WO2023222144A1 - 一类硫代糖苷列净类似物及其制备方法和应用 - Google Patents

一类硫代糖苷列净类似物及其制备方法和应用 Download PDF

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WO2023222144A1
WO2023222144A1 PCT/CN2023/107675 CN2023107675W WO2023222144A1 WO 2023222144 A1 WO2023222144 A1 WO 2023222144A1 CN 2023107675 W CN2023107675 W CN 2023107675W WO 2023222144 A1 WO2023222144 A1 WO 2023222144A1
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thioglycoside
gliflozin
analogues
preparation
class
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French (fr)
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董海
冯广京
王世运
李海林
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上海科利生物医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • the invention belongs to the technical fields of medicine and sugar chemical synthesis, and specifically relates to a class of thioglycoside gliflozin analogs and their preparation methods and applications.
  • Gliflozin drugs are drugs developed based on SGLT-2 inhibitors for the treatment of type 2 diabetes. These drugs reduce blood sugar by inhibiting the transport of glucose by SGLT-2 in the kidneys, causing glucose to be excreted in the urine. These drugs not only have the advantage of reducing weight and reducing the risk of hypoglycemia when used, but new research has found that they also have protective effects on the kidneys and cardiovascular system.
  • the FDA has approved 7 gliflozin drugs.
  • the structures of Empagliflozin, Dapagliflozin, and Canagliflozin which have been marketed in China, are as follows:
  • phloridzin and the glucosinolate derivatives subsequently developed and synthesized based on the phloridzin structure are easily hydrolyzed by ⁇ -glucosidase in the small intestine, and ultimately cannot be made into medicines.
  • the currently marketed leucoside drugs are carbon glycosides with stable structures and cannot be hydrolyzed by ⁇ -glucosidase in the small intestine.
  • the glycosides and aglycones of thioglycosides are linked by sulfur atoms and are often used as enzyme inhibitors because they are stable to hydrolysis under acidic and enzymatic conditions.
  • the inventor of the present application has disclosed the synthesis of thioglycoside analogs for the first time (application number: 202011424879.4), and prepared thioglycoside analogs A and B using the molecular structures of dapagliflozin and canagliflozin as templates. , but biological activity tests show that although A and B can tolerate hydrolysis by ⁇ -glucosidase, the inhibition rate of SGLT2 is only about 50-60% at a high concentration of 100 ⁇ M.
  • the inventor of the present application also synthesized the thioglycoside analog C using the molecule of empagliflozin as a template. The biological activity test showed that at a concentration of 100 ⁇ M, the inhibitory rate of C on SGLT2 was only about 60%.
  • the object of the present invention is to provide a class of thioglycoside gliflozin analogs and their preparation methods and applications.
  • the thioglycoside analogue shows a good half-inhibitory concentration of SGLT2, is non-cytotoxic, and can tolerate hydrolysis by ⁇ -glucosidase, so it is very likely to be developed into a class of drugs for the treatment of type 2 diabetes. New class of drugs.
  • R 1 is one or more of hydrogen, alkyl, and halogen
  • R 2 is an aryl group
  • the alkyl group is one or more of methyl, ethyl, halomethyl, and haloethyl.
  • the halogen is one or more of iodine, bromine, chlorine and fluorine.
  • the aryl group is:
  • a method for preparing a class of thioglycoside gliflozin analogues including the following steps:
  • glucosinolate analogs Dissolve the tetraacetyl-protected glucosinolate analogue obtained in step (1) into the prepared sodium hydroxide methanol solution, and stir at room temperature for 2 to 4 hours. Add hydrogen ion exchange resin to neutralize, concentrate and purify through column chromatography to prepare glucosinolate analogues.
  • the concentration of tetraacetylglucose 1-thiol is 0.1 ⁇ 0.2mmol/L.
  • the molar ratio of tetraacetylglucose 1-thiol, iodoaryl derivative, and triethylamine is 1:0.5 ⁇ 1.5:0.5 ⁇ 1.5.
  • the ratio of tetraacetylglucose 1-thiol and palladium catalyst is 1:0.04 ⁇ 0.08.
  • the amount of methanol in the sodium hydroxide methanol solution is 5 mL/mmol of the acetyl-protected glioglutide analogue.
  • the concentration of the sodium hydroxide methanol solution is 0.01 mol/L.
