Nullscript_HDAC抑制剂scriptaid的阴性对照_300816-11-9_Apexbio
PF-03084014_γ-分泌酶抑制剂_865773-15-5_Apexbio
参考文献: [1]. Li YM, Lai MT, Xu M, Huang Q, DiMuzio-Mower J, Sardana MK, Shi XP, Yin KC, Shafer JA, Gardell SJ. Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state. Proc Natl Acad Sci U S A. 2000 May 23;97(11):6138-43. [2]. Wei P, Walls M, Qiu M, et al. Evaluation of selective γ-secretase inhibitor PF-03084014 for its antitumor efficacy and gastrointestinal safety to guide optimal clinical trial design. Molecular cancer therapeutics, 2010, 9(6): 1618-1628.
参考文献: 1. Wei P, Walls M, Qiu M, et al. Evaluation of selective γ-secretase inhibitor PF-03084014 for its antitumor efficacy and gastrointestinal safety to guide optimal clinical trial design. Molecular cancer therapeutics, 2010, 9(6): 1618-1628. 2. Arcaroli J J, Quackenbush K S, Purkey A, et al. Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor, in a preclinical colorectal explant model. British journal of cancer, 2013, 109(3): 667-675. 3. López-Guerra M, Xargay-Torrent S, Rosich L, et al. The γ-secretase inhibitor PF-03084014 combined with fludarabine antagonizes migration, invasion and angiogenesis in NOTCH1-mutated CLL cells. Leukemia, 2014. 4. Yabuuchi S, Pai S G, Campbell N R, et al. Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer. Cancer letters, 2013, 335(1): 41-51.
生物制剂控制软件 biocommand
BioCommand ® bioprocess control software for fermentation and cell cultureSeamlessUp to eight parameters can be displayed per window; and multiple windows can be opened at a time. Track multiple parameters from one or more process runs; compare historical vs. current values; and export it all to a report if desired. Compress several day‘s data onto one screen for an overview of events, or “zoom in“ to view the details. Our new customizable “add event“ feature allows you to easily add notes or tags to your batch record to track events or conditions — such as inoculation or glucose addition — that may have transpired.BioCommand Comparison Recipe Features Track & Trend Batch Control Batch Control Plus Recipe Info • • •Loop Info • • •Alarms• • •Trend Graphs •• •Programming • •Synoptic Display • •Batch FeaturesTrack & TrendBatch Control BatchControl Plus Batch Info • • •User-Defined Event Log • • •Summary • • •Loop Info • • •Alarms • • •Trend Graphs • • •Data Log • • •Reports • • •Programming • •Synoptic Display • •Equipment Lock-Out ••Audit Trail •Security•Added ConvenienceW hich BioCommand is best for you?Three distinct BioCommand packages are offered,providing the tools needed for research, optimization, and, if needed, security and audit trails to meet your regulatory requirements:BioCommand Track and Trend >Ideal for basic lab management>This package provides full monitoring and historical record keeping capabilities; with control of setpoints, alarm settings and trend displaysBioCommand Batch Control>Includes all the features of Track and Trend, plus equipment lock-out and additional recipe featuressuch as programming capability and a customizable synoptic display>The right choice to optimize and control your process BioCommand Batch Control Plus>Adds three levels of security (operator, supervisor and administrator), event logs, audit trails and a database structure to help comply with 21 CFR Part 11 guidelines regarding electronic record keeping>Allows the powerful control capabilities of BioCommand to be utilized in FDA-validated processesBioCommand software packages are designed to enhance your ability to monitor and control your fermentation and cell culture processes through your personal computer.BioCommand Track and Trend, Batch Control, and Batch Control Plus supervisory control and data acquisition (SCADA) software packages provide: >Automatic data logging>Remote monitoring and control capabilities>OPC-compatibility; data from other OPC-compatibledevices in your lab or production facility can be integrated into your control strategies>Ability to monitor and supervise several fermentors and bioreactors from a single PC>Connectivity with our current and past PC-compatible fermentors and bioreactors, as well as with Windows ® 10 operating systems>Batch Control and Batch Control Plus provide advanced programming capabilities with easy-to-use tools intuitive to most biological scientists and techniciansThe improved recipe wizard, standard in all three packages, provides easy-to-follow onscreen instructions that walk you through the set-up of recipes, start to finish. Check marks indicate which parts of the recipe you have already created and saved, and which steps you still need to complete.1. Batch and recipe explorer screens, included in all three BioCommand packages, provide at-a-glance viewing and easy navigation to all your recipe information, batch runs, alarm settings, control loops and more. They eliminate the need to close your process window to hunt for a stored recipe or file. Scan the list, locate the batch, double-click to open.The BioCommand powerful programming feature has been designed to help scientists and process technicians write sophisticated control strategies to automate setpoint changes based on culture conditions and time. No programming skills needed. Simply drag and drop the logic blocks intoplace to create “If-Then” statements. In the example (right, top), if the DO is greater than 40, the software automatically adjusts the setpoint of Pump 1 to 100. If the statement is false, the setpoint is set to 0. Programs can also be used to calculate values which are not already present as loops based on already-available information (right, bottom). Here, tip speed is calculated from a mathematical formula and agitator speed. This value becomes a new loop and can be recorded as part of the batch information. For advanced programmers, Visual Basic script can also be used.12342. Trend graph windows: Custom trend screens allow you to compare and track all of your process data. Multiply trend graph windows can be simultaneously opened.3. Batch summary screen displays setpoints, current values, and more. Provided in all three BioCommand packages.For infinite convenience you can open and view multiple screens at a time; simply click on the blue header to collapse individual screen views:4. Program screen displays user-defined programs as described above.Windows is a registered trademark of Microsoft Corporation, USA. Eppendorf and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Germany. BioCommand and BioFlo are registered trademarks of Eppendorf, Inc., USA. All rights reserved, including graphics and images. Copyright © 2022 by Eppendorf SE. AN01 611 020/GB4/0.5T/0422/EBC/STEFF. Carbon neutrally printed in Germany.BioCommand Specifications*System RequirementsSupported OSWindows 10 (Professional, Enterprise) – English OS Only Required Hardware CD-ROM and Unshared Serial Port (RS-232)32-Bit SystemsComputer with Intel or compatible 1GHz or faster processor (2 GHz or faster is recommended. Only a single processor is supported.)Minimum of 512 MB of RAM (1 GB or more is recommended)4 GB of free hard disk space64-Bit Systems 1.4 GHz or higher processor (2 GHz or faster is recommended. Only a single processor issupported.)Minimum of 512MB of RAM (1 GB or more is recommended)4 GB of free hard disk spaceSupported EquipmentAll instruments using the NBS AFS, NBS MODBUS, TCP MODBUS and Allen Bradley MODBUS communication protocols. These include: BioFlo ® 120, BioFlo 320, BioFlo/CelliGen 510, BioFlo 610, BioFlo 720 and BioFlo Pro; Selected legacy controllers are also supported.CommunicationsProtocol: NBS AFS / NBS MODBUS / AB MODBUS / TCP MODBUS Fermentor to MCA: RS-422 Multidrop MCA to Fermentor: RS-232Maximum of four systems per com port recommendedData Logging Automatically initiated; User-selected interval; Independent data logs for each process; Secure database DisplaysTrend Graphs: Up to 8 x-y plots on one window Batch Summary: Alphanumeric table of loop valuesSynoptic: Graphical representation of process information Programs: Displays current control diagrams Alarms: Current alarms, alarm log, alarm setupReports: Can be run any time for current or historical batchesEvent Log: Displays all batch events which have occurred (Batch Control Plus only)User-Defined Event Log: Displays all “Add-Events” marked on trend screens Data Log: Includes all loop data for the batchUser Authorization LevelsAdministrator, Supervisor and Operator (Batch Control Plus package only)Ordering InformationItemPart No.DescriptionBioCommand ® Track and Trend M1326-0000Includes Program on CD-ROM, one Multi-Controller Connection Kit and BioCommand ® User Guide. Additional kits and/or cables may be purchased separately, as listed below.BioCommand ® Batch Control M1326-0010BioCommand ® Batch Control Plus M1326-0020Multi-Controller Connection Kit M1286-0100Contains one 50’ / 16 meter RS-422 Serial Cable, one RS-232/RS-422 Converter and one BioCommand ® Cable.RS-232/USB Interface BoxM1287-0020Converts up to eight RS-232 COM ports into USB for customers whose computers lack an RS-232 connection* Specifications and catalog numbers subject to change without notice.Your local distributor: /contactEppendorf SE · Barkhausenweg 1 · 22339 Hamburg · Germany ***********************·。
食品中那非类物质的测定(90种)
附件食品中那非类物质的测定（BJS 201805）1 范围本标准规定了食品（含保健食品）基质中90种那非类物质的超高效液相色谱-串联质谱测定方法。
标准中方法一超高效液相色谱-三重四极杆串联质谱法，适用于饮料、果冻、蛋白粉、牡蛎粉、饼干、糖果、酒类及咖啡等食品（含与上述基质相同的保健食品及片剂、胶囊、软胶囊等剂型）中90种那非类物质的定性和定量测定。
标准中方法二超高效液相色谱-串联高分辨质谱法，适用于饮料、果冻、蛋白粉、牡蛎粉、饼干、糖果、酒类及咖啡等食品（含与上述基质相同的保健食品及片剂、胶囊、软胶囊等剂型）中90种那非类物质的筛查和定性确证。
方法一超高效液相色谱-三重四极杆串联质谱法2原理试样经甲醇超声提取，过滤后，滤液供超高效液相色谱—三重四极杆串联质谱仪测定，外标法定量。
3试剂和材料注：水为GB/T 6682规定的一级水。
3.1 试剂3.1.1 甲醇（CH3OH）：质谱级。
3.1.2 甲酸（HCOOH）：质谱级。
3.1.3 乙酸乙酯（C4H8O2）：分析纯。
3.1.4 盐酸（HCl）：分析纯。
3.2 试剂配制3.2.1 0.1%甲酸水溶液：取甲酸1mL用水稀释至1000mL，用滤膜（4.3）过滤后备用。
3.2.2 盐酸甲醇溶液（1+99）：取盐酸1mL用甲醇稀释至100mL。
3.2.3 甲醇水溶液（1+1）：将甲醇与水等体积混合。
3.3 标准品西地那非等90种物质标准品的中文名称、英文名称、CAS登录号、分子式、相对分子量见附录A表A.1，纯度≥98%。
3.4 标准溶液配制—1 —3.4.1标准储备液（200 μg/mL）：分别精密称取Lodenafil carbonate（46罗地那非碳酸酯）、Sildenafil dimer impurity（63西地那非二聚体杂质）、Vardenafil dimer（69伐地那非二聚体）标准品（3.3）各10mg，用盐酸甲醇溶液（1+99）溶解并稀释至50mL，摇匀；分别精密称取其余87种标准品（3.3）各10 mg，用甲醇溶解并稀释至50mL，摇匀，制成浓度为200 μg/mL标准储备液。
美罗培南_美国药典
