 ⁇ M; Figure (C) is DMSO, fluconazole, 1, 2 A microscopic observation of the inhibition of hyphal formation at the final concentration of 100 ⁇ M for compounds No. 3 and No. 8; this data shows the average results of three biological experiments, and the error bars reflect the standard deviation.
  • Figure (A) is the formation of 13 synthetic diaryl chalcogenides at a final concentration of 100 ⁇ M against Candida albicans hyphae
  • Figure (B) shows the inhibition effect of compounds No. 1, 2, 3, and 8 at different concentrations of 6.25 ⁇ M to 100 ⁇ M
  • Figure (C) is DMSO
  • Figure 8 is a graph showing the effect of diaryl chalcogenide on the growth rate of Candida albicans; DMSO as a control; the data shows the average of three biological replicates, and the error bars reflect the standard deviation.
  • Figure 9 is the effect of diaryl chalcogenide on the pathogenicity of Candida albicans in A549 cells;
  • A cytotoxicity of 13 diaryl chalcogenides at a final concentration of 100 ⁇ M on A549 cells;
  • B Effects of 13 diaryl chalcogenides at a final concentration of 100 ⁇ M on Candida albicans infection;
  • C Compounds 1, 2, 3, and 8 against Candida albicans at different concentrations ranging from 6.25 ⁇ M to 100 ⁇ M The effect after staining the cells.
  • the compounds A-1 to A-6 are: tetrahydroquinoline, indoline, 4-cyanoindoline, methyl 6-dihydroporphyrinate, 7-azaindoline Ruthenium, 7,8-benzotetrahydroquinoline (see Figure 1 for structural formula);
  • Compounds B-1 to B-6 are: methyl 2,2'-diphenyl sulfide, 2,2'-dipyridine sulfide, diphenyl sulfide, 4,4'-dimethyldiphenyl sulfide Ether, diphenyl selenide, 4,4'-dimethoxydiphenyl selenide (see Figure 1 for structural formula).
  • Candida albicans standard strain SC5314 was activated in LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L), and placed at 30 Incubate overnight in a °C incubator.
  • the 96-well plate was incubated at 37 ° C, and after 4 hours, the bacterial solution was discarded, and 50 ⁇ L of 0.5% crystal violet was added thereto, and the mixture was allowed to stand at room temperature for 45 minutes.
  • the crystal violet was discarded and washed 10 times with ice ddH 2 O, 200 ⁇ L of 75% ethanol was added, and left at room temperature for 30 minutes, and OD 590 was measured, and the data was processed by GraphPad Prism6 software.
  • Take 1 mL of the bacterial solution in a 1.5 ml EP tube add the compound with a final concentration of 100 ⁇ M in turn, shake and mix, add 300 ⁇ L each to the 100-well plate, set 4 replicates for each treatment, and set only DMSO plus fluconazole. Processing.
  • the results were placed in a growth curve analyzer at 30 ° C, 200 rpm, and the OD 600 value was measured every 2 hours. After 2 days, the experimental results were observed, and GraphPad Prism 6 was used to process the data.
  • A549 cell preparation A549 cells were cultured in a 96-well plate at a cell concentration of 1.5 ⁇ 10 4 cells/well in a high-sugar medium DMEM containing 10% fetal calf serum overnight. When the cells were covered with 80% of the bottom of the 96-well plate, the culture solution was discarded, and the cells were washed 3 times with 1 ⁇ PBS.
  • Cytotoxicity assay a final concentration of 100 ⁇ M compound was added to the cell-containing maintenance medium, 100 ⁇ L was added to the prepared A549 cells, and cultured at 37 ° C in a 5% CO 2 cell incubator for 8 h. Four replicates were processed, and only DMSO, BDSF, and fluconazole were added as controls, and the virulence of the compound to the cells themselves was measured, that is, the compound having a final concentration of 100 ⁇ M was added to the cell-free maintenance solution, and treated as above. Refer to Promega's CytoTox The NonRadioactive Cytotoxicity Assay method was used to determine cellular LDH activity, followed by processing data using GraphPad Prism 6.