  • step (1) the structure of the palladium catalyst is:
  • the structure of the iodoaryl derivative is , where R 1 is one or more of hydrogen, alkyl (methyl, ethyl, halomethyl, haloethyl, etc.), halogen (iodine, bromine, chlorine, fluorine, etc.), R 2 for , , , or one or more of them.
  • the present invention claims the use of the prepared thioglycoside gliflozin analog in the preparation of drugs for treating type 2 diabetes.
  • the present invention has the following beneficial effects:
  • the present invention provides a class of thioglycoside gliflozin analogues with novel molecular structures, in which glucose 1-thiol is connected to the ortho position of the aglycone phenylcycloalkyl group; substances D, E, F and G provided by the invention At a concentration of 100 ⁇ M, the inhibition rate of SGLT2 reached almost 100%, and the measured half-inhibitory concentration was comparable to that of gliflozin drugs, making it a very good SGLT2 inhibitor; while the corresponding meta-structure substances A, B and The inhibitory rate of C on SGLT2 does not exceed 62% at a concentration of 100 ⁇ M, which is a very poor inhibitor of SGLT2. Therefore, the present invention has made significant progress in improving the inhibitory activity of thioglycosides analogues on SGLT2.
  • the thioglycoside gliflozin analog provided by the present invention has no cytotoxicity, is resistant to ⁇ -glucosidase hydrolysis, has high inhibitory activity on SGLT2, and has great potential to be developed as a new glucoside drug for the treatment of type 2 diabetes.
  • the reaction conditions are mild, the synthesis steps are fewer, the total yield is high, the synthesis cost is reduced, and it has good development prospects. .
  • the chemical reagents and materials in the present invention are all purchased from the market or synthesized from raw materials purchased from the market.
  • a method for preparing a class of thioglycoside sergliflozin analogues including the following steps:
  • step A The product obtained in step A (201 mg, 0.36 mmol) was dissolved in sodium hydroxide methanol solution (1 mL, 0.01 M). The reaction mixture was stirred at room temperature for 2 hours under nitrogen protection. The mixture was then neutralized with Amberlite IR-120 (H + ) ion exchange resin and filtered. After column chromatography, the thioglycoside sergliflozin analog D (97%, 136 mg) was obtained.
  • a method for preparing a thioglycoside dapagliflozin analogue including the following steps:
  • step A The product obtained in step A (207 mg, 0.34 mmol) was dissolved in sodium hydroxide methanol solution (1 mL, 0.01 M). The reaction mixture was stirred at room temperature for 3 hours under nitrogen protection. The mixture was then neutralized with Amberlite IR-120 (H + ) ion exchange resin and filtered. Dapagliflozin thioglycoside analog E (99%, 147 mg) was obtained after column chromatography.
  • a method for preparing a class of thioglycoside empagliflozin analogs including the following steps:
  • step A The product obtained in step A (229 mg, 0.35 mmol) was dissolved in sodium hydroxide methanol solution (1 mL, 0.01 M). The reaction mixture was stirred at room temperature for 3 hours under nitrogen protection. The mixture was then neutralized with Amberlite IR-120 (H + ) ion exchange resin and filtered. After column chromatography, the thioglycoside empagliflozin analog F (99%, 169 mg) was obtained.
  • a method for preparing a class of thioglycoside canagliflozin analogs including the following steps:
  • step A The product obtained in step A (346 mg, 0.54 mmol) was dissolved in sodium hydroxide methanol solution (1 mL, 0.01 M). The reaction mixture was stirred at room temperature for 3 hours under nitrogen protection. The mixture was then neutralized with Amberlite IR-120 (H + ) ion exchange resin and filtered. After column chromatography, the thioglycoside canagliflozin analog G (97%, 251 mg) was obtained.
  • iodobenzene derivative K and thioglycoside canagliflozin analog G are as follows:
  • PNPG 4-nitrophenyl- ⁇ -D-glucopyranoside
  • PNPG and ⁇ -glucosidase substrate are mixed in Tri-hydrochloride buffer solution and placed The reaction was carried out in a constant temperature shaker at 37°C for 10 days. After the reaction, the mixture is eluted by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • PNPG was all hydrolyzed by ⁇ -glucosidase, indicating that the ⁇ -glucosidase used had good activity; while none of the glucosinolate analogues prepared in the present invention was hydrolyzed, indicating that they have good resistance to ⁇ -glucosidase hydrolysis.