USP 35Official Monographs / Meropenem3813C U= nominal concentration of mercaptopurine in the essary correction. Each mL of 0.1 N hydrochloric acid is equiva-Sample solution (mg/mL)lent to 12.60 mg of Hg(NH2)Cl.F= relative response factor for each individualimpurity (see Table 2)Acceptance criteriaIndividual impurities: See Table 2. [N OTE—Disregard any Meropenemimpurity peak less than 0.05%.]Table 2Relative Relative AcceptanceRetention Response Criteria,Name Time Factor NMT (%)Didanosine relatedC17H25N3O5S·3H2O437.52compound A a0.54 6.30.31-Azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, 3-[[5-Mercaptopurine 1.00——[(dimethylamino)carbonyl]-3-pyrrolidinyl]thio]-6-(1-hydroxy-Mercaptopurine ethyl)-4-methyl-7-oxo, trihydrate, [4R-disulfide b 2.90 4.40.4[3(3S*,5S*),4α,5β,6β(R*)]]-.Any unspecified—(4R,5S,6S)-3-[[(3S,5S)-5-(Dimethylcarbamoyl)-3-pyrrolidinyl]thio] impurity 1.00.2-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0] Total impurities——0.6hept-2-ene-carboxylic acid, trihydrate [119478-56-7].a Hypoxanthine.Anhydrous383.47 [96036-03-2].b1,2-Di(9H-purin-6-yl)disulfane.» Meropenem contains not less than 98.0 per-v USP35cent and not more than 101.0 percent ofC17H25N3O5S, calculated on the anhydrous basis. ADDITIONAL REQUIREMENTS•P ACKAGING AND S TORAGE: Preserve in well-closed containers.Packaging and storage—Preserve in tight containers. Store •L ABELING: When more than one Dissolution test is given, thethe dry powder at controlled room temperature.labeling states the Dissolution test used only if Test 1 is notLabeling—Where it is intended for use in preparing injectable used.dosage forms, the label states that it is sterile or must be sub-•USP R EFERENCE S TANDARDS〈11〉jected to further processing during the preparation of injectable USP Mercaptopurine RSdosage forms.USP Reference standards 〈11〉—USP Endotoxin RSAmmoniated MercuryUSP Meropenem RSIdentification—Hg(NH2)Cl252.07A: Infrared Absorption 〈197K〉.Mercury amide chloride.Mercury amide chloride [10124-48-8].B: Ultraviolet Absorption 〈197U〉—Solution:30 µg per mL.» Ammoniated Mercury contains not less thanMedium:water.98.0 percent and not more than 100.5 percentSpecific rotation 〈781〉:between −17° and −21°, measured of Hg(NH2)Cl.at 20°.Test solution: 5 mg per mL, in water.Packaging and storage—Preserve in well-closed, light-resis-tant containers.pH 〈791〉: between 4.0 and 6.0, in a solution (1 in 100). Identification—Water, Method Ic 〈921〉:between 11.4% and 13.4%.A: A 0.1-g portion is soluble, with the evolution of ammo-Residue on ignition 〈281〉: not more than 0.1%, igniting at nia, in a cold solution of 1g of sodium thiosulfate in 2 mL of500±50°, instead of at 800±25°. Use a desiccator containing water. When this solution is heated gently, a rust-colored mix-silica gel.ture is formed, from which a red precipitate is obtained on Heavy metals—centrifugation. If the solution is strongly heated, a black mixture Sodium sulfide reagent—Dissolve 5g of sodium sulfide in a forms.mixture of 10 mL of water and 30 mL of glycerin. Preserve in B: When heated with 1N sodium hydroxide, it becomes yel-well-filled, light-resistant bottles, and use within 3 months. low, and ammonia is evolved.Test solution—Transfer 1.0 g of Meropenem to a quartz or C: A solution in warm acetic acid yields with potassium io-porcelain crucible, cover loosely with a lid, and carbonize by dide TS a red precipitate, which is soluble in an excess of the gentle ignition. After cooling, add 2 mL of nitric acid and 5 reagent. The solution yields a white precipitate with silver ni-drops of sulfuric acid, heat cautiously until white fumes evolve, trate TS.and incinerate by ignition at 500° to 600°. Cool, add 2 mL of Residue on ignition 〈281〉: not more than 0.2%.hydrochloric acid, and evaporate on a water bath to dryness.Moisten the residue with 3 drops of hydrochloric acid, add 10 Mercurous compounds—Dissolve 2.5 g in 25 mL of warmmL of hot water, and warm for 2 minutes. Add 1 drop of phe-hydrochloric acid, filter through a tared filtering crucible, washnolphthalein TS, add ammonia TS, dropwise, until the solution with water, and dry at 60° to constant weight: the weight ofdevelops a pale red color, and add 2 mL of 1N acetic acid.the residue does not exceed 5 mg (0.2%).Filter, if necessary, to obtain a clear solution, washing the filter Assay—Mix about 0.25 g of Ammoniated Mercury, accuratelywith 10 mL of water. Transfer the filtrate and the washing to a weighed, with about 10 mL of water. Add 3g of potassium50-mL color-comparison tube, and add water to obtain a vol-iodide, mix occasionally until dissolved, and add about 40 mLume of 50 mL.of water. Add methyl red TS, and titrate with 0.1 N hydrochlo-Standard solution—Evaporate a mixture of 2 mL of nitricric acid VS. Perform a blank determination, and make any nec-acid, 5 drops of sulfuric acid, and 2 mL of hydrochloric acid ona water bath, further evaporate to dryness on a hot sand bath,3814Meropenem / Official Monographs USP 35and moisten the residue with 3 drops of hydrochloric acid. Pro-than 1.5; and the relative standard deviation for replicate injec-ceed as directed for Test solution, beginning with “add 10 mL tions is not more than 2.0%.of hot water,” except add water to obtain a volume of 49 mL.Procedure—Separately inject equal volumes (about 10 µL) of Add 1.0 mL of Standard Lead Solution (see Heavy Metals 〈231〉).the Standard solution and the Test solution into the chromato-Procedure—To the tubes containing the Test solution and the graph, record the chromatograms, using a period of chroma-Standard solution, add 1 drop of Sodium sulfide reagent, mix,tography for the Test solution that is about 3 times the retention and allow to stand for 5 minutes. The color in the tube contain-time of meropenem, and measure the peak responses. Major ing the Test solution is not darker than the color in the tube impurity peaks may be observed at retention times of about containing the Standard solution (0.001%).0.45 and 1.9 in relation to the retention time of meropenem.Calculate the percentage of each impurity in the chromatogram Limit of acetone—obtained from the Test solution by the formula: Internal standard solution—Prepare a solution in dimethyl-formamide containing 0.05 µL of ethyl acetate per mL.(CS/C U)(P)(r i/r S) Standard solution—Transfer about 50 mg of acetone, accu-rately weighed, to a 100-mL volumetric flask, dilute with di-in which CS is the concentration, in mg per mL, of USP Mer-methylformamide to volume, and mix. To 1.0 mL of this solu-openem RS in the Standard solution; CU is the concentration, in tion, add 10.0 mL of the Internal standard solution, and mix.mg per mL, of Meropenem in the Test solution; P is the statedTest solution—Dissolve 100 mg of Meropenem, accurately percentage, calculated on the anhydrous basis, of meropenem weighed, in 0.2 mL of dimethylformamide and 2.0 mL of Inter-in USP Meropenem RS; r i is the peak response of any individual nal standard solution.impurity obtained from the Test solution; and r S is the peak re-sponse of meropenem obtained from the Standard solution. Not Chromatographic system (see Chromatography 〈621〉)—Themore than 0.3% of any of two major impurities is found, calcu-gas chromatograph is equipped with a flame-ionization detectorlated on the anhydrous basis; not more than 0.1% of any other and a 3-mm × 2-m column that contains support S2 and isimpurity is found, calculated on the anhydrous basis; and the maintained at a constant temperature of about 150°. The injec-sum of all such other impurities is not more 0.3%.tion port temperature is maintained at about 170°. Nitrogen isthe carrier gas, with the flow rate adjusted so that the retention Other requirements—Where the label states that Mer-time for acetone is about 3 minutes.openem is sterile, it meets the requirements for Sterility 〈71〉and for Bacterial endotoxins under Meropenem for Injection.Procedure—Separately inject equal volumes (about 2 µL) ofWhere the label states that Meropenem must be subjected to the Standard solution and the Test solution into the chromato-further processing during the preparation of injectable dosage graph, record the chromatograms, and measure the peak re-forms, it meets the requirements for Bacterial endotoxins under sponses for the acetone peak and the internal standard peak.Meropenem for Injection.Calculate the percentage of acetone in the portion of Mer-openem taken by the formula:Assay—Diluted phosphoric acid—Dilute 10 mL of phosphoric acid (W A/5W U)(R U/R S)with water to make 100 mL of solution.Solvent—Transfer 1.0 mL of triethylamine to a 1000-mL volu-in which W A is the weight, in mg, of acetone in the Standard metric flask containing 900 mL of water. Adjust with Dilutedsolution; W U is the quantity, in mg, of Meropenem in the Test phosphoric acid to a pH of 5.0 ± 0.1, dilute with water to vol-solution; and R U and R S are the peak area ratios of acetone to ume, and mix.the internal standard obtained from the Test Solution and theMobile phase—Prepare a mixture of Solvent and methanol Standard solution, respectively. Not more than 0.05% is found.(5:1). Make adjustments if necessary (see System Suitability Chromatographic purity—under Chromatography 〈621〉).Diluted phosphoric acid—Dilute 10 mL of phosphoric acid Standard preparation—Transfer about 25 mg of USP Mer-with water to make 100 mL of solution.openem RS, accurately weighed, to a 50-mL volumetric flask, Solvent—Transfer 1.0 mL of triethylamine to a 1000-mL volu-add Solvent, swirl to dissolve, dilute with Solvent to volume, and metric flask containing 900 mL of water. Adjust with Diluted mix. [NOTE—Immediately after preparation, store this solution in phosphoric acid to a pH of 5.0 ± 0.1, dilute with water to vol- a refrigerator. It may be used for 24 hours.]ume, and mix.Assay preparation—Transfer about 25 mg of Meropenem, ac-Mobile phase—Transfer 1.0 mL of triethylamine to a 1000-mL curately weighed, to a 50-mL volumetric flask, add Solvent, swirl volumetric flask containing 900 mL of water. Adjust with Diluted to dissolve, dilute with Solvent to volume, and mix. Use this phosphoric acid to a pH of 5.0 ± 0.1, dilute with water to vol-solution immediately after preparation.ume, and mix. Mix this solution with 70 mL of acetonitrile.Chromatographic system (see Chromatography 〈621〉)—The Make adjustments if necessary (see System Suitability under liquid chromatograph is equipped with a 300-nm detector and Chromatography 〈621〉). a 4.6-mm × 25-cm column that contains 5-µm packing L1. Ad-Standard solution—Prepare a solution of USP Meropenem RS just the flow rate so that the retention time for meropenem is in Solvent having a known concentration of about 0.025 mg of about 6 to 8 minutes. The flow rate is about 1.5 mL per min-USP Meropenem