  • mice tail vein injection Compounds 3 and 8 at a final concentration of 100 ⁇ M were added to the PBS containing bacteria, and mixed for use. This trial was conducted in accordance with the National Institutes of Health's Health Guidance for the Care and Use of Laboratory Animals (NIH 8023 publication, revised 1978). The mice were weighed, injected into the tail vein, 100 ⁇ L/10 g, and simultaneously injected with 1 ⁇ PBS and 100 ⁇ M fluconazole as controls. The survival of the mice was continuously observed, and the data was recorded after 20 days, and the data was processed with GraphPad Prism 6.
  • the compound can inhibit the virulence of Candida albicans strain SC5314 on cells
  • mice 1, 2, 3, and 8 Based on the results of all aspects of the diaryl chalcogenide, we selected mice 1, 2, 3, and 8 that performed well in all aspects, and the experiment was continued for 20 days.
  • the results of the experiment show that compounds No. 3 and No. 8 are new candidates for Candida albicans drugs.
  • the present invention synthesizes a series of diaryl chalcogenides and screens their ability to combat fungal pathogens. Some of these compounds show excellent inhibition of Candida albicans cell adhesion, hyphal formation and pathogenicity; although they are not toxic to Candida albicans cells themselves, they are also not toxic to human cells. Experimental results show that some of these compounds may be developed as novel drugs against Candida albicans infection.

Abstract

本发明公开了一种抗白色念珠菌的二芳基硫族化合物及其制备和应用。所述二芳基硫族化合物的结构如式(I)所示：其中，Y为S或Se。该二芳基硫族化合物对白色念珠菌的粘附性、菌丝形态转换和致病性具有很好的抑制作用。而且，该化合物本身毒性较小，不影响人类细胞的生长，不易产生耐药性，在新型抗真菌药物的开发，尤其是抗白色念珠菌感染药物的开发方面具有很好的应用前景。

Description

一种抗白色念珠菌的二芳基硫族化合物及其制备和应用 技术领域
本发明属于生物医药技术领域。更具体地，涉及一种抗白色念珠菌的二芳基硫族化合物及其制备和应用。
背景技术