  • Test method MTT colorimetric method is used to determine the in vitro cytotoxicity of the compound.
  • Sample settings are available in 7 quantities (10, 50, 100, 200, 300, 500 and 1000 ⁇ M).
  • HEK293 cells were seeded on a 96-well plate, with the number of cells in each well being approximately 1 ⁇ 10 4 , and placed in a constant temperature incubator at 37°C and 5% CO2 for 24 h.
  • Aspirate and discard the supernatant in the wells wash twice with PBS, set up 3 duplicate wells in each group, add a series of concentration samples diluted with MEM culture medium, and set up a negative control group (0 ⁇ mol/L) and a blank control group.
  • the cytotoxicity evaluation level is 0 ⁇ 1.
  • the concentration reaches 500 ⁇ M or more, it shows a certain degree of cytotoxicity, and the toxicity evaluation level is 2.
  • Level when the concentration is 100 ⁇ M and below, the cell proliferation rate is above 99%, and the toxicity grade is level 0.
  • 2-deoxyglucose (2-DG) is a natural glucose derivative that enters cells through glucose transporters. Fluorescently labeled 2-deoxyglucose (2-(N-7-nitro-2,1,3-benzoxadiazole-4-amino)-2-deoxy-D-glucose, 2-NBDG) was confirmed Similar to 2-DG, it can also enter living cells through glucose transporters. 2-NBDG has an excitation wavelength of 460 ⁇ 490 nm and an emission wavelength of 530 ⁇ 550 nm, which can be detected by fluorescence microplate readers.
  • the HEK293 cells used in the experiment are a cell line derived from human embryonic kidney cells.
  • the two glucose transport proteins contained in HEK293 cells are mainly SGLT2 and GLUT. Therefore, it is necessary to rule out the decrease in glucose uptake caused by inhibiting GLUT protein in the experiment.
  • relaxin B is a specific inhibitor of GLUT.
  • a relaxin B control group was set up in the experiment to subtract the reduction in glucose transport caused by inhibiting GLUT.
  • the inhibitory activity of glucosinolate analogues on SGLT2 was tested respectively, and a canagliflozin positive control group was set up.