RS per mL. [NOTE—Immediately after prepara-ute. Chromatograph the Standard preparation, and record the tion, store this solution in a refrigerator and use within 24peak responses as directed for Procedure: the column efficiency hours.]is not less than 2500 theoretical plates; the tailing factor is not Test solution—Dissolve an accurately weighed quantity of more than 1.5; and the relative standard deviation for replicate Meropenem quantitatively in Solvent to obtain a solution having injections is not more than 2.0%.a known concentration of about 5 mg per mL. Use this Test Procedure—Separately inject equal volumes (about 5 µL) of solution immediately.Standard preparation and Assay preparation into the chromato-Chromatographic system (see Chromatography 〈621〉)—The graph, record the chromatograms, and measure the areas for liquid chromatograph is equipped with a 220-nm detector and the major peaks. Calculate the quantity, in mg, of C17H25N3O5S a 4.6-mm × 25-cm column that contains 5-µm packing L1 and in the portion of Meropenem taken by the formula:is maintained at a constant temperature of about 40°. The flowrate is about 1.6 mL per minute, and is adjusted so that the(W S/W U)(P)(r U/r S)retention time of meropenem is between 5 and 7 minutes.Chromatograph the Standard solution, and record the peak re-in which W S is the weight, in mg, of USP Meropenem RS taken sponses as directed for Procedure: the column efficiency is not to prepare the Standard preparation, calculated on the anhy-less than 2500 theoretical plates; the tailing factor is not more drous basis; W U is the weight, in mg, of Meropenem taken toprepare the Assay preparation; P is the stated percentage, calcu-USP 35Official Monographs / Meropenem 3815lated on the anhydrous basis, of meropenem in USP Mer-than 0.8% of the impurity, if any, with a retention time of openem RS; and r U and r S are the meropenem peak responses about 0.45 relative to that of meropenem, is found; and not obtained from the Assay preparation and the Standard prepara-more than 0.6% of the impurity, if any, with a retention time tion, respectively.of about 1.9 relative to that of meropenem, is found.Content of sodium—Potassium chloride solution—Transfer 38.1 g of potassium chloride to a 1000-mL volumetric flask, dissolve in and dilute with water to volume, and mix.Meropenem for InjectionStandard sodium solution—Dissolve 25.42 mg of sodium chloride, previously dried at 105° for 2 hours and accurately » Meropenem for Injection is a sterile dry mixture weighed, quantitatively in water to obtain a solution having a of Meropenem and Sodium Carbonate. It con-concentration of 25.42 µg of sodium chloride per mL. Transfer tains not less than 90.0 percent and not more 5.0 mL of this solution to a 50-mL volumetric flask, add 5.0 mL of Potassium chloride solution , dilute with water to volume, and than 120.0 percent of the labeled amount of mix.meropenem (C 17H 25N 3O 5S).Test solution—Transfer an accurately measured volume of the Packaging and storage—Preserve in tight Containers for stock solution used to prepare Assay preparation 1 or Assay Sterile Solids as described under Injections 〈1〉. Store at con-preparation 2, as appropriate, equivalent to about 25 mg of trolled room temperature.meropenem, to a 200-mL volumetric flask, dilute with water to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL Labeling—It meets the requirements for Labeling under Injec-volumetric flask, add 5.0 mL of Potassium chloride solution , di-tions 〈1〉. Label it to state the quantity, in mg, of sodium (Na) in lute with water to volume, and mix.a given dosage of meropenem.Blank solution—Transfer 5.0 mL of Potassium chloride solution USP Reference standards 〈11〉—to a 50-mL volumetric flask, dilute with water to volume, and USP Endotoxin RS mix.USP Meropenem RSProcedure—Concomitantly determine the absorbances of the Constituted solution—At the time of use, it meets the re-Standard sodium solution and the Test solution at the sodium quirements for Constituted Solutions under Injections 〈1〉.emission line at 589.6 nm with an atomic absorption spectro-Identification—The retention time for the meropenem peak photometer (see Spectrophotometry and Light-Scattering 〈851〉),in the chromatogram of the Assay preparation corresponds to equipped with a sodium hollow-cathode lamp and a single-slot that in the chromatogram of the Standard preparation, as ob-burner, using an air–acetylene flame and the Blank solution as tained in the Assay.the blank. Calculate the quantity, in mg, of sodium (Na) in the constituted Meropenem for Injection by the formula:Bacterial endotoxins 〈85〉—It contains not more than 0.125USP Endotoxin Unit per mg of meropenem.(22.99/58.44)(C )(2000V/vM )(A U /A S )Sterility 〈71〉—It meets the requirements when tested as di-rected for Membrane Filtration under Test for Sterility of the Prod-in which 22.99 and 58.44 are the atomic weight of sodium and uct to be Examined.the molecular weight of sodium chloride, respectively; C is the Uniformity of dosage units 〈905〉: meets the requirements.concentration, in µg per mL, of sodium chloride in the Standard pH 〈791〉: between 7.3 and 8.3, in a solution (1 in 20).sodium solution; V is the volume, in mL, of the stock solution obtained in Assay preparation 1 or Assay preparation 2, as ap-Loss on drying 〈731〉—Dry it in vacuum at 65° for 6 hours: it propriate; v is the volume, in mL, of the portion of the stock loses between 9.0% and 12.0% of its weight.solution taken to prepare the Test solution; M is the total quan-Particulate matter 〈788〉: meets the requirements for small-tity, in mg, of meropenem in the stock solution obtained in volume injections.Assay preparation 1 or Assay preparation 2, as appropriate,Chromatographic purity—based on the result of the Assay; and A U and A S are the ab-Diluted phosphoric acid , Solvent , Mobile phase , and Chromato-sorbances of the Test solution and the Standard sodium solution,graphic system—Proceed as directed in the test for Chromato-respectively: it contains between 80% and 120% of the labeled graphic purity under Meropenem .amount of sodium.Standard solution—Prepare a solution of USP Meropenem RS Assay—in Solvent having a known concentration of about 0.029 mg of Mobile phase—Dilute 15 mL of tetrabutylammonium hydrox-USP Meropenem RS per mL. [NOTE —Immediately after prepara-ide solution (25% in water) with water to 750 mL. Adjust with tion, store this solution in a refrigerator. It may be used for 24dilute phosphoric acid (1 in 10) to a pH of 7.5 ± 0.1. Add 150hours.]mL of acetonitrile and 100 mL of methanol, mix, and degas.Test solution—Transfer an accurately weighed portion of Mer-Make adjustments if necessary (see System Suitability under openem for Injection, equivalent to about 50 mg of mer-Chromatography 〈621〉).openem, based on the labeled amount, to a 10-mL volumetric Standard preparation—Dissolve an accurately weighed por-flask, dilute with Solvent to volume, and mix. Use this Test solu-tion of USP Meropenem RS quantitatively in Mobile phase to tion immediately.obtain a solution having a known concentration of about 0.11Procedure—Proceed as directed in the test for Chromato-mg per mL. This solution contains the equivalent of about 0.1graphic purity under Meropenem . Calculate the percentage of mg of meropenem per mL. [NOTE —Immediately after prepara-each impurity in the portion of Injection taken by the formula:tion, store this solution in a refrigerator and use within 24hours.]10(CP /m )(r i /r S )Assay preparation 1 (where it is represented as being a sin-gle-dose container)—Constitute a container of Meropenem for in which C is the concentration, in mg per mL, of USP Mer-Injection with a volume of water, accurately measured, corre-openem RS in the Standard solution; P is the stated percentage,sponding to the amount of solvent specified in the labeling.calculated on the anhydrous basis, of meropenem in USP Mer-Withdraw all of the withdrawable contents, using a suitable hy-openem RS; m is the amount, in mg, of meropenem in the podermic needle and syringe, and transfer to a 100-mL volu-portion of Injection taken to prepare the Test solution , based on metric flask. Dilute with water to volume, and mix. Dilute an the label claim; r i is the peak response of any individual impu-accurately measured volume of this stock solution quantitatively rity obtained from the Test solution; and r S is the peak response with Mobile phase to obtain a solution having a concentrationof meropenem obtained from the Standard solution . Not more。
G418筛选实验
G418筛选实验相关问题点击次数：1011 作者：guodengjun12 zhaolifei sandy99等发表于：2008-08-06 23:39转载请注明来自丁香园来源：丁香园问：G418筛选预实验用未转染的细胞做，选10-14天内细胞死亡的最小浓度，然后将更高一级别的浓度应用于转染的细胞，是这样吗？答：G418是一种细胞毒性药物（氨基糖苷类抗生素），依不同浓度，对细胞有一定的抑制或杀伤作用，而拟转染的质粒上带有G4 18的药代酶，能降解G418，从而使细胞获得对G418的耐药性。
也就是说转染后的细胞可以耐受G418，而未转染成功的细胞不能耐受G418，因而被杀灭或抑制。
通过G418筛选体系，使得未转染成功的细胞比例不断减少，转染成功的细胞比例不断增高。
如果G418浓度太高则会杀伤所有的细胞，如果G418浓度太低则没有什么毒性，两种情况都不能起到筛选作用。
G418筛选预实验则是先用未转染的细胞做，选14天内细胞死亡的最小浓度应用于转染的细胞，因为不同的细胞对G418的耐受性也不一样，这样的目的是找一个实验细胞能耐受G418的工作浓度，从而为筛选转染的细胞打下基础，因为这种浓度只能抑制未转染的细胞，而不抑制转染成功的细胞的！从而起到筛选作用。
建议你再多查找一下国内外相关文献，实验动手之前一定要明白其原理，要不然会很盲目，充分的准备可以避免走很多不必要的弯路！问：第二次筛选要怎么做？答：第二次筛选就是将转染好的细胞接种培养后，向培养液中加入G418以杀伤或抑制其中的未转染成功的细胞，因为不管转染效率多么高，都不可能是100%，所以要通过G418筛选体系（或者其他筛选体系）来筛掉那些没有成功转染的细胞，此时加入G418的浓度可以略高于你第一次预实验时摸索出来的“最小有效浓度”，实验中可以再根据细胞生长情况进行浓度的调整！问：第二次筛选是经过第一次筛选以后，已经筛选出一部分转染细胞了，但是因为有些细胞还是会丢失目的基因所以要得到稳定表达的细胞还要进行2-3次筛选，这时候还是用原来预实验的浓度，还是要再提高一个浓度？答：你要在荧光显微镜下观察一下，判断一下细胞转染的效率，也就是转染成功的细胞的比例，再根据情况选用或高或低的浓度！转染成功的细胞的比例越高，则加入G418的浓度就可以越低。
LY 294002_PI3K抑制剂_154447-36-6_Apexbio
参考文献: [1] Hu L, Zaloudek C, Mills G B, et al. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clinical Cancer Research, 2000, 6(3): 880-886.
参考文献: [1]Maira et al. (2009). PI3K inhibitors for cancer treatment: where do we stand?. doi:10.1042/BST0370265. [2]Vlahos CJ. , et al. (1994). A Specific Inhibitor of Phosphatidylinositol 3-Kinase, 2-(4-Morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002). Journal of Biological Chemistry 269 (7): 5241–5248. [3]Chaussade C, et al. Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem J, 2007, 404(3), 449-58. [4]Hu L, et al. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res, 2000, 6(3), 880-6. [5]Blommaart EF. , et al., (1997). The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243: 240–246. [6]Dittmann, A. , et al. (2013). The Commonly Used PI3-Kinase Probe LY294002 is an Inhibitor of BET Bromodomains. ACS Chemical Biology: 131210150813004.