白色念珠菌(Candida albicans)是在人类中广泛传播的真菌疾病，是一种重要的条件致病真菌，通常会引起急性、亚急性或慢性感染，也是现在医院获得性感染最重要的病原之一。在健康人体黏膜表面，如口腔、肠道，白色念珠菌通常不会引起病害，但在免疫系统受到损害或抑制的病人体内，如化疗病人、器官移植病人或艾滋病人中，会引起严重的系统性感染，其致死率高达40％。
目前在临床上抗真菌药物种类有限，其中唑类药物(氟康唑)应用广泛，氟康唑是通过抑制真菌复制起到抑菌的作用，但是随着抗生素的滥用，耐药性的现象越来越严重。
发明内容
本发明要解决的技术问题是克服现有抗白色念珠菌药物的缺陷和不足，利用新型抑菌策略设计抗白色念珠菌的化合物，从白色念珠菌特有的酵母-菌丝二相性入手，针对性地设计出了高效、低毒、不易产生耐药性的新型化合物，具有重要的科学意义和应用前景。
本发明的目的是提供一种抗白色念珠菌的二芳基硫族化合物。
本发明另一目的是提供所述二芳基硫族化合物的制备方法。
本发明的再一目的是提供所述二芳基硫族化合物的应用。
本发明上述目的通过以下技术方案实现：
一种抗白色念珠菌的二芳基硫族化合物，其结构如式(I)所示：
Figure PCTCN2017105400-appb-000001
其中，Y为S或Se。
所述二芳基硫族化合物的制备方法为：将氢化含氮杂环化合物和二芳基硫/ 硒醚化合物混合，加入二甲基亚砜作为溶剂和碘单质作为促进剂，搅拌反应结束后冷却至室温，用乙酸乙酯稀释反应液，然后洗涤干燥，减压蒸除溶剂得粗产物，最后经柱层析提纯得到所述二芳基硫族化合物。
其中，优选地，所述氢化含氮杂环化合物、二芳基硫/硒醚化合物和碘单质的摩尔比为0.5～1.5：0.5～1.5：1～2。
更优选地，所述氢化含氮杂环化合物、二芳基硫/硒醚化合物和碘单质的摩尔比为1：1：1.5。
优选地，所述氢化含氮杂环化合物为二氢吲哚。
优选地，所述二芳基硫/硒醚化合物为二苯硫醚或二苯硒醚。
优选地，所述的溶剂二甲基亚砜和碘单质的用量比为1～2mL：1～2mmol。
更优选地，所述的溶剂二甲基亚砜和碘单质的用量比为2mL：1.5mmol。
优选地，所述搅拌反应的条件为在100℃下搅拌反应4～20小时。
优选地，所述洗涤是用水洗涤数遍。
优选地，用水洗涤3遍。
酵母-菌丝二相性是白色念珠菌特有的特性。游离的酵母态在感染期间对于宿主是没有毒性的，主要行使粘附于受体组织的作用，随后进行由酵母态向菌丝态的转变以促进入侵，之后以菌丝态为病原进入侵染组织，进一步发挥毒性作用。酵母-菌丝的形态转变是白色念珠菌发挥毒力的重要过程。因此，本发明利用新型抑菌策略设计抗白色念珠菌的化合物，从白色念珠菌特有的酵母-菌丝二相性入手，针对性地设计高效、低毒、不易产生耐药性的新型化合物。然后以白色念珠菌(Candida albicans)为供试对象，考察了本发明设计合成的二芳基硫族化合物对白色念珠菌的粘附性、菌丝形成率、细胞毒性以及小鼠存活率的影响作用，目的是通过检测化合物对白色念珠菌毒力形成因素的干扰，进一步影响白色念珠菌在哺乳动物体内的毒性作用。结果显示，该化合物对白色念珠菌的粘附性、菌丝形态转换和致病性具有很好的抑制作用。而且，该化合物本身毒性较小，不影响白色念珠菌及人类细胞的生长。
因此，上述二芳基硫族化合物在制备抗白色念珠菌感染的药物中的应用，以及在制备预防和/或治疗白色念珠菌引起的感染性疾病的药物中的应用，均应在本发明的保护范围之内。
具体地，所述抗白色念珠菌是指抑制白色念珠菌的粘附性、菌丝形态转换、 致病性和/或对细胞的毒力作用。
一种含有所述二芳基硫族化合物的抗白色念珠菌的药物或防治白色念珠菌引起的感染性疾病的药物，也应在本发明的保护范围之内。
本发明具有以下有益效果：
本发明提供了一种新的二芳基硫族化合物，对白色念珠菌的粘附性、菌丝形态转换和致病性具有很好的抑制作用。而且，该化合物本身毒性较小，不影响人类细胞的生长，在新型抗真菌药物的开发，尤其是抗白色念珠菌感染药物的开发方面具有很好的应用前景。
本发明还研究发现，该二芳基硫族化合物对白色念珠菌的生长只是有一定的抑制作用，表明该化合物对白色念珠菌菌株的作用并不主要是依靠杀死细菌，而是通过抑制菌的粘附性、菌丝形态转换和致病性，因此不易产生耐药性。
附图说明
图1是二芳基硫族化合物的合成过程图。
图2是3号化合物的核磁氢谱。
图3是3号化合物的碳谱。
图4是8号化合物的核磁氢谱。