  • 2-NBDG was used as the substrate to conduct glucose transport experiments.
  • the specific experimental methods are as follows:
  • sample/2-NBDG mother solution Dissolve sample/2-NBDG in DMSO to prepare a solution with a concentration of 100 mM, and store it in a -20°C refrigerator. When used, dilute with serum-free culture medium to the required concentration, and DMSO ⁇ 0.1%.
  • HEK293 cells Inoculate HEK293 cells on a 96-well plate, with the number of cells in each well being approximately 2 ⁇ 104, and place them in a constant temperature incubator at 37°C and 5% CO2 for 12 hours. Aspirate and discard the supernatant from the wells, and wash twice with glucose-free and serum-free medium.

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Abstract

一类分子结构新颖的SGLT2（2型钠葡萄糖转运蛋白）抑制剂及其制备方法和应用。所述SGLT2抑制剂为硫代糖苷列净类似物，以碳苷和氧苷列净类分子为模板设计，由糖基与苷元部分经硫原子联接而成。经生物活性测试，所述硫代糖苷列净类似物表现出低细胞毒性且耐β-葡萄糖苷酶水解，对SGLT-2的抑制活性与相应的列净类药物相当，因而是潜在的治疗2型糖尿病的药物。所述硫代糖苷列净类似物的结构通式如下：（I）。

Description

一类硫代糖苷列净类似物及其制备方法和应用 技术领域
本发明属于医药和糖化学合成技术领域，具体涉及一类硫代糖苷列净类似物及其制备方法和应用。
 背景技术
列净（gliflozin）类药物是基于SGLT-2抑制剂开发的用于治疗2型糖尿病的药物。这些药物通过抑制肾脏中SGLT-2对葡萄糖的转运，使葡萄糖随尿液排出体外，从而达到降低血糖的目的。这些药物在使用时不但具有降低体重和减少低血糖发生风险的优点，新研究发现它们还对肾脏和心血管具有保护作用。目前，FDA已经批准了7个列净类药物，其中已在国内上市的恩格列净 (Empagliflozin)、达格列净（Dapagliflozin）、和坎格列净(Canagliflozin)的结构如下所示：
恩格列净、达格列净和坎格列净是目前世界上治疗2-型糖尿病三种最畅销药物，文献报道的它们对SGLT2的半抑制浓度分别为3.1 nM，1.2 nM和2.7 nM。在2021年，它们在全球销售额分别约为41亿美元，21亿美元和8亿美元，分别排在全球小分子药物销售额排行榜第9位，第32位和第105位。列净类药物都是基于根皮苷结构发展而来。根皮苷是最早发现的天然的SGLT2抑制剂，对SGLT2的半抑制浓度为21 nM。然而根皮苷及随后基于根皮苷结构发展合成的葡萄糖氧苷衍生物，都易于被小肠内的β-葡萄糖苷酶水解，最终无法成药。目前上市的列净类药物都是碳苷，结构稳定，不能被小肠内β-葡萄糖苷酶水解。硫代糖苷的糖基和苷元由硫原子联接而成，由于在酸性和酶促条件下对水解稳定，常用作酶的抑制剂。
本申请发明人曾首次公开了硫代糖苷列净类似物的合成（申请号为：202011424879.4），以达格列净和坎格列净的分子结构为模板制备了硫代糖苷类似物A和B，但生物活性测试表明，尽管A和B可以耐受β-葡萄糖苷酶的水解，但在100 μM的高浓度下，对SGLT2的抑制率仅在50-60%左右。本申请发明人又以恩格列净的分子为模板合成了硫代糖苷类似物C, 生物活性测试表明，在100 μM浓度下，C对SGLT2的抑制率也仅在60%左右。
 发明内容
为了解决现有技术存在的不足，本发明的目的在于提供一类硫代糖苷列净类似物及其制备方法和应用。所述硫代糖苷列净类似物表现出了良好的SGLT2的半抑制浓度，且无细胞毒性，能耐受β-葡萄糖苷酶的水解，因而极有可能被发展为一类治疗2型糖尿病的新列净类药物。