去垢剂
在生物学或生物化学实验室使用的去污剂都是作用比较温和的表面活性剂（=表面活性成分），是用来破坏细胞膜（裂解细胞）以释放细胞内的可溶性物质。
它们可以破坏蛋白质-蛋白质、蛋白质-脂质、脂质-脂质之间的连接，使蛋白质发生结构上的变性，防止蛋白质结晶，另外在免疫学实验中还可避免非特异性吸附。
去污剂根据其特性可以分为好几类，因此科学研究中去污剂的选择很关键，取决于后续研究的具体内容。
实际应用中有众多不同的去污剂可以选择。
为了某些特殊的应用，新的去污剂被不断开发出来[]。
在这篇综述中，对一些最常用的去污剂的特点和应用进行了论述。
去污剂是由一个疏水尾端基团和一个极性亲水头端基团组成的有机化合物（图一A）。
在一定的温度条件下，以特定浓度溶解于水时，去污剂分子会形成胶束，疏水基团部分位于胶束内部，而极性亲水基团则在其外部（图一B）。
因此，胶束的疏水中心会结合到蛋白的疏水区域。
一个胶束中，去污剂分子的聚集数目，是用来评价膜蛋白溶解度的一个重要参数[]。
去污剂分子疏水区域的长度和其疏水性成正比，且去污剂的疏水区域非常恒定，而极性头端亲水基团是可变的，可据其特点，把去污剂分为三类：离子型（阴离子或阳离子型），两性离子型和非离子型（见表一）。
在特定的温度下，表面活性剂分子缔合形成胶束的最低浓度，称之为临界胶束浓度（CMC）。
当去污剂低于临界胶束浓度时，只有单体存在；当高于临界胶束浓度时，胶束、单体以及其余不溶于水的非胶束相共存。
同样，胶束形成的最低温度称为临界胶束温度（CMT）。
因此，温度和浓度是去污剂两相分离和溶解性的重要参数。
一般来说，低亲脂或憎油的去污剂的临界胶束浓度会较高。
种类化合物离子去污剂十二烷基硫酸钠（SDS），脱氧胆酸钠，胆酸钠，肌氨酸非离子去污剂tritonX-100，十二烷基麦芽糖苷，洋地黄皂苷，tween20，tween80两性离子去污剂CHAPS离液剂尿素表一：去污剂的分类。
离子去污剂离子去污剂是由一个亲水链和一个阳离子或阴离子的极性头端基团组成。
噬菌体基因敲除
噬菌体侵染分支杆菌基因敲除实验实验材料与试剂7H9、7H10 培养基、Top Agar 、牛血清白蛋白（BSA）、潮霉素、胰蛋白胨、酵母提取物、琼脂粉、Tris、SDS、卡那霉素、西班牙琼脂糖（Biowest）、Glodview DNA 染料Van91I、AlwNI、PflMI、PacI、Tween-80、T4 DNA 连接酶、EcoRI、HindⅢ、BamHI、TransTaq HiFi DNA 酶（含dNTPS）、Trans2K Plus DNA Marker、λDNA/Hind ⅢDNA Marker、DNA Marker Ⅲ、普通质粒小提试剂盒、琼脂糖凝胶DNA 回收试剂盒、冰醋酸、氯仿、无水乙醇、甘油、乙二胺四乙酸（EDTA）、CaCl2、MgCl2、HCl、NaCl、葡萄糖、异丙醇。
主要试剂配置方法：①ADS：50g BSA，20g 葡萄糖，8.5g NaCl 定容到1L 蒸馏水中，用0.22μm Millipore 滤膜过滤后，放于4℃冰箱备用。
②7H9 液体培养基：4.7g 7H9 溶于900ml 蒸馏水中，混匀，加入10ml 50%（质量体积比）甘油，120℃高压灭菌20min，冷却后加入100mlADS，放于4℃冰箱备用。
使用时每40ml 7H9 培养基需加入100μl 20% Tween-80。
③7H10 固体培养基：19g 7H10 溶于900ml 蒸馏水中，混匀，加入10ml 50%（质量体积比）甘油，120℃高压灭菌20min，冷却后加入100mlADS，到平板。
④Top Agar：0.4g 7H9，0.75g agar 溶于100ml 蒸馏水中，120℃高压灭菌20min，冷却后加入1ml 20%葡萄糖（0.22μm Millipore 滤膜过滤），备用。
⑤MP buffer：pH 7.6 Tris-Cl 50mM，Nacl 150mM，MgCl2 10mM，CaCl2 2mM。
DIBAL-H选择还原酯基而不影响苄溴
1250J.Med.Chem.2010,53,1250–1260DOI:10.1021/jm901530bSynthesis and Structure-Activity Relationships of Azamacrocyclic C-X-C Chemokine Receptor4 Antagonists:Analogues Containing a Single Azamacrocyclic Ring are Potent Inhibitors of T-Cell Tropic(X4)HIV-1ReplicationGary J.Bridger,*,†Renato T.Skerlj,†,)Pedro E.Hernandez-Abad,‡David E.Bogucki,†Zhongren Wang,†Yuanxi Zhou,†Susan Nan,†Eva M.Boehringer,†Trevor Wilson,†Jason Crawford,†Markus Metz,†,)Sigrid Hatse,§Katrien Princen,§Erik De Clercq,§and Dominique Schols§†AnorMED Inc.now Genzyme Corporation,500Kendall Street,Cambridge,Massachusetts02142,‡Johnson Matthey Pharmaceutical Research,1401King Road,West Chester,Pennsylvania19380,and§Rega Institute for Medical Research,Katholieke Universiteit Leuven, Minderbroedersstraat10,B-3000Leuven,Belgium.)Genzyme Corp.,153Second Avenue,Waltham,Massachusetts02451.Received October15,2009Bis-tetraazamacrocycles such as the bicyclam AMD3100(1)are a class of potent and selective anti-HIV-1agents that inhibit virus replication by binding to the chemokine receptor CXCR4,the coreceptor for entryof X4viruses.By sequential replacement and/or deletion of the amino groups within the azamacrocyclic ringsystems,we have determined the minimum structural features required for potent antiviral activity in thisclass of compounds.All eight amino groups are not required for activity,the critical amino groups on a perring basis are nonidentical,and the overall charge at physiological pH can be reduced without compromisingpotency.This approach led to the identification of several single ring azamacrocyclic analogues such asAMD3465(3d),36,and40,which exhibit EC50’s against the cytopathic effects of HIV-1of9.0,1.0,and4.0nM,respectively,antiviral potencies that are comparable to1(EC50against HIV-1of4.0nM).Moreimportantly,however,the key structural elements of1required for antiviral activity may facilitate the designof nonmacrocyclic CXCR4antagonists suitable for HIV treatment via oral administration.IntroductionThe development of antiviral agents that inhibit alternative targets in the HIV a-replicative cycle remains an important goal in order to alleviate the side effects of currently approved agents or to overcome the problem of drug resistance.In this regard,we have focused on the development of compounds that inhibit CXCR4,the coreceptor used by T-tropic(T-cell tropic)viruses for fusion and entry of HIV into target cells of the immune system.The corresponding chemokine receptor CCR5is used by M-tropic(macrophage tropic)viruses and has been associated with the early stages of infection and replication in HIV-positive patients.1,2The transition from M-tropic to T-tropic(or dual/mixed-tropic)virus during the course of HIV infection in approximately50%of patients is associated with a faster CD4þT-cell decline and a more rapid disease progression.3-5Recently,we reported the results of clinical trials with our prototype CXCR4antagonist AMD31006-8(1)and an orally bioavailable CXCR4antagonist,(S)-N0-(1H-benzimidazol-2-ylmethyl)-N0-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-dia-mine(AMD070).9-11When administered to HIV positive patients whose virus was confirmed to use CXCR4for viral entry,both agents were able to suppress the replication of CXCR4and dual-tropic strains of HIV.Similarly,the CCR5 antagonist Maraviroc suppresses replication of HIV-1that exclusively uses CCR5for entry12and was recently approved by the FDA for combined antiretroviral therapy in treatment-experienced patients.13A combination of CCR5and CXCR4 antagonists for treatment of dual/mixed-tropic HIV infection is therefore highly desirable.Beyond its use as a coreceptor for HIV,the CXCR4 chemokine receptor has a more fundamental role in the trafficking of white blood cells,which broadly express CXCR4.14,15A member of the superfamily of G-protein coupled receptors,the interaction of CXCR4and its ligand, stromal cell-derived factor-1(SDF-1),plays a central role in the homing and retention of cells within the bone marrow microenvironment.16Consistent with these observations,ad-ministration of1to healthy volunteers caused a dose-depen-dent leukocytosis6,7that in subsequent studies was shown to include the mobilization of CD34þstem and progenitor cells suitable for hematopoietic stem cell transplantation.17-20The ability of analogues of1to mobilize progenitors correlated with their in vitro capacity to inhibit SDF-1binding to CXCR4.21Because of the need for parenteral administration, 1was developed in combination with granulocyte colony-stimulating factor(G-CSF)to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin’s lymphoma(NHL)and multiple myeloma(MM).22-25Plerix-afor(1)was approved by the FDA in December2008.We have previously reported the structure-activity rela-tionships of anti-HIV bis-azamacrocycles and their transition*To whom correspondence should be addressed.Phone:617-429-7994.Fax:617-768-9809.E-mail:gary.bridger@.Ad-dress:Gary J.Bridger,Genzyme Corporation,55Cambridge Parkway,Cambridge MA02142.a Abbreviations:HIV,Human Immunodeficiency Virus;CXCR4,C-X-C chemokine receptor4;CCR5,C-C-R chemokine receptor5./jmc Published on Web12/31/2009r2009American Chemical SocietyArticle Journal of Medicinal Chemistry,2010,Vol.53,No.31251 metal complexes in detail.26-28Because of the commonstructural features between a doubly protonated cyclam(1,4,8,11-tetraazacyclotetradecane)ring present in1(at phy-siological pH)and a kinetically labile transition metal com-plex of cyclam with an overall charge ofþ2,we proposed thatboth structural motifs may bind to the CXCR4receptorthrough interactions with amino acid residues containingcarboxylate groups.29We have subsequently shown via direc-ted mutagenesis of the aspartate and glutamic acid residues inCXCR4that binding of1and related analogues to the seventransmembrane,G-protein coupled receptor is highly depen-dent upon the amino acids Asp171and Asp262,located intransmembrane region(TM)-IV and TM-VI at each end ofthe main ligand binding crevice of the receptor.30-35Mutationof either aspartic acid to aspargine significantly reduced theability of1to inhibit binding of radiolabeled stromal cellderived factor-1R(125I-Met-SDF-1R).More importantly,however,U87cells stably transfected with CD4and themutant coreceptors CXCR4[D171N]and CXCR4[D262N]were less effective at supporting infection of the CXCR4-usingHIV-1strain NL4.3compared to the wild-type receptor andthe double mutant CXCR4[D171N,D262N]completely failedas a coreceptor for HIV infection.31Correspondingly,theability of1to inhibit HIV-1infection via CXCR4[D171N]andCXCR4[D262N]was also diminished,thereby confirmingthat1binds in a region of the receptor that is critical for X4HIV-1coreceptor function.We have also reported that binding of the bis-Zn,Ni,andCu complexes of1were also dependent upon D171and D262of the receptor.36In a similar manner to1,the transitionmetal complexes were found to be less effective inhibitors of125I-Met-SDF-1R binding to the mutant receptors CXCR4-[D171N]and CXCR4[D262N]compared to the wild-typereceptor.Incorporation of Zn,Ni,or Cu into the cyclam ringsof1increased the affinity to the wild-type CXCR4receptor,but the enhancement was selectively eliminated by substitu-tion of Asp262.Supporting physiochemical evidence for theinteraction of acetate(carboxylates)with metal complexes ofazamacrocycles,including1,has been recently reported.37,38In the current study,we determine the minimum struc-tural features of1required for potent antiviral activity, leading to the identification of the single azamacrocyclic ring analogue AMD346532,33,39,40(3d)and ultimately the design of nonmacrocyclic,orally biovailable CXCR4an-tagonists.11,41,42Given the growing body of evidence that the CXCR4/SDF-1interaction is involved in regulating several human malignancies,43-45CXCR4antagonists may have additional therapeutic applications in addition to HIV treatment.ChemistryAnalogues containing a single1,4,8,11-tetraazacyclotetra-decane(cyclam)ring were prepared by modifications to previously published routes26,29as shown in Scheme1.Reac-tion of the selectively protected tris-diethylphosphoramidate (Dep)cyclam ring(2a)with R,R-dibromo-p-xylene in aceto-nitrile containing potassium carbonate gave the desired bro-momethyl intermediate(2b).Reaction of the bromide with an excess of the requisite amine,followed by deprotection of the Dep-groups with a saturated solution of hydrogen bromide in acetic acid at room temperature.gave analogues3a-i as the corresponding hydrobromide salts.To prepare analogues of3d in which the cyclam ring was replaced by a series of14-membered azamacrocyclic rings,we prepared a series of selectively protected macrocyclic ring systems containing a single(unprotected)secondary amine. This approach ensures the regiochemical outcome of the reaction with a benzylic halide during final construction (as shown in Scheme6).The syntheses of appropriate pre-cursors are shown in Schemes2-5.To incorporate fluorine groups at the desired position in the macrocyclic ring,suitably fluorinated bis-electrophiles were prepared,starting from 4-oxo-heptanedioic acid diethyl ester(4)and heptane-1,4,7-triol(8)as depicted in Scheme2.Reaction of the ketone(4) with neat(diethylamino)-sulfur trifluoride46,47(DAST)at room temperature for12days gave the corresponding di-fluoro-intermediate(5)in43%yield.Reduction of the ester groups with LAH(to give the diol6),followed by derivatiza-tion with toluenesulfonyl chloride,gave the bis-electrophile (7)required for the impending macrocyclization reaction.The corresponding monofluorinated intermediate was prepared in a similar manner.Protection of the primary alcohols in8as the acetyl group using acetic anhydride gave the secondary alcohol9,which was rapidly(and virtually quantitatively) converted to the fluorinated intermediate(10)with DAST (2.0equiv)in dichloromethane.Removal of the acetyl pro-tecting groups with saturated ammonia in methanol,followed by reaction of the diol(11)with p-toluenesulfonyl chloride, Scheme1aa Reagents:(a)R,R0-dibromo-p-xylene,K2CO3,CH3CN,reflux;(b)amine,K2CO3,CH3CN,reflux;(c)HBr,acetic acid,room temp. Scheme2aa Reagents:(a)Et2NSF3(neat),room temp;(b)LAH,Et2O;(c)Ts-Cl,Et3N,CH2Cl2;(d)acetic anhydride,pyridine;(e)Et2NSF3, CH2Cl2,-78°C,then room temp;(f)NH3/MeOH,room temp;(g)Ts-Cl,Et3N,CH2Cl2.1252Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.gave the desired bis-electrophile 12containing a single fluorine group.The selectively protected azamacrocyclic rings were pre-pared via directed combinatorial macrocyclization