图5是8号化合物的碳谱。
图6是二芳基硫族化合物对白色念珠菌粘附性的影响；其中，图(A)为终浓度为100μM的13种化合物对白色念珠菌细胞粘附于聚苯乙烯上的影响结果图；图(B)为1、2、3、8号共4种化合物在6.25μM到100μM的不同浓度下的抑制率的结果图；DMSO作为对照；数据显示的是8个生物学重复的平均结果，误差棒反映了标准差。
图7是二芳基硫族化合物对白色念珠菌菌丝形成的影响结果图；其中，图(A)是终浓度为100μM的13种合成的二芳基硫族化合物对白色念珠菌菌丝形成的抑制率的测定结果图；图(B)是1、2、3、8号化合物在6.25μM到100μM的不同浓度下的抑制效果图；图(C)是DMSO，氟康唑，1、2、3、8号化合物在终浓度为100μM时对菌丝生成抑制的显微镜观察图片图；此数据显示的是3次生物学实验的平均结果，误差棒反映了标准差。
图8是二芳基硫族化合物对白色念珠菌生长速率的影响结果图；DMSO作为对照；数据显示的是3个生物学重复的平均结果，误差棒反映了标准差。
图9是二芳基硫族化合物对白色念珠菌在A549细胞的致病性方面的影响；(A)终浓度为100μM的13种二芳基硫族化合物对A549细胞的细胞毒性；(B)终浓度为100μM的13种二芳基硫族化合物对白色念珠菌侵染细胞后的影响；(C)1、2、3、8号化合物在6.25μM到100μM的不同浓度下对白色念珠菌侵染细胞后的影响。通过检测LDH的释放量来检测细胞的毒性，在检测白色念珠菌的细胞毒性时，我们将加入了DMSO组的LDH释放量作为100％，并由此来规范其他加入二芳基硫族化合物组的LDH释放比例。数据显示的是4个生物学重复的平均结果，误差棒反映了标准差。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1二芳基硫族化合物的合成
1、二芳基硫族化合物的合成示意图如图1所示，具体合成方法包括以下步骤：
(1)在反应器中，加入1mmol氢化含氮杂环化合物(化合物A-1～A-6)和1mmol二芳基硫/硒醚化合物(化合物B-1～B-6)，再加入2mL二甲基亚砜作为溶剂和1.5mmol的碘单质为促进剂，在100℃下搅拌反应4～20小时；
其中，化合物A-1～A-6分别为：四氢喹啉、二氢吲哚、4-氰基二氢吲哚、6-二氢吲哚啉酸甲酯、7-氮杂二氢吲哚、7，8-苯并四氢喹啉(结构式见图1)；
化合物B-1～B-6分别为：2，2’-二苯硫醚酸甲酯、2，2’-二吡啶硫醚、二苯硫醚、4，4’-二甲基二苯硫醚、二苯硒醚、4，4’-二甲氧基二苯硒醚(结构式见图1)。
(2)上述反应结束后冷却至室温，用乙酸乙酯稀释反应液，接着水洗三遍，干燥，减压蒸除溶剂得到粗产物，最后经柱层析提纯得到二芳基硫族化合物1-10号化合物。
Figure PCTCN2017105400-appb-000002
2、其中，化合物3和化合物8的核磁氢谱和碳谱分别如图2～4所示。
实施例2二芳基硫族化合物的效果检测
1、试验方法：
(1)白色念珠菌菌株的活化：将白色念珠菌标准菌株SC5314于LB培养基活化(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L)，置于30℃培养箱培养过夜。
(2)化合物对白色念珠菌菌株SC5314粘附性的影响：
挑取LB固体平板上的SC5314菌株，接种于YNB培养液(6.7g/L，使用之前加入0.2％葡萄糖)中，30℃，200rpm振荡培养过夜，测定菌液OD600。随后用GMM将菌液稀释至OD600＝0.5，取1mL菌液于1.5ml EP管中，依次加入终浓度为100μM的化合物，震荡混匀，各取200μL加入96孔板中，每个处理设置4个重复，同时设置只加DMSO的处理。将96孔板静置于37℃中孵育，4h后弃掉菌液，加入50μL 0.5％结晶紫，室温作用45min。将结晶紫弃掉，并用冰ddH2O洗10次，加入200μL 75％乙醇，室温放置30分钟，测定OD590，用GraphPad Prism6软件处理数据。
(3)化合物对白色念珠菌菌株SC5314菌丝的影响：