为了实现上述目的，本发明采用如下技术方案：
一类硫代糖苷列净类似物，其特征在于，分子结构如下：
。
其中，R 1为氢、烷基、卤素中的一种或多种；R 2为芳基；
优选的，所述烷基为甲基、乙基、卤代甲基、卤代乙基中的一种或多种。
优选的，所述卤素为碘、溴、氯、氟中的一种或多种。
优选的，所述芳基为：
， ， ， 或 中的一种或多种。
一类硫代糖苷列净类似物的制备方法，包括如下步骤：
（1）四乙酰基保护的硫苷列净类似物的制备：将四乙酰基葡萄糖1-硫醇、碘代芳基衍生物和钯催化剂按比例溶于四氢呋喃，在充分搅拌下加入三乙胺，然后在室温下反应1~4小时，浓缩后用二氯甲烷和水萃取，再将有机相浓缩，柱层析纯化，制得四乙酰基保护的硫苷列净类似物；
（2）硫苷列净类似物的制备：将步骤（1）中所得四乙酰基保护的硫苷列净类似物溶于配制好的氢氧化钠甲醇溶液中，常温下搅拌2~4小时，加入氢离子交换树脂中和，浓缩后经柱层析纯化，制得硫苷列净类似物。
优选的，所述步骤（1）中，四乙酰基葡萄糖1-硫醇的浓度为0.1~0.2mmol/L。
优选的，所述步骤（1）中，四乙酰基葡萄糖1-硫醇、碘代芳基衍生物、三乙胺的摩尔比为1：0.5~1.5：0.5~1.5。
优选的，所述步骤（1）中，四乙酰基葡萄糖1-硫醇与钯催化剂的比例为1：0.04~0.08。
优选的，所述步骤（2）中，氢氧化钠甲醇溶液中甲醇的用量为5mL/mmol的乙酰基保护的硫苷列净类似物。
优选的，所述步骤（2）中，氢氧化钠甲醇溶液的浓度为0.01mol/L。
更优选的，所述步骤（1）中，所述钯催化剂的结构为：
。
更优选的，所述步骤（1）中，所述所述碘代芳基衍生物的结构为 ，其中R 1为氢、烷基（甲基、乙基、卤代甲基、卤代乙基等）、卤素（碘、溴、氯、氟等）中的的一种或多种，R 2为 、 、 、 或 中的一种或多种。
同时，本发明要求保护制备得到的所述硫代糖苷列净类似物在制备治疗2型糖尿病药物中的应用。
与现有技术相比，本发明具有如下有益效果：
1、本发明提供了一类分子结构新颖的硫代糖苷列净类似物，其中葡萄糖1-硫醇连接在苷元苯环烷基的邻位；本发明提供的物质D、E、F和G在浓度为100 μM时对SGLT2的抑制率几乎达到了100％，测得的半抑制浓度与列净类药物相当，是非常好的SGLT2抑制剂；而相对应间位结构的物质A、B和C在浓度为100 μM时对SGLT2的抑制率不超过62％，是很差的SGLT2抑制剂，所以本发明在提高硫代糖苷列净类似物对SGLT2的抑制活性方面有着显著的进步。
2、本发明提供的硫代糖苷列净类似物无细胞毒性，耐β-葡萄糖苷酶水解，对SGLT2的抑制活性高，有极大潜力被开发为治疗2型糖尿病的新列净类药物。
3、本发明提供的合成硫代糖苷列净类似物的方法与合成碳苷列净的方法相比，反应条件温和，合成步骤少，总收率高，降低了合成成本，有着良好的发展前景。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚，以下结合实施例，对本发明作进一步的详细说明。当然，此处所描述的具体实施例仅仅用于解释本发明。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
本发明中的步骤虽然用标号进行了排列，但并不用于限定步骤的先后次序，除非明确说明了步骤的次序或者某步骤的执行需要其他步骤作为基础，否则步骤的相对次序是可以调整的。可以理解，本文中所使用的术语“和/或”涉及且涵盖相关联的所列项目中的一者或一者以上的任何和所有可能的组合。
如无特殊说明外，本发明中的化学试剂和材料均通过市场途径购买或通过市场途径购买的原料合成。
实施例1
一类硫代糖苷舍格列净类似物的制备方法，包括如下步骤：
（1）氮气保护条件下向瓶内加入四乙酰化硫代葡萄糖（150 mg，0.41 mmol），碘苯衍生物H（133 mg，0.41 mmol）和钯催化剂（15 mg，0.016 mmol），再加入四氢呋喃（2 mL）。 混合物搅拌均匀后，滴加三乙胺（56μL，0.41 mmol）至瓶内，室温反应4小时。将反应混合物减压浓缩柱层析，得到乙酰基保护的硫代糖苷舍格列净类似物（87％，201 mg）。