of bis-2-nitrobenzenesulfonamides 48(Ns)(15a -c ,16a -c ,18)with bis-electrophiles (7,12,17)using previously optimized condi-tions 28(Scheme 3).To incorporate a phenyl or heterocyclic ring into the macrocycle,the corresponding bis-2-nitrobenze-nesulfonamide (15a -c )was prepared from the bis-aminoethyl intermediates 28(13a -c )by reaction with nosyl chloride (Et 3N,CH 2Cl 2).Similarly,16a ,b were obtained by reac-tion of commercially available intermediates 14a ,b with nosyl chloride or in the case of 16c (X=S)by reduction of 3,30-thiodipropionitrile with BH 33Me 2S and reaction of the intermediate diamine (14c )with nosyl chloride to give 16c .Macrocyclization was accomplished by dropwise addition of a DMF solution of the bis-electrophile to a DMF solution of the bis-2-nitrobenzenesulfonamide containing Cs 2CO 3maintained at a temperature of 80°C.Standard workup,followed by purification of the crude product by column chromatography on silica gel,gave the desired macrocycles 19a -c ,20a -c ,and 21a ,b in yields of 19-55%.Reaction of theintermediates from above with HBr/acetic acid at room temperature gave 22a -c ,23a -c ,and 24a ,b ,respectively.Because of synthetic convenience,we also prepared the selectively protected “isomers”of 22a ,b and 23a in which the alternative secondary amine was available for the alkylation reaction.We reasoned that reaction of 19a ,b and 20a with approximately 1equiv of thiophenol 49(our reagent of choice for nosyl deprotections)may allow pseudoselective deprotec-tion of a single nosyl group,leaving the Dep group intact.After some optimization,we found that reaction of 19a ,b and 20a with 0.8equiv of thiophenol and potassium carbonate in DMF (or acetonitrile)gave the precursors 25and 26a ,b in manageable,albeit modest yields (20-50%)following col-umn purification on silica gel (Scheme 4).Finally,the inter-mediates 27a ,b and 28(Scheme 5)were synthesized as recently described by palladium(0)catalyzed coupling of organozinc iodide reagents with bromopyridines.50Having completed the series of selectively protected aza-macrocycles,we proceeded to completion of the desired analogues by straightforward installation of the right-hand portion containing the aminomethyl pyridine moiety.As shown in Scheme 6,this was accomplished in all cases by direct alkylation of the available secondary amine of the macrocycle with the benzylic chlorides 34a ,b .Intermediate 34a was prepared in four steps from 4-bromomethyl benzoic acid methyl ester (29)and 2-aminomethylpyridine (31):con-version of 31to the 2-nitrobenzenesulfonamide 32,followed by alkylation with the benzyl bromide 30(obtained by reduc-tion of 29with DIBAL-H)gave the desired alcohol 33.As previously reported,28reaction of benzylic alcohols such as 33with methanesulfonyl chloride gave the chloride 34a rather than the corresponding mesylate,presumably via in situ nucleophilic substitution of the initially formed mesylate with chloride.Intermediate 34b (Scheme 6)containing a Dep-protecting group was prepared by an alternative synthesisScheme 3aaReagents:(a)Ns-Cl,Et 3N,CH 2Cl 2;(b)Cs 2CO 3,DMF,80°C;(c)HBr(g),AcOH,room temp.Scheme4Scheme5Article Journal of Medicinal Chemistry,2010,Vol.53,No.31253(procedures in Supporting Information).Alkylation of the available secondary amine of the macrocycles with 34a (or 34b )in CH 3CN in the presence of K 2CO 3gave the penultimate intermediates 35a -n .Deprotection of the nosyl groups with thiophenol and K 2CO 3in DMF gave the free base of the desired analogues,which in the vast majority of cases were converted to the corresponding hydrobromide salts.For analogues derived from the macrocyclic precursors 25and 26a ,b ,the intermediates isolated prior to the deprotection also contained a residual Dep group in addition to nosyl groups.For compound 45,we found that conversion to the hydro-bromide salt using a saturated solution of HBr in acetic acid resulted in concomitant deprotection of the remaining Dep group to obtain compound 45.For compounds 44and 46,the residual Dep group was removed prior to nosyl deprotection and salt formation.The thioether analogue 41a was also used to prepare the corresponding sulfoxide and sulfone analogues for antiviral evaluation as shown in Scheme 7.Initially,we globally protected the amino groups of 41a with Boc and subjected this intermediate to oxidation with oxone in MeOH 51at -10°C to give a mixture of the sulfoxide and sulfone that were separated by column chromatography on silica gel.However,while deprotection of the Boc groups with simulta-neous conversion to the hydrobromide salt proceeded without incident for the sulfone (to give 41c ),we found that deprotec-tion of the corresponding sulfoxide led to substantial reduc-tion and hence recovery of the starting analogue 41a .To overcome this problem,the sulfoxide was synthesized by direct oxidation of 41a with 1equiv of oxone in MeOH to give 41b in a 21%isolated yield and was subsequently tested as the free base in antiviral assays.Finally,we prepared a short series of analogues containing a carbon atom in place of a tertiary nitrogen group at the ring junction.To economize on the number of synthetic steps,weelected to synthesize the dimesylate 54(Scheme 8),an inter-mediate that could be commonly used for the synthesis of multiple analogues via macrocylization with the bis-2-nitro-benzenesulfonamide precursors already in our possession (namely 15a ,16a ,b from Scheme 3).Intermediate 54was prepared from the commercially available starting material bromo-p -tolunitrile via a double one-carbon homologation of the malonate 51,followed by derivatization to gave the requisite bis-methanesulfonate 54.Macrocyclizations of 54with bis-sulfonamides 15a and 16a ,b were performed as described above.Deprotection of the nosyl groups followed by conversion to the corresponding hydrobromide salts gave analogues 56and 58a ,b .DiscussionHaving previously established the optimum ring size and distance between the amines of both aliphatic andScheme 6a aReagents:(a)DIBAL-H,CH 2Cl 2;(b)Ns-Cl,Et 3N,CH 2Cl 2;(c)K 2CO 3,CH 3CN,60°C;(d)Ms-Cl,Et 3N,CH 2Cl 2;(e)K 2CO 3,CH 3CN,80°C;(f)R =Ns:thiophenol,K 2CO 3,DMF,or R =Dep:HBr(g),AcOH,room temp.Scheme 7aaReagents:(a)oxone,MeOH,-10°C;(b)(Boc)2O,THF;(c)HBr(g),AcOH,room temp.Scheme 8aaReagents:(a)NaH,R -bromo-tolunitrile,THF;(b)LiAlH 4,THF;(c)Ns-Cl,Et 3N,CH 2Cl 2;(d)2-picolyl chloride,Et 3N,K 2CO 3,KBr,CH 3CN,reflux;(e)Ms-Cl,Et 3N,CH 2Cl 2;(f)cetyltrimethyammonium bromide,NaCN,benzene,H 2O,reflux;(g)conc HCl/AcOH (4:1),reflux;(h)BH 3.Me 2S,THF;(i)Ms-Cl,Et 3N,CH 2Cl 2;(j)Cs 2CO 3,DMF,80°C;(k)thiophenol,K 2CO 3,CH 3CN (or DMF),40°C.1254Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.pyridine-fused bis-tetraazamacrocycles required for potent X4anti-HIV activity,we designed a series of compounds to address the question of structural redundancy.The prototype bis-macrocycle 1has a center of symmetry and contains eight amino groups,of which four are positively charged at phy-siological pH.In the current study,we aimed to answer two specific questions:(1)Are all four positive charges required for potent anti-HIV activity?(2)On a per ring basis,what are the minimum structural requirements for activity?Assuming that the structural requirements are not iden-tical for both rings of 1,we reasoned that the simplest replacement for a single tetraaza-macrocyclic ring would be a pseudo diamine-segment,representing the first two amino groups of the macrocyclic ring from the point of attachment at the benzylic position.A judicious choice of “diamine”would also reduce the overall charge to þ1.Having previously established that the optimum distance between the first two amino groups was a two-carbon unit,we prepared a series of aminomethyl-substituted analogues in which the second amino group was a substituent upon an aromatic ring or part of a heterocyclic ring.In either case,the second p K a would be sufficiently low to prevent a second protonation at physiological pH.The compounds were tested for their ability to inhibit replication of HIV-1III B in MT-4cells,a strain of HIV-1that uses exclusively CXCR4for fusion and viral entry into target cells.The results are shown in Table 1.Compared to 1,the introduction of a benzylamine group (3a )in place of the azamacrocyclic ring substantially reduced anti-HIV potency,although the compound remained active at submicromolar concentrations.The concentration of 3a re-quired to inhibit HIV-1replication by 50%(the EC 50)was 0.49μM,which was approximately 100-fold higher than the 50%inhibitory concentration of 1.Aromatic amino groups at the 2-position (3b )or 4-position (3c )did not affect antiviral potency.Both 3b ,c exhibited comparable EC 50’s to the un-substituted benzyl group (3a ).However,we observed a sub-stantial increase in anti-HIV potency when the benzyl group was replaced by a pyridyl group (3d ).Compound 3d exhibited a 50%inhibitory concentration of 0.009μM,which was only ca.2-fold higher than the EC 50of 1.Furthermore,the 50%cytotoxic concentration (CC 50)of compound 3d in MT-4cells was greater than 112μM.Thus 3d exhibits a selectivity index of greater than 12000.The positional specificity of the pyridine-N in 3d was also examined.Replacement of the 2-pyridyl group with the 3-pyridyl (3e )or 4-pyridyl (3f )group had a detrimental effect on anti-HIV potency.For example,the EC 50’s of analogues 3e ,f were approximately 3orders of magnitude higher than the concentration of 3d required to inhibit HIV-1replication by 50%(the EC 50’s of 3e and 3f were 8.470and 9.977μM,respectively).Methylation of the amine in 3d (to give 3g )or extension of the connectivity to an aminoethyl pyridine group (to give 3h )also adversely affected the anti-HIV potency.Finally,we replaced the pyridine moiety with a comparable heterocycle of lower p K a than pyridine,namely the pyrazine group (3i ).Perhaps not surprisingly,the antiviral potency of analogue 3i was approximately comparable to the benzyl analogue 3a ,which did not contain a vicinal heterocycle nitrogen atom.With the optimized “right-hand”replacement for the aza-macrocycle ring of 1fixed as the 2-aminomethyl pyridine group,we then turned our attention to the “left-hand”ring.Needless to say,the mandatory synthesis of the symmetrical analogue in which both rings were replaced by a 2-amino-methyl pyridine group turned out to be a predictably fruitless exercise (EC 50was >250μM,data not shown).We therefore focused on systematically replacing individual amine groups of the left ring.As shown in Table 2,we first prepared an analogue in which the [14]aneN 4(cyclam)ring had been replaced by the optimized and equally suitable,py[iso -14]-aneN 4ring (to give compound 36).Consistent with the structure -activity relationship of py[iso -14]aneN 4bis-azama-crocycles,compound 36proved to be a potent inhibitor of HIV-1replication,exhibiting an EC 50of 0.001μM,that is,around 9-fold and 4-fold lower,respectively,than the con-centration of 3d or 1required to inhibit viral replication by 50%.Although the pyridine-N of the macrocyclic ring in 36was previously found to be critical for high antiviral potency,we reasoned that a precise determination of the pyridine-N contribution to potency could help redesign a less basic pounds 37and 38were then prepared to answer this question.Both analogues 37,containing a phenyl replacement and 38,containing an “exocyclic”pyridine fused group,retained reasonable anti-HIV potency (the EC 50’s of 37and 38were 0.040and 0.104μM,respectively)but were at least 40-to 100-fold less potent than analogue 36.So what role does the pyridine group play?At physiological pH,the overall charge of the py[iso -14]-aneN 4ring in 36is also þ2(in a similar manner to cyclam 52)and the likely protonation sequence is indicated in Figure 1A,based on the sequence reported by Delgado et al.53for similar 14-membered tetraazamacrocyclic rings contain-ing pyridine.Presumably,the secondary amino groups are predominantly protonated and the overall structure is stabi-lized by intramolecular hydrogen bond interactions from the adjacent hydrogen-bond acceptors,the pyridine and tertiary benzylic amine groups (while minimizing the elec-trostatic repulsion of two positive charges in a confined macrocyclic ring).This is