挑取LB固体平板上的SC5314菌株，接种于GMM培养液中，30℃，200rpm振荡培养过夜，测定菌液OD600，用GMM将菌液稀释至OD600＝0.1。取500μL 菌液于1.5mL EP管中，分别加入终浓度为100μM化合物，同时设置DMSO、BDSF(B.cenocepacia diffusible signal factor，对SC5314菌丝形成有很好的抑制作用)分别为阳性、阴性对照。震荡混匀，置于37℃水浴锅中孵育，6h后，离心5000rpm，10min，弃上清，加入40μL GMM培养液重悬菌体，于Zeiss Axioplan2显微镜下观察菌丝的形成，取不同视野拍摄照片。
(4)化合物对白色念珠菌菌株SC5314生长影响的测定：
挑取菌株SC5314单菌落接种于GMM培养液，30℃，200rpm振荡培养过夜，测定菌液OD600，用GMM将菌液稀释至OD600＝0.05。取1mL菌液于1.5mlEP管中，依次加入终浓度为100μM的化合物，震荡混匀，各取300μL加入100孔板中，每个处理设置4个重复，同时设置只加DMSO、加氟康唑的处理。置于生长曲线测定仪中，30℃，200rpm，每2h测定一次OD600值，2d后观察实验结果，GraphPad Prism 6处理数据。
(5)化合物对白色念珠菌菌株SC5314细胞毒力的影响：
(a)A549细胞的复苏及培养：将冻融的A549细胞转移至含10％FBS的DMEM培养基(Gioco公司)中，37℃，5％CO2条件下过夜培养。
(b)A549细胞准备：A549细胞在含10％胎牛血清的高糖培养基DMEM中，以1.5×104个/孔的细胞浓度于96孔板中培养过夜。待细胞布满96孔板底部80％的时候，弃去培养液，用1×PBS清洗细胞3次。
(c)白色念珠菌准备：挑取新鲜SC5314接种于GMM培养液中，于30℃，200rpm条件下振荡培养过夜；用含1％FBS的DMEM细胞维持液调节至OD600＝1.0，再用DMEM(1％FBS)稀释10倍(≈108cfu/mL)待用。
(d)细胞毒力测定：将终浓度为100μM化合物加入含菌的细胞维持液中，取100μL加入准备好的A549细胞中，置于37℃，5％CO2细胞培养箱内培养8h，每处理4个重复，同时设置只加DMSO、加BDSF和氟康唑作为对照，同时测定化合物对细胞本身的毒力作用，即将终浓度为100μM化合物加入不含菌的细胞维持液中，同上处理。参照Promega公司CytoTox
Figure PCTCN2017105400-appb-000003
NonRadioactive Cytotoxicity Assay操作方法测定细胞LDH活性，随后用GraphPad Prism 6处理数据。
(6)化合物对白色念珠菌菌株SC5314小鼠体内侵染的影响：
(a)小鼠饲养：将购于广东省实验动物中心的6–8周龄的雄性BALB/c小 鼠，于华南农业大学实验动物中心饲养。随机分配8只/组，称重记录并给每只小鼠做好标记。
(b)白色念珠菌准备：挑取新鲜SC5314接种于GMM培养液中，于30℃，200rpm条件下振荡培养过夜，5000rpm，5min离心收集菌体，用1×PBS清洗菌体3次，以5×108cfu/mL的浓度分散在含PBS中。
(c)小鼠尾静脉注射：将终浓度为100μM化合物3、8号加入含菌的PBS中，混匀待用。本试验是按照美国国立卫生研究院的健康指导中实验动物的护理和使用条例(NIH 8023号出版物，1978年修订)进行。小鼠称重，尾静脉注射，100μL/10g，同时注射1×PBS，100μM氟康唑分别作为对照。持续观察小鼠存活情况，记录，20d后统计数据，用GraphPad Prism 6处理数据。
2、实验结果
(1)化合物抑制白色念珠菌菌株SC5314的粘附性
如附图6中A图所示，以DMSO组中白色念珠菌的粘附性作为参照，结果显示，10个化合物中，3号和8号化合物处理后的细胞粘附性降低在35％左右，起到了显著抑制白色念珠菌SC5314的粘附性的作用。
结合化合物在菌丝形成率以及细胞毒性方面的生物活性，我们选择1号、2号、3号及8号化合物进行浓度梯度测试。结果显示(附图6中的B图)，3号和8号化合物呈现出了依赖浓度梯度的粘附抑制作用，随着化合物浓度的降低，对白色念珠菌的黏附性抑制作用也会随之降低。
(2)化合物抑制白色念珠菌菌株SC5314菌丝的形成