（2）步骤A得到产物（201 mg，0.36 mmol）溶于氢氧化钠甲醇溶液（1 mL, 0.01 M）。 将反应混合物在氮气保护下于室温搅拌2小时。然后将混合物用Amberlite IR-120（H +）离子交换树脂中和并过滤。柱层析后得到硫代糖苷舍格列净类似物D（97％，136 mg）。1H NMR (400 MHz, CD3OD): δ 7.70 – 7.68 (m, 1H), 7.19 – 7.17 (m, 2H), 7.13 – 7.10 (m, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 8.7 Hz, 2H), 4.59 (d, J = 9.7 Hz, 1H), 4.14 (S, 2H), 3.85 (dd, J = 12.1, 2.1 Hz, 1H), 3.74 (S, 3H), 3.66 (dd, J = 12.1, 5.2 Hz, 1H), 3.40 - 3.33 (m, 2H), 3.29 – 3.25 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 159.4, 143.8, 135.1, 134.1, 133.2, 131.1, 131.0, 128.4, 127.9, 114.7, 89.7, 81.9, 79.7, 78.5, 74.1, 71.3, 62.8, 55.6, 39.8 ppm。
其中，碘苯衍生物H和硫代糖苷舍格列净类似物D的结构如下所示：
实施例2
一类硫代糖苷达格列净类似物的制备方法，包括如下步骤：
（1）氮气保护条件下向瓶内加入四乙酰化硫代葡萄糖（150 mg，0.41 mmol），碘苯衍生物I（153 mg，0.41 mmol）和钯催化剂（30 mg，0.032mmol），再加入四氢呋喃（2 mL）。 混合物搅拌均匀后，滴加三乙胺（56μL，0.41 mmol）至瓶内，室温反应2小时。将反应混合物减压浓缩柱层析，得到乙酰基保护的硫代糖苷达格列净类似物（83％，207 mg）。
（2）步骤A得到产物（207 mg，0.34 mmol）溶于氢氧化钠甲醇溶液（1 mL, 0.01 M）。 将反应混合物在氮气保护下于室温搅拌3小时。然后将混合物用Amberlite IR-120（H +）离子交换树脂中和并过滤。柱层析后得到达格列净硫代糖苷类似物E（99％，147 mg）。1H NMR (400 MHz, CD3OD): δ 7.68 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.3 Hz, 2H), 6.74 (d, J = 8.6 Hz, 2H), 4.61 (d, J = 9.7 Hz, 1H), 4.42 – 4.31 (m, 2H), 3.99 – 3.94 (m, 2H), 3.83 (d, J = 12.4 Hz, 1H), 3.64 (dd, J = 12.1, 4.9 Hz, 1H), 3.38-3.22 (m, 4H), 1.33 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 158.6, 140.4, 138.7, 136.3, 132.2, 131.4, 130.4, 129.4, 129.0, 115.3, 89.5, 82.0, 79.7, 74.0, 71.2, 64.4, 62.7, 37.1, 15.2 ppm。
其中，碘苯衍生物I和硫代糖苷达格列净类似物E的结构如下：
实施例3
一类硫代糖苷恩格列净类似物的制备方法，包括如下步骤：
（1）氮气保护条件下向瓶内加入四乙酰化硫代葡萄糖（150 mg，0.41 mmol），碘苯衍生物J（170 mg，0.41 mmol）和钯催化剂（30 mg，0.032 mmol），再加入四氢呋喃（2 mL）。混合物搅拌均匀后，滴加三乙胺（56μL，0.41 mmol）至瓶内，室温反应4小时。将反应混合物减压浓缩柱层析，得到乙酰基保护的硫代糖苷恩格列净类似物（86％，229 mg）。
（2）步骤A得到产物（229 mg，0.35 mmol）溶于氢氧化钠甲醇溶液（1 mL, 0.01 M）。 将反应混合物在氮气保护下于室温搅拌3小时。然后将混合物用Amberlite IR-120（H +）离子交换树脂中和并过滤。柱层析后得到硫代糖苷恩格列净类似物F（99％，169 mg）。1H NMR (400 MHz, CD3OD): δ 7.69 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 8.2 Hz, 2H), 6.75 (d, J = 8.7 Hz, 2H), 4.93 (s, 1H), 4.61 (d, J = 9.7 Hz, 1H), 4.42 - 4.31 (m, 2H), 3.95 - 3.81 (m, 5H), 3.65 (dd, J = 12.0, 4.9 Hz, 1H), 3.38 - 3.22 (m, 4H), 2.23 - 2.04 (m, 1H). 13C NMR (101 MHz, CD3OD) δ 157.1, 140.3, 138.7, 136.3, 132.8, 131.5, 130.5, 129.5, 129.1, 116.3, 89.5, 82.0, 79.7, 78.5, 74.0, 74.0, 71.2, 68.1, 62.8, 37.1, 33.8 ppm。