confirmed by a conformational analysis of 36on B3LYP/6-31G*level followed by single point energy calculations.In the energetically most stable ring conformation (LMP2/6-311þG*þZPE),the pyridine nitro-gen forms two six-membered intramolecular hydrogen bond interactions with the two adjacent protonated nitrogens as shown in Figure 2.Potential five-membered intramolecular hydrogen bond interactions are formed with the tertiary amine.Table 1.Antiviral Activity of Single RingAzamacrocyclesnR 1R 2HIV-1(III B )EC 50(μM)MT-4cells CC 50(μM)3a 1H Ph0.4911603b 1H 2-amino-Ph 1.825243c 1H 4-amino-Ph 0.7172273d 1H 2-pyridine 0.009>1123e 1H 3-pyridine 8.470373f 1H 4-pyridine 9.977>2793g 1Me 2-pyridine 0.416383h 2H 2-pyridine 49.135>1103I 1H5-Me-pyrazine1.8957810.004>421ArticleJournal of Medicinal Chemistry,2010,Vol.53,No.31255The stabilization provided by this “shared”protonated structure could account for the high basicity of azamacrocyc-lic rings,as suggested by Kimura et al.54It did not seem unreasonable,therefore,that a potential role of the pyridine group is the contribution of a single intramolecular hydrogen-bond,which locks the conformation of the protonated aza-macrocyclic ring in manner that is beneficial to antiviral potency.To test this hypothesis,we prepared a series of analogues (depicted in Figure 1B,data in Table 2)in which the fused aromatic group had been removed and replaced by an aliphatic group,in some cases containing a hydrogen-bondacceptor at the key position “x,”the position occupied by the pyridine nitrogen in compound 36.Consistent with the hydrogen-bonding hypothesis,the alkyl analogue 39exhibited an anti-HIV potency that was compar-able to the phenyl and exocyclic pyridine analogues 37and 38(the EC 50’s of 37and 39,were 0.040and 0.043μM,re-spectively).This result categorically rules out the possibility that the conformational restrictions imposed by the fused aromatic groups in compounds 37,38were even partially responsible for the high potency of 36.However,incorpora-tion of a hydrogen-bond acceptor at position x (Figure 1B)in some cases restored activity comparable to 36.For example,the oxygen analogue 40exhibited an EC 50that was only 4-fold higher than the concentration of 36required to inhibit HIV-1replication by 50%(the EC 50of 40was 0.004μM).The corresponding thioether analogue 41a exhibited an EC 50of 0.013μM,which is approximately 3-fold higher than com-pound 40.Although the antiviral potency of the thioether analogue 41a compared to the ether analogue 41is greater than one would predict from the strength of the hydrogen-bond acceptor acceptor capabilities (thioether groups are considerably weaker H-bond acceptors than the oxygen inTable 2.Antiviral Activity of Single RingAzamacrocyclesFigure 1.Proposed hydrogen-bond structure of protonated aza-macrocycles.1256Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.40),this result can be reconciled by considering the nature of the H-bond required;a six-membered intramolecular H-bond constrained by the macrocyclic ring (Figure 2).With the thioether compound 41a in hand,we also pre-pared the sulfoxide (41b )and sulfone (41c )analogues by direct oxidation of 41a .We reasoned that the oxygen atoms of the sulfoxide and sulfone are stronger H-bond acceptors than the sulfur atom of 41a and may consequently improve the anti-HIV potency.However,both 41b and 41c were considerably weaker antiviral agents,exhibiting 50%effective concentra-tions for inhibition of HIV-1replication that were at least 79-fold higher than the EC 50of 41a (the EC 50’s of 41b and 41c were 0.485and 11.878μM,respectively).The precise reason for the poor antiviral activity exhibited by analogues 41b ,c was unclear;although the sulfoxide and sulfone are more sterically demanding than the thioether and could induce a ring conformation that is detrimental to antiviral activity,we could not rule out the possibility that the H-bond acceptor oxygen is now “one-bond”outside of the ring,and the intramolecular H-bond itself induces an unfavorable confor-mation (a seven-membered ring H-bond in 41b ,c (Figure 2)compared to a six-membered in 41a ).To complete this series of compounds therefore,we decided to introduce the fluoro and difluoro substituents at position x (Figure 1B).Several reports have demonstrated that the fluoro group can partici-pate as an acceptor for intramolecular H-bonds,particularly within highly constrained ring structures.55-57This is also confirmed by our calculations,as shown in Figure 2.The fluoro (43)and difluoro (42)analogues were also attractive substituents for two other reasons:(1)the substituents would be situated at the fourth carbon from the adjacent amine group,thereby minimizing the affect on p K a ;(2)in a similar manner to the sulfoxide and sulfone,the H-bond acceptor would be one-bond outside of the macrocyclic ring.However in this case,because the fluorine atom in C -F groups is isostructural with hydrogen,a negative effect of the fluoro substituents on antiviral activity can only be attributed to an inappropriately positioned H-bond rather than steric requirements (that is,in the absence of an H-bond,we would expect the fluoro or difluoro analogues to exhibit an EC 50comparable to the methylene analogue 39).In antiviral test-ing,the fluoro (43)and difluoro (42)analogues displayed EC 50’s that were greater than 20-fold higher than the methy-lene analogue 39(the EC 50’s of 39,42,and 43were 0.043,0.920,and 1.239μM,respectively),confirming the negative consequences of an incorrectly positioned hydrogen-bond (Figure 2).Next,we focused on the sequence of aliphatic amine groups in the macrocyclic ring required for potent antiviral activity.By straightforward synthetic manipulation of our collection of ring systems,we prepared the structural isomers of analo-gues 36,37,and 39in which the side-chain (R,in Table 2)was connected to the alternative secondary amine group to give compounds 44,45,and 46.In antiviral testing,analogue 44was substantially less potent than its corresponding regioi-somer 39:the EC 50of 44was 11.131μM,which was approxi-mately 260-fold higher than the EC 50of 39.A similar loss of antiviral potency was observed with the phenyl analogue 46and its isomer 37(the EC 50’s of 46and 37were 14.106and 0.040μM,respectively).Interestingly,the loss of antiviral potency with the pyridine-fused isomer 45compared to 36was significant but not as substantial;the EC 50of 45was 0.063μM,around 60-fold higher than the concentration of 36required to inhibit HIV-1replication by 50%.There was a possibility,therefore,that while the “tri-aza”ring configura-tion required for potent antiviral activity is clearlyrepresentedFigure 2.Lowest energy conformations of compounds 36,40,41c ,and 42.View from top on a plane defined by three nitrogens and X (see Figure 1).Dashed lines indicate hydrogen bond interactions:the hydrogen bond acceptors in 36and 40are in one plane with the three nitrogens.This is not the case for 41c and 42.Bond angles:36:—(N 333H -N þ)=140.5°,122.4°,102.1°,108.4°.40:—(O 333H -N þ)=135.1°,141.5°;—(N 333H -N þ)=104.6°,102.8°.41c :—(O 333H -N þ)=112.8°,112.8°;—(N 333H -N þ)=108.2°,108.0°.42:—(F 333H -N þ)=142.2°,142.2°;—(N 333H -N þ)=114.7°,114.7°.。
8082方法
METHOD 8082POLYCHLORINATED BIPHENYLS (PCBs)BY GAS CHROMATOGRAPHY1.0SCOPE AND APPLICATION1.1Method 8082 is used to determine the concentrations of polychlorinated biphenyls (PCBs) as Aroclors or as individual PCB congeners in extracts from solid and aqueous matrices. Open-tubular, capillary columns are employed with electron capture detectors (ECD) or electrolytic conductivity detectors (ELCD). When compared to packed columns, these fused-silica, open-tubular columns offer improved resolution, better selectivity, increased sensitivity, and faster analysis. The target compounds listed below may be determined by either a single- or dual-column analysis system. The PCB congeners listed below have been tested by this method, and the method may be appropriate for additional congeners.Compound CAS Registry No.IUPAC #Aroclor 101612674-11-2-Aroclor 122111104-28-2-Aroclor 123211141-16-5-Aroclor 124253469-21-9-Aroclor 124812672-29-6-Aroclor 125411097-69-1-Aroclor 126011096-82-5-2-Chlorobiphenyl2051-60-712,3-Dichlorobiphenyl16605-91-752,2',5-Trichlorobiphenyl37680-65-2182,4',5-Trichlorobiphenyl16606-02-3312,2',3,5'-Tetrachlorobiphenyl41464-39-5442,2',5,5'-Tetrachlorobiphenyl35693-99-3522,3',4,4'-Tetrachlorobiphenyl32598-10-0662,2',3,4,5'-Pentachlorobiphenyl38380-02-8872,2',4,5,5'-Pentachlorobiphenyl37680-73-21012,3,3',4',6-Pentachlorobiphenyl38380-03-91102,2',3,4,4',5'-Hexachlorobiphenyl35065-28-21382,2',3,4,5,5'-Hexachlorobiphenyl52712-04-61412,2',3,5,5',6-Hexachlorobiphenyl52663-63-51512,2',4,4',5,5'-Hexachlorobiphenyl35065-27-11532,2',3,3',4,4',5-Heptachlorobiphenyl35065-30-61702,2',3,4,4',5,5'-Heptachlorobiphenyl35065-29-31802,2',3,4,4',5',6-Heptachlorobiphenyl52663-69-11832,2',3,4',5,5',6-Heptachlorobiphenyl52663-68-01872,2',3,3',4,4',5,5',6-Nonachlorobiphenyl40186-72-9206CD-ROM8082 - 1Revision 0December 19961.2Aroclors are multi-component mixtures. When samples contain more than one Aroclor,a higher level of analyst expertise is required to attain acceptable levels of qualitative and quantitative analysis. The same is true of Aroclors that have been subjected to environmental degradation ("weathering") or degradation by treatment technologies. Such weathered multi-component mixtures may have significant differences in peak patterns than those of Aroclor standards.1.3Quantitation of PCBs as Aroclors is appropriate for many regulatory compliance determinations, but is particularly difficult when the Aroclors have been weathered by long exposure in the environment. Therefore, this method provides procedures for the determination of selected individual PCB congeners. The 19 PCB congeners listed above have been tested by this method.1.4The PCB congener approach potentially affords greater quantitative accuracy when PCBs are known to be present. As a result, this method may be used to determine Aroclors, some PCB congeners, or "total PCBs," depending on regulatory requirements and project needs. The congener method is of particular value in determining weathered Aroclors. However, analysts should use caution when using the congener method when regulatory requirements are based on Aroclor concentrations.1.5Compound identification based on single-column analysis should be confirmed on a second column, or should be supported by at least one other qualitative technique. This method describes analytical conditions for a second gas chromatographic column that can be used to confirm the measurements made with the primary column. GC/MS Method 8270 is also recommended as a confirmation technique when sensitivity permits (Sec. 8.0).1.6This method also describes a dual-column option. The option allows a hardware configuration of two analytical columns joined to a single injection port. The option allows one injection to be used for dual-column analysis. Analysts are cautioned that the dual-column option may not be appropriate when the instrument is subject to mechanical stress, many samples are to be run in a short period, or when highly contaminated samples are analyzed.1.7The analyst must select columns, detectors and calibration procedures most appropriate for the specific analytes of interest in a study. Matrix-specific performance data must be established and the stability of the analytical system and instrument calibration must be established for each analytical matrix (e.g., hexane solutions from sample extractions, diluted oil samples, etc.). Example chromatograms and GC conditions are provided as guidance.1.8The MDLs for Aroclors vary in the range of 0.054 to 0.90 µg/L in water and 57 to 70 µg/kg in soils. Estimated quantitation limits may be determined using the data in Table 1.1.9This method is restricted to use by, or under the supervision of, analysts experienced in the use of gas chromatographs (GC) and skilled in the interpretation of gas chromatograms. Each analyst must demonstrate the ability to generate acceptable results with this method.2.0SUMMARY OF METHOD2.1 A measured volume or weight of sample (approximately 1 L for liquids, 2 g to 30 g for solids) is extracted