以DMSO及BDSF处理过的菌丝形成率作为对照，如附图7中A图所示，10个化合物中有6个对于白色念珠菌SC5314有显著的抑制作用，菌丝形成率均降低在了35％以下。附图7中C图显示出在显微镜下观察的菌丝形成结果。
同样，结合其他方面的活性，我们检测了1、2、3、8号化合物在6.25μM到100μM浓度梯度下，白色念珠菌菌株SC5314的菌丝形成率。结果显示(附图7中B图)，四种化合物对白色念珠菌的菌丝抑制作用均呈现出浓度依赖的特性，并且8号化合物在6.25μM时，也可以使得菌丝形成率降低在40％左右。
(3)化合物对白色念珠菌菌株SC5314的生长有一定影响
结果如附图8中A图所示，以DMSO和氟康唑为对照，100μM的1、2、3、8号化合物对白色念珠菌菌株SC5314的生长有一定的抑制作用。同时，我们测 试3号和8号化合物不同浓度梯度对白色念珠菌的影响。结果显示(附图8中B图和C图)，3号化合物影响较小，8号化合物相对影响较大，对SC5314的生长有一定抑制作用。
(4)化合物可以抑制白色念珠菌菌株SC5314对细胞的毒力作用
细胞毒力实验结果显示(如附图9中A图)，以DMSO对照，在没有菌的条件下，10个化合物中，均对细胞没有毒性。
在加白色念珠菌SC5314的条件下，以DMSO，BDSF处理后的细胞毒性为对照，附图9中B图和C图显示，化合物1、2、3、8号在抑制菌株SC5314对细胞的侵染是具有保护作用的，均可以使白色念珠菌的毒力降低在30％以下。
(5)化合物对白色念珠菌菌株SC5314侵染的小鼠有保护作用
综合二芳基硫族化合物各方面表现结果，我们挑选出各方面表现较好的1、2、3、8号进行小鼠实验，实验持续观察20d。
结果显示，1号和2号化合物在体内实验没有保护作用，而3号和8号化合物处理后的小鼠存活率均在100％，如表1所示。
表1老鼠感染模型中化合物对白色念珠菌致病性的影响
化合物 存活率(％)
DMSO 0
氟康唑 100
3号 100
8号 100
综上实验结果可以看出，3号和8号化合物是抗白色念珠菌药物的新型候选者。总的来说，本发明合成了一系列二芳基硫族化合物，并筛选了他们对抗真菌病原体的能力。它们当中的一些化合物显示出了很好的抑制白色念珠菌细胞粘附、菌丝形成和致病性的特性；虽然它们对白色念珠菌细胞本身没有毒性，但是同时也对人体细胞没有毒性。实验结果显示，这些化合物中的有些化合物可能被开发为新型抗白色念珠菌感染的药物。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种抗白色念珠菌的二芳基硫族化合物，其特征在于，其结构如式(I)所示：
    Figure PCTCN2017105400-appb-100001
    其中，Y为S或Se。
  2. 权利要求1所述二芳基硫族化合物的制备方法，其特征在于，包括以下步骤：将氢化含氮杂环化合物和二芳基硫/硒醚化合物混合，加入二甲基亚砜作为溶剂和碘单质作为促进剂，搅拌反应结束后冷却至室温，用乙酸乙酯稀释反应液，然后洗涤干燥，减压蒸除溶剂得粗产物，最后经柱层析提纯得到所述二芳基硫族化合物。
  3. 根据权利要求2所述的制备方法，其特征在于，所述氢化含氮杂环化合物、二芳基硫/硒醚化合物和碘单质的摩尔比为0.5～1.5：0.5～1.5：1～2。
  4. 根据权利要求2所述的制备方法，其特征在于，所述氢化含氮杂环化合物为二氢吲哚，所述二芳基硫/硒醚化合物为二苯硫醚或二苯硒醚。
  5. 根据权利要求2所述的制备方法，其特征在于，所述搅拌反应的条件为在100℃下搅拌反应4～20小时。
  6. 根据权利要求2所述的制备方法，其特征在于，所述洗涤是用水洗数遍。
  7. 权利要求1所述二芳基硫族化合物在制备抗白色念珠菌的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述抗白色念珠菌是指抑制白色念珠菌的粘附性、菌丝形态转换、致病性和/或对细胞的毒力作用。
  9. 权利要求1所述二芳基硫族化合物在制备预防和/或治疗白色念珠菌引起的感染性疾病的药物中的应用。
  10. 一种抗白色念珠菌的药物或防治白色念珠菌引起的感染性疾病的药物，其特征在于，含有权利要求1所述二芳基硫族化合物。
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