其中，碘苯衍生物J和硫代糖苷恩格列净类似物F的结构如下所示：
实施例4
一类硫代糖苷坎格列净类似物的制备方法，包括如下步骤：
（1）氮气保护条件下向瓶内加入四乙酰化硫代葡萄糖（225 mg，0.63 mmol），碘苯衍生物K（234 mg，0.57 mmol）和钯催化剂（63 mg，0.046 mmol），再加入四氢呋喃（1 mL）。混合物搅拌均匀后，滴加三乙胺（80μL，0.63 mmol）至瓶内，室温反应4小时。将反应混合物减压浓缩柱层析，得到乙酰基保护的硫代糖苷恩格列净类似物（93％，346 mg）。
（2）步骤A得到产物（346 mg，0.54 mmol）溶于氢氧化钠甲醇溶液（1 mL, 0.01 M）。 将反应混合物在氮气保护下于室温搅拌3小时。然后将混合物用Amberlite IR-120（H +）离子交换树脂中和并过滤。柱层析后得到硫代糖苷坎格列净类似物G（97％，251 mg）。1H NMR (400 MHz, CD3OD): δ7.66 (dd, J = 7.2, 2.1 Hz, 1H), 7.55 – 7.48 (m, 2H), 7.21 – 7.13 (m, 2H), 7.10 – 7.02 (m, 3H), 6.61 (d, J = 3.5 Hz, 1H), 4.66 – 4.45 (m, 2H), 3.85 (dd, J = 12.1, 2.2 Hz, 1H), 3.67 (dd, J = 12.1, 5.4 Hz, 1H), 3.42 – 3.21 (m, 5H), 2.35 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 143.24, 140.78, 139.74, 137.54, 134.11, 131.13, 131.09, 130.78, 129.72, 126.96, 126.75, 126.67, 125.49, 122.38, 115.32, 115.10, 88.88, 80.56, 78.35, 72.66, 69.87, 61.42, 31.12, 19.07 ppm。
其中，碘苯衍生物K和硫代糖苷坎格列净类似物G的结构如下所示：
对于实施例1~4中所得产品的耐β-葡萄糖苷酶水解性、细胞毒性和体外细胞水平的抗糖尿病活性进行测试，如下所示：
（1）耐β-葡萄糖苷酶水解性
测试方法：
1）硫苷类似物分别与β-葡萄糖苷酶在 Tri-盐酸缓冲溶液中混匀后放置于37 ℃恒温摇床，振荡反应10天；
2）设置4-硝基苯基-β-D-吡喃葡萄糖苷（PNPG，β-葡萄糖苷酶底物）对照组， PNPG与β-葡萄糖苷酶在 Tri-盐酸缓冲溶液中混匀后放置于37 ℃恒温摇床，振荡反应10天。反应结束后的混合液通过高效液相色谱（HPLC）洗脱。
结果分析：PNPG均被β-葡萄糖苷酶水解，表明所用β-葡萄糖苷酶活性良好；而本发明制备的硫苷类似物均未被水解，表明它们具有良好耐β-葡萄糖苷酶水解性。
（2）细胞毒性
测试方法：采用MTT比色法测定化合物的体外细胞毒性。样品设置7个数量值（10, 50, 100, 200, 300, 500和1000 μM）。将HEK293细胞接种在96孔板上，每孔细胞数约为1×10 4，放置于37 ℃、5% CO 2恒温培养箱内培养24 h。吸弃孔中上清液，用PBS洗2遍，每组设3个复孔，分别加入MEM培养基稀释得到的一系列浓度样品，另外设阴性对照组（0 μmol/L）和空白对照组。将细胞板继续放入培养箱内孵育24 h，吸弃上清液，用PBS洗2遍。向每孔加入20 μL MTT溶液（5 mg/mL，即0.5% MTT）和80 μL无血清培养基，培养4 h后终止培养。吸弃上清液，每孔加入150 μL DMSO，放置在摇床上低速震荡10 min。用酶标仪测定各孔在490 nm处的吸光度值（OD值）。按以下公式计算细胞的相对增殖率（relative growth rate，RGR）：RGR% = （OD样品- OD空白）/（OD对照- OD空白）×100%。并根据美国药典评定各浓度药液的细胞毒性。
结果分析：本发明制备的硫苷类似物在浓度低于500 μM时，对细胞毒性的评价等级在0~1级，浓度达到500 μM以上时，表现出一定的细胞毒性，毒性评价等级为2级，浓度在100 μM及以下时，细胞增值率均在99%以上，毒性分级为0级。
（3）体外细胞水平的SGLT2抑制活性