using the appropriate matrix-specific sample extraction technique.2.2Aqueous samples are extracted at neutral pH with methylene chloride using Method 3510 (separatory funnel), Method 3520 (continuous liquid-liquid extractor), or other appropriate technique. CD-ROM8082 - 2Revision 0December 19962.3Solid samples are extracted with hexane-acetone (1:1) or methylene chloride-acetone (1:1) using Method 3540 (Soxhlet), Method 3541 (automated Soxhlet), or other appropriate technique.2.4Extracts for PCB analysis may be subjected to a sulfuric acid/potassium permanganate cleanup (Method 3665) designed specifically for these analytes. This cleanup technique will remove (destroy) many single component organochlorine or organophosphorus pesticides. Therefore, Method 8082 is not applicable to the analysis of those compounds. Instead, use Method 8081.2.5After cleanup, the extract is analyzed by injecting a 2-µL aliquot into a gas chromatograph with a narrow- or wide-bore fused silica capillary column and electron capture detector (GC/ECD).2.6The chromatographic data may be used to determine the seven Aroclors in Sec. 1.1, individual PCB congeners, or total PCBs.3.0INTERFERENCES3.1Refer to Methods 3500 (Sec. 3.0, in particular), 3600, and 8000 for a discussion of interferences.3.2Interferences co-extracted from the samples will vary considerably from matrix to matrix. While general cleanup techniques are referenced or provided as part of this method, unique samples may require additional cleanup approaches to achieve desired degrees of discrimination and quantitation. Sources of interference in this method can be grouped into three broad categories.3.2.1Contaminated solvents, reagents, or sample processing hardware.3.2.2Contaminated GC carrier gas, parts, column surfaces, or detector surfaces.3.2.3Compounds extracted from the sample matrix to which the detector will respond.3.3Interferences by phthalate esters introduced during sample preparation can pose a major problem in PCB determinations.3.3.1Common flexible plastics contain varying amounts of phthalate esters which areeasily extracted or leached from such materials during laboratory operations. Interferences from phthalate esters can best be minimized by avoiding contact with any plastic materials and checking all solvents and reagents for phthalate contamination.3.3.2Exhaustive cleanup of solvents, reagents and glassware may be required toeliminate background phthalate ester contamination.3.3.3These materials can be removed through the use of Method 3665 (sulfuricacid/permanganate cleanup).3.4Cross-contamination of clean glassware routinely occurs when plastics are handled during extraction steps, especially when solvent-wetted surfaces are handled. Glassware must be scrupulously cleaned.Clean all glassware as soon as possible after use by rinsing with the last solvent used. This should be followed by detergent washing with hot water, and rinses with tap water and organic-free CD-ROM8082 - 3Revision 0December 1996CD-ROM 8082 - 4Revision 0December 1996reagent water. Drain the glassware, and dry it in an oven at 130E C for several hours, or rinse with methanol and drain. Store dry glassware in a clean environment. NOTE:Oven-drying of glassware used for PCB analysis can increase contaminationbecause PCBs are readily volatilized in the oven and spread to other glassware.Therefore, exercise caution, and do not dry glassware from samples containinghigh concentrations of PCBs with glassware that may be used for trace analyses.3.5Elemental sulfur (S ) is readily extracted from soil samples and may cause 8chromatographic interferences in the determination of PCBs. Sulfur can be removed through the use of Method 3660.4.0APPARATUS AND MATERIALS4.1Gas chromatograph - An analytical system complete with gas chromatograph suitable for on-column and split-splitless injection and all required accessories including syringes, analytical columns, gases, electron capture detectors (ECD), and recorder/integrator or data system.4.2GC columnsThis method describes procedures for both single-column and dual-column analyses. The single-column approach involves one analysis to determine that a compound is present, followed by a second analysis to confirm the identity of the compound (Sec. 8.4 describes how GC/MS confirmation techniques may be employed). The single-column approach may employ either narrow-bore (# 0.32 mm ID) columns or wide-bore (0.53 mm ID) columns. The dual-column approach involves a single injection that is split between two columns that are mounted in a single gas chromatograph. The dual-column approach employs only wide-bore (0.53 mm ID) columns. A third alternative is to employ dual columns mounted in a single GC, but with each column connected to a separate injector and a separate detector.The columns listed in this section were the columns used to develop the method performance data. Listing these columns in this method is not intended to exclude the use of other columns that may be developed. Laboratories may use other capillary columns provided that they document method performance (e.g., chromatographic resolution, analyte breakdown, and MDLs) that equals or exceeds the performance specified in this method.4.2.1Narrow-bore columns for single-column analysis (use both columns to confirmcompound identifications unless another confirmation technique such as GC/MS is employed).Narrow bore columns should be installed in split/splitless (Grob-type) injectors.4.2.1.130 m x 0.25 or 0.32 mm ID fused silica capillary column chemicallybonded with SE-54 (DB-5 or equivalent), 1 µm film thickness.4.2.1.230 m x 0.25 mm ID fused silica capillary column chemically bonded with35 percent phenyl methylpolysiloxane (DB-608, SPB-608, or equivalent), 2.5 µm coatingthickness, 1 µm film thickness.4.2.2Wide-bore columns for single-column analysis (use two of the three columnslisted to confirm compound identifications unless another confirmation technique such as GC/MS is employed). Wide-bore columns should be installed in 1/4 inch injectors, with deactivated liners designed specifically for use with these columns.4.2.2.130 m x 0.53 mm ID fused silica capillary column chemically bonded with35 percent phenyl methylpolysiloxane (DB-608, SPB-608, RTx-35, or equivalent), 0.5 µmor 0.83 µm film thickness.4.2.2.230 m x 0.53 mm ID fused silica capillary column chemically bonded with14% cyanopropylmethylpolysiloxane (DB-1701, or equivalent), 1.0 µm film thickness.4.2.2.330 m x 0.53 mm ID fused silica capillary column chemically bonded withSE-54 (DB-5, SPB-5, RTx-5, or equivalent), 1.5 µm film thickness.4.2.3Wide-bore columns for dual-column analysis (choose one of the two pairs ofcolumns listed below).4.2.3.1Column pair 130 m x 0.53 mm ID fused silica capillary column chemically bonded with SE-54(DB-5, SPB-5, RTx-5, or equivalent), 1.5 µm film thickness.30 m x 0.53 mm ID fused silica capillary column chemically bonded with 14%cyanopropylmethylpolysiloxane (DB-1701, or equivalent), 1.0 µm film thickness.Column pair 1 is mounted in a press-fit Y-shaped glass 3-way union splitter (J&W Scientific, Catalog No. 705-0733) or a Y-shaped fused-silica connector (Restek, CatalogNo. 20405), or equivalent.4.2.3.2Column pair 230 m x 0.53 mm ID fused silica capillary column chemically bonded with SE-54(DB-5, SPB-5, RTx-5, or equivalent), 0.83 µm film thickness.30 m x 0.53 mm ID fused silica capillary column chemically bonded with 14%cyanopropylmethylpolysiloxane (DB-1701, or equivalent), 1.0 µm film thickness.Column pair 2 is mounted in an 8 in. deactivated glass injection tee (Supelco, Catalog No. 2-3665M), or equivalent.4.3Column rinsing kit - Bonded-phase column rinse kit (J&W Scientific, Catalog No. 430-3000), or equivalent.4.4Volumetric flasks - 10-mL and 25-mL, for preparation of standards.5.0REAGENTS5.1Reagent grade or pesticide grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all reagents shall conform to specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening the accuracy of the determination.NOTE:Store the standard solutions (stock, composite, calibration, internal, and surrogate standards) at 4E C in polytetrafluoroethylene (PTFE)-sealed containers CD-ROM8082 - 5Revision 0December 1996CD-ROM 8082 - 6Revision 0December 1996in the dark. When a lot of standards is prepared, it is recommended that aliquotsof that lot be stored in individual small vials. All stock standard solutions mustbe replaced after one year or sooner if routine QC (Sec. 8.0) indicates a problem.All other standard solutions must be replaced after six months or sooner if routineQC (Sec. 8.0) indicates a problem.5.2Sample extracts prepared by Methods 3510, 3520, 3540, 3541, 3545, or 3550 need to undergo a solvent exchange step prior to analysis. The following solvents are necessary for dilution of sample extracts. All solvent lots should be pesticide quality or equivalent and should be determined to be phthalate-free.5.2.1n-Hexane, C H 6145.2.2Isooctane, (CH )CCH CH(CH )332325.3The following solvents may be necessary for the preparation of standards. All solvent lots must be pesticide quality or equivalent and should be determined to be phthalate-free.5.3.1Acetone, (CH )CO 325.3.2Toluene, C H CH 6535.4Organic-free reagent water - All references to water in this method refer to organic-free reagent water as defined in Chapter One.5.5Stock standard solutions (1000 mg/L) - May be prepared from pure standard materials or can be purchased as certified solutions.5.5.1Prepare stock standard solutions by accurately weighing about 0.0100 g of purecompound. Dissolve the compound in isooctane or hexane and dilute to volume in a 10-mL volumetric flask. If compound purity is 96 percent or greater, the weight may be used without correction to calculate the concentration of the stock standard solution.5.5.2Commercially-prepared stock standard solutions may be used at any concentration if they are certified by the manufacturer or by an independent source.5.6Calibration standards for Aroclors5.6.1 A standard containing a mixture of Aroclor 1016 and Aroclor 1260 will includemany of the peaks represented in the other five Aroclor mixtures. As a result, a multi-point initial calibration employing a mixture of Aroclors 1016 and 1260 at five concentrations should be sufficient to demonstrate the linearity of the detector response without the necessity of performing initial calibrations for each of the seven Aroclors. In addition, such a mixture can be used as a standard to demonstrate that a sample does not contain peaks that represent any one of the Aroclors. This standard can also be used to determine the concentrations of either Aroclor 1016 or Aroclor 1260, should they be present in a sample. Prepare a minimum of five calibration standards containing equal concentrations of both Aroclor 1016 and Aroclor 1260by dilution of the stock standard with isooctane or hexane. The concentrations should correspond to the expected range of concentrations found in real samples and should bracket the linear range of the detector.5.6.2Single standards of each of the other five Aroclors are required to aid the analystin pattern recognition. Assuming that the Aroclor 1016/1260 standards described in Sec. 5.6.1 have been used to demonstrate the linearity of the detector, these single standards of the remaining five Aroclors are also used to determine the calibration factor for each Aroclor.Prepare a standard for each of the other Aroclors. The concentrations should correspond to the mid-point of the linear range of the detector.5.7Calibration standards for PCB congeners5.7.1If results are to be determined for individual PCB congeners, then standards forthe pure congeners must be prepared. The table in Sec. 1.1 lists 19 PCB congeners that have been tested by this method along with the IUPAC numbers designating these congeners. This procedure may be appropriate for other congeners as well.5.7.2Stock standards may be prepared in a fashion similar to that described for theAroclor standards, or may be purchased as commercially-prepared solutions. Stock standards should be used to prepare a minimum of five concentrations by dilution of the stock standard with isooctane or hexane. The concentrations should correspond to the expected range