测试方法：2-脱氧葡萄糖（2-DG）是天然的葡萄糖衍生物，通过葡萄糖转运蛋白进入细胞。荧光标记的2-脱氧葡萄糖（2-(N-7-硝基-2,1,3-苯并恶二唑-4-氨基)-2-脱氧-D-葡萄糖，2-NBDG）被证实其与2-DG相似，也可通过葡萄糖转运蛋白进入活细胞。2-NBDG激发波长460~490 nm，发射波长530~550 nm，能够被荧光酶标仪。实验使用的HEK293细胞是一个衍生自人胚胎肾细胞的细胞系。HEK293细胞中包含的转运葡萄糖的蛋白主要是SGLT2和GLUT这两种，因此需要排除实验中因抑制GLUT蛋白而导致的葡萄糖摄取量下降。已知松弛素B是GLUT的特异性抑制剂，在实验中设置松弛素B对照组，扣除因抑制GLUT导致的葡萄糖转运的减少。分别测试硫苷类似物对SGLT2的抑制活性，并设置坎格列净阳性对照组。采用2-NBDG作为底物进行葡萄糖转运实验，具体实验方法如下：
1）配制样品/2-NBDG母液：样品/2-NBDG溶于DMSO配制成浓度为100 mM的溶液，低温保存于-20 ℃冰箱。使用时用无血清培养基稀释至所需浓度，且DMSO﹤0.1%。
2）用无葡萄糖、无血清培养基将细胞松弛素B原液稀释至20 μM浓度，待用。
3）将HEK293细胞接种在96孔板上，每孔细胞数约为2×104，放置于37 ℃、5% CO 2恒温培养箱内培养12 h。吸弃孔中上清液，用无葡萄糖、无血清培养基洗2遍。
4）每孔加入100 μL不同浓度的样品，每组设5个复孔，另外设空白对照组、阴性对照组（0 μM）和细胞松弛素B对照组。置于37 ℃、5% CO 2恒温培养箱内孵育6 h。吸弃孔中上清液，用无葡萄糖、无血清培养基洗2遍。
5）每孔加入100 μL稀释至100 μM浓度的2-NBDG，避光在37 ℃、5% CO 2恒温培养箱内孵育30 min。吸弃孔中上清液，用冷的PBS洗2遍。
6）每孔加入70 μL的0.1 M磷酸钾缓冲溶液（PPS），PPS的pH = 10.0，在暗处孵育10 min。
7）每孔加入70 μL DMSO，吹打均匀。用酶标仪测定各孔的吸光度值（OD值），筛选的最佳波长：激发波长467 nm，发射波长543 nm。利用以下公式计算样品抑制率：Inhibition% = （OD样品- OD空白）/（OD对照- OD空白）×100%。
结果分析：硫苷类似物A、B和C浓度为100 μM时，对SGLT2的抑制率分别是54%、57%和62%，抑制效果不理想。但是，浓度为100 μM时，化合物D、E、F和G对SGLT2的抑制率都近似100%。通过测试D、E、F和G在不同浓度下对SGLT2的抑制率得到它们的半抑制浓度（IC50），分别为6.5 nM、3.7 nM、3.4 nM和3.5 nM。以坎格列净的测试为对照组，测得对SGLT2的IC50值为3.4 nM，文献报道值为2.7 nM，说明测定方法可靠。
所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (8)

  1. 一类硫代糖苷列净类似物，其特征在于，分子结构通式如下：
    ；
    其中，R 1为氢、烷基、卤素中的一种或多种；R 2为芳基。
  2. 一类权利要求1所述的硫代糖苷列净类似物的制备方法，其特征在于，包括如下步骤：
    （1）四乙酰基保护的硫苷列净类似物的制备：将四乙酰基葡萄糖1-硫醇、碘代芳基衍生物和钯催化剂按比例溶于四氢呋喃，在充分搅拌下加入三乙胺，然后在室温下反应1~4小时，浓缩后用二氯甲烷和水萃取，再将有机相浓缩，柱层析纯化，制得四乙酰基保护的硫苷列净类似物；
    （2）硫苷列净类似物的制备：将步骤（1）中所得四乙酰基保护的硫苷列净类似物溶于配制好的氢氧化钠甲醇溶液中，常温下搅拌2~4小时，加入氢离子交换树脂中和至中性，浓缩后经柱层析纯化，制得硫苷列净类似物。
  3. 根据权利要求2所述的一类硫代糖苷列净类似物的制备方法，其特征在于，所述步骤（1）中，四乙酰基葡萄糖1-硫醇的浓度为0.1~0.2mmol/L。
  4. 根据权利要求2所述的一类硫代糖苷列净类似物的制备方法，其特征在于，所述步骤（1）中，四乙酰基葡萄糖1-硫醇、碘代芳基衍生物、三乙胺的摩尔比为1：0.5~1.5：0.5~1.5。
  5. 根据权利要求2所述的一类硫代糖苷列净类似物的制备方法，其特征在于，所述步骤（1）中，四乙酰基葡萄糖1-硫醇与钯催化剂的质量比为1：0.04~0.08。
  6. 根据权利要求2所述的一类硫代糖苷列净类似物的制备方法，其特征在于，所述步骤（2）中，氢氧化钠甲醇溶液中甲醇的用量为5mL/mmol的乙酰基保护的硫苷列净类似物。
  7. 根据权利要求2所述的一类硫代糖苷列净类似物的制备方法，其特征在于，所述步骤（2）中，氢氧化钠甲醇溶液的浓度为0.007~0.013mol/L。
  8. 一类由权利要求1所述的硫代糖苷列净类似物在制备治疗2型糖尿病药物中的应用。
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