of concentrations found in real samples and should bracket the linear range of the detector.5.8Internal standard5.8.1When PCB congeners are to be determined, the use of an internal standard ishighly recommended. Decachlorobiphenyl may be used as an internal standard, added to each sample extract prior to analysis, and included in each of the initial calibration standards.5.8.2When PCBs are to be determined as Aroclors, an internal standard is not used,and decachlorobiphenyl is employed as a surrogate (see Sec. 5.8).5.9Surrogate standards5.9.1When PCBs are to be determined as Aroclors, decachlorobiphenyl is used as asurrogate, and is added to each sample prior to extraction. Prepare a solution of decachlorobiphenyl at a concentration of 5 mg/L in acetone.5.9.2When PCB congeners are to be determined, decachlorobiphenyl is recommendedfor use as an internal standard, and therefore, cannot also be used as a surrogate. Therefore, tetrachloro-meta-xylene may be used as a surrogate for PCB congener analysis. Prepare a solution of tetrachloro-meta-xylene at a concentration of 5 mg/L in acetone.6.0SAMPLE COLLECTION, PRESERVATION, AND HANDLING6.1See Chapter Four, Organic Analytes for sample collection and preservation instructions.6.2Extracts must be stored under refrigeration in the dark and analyzed within 40 days of extraction.CD-ROM8082 - 7Revision 0December 19967.0PROCEDURE7.1Sample extraction7.1.1Refer to Chapter Two and Method 3500 for guidance in choosing the appropriateextraction procedure. In general, water samples are extracted at a neutral pH with methylene chloride using a separatory funnel (Method 3510) or a continuous liquid-liquid extractor (Method 3520) or other appropriate procedure. Solid samples are extracted with hexane-acetone (1:1) or methylene chloride-acetone (1:1) using one of the Soxhlet extraction (Method 3540 or 3541) procedures, ultrasonic extraction (Method 3550), or other appropriate procedure.NOTE:Use of hexane-acetone generally reduces the amount of interferences that are extracted and improves signal-to-noise.7.1.2Reference materials, field-contaminated samples, or spiked samples should beused to verify the applicability of the selected extraction technique to each new sample type.Such samples should contain or be spiked with the compounds of interest in order to determine the percent recovery and the limit of detection for that sample type (see Chapter One). When other materials are not available and spiked samples are used, they should be spiked with the analytes of interest, either specific Aroclors or PCB congeners. When the presence of specific Aroclors is not anticipated, the Aroclor 1016/1260 mixture may be an appropriate choice for spiking. See Methods 3500 and 8000 for guidance on demonstration of initial method proficiency as well as guidance on matrix spikes for routine sample analysis.7.2Extract cleanupRefer to Methods 3660 and 3665 for information on extract cleanup.7.3GC conditionsThis method allows the analyst to choose between a single-column or a dual-column configuration in the injector port. Either wide- or narrow-bore columns may be used. See Sec.7.7 for information on techniques for making positive identifications of multi-componentanalytes.7.3.1Single-column analysisThis capillary GC/ECD method allows the analyst the option of using 0.25-0.32 mm ID capillary columns (narrow-bore) or 0.53 mm ID capillary columns (wide-bore). The use of narrow-bore (0.25-0.32 mm ID) columns is recommended when the analyst requires greater chromatographic resolution. Use of narrow-bore columns is suitable for relatively clean samples or for extracts that have been prepared with one or more of the clean-up options referenced in the method. Wide-bore columns (0.53 mm ID) are suitable for more complex environmental and waste matrices.7.3.2Dual-column analysisThe dual-column/dual-detector approach involves the use of two 30 m x 0.53 mm ID fused-silica open-tubular columns of different polarities, thus different selectivities towards the target compounds. The columns are connected to an injection tee and ECD detectors. CD-ROM8082 - 8Revision 0December 19967.3.3GC temperature programs and flow rates7.3.3.1Table 2 lists GC operating conditions for the analysis of PCBs asAroclors for single-column analysis, using either narrow-bore or wide-bore capillarycolumns. Table 3 lists GC operating conditions for the dual-column analysis. Use theconditions in these tables as guidance and establish the GC temperature program andflow rate necessary to separate the analytes of interest.7.3.3.2When determining PCBs as congeners, difficulties may be encounteredwith coelution of congener 153 and other sample components. When determining PCBsas Aroclors, chromatographic conditions should be adjusted to give adequate separationof the characteristic peaks in each Aroclor (see Sec. 7.4.6).7.3.3.3Tables 4 and 5 summarize the retention times of up to 73 Aroclor peaksdetermined during dual-column analysis using the operating conditions listed in Table 2.These retention times are provided as guidance as to what may be achieved using theGC columns, temperature programs, and flow rates described in this method. Note thatthe peak numbers used in these tables are not the IUPAC congener numbers, butrepresent the elution order of the peaks on these GC columns.7.3.3.4Once established, the same operating conditions must be used for theanalysis of samples and standards.7.4Calibration7.4.1Prepare calibration standards as described in Sec. 5.0. Refer to Method 8000(Sec. 7.0) for proper calibration techniques for both initial calibration and calibration verification.When PCBs are to be determined as congeners, the use of internal standard calibration is highly recommended. Therefore, the calibration standards must contain the internal standard (see Sec. 5.7) at the same concentration as the sample extracts. When PCBs are to be determined as Aroclors, external standard calibration should be used.NOTE:Because of the sensitivity of the electron capture detector, the injection port and column should always be cleaned prior to performing the initialcalibration.7.4.2When PCBs are to be quantitatively determined as congeners, an initial five-pointcalibration must be performed that includes standards for all the target analytes (congeners).7.4.3When PCBs are to be quantitatively determined as Aroclors, the initial calibrationconsists of two parts, described below.7.4.3.1 As noted in Sec. 5.6.1, a standard containing a mixture of Aroclor 1016and Aroclor 1260 will include many of the peaks represented in the other five Aroclormixtures. Thus, such a standard may be used to demonstrate the linearity of thedetector and that a sample does not contain peaks that represent any one of theAroclors. This standard can also be used to determine the concentrations of eitherAroclor 1016 or Aroclor 1260, should they be present in a sample. Therefore, an initialfive-point calibration is performed using the mixture of Aroclors 1016 and 1260 describedin Sec. 5.6.1.CD-ROM8082 - 9Revision 0December 1996RF 'A s ×C isA is ×C sCD-ROM 8082 - 10Revision 0December 19967.4.3.2Standards of the other five Aroclors are necessary for patternrecognition. These standards are also used to determine a single-point calibration factorfor each Aroclor, assuming that the Aroclor 1016/1260 mixture in Sec. 7.3.4.1 has beenused to describe the detector response. The standards for these five Aroclors shouldbe analyzed before the analysis of any samples, and may be analyzed before or after theanalysis of the five 1016/1260 standards in Sec. 7.3.4.1.7.4.3.3In situations where only a few Aroclors are of interest for a specificproject, the analyst may employ a five-point initial calibration of each of the Aroclors ofinterest (e.g., five standards of Aroclor 1232 if this Aroclor is of concern) and not use the1016/1260 mixture described in Sec. 7.4.3.1 or the pattern recognition standardsdescribed in 7.4.3.2.7.4.4Establish the GC operating conditions appropriate for the configuration (single-column or dual column, Sec. 7.3). Optimize the instrumental conditions for resolution of the target compounds and sensitivity. A final temperature of 240-270E C may be required to elute decachlorobiphenyl. Use of injector pressure programming will improve the chromatography of late eluting peaks.NOTE:Once established, the same operating conditions must be used for bothcalibrations and sample analyses.7.4.5 A 2-µL injection of each calibration standard is recommended. Other injectionvolumes may be employed, provided that the analyst can demonstrate adequate sensitivity for the compounds of interest.7.4.6Record the peak area (or height) for each congener or each characteristic Aroclorpeak to be used for quantitation.7.4.6.1 A minimum of 3 peaks must be chosen for each Aroclor, and preferably5 peaks. The peaks must be characteristic of the Aroclor in question. Choose peaks inthe Aroclor standards that are at least 25% of the height of the largest Aroclor peak. Foreach Aroclor, the set of 3 to 5 peaks should include at least one peak that is unique tothat Aroclor. Use at least five peaks for the Aroclor 1016/1260 mixture, none of whichshould be found in both of these Aroclors.7.4.6.2Late-eluting Aroclor peaks are generally the most stable in theenvironment. Table 6 lists diagnostic peaks in each Aroclor, along with their retentiontimes on two GC columns suitable for single-column analysis. Table 7 lists 13 specificPCB congeners found in Aroclor mixtures. Table 8 lists PCB congeners withcorresponding retention times on a DB-5 wide-bore GC column. Use these tables asguidance in choosing the appropriate peaks.7.4.7When determining PCB congeners by the internal standard procedure, calculatethe response factor (RF) for each congener in the calibration standards relative to the internal standard, decachlorobiphenyl, using the equation that follows.where:。
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产品名: Nullscript 修订日期: 6/30/2016产品说明书
化学性质
产品名:
Nullscript
Cas No.:
300816-11-9 分子量:
298.3 分子式: C16H14N2O4
化学名:
N-hydroxy-1,3-dioxo-1H-benz[de]isoquinoline-2(3H)-butanamide SMILES:
O=C(C1=CC=CC2=CC=CC3=C12)N(CCCC(NO)=O)C3=O 溶解性:
Soluble in DMSO 储存条件:
Store at -20°C 一般建议: For obtaining a higher solubility , please warm the tube at 37°C
and shake it in the ultrasonic bath for a while.Stock solution can be
stored below -20°C for several months.
运输条件:
Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request
生物活性
靶点 :
DNA Damage/DNA Repair 信号通路:
HDAC 产品描述:
Nullscript is an HDAC inhibitor.
Histone deacetylase inhibitors (HDIs) have been used in psychiatry and neurology as mood stabilizers and anti-epileptics, such as valproic acid. Recently, HDIs are being studied as a
mitigator or treatment for neurodegenerative diseases. Moreover, there has been an effort to develop HDIs for cancer therapy.
In vitro: Nullscript, a close analog of scriptaid, was found to be inactive in transcriptional
facilitation at corresponding concentrations, which confirmed a minimal requirement for the length of the linker chain expected for this class of HDAC inhibitors. In addition, nullscript was not able to induce the p6SBE-luc reporter construct, which was identified from the library using ChemFinder by its structural similarity to scriptaid [1].
In vivo: A standard in vivo model of cardiac I/RWe was utilized to examine the in vivo consequences of HDAC inhibition in the intact heart. Results showed that the treatment with scriptaid led to a nearly identical effect when compared to nullscript, with a 46.8% reduction in infarct size. Such results strongly suggested that in murine models, HDACIs could reverse the induction of ischemia-induced HDAC activity and reduced myocardial infarct size by more than 50% [2].
Clinical trial: So far, no clinical study has been conducted.
参考文献:
[1] G. H. Su, T. A. Sohn, B. Ryu, et al. A novel histone deacetylase inhibitor identified by
high-throughput transcriptional screening of a compound library. Cancer Research 60, 3137-3142 (2000).
[2] Anne Granger et al. Histone deacetylase inhibition reduces myocardial ischemia-reperfusion injury in mice. FASEB J. 2008 Oct; 22(10): 3549–3560.
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收到产品后，按照产品说明书上的要求进行储存。