Nolatrexed 2HCl   中文名称: 盐酸诺拉曲塞

Human Thymidylate Synthase (Ki = 11 nM)

3,4-二氢-2-氨基-6甲基-4-氧代-5-（4-吡啶硫基）-喹唑啉二盐酸盐（AG337）是一种水溶性、亲脂性的胸苷酸合成酶（TS）抑制剂，使用基于X射线结构的方法设计，在该酶的叶酸辅因子结合位点相互作用。AG337以11nM的Ki抑制纯化的重组人TS，并显示出非竞争性抑制动力学。在一组鼠源和人源的细胞系中进一步显示其抑制细胞生长，显示出0.39微M至6.6微M的IC50。通过胸苷拮抗细胞生长抑制的能力以及使用氚释放测定法在全细胞中直接证明TS抑制，TS被认为是AG337的作用位点。通过流式细胞术证明，AG337处理的L1210细胞被阻滞在细胞周期的S期，这也与TS的阻断相一致，在AG337治疗的细胞中，核糖核苷酸和脱氧核糖核苷酸库的调节模式也一致，显示TTP水平显著降低。AG337的作用在药物去除后迅速逆转，这表明，正如亲脂性试剂所预期的那样，细胞内有快速的流入和流出，而没有细胞内代谢为具有增强保留的衍生物。[1]
使用连续或间歇性药物暴露测定亲脂性胸苷酸合成酶（TS）抑制剂AG337对人头颈部鳞状细胞癌（HNSCC）细胞系和具有获得性甲氨蝶呤（MTX）耐药性的CCRF-CEM人白血病细胞及其亚系的影响。在120-h连续暴露期间，HNSCC细胞系A253和FaDu对MTX同样敏感（EC50等于约15nM）；AG337的效力较低（EC50大约等于1微M）。A253对24小时间歇性MTX暴露具有内在抗性（EC50约等于17微M；FaDu，EC50约为0.3微M）；两种HNSCC细胞系都对24小时AG337暴露具有抗性（EC50＞100微M）。CCRF-CEM在类似于HNSCC的连续暴露中显示MTX（EC50＝14nM）和AG337（EC50等于约0.6微米）敏感性；然而，AG337在间歇暴露中保持对CCRF-CEM细胞的效力（24小时，EC50等于约2微M；6小时，EC50=约48微M）。＞或等于0.1μM的还原叶酸亚叶酸（LV）可完全保护连续暴露于MTX的细胞不受生长抑制，但AG337的生长抑制仅在＜或等于100μM的LV时轻微逆转。胸苷可完全保护A253和FaDu不受AG337生长抑制，而次黄嘌呤单独作用则无效，表明抑制是TS特异性的。具有由DHFR过表达、MTX转运缺陷或MTX多聚谷氨酰化缺陷引起的获得性MTX抗性的CCRF-CEM亚系在连续暴露中保持对AG337的完全敏感性（所有EC50=0.4μM）。这些数据表明，AG337可用于通过最常见的机制治疗对MTX具有获得性耐药性的肿瘤。[2]

在体内，AG337对胸苷激酶缺陷的小鼠L5178Y/TK淋巴瘤具有高活性，该淋巴瘤在腹膜内或口服后腹膜内或腹膜内植入。活动需要延长5或10天的给药期，每天两次给药可提高疗效。剂量水平为25mg/kg，每天两次，持续10天，50mg/kg，每天一次，持续5天，引起对腹膜内肿瘤的100%治愈。抗腹膜内肿瘤活性所需的剂量更高（100mg/kg，每天两次，持续5天或10天），但证明了AG337穿透固体组织屏障的能力。口服给药需要剂量大于或等于150 mg/kg，每天两次，持续5-10天，以产生针对腹膜内和腹膜内植入肿瘤的100%治愈率。这些结果与大鼠体内确定的药代动力学参数一致，大鼠口服生物利用度为30-50%，消除半衰期相对较短，为2h。AG337的临床研究目前正在进行中[1]。

酶动力学[1]
TS活性通过改良的氚释放方法使用纯化的重组人TS进行测量。通过对辅因子（6R，6S）5，10-亚甲基四氢叶酸的稳态分析来确定抑制常数，该辅因子是通过四氢叶酸与甲醛在基本饱和[5-3H]dUMP的条件下反应原位产生的。测定含有50mM TRIS Cl pH 7.6、10mM二硫苏糖醇、1mM乙二胺四乙酸、25mM MgCl2、15mM甲醛、1%DMSO、25lM[5-3H]dUMP（比活性2]108cpm/lmol）、四氢叶酸（10-80lM）、AG337（0-100nM）、TS（4nM）和1-5lg/ml牛血清白蛋白以稳定酶。反应在25°C下进行6分钟，并通过添加木炭来猝灭，然后离心以除去未反应的dUMP。通过上清液的液体闪烁测定从dUMP的5个位置释放的氚。表观K*（K*，！11）是通过计算机辅助非线性最小二乘法将数据拟合到Morrison[1]的紧密结合抑制方程中，在每种浓度的5,10-亚甲基四氢叶酸下测定的

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全细胞TS测定[1]
TS活性通过氚释放测定来测量，其中细胞与[5-3H]脱氧尿苷孵育，脱氧尿苷在细胞摄取后转化为TS底物dUMP。酶活性定量为TS催化反应期间[5-3H]dUMP释放的氚。每个反应混合物包含30lM dUdR、5uCi/ml 5-3HdUdR和8]105个细胞。在AG337存在和不存在的情况下，在37°C和5%CO2下进行反应，并通过添加细胞引发反应。在不同的孵育时间后，通过加入0.4ml冰冷的1M高氯酸将它们停止，并如前所述进行处理。

细胞系在含有10%热灭活胎牛血清的RPMI-1640培养基（Hyclone Laboratories，Logan，Utah）中不含抗生素地维持，但L1210、CCRF-CEM和L1210/R6细胞除外，它们在5%血清中传代。R6系在2mM甲氨蝶呤存在下进一步保持，但在其不存在下传代至少两次，然后用于测定。生长抑制研究是通过在96孔平底微量滴定板中连续暴露于AG337进行的，使用Mosmann[20]的比色法的修改。活细胞数的测定基于MTT（Sigma Chemical，St.Louis，Mo）还原为甲赞，这是活细胞线粒体中脱氢酶活性的结果。在溶解于DMSO中之后，通过在540nm处的吸光度测量对甲zan产物进行定量。在每次测定中，在一式四份的微培养物中测试八份或九份连续两倍稀释的测试试剂的效果。IC50值表示将光密度降低到对照培养物中观察到的光密度的50%的AG337的浓度，其来源于细胞生长（以对照的百分比表示）与AG337浓度的半对数图。对于旨在解决AG337细胞内活性位点的保护性研究，胸苷、次黄嘌呤和亚叶酸（Sigma Chemical，St.Louis，Mo）分别以10、35和0.5μM的浓度存在于测定中。[1]

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核糖核苷酸和脱氧核糖核苷酸库水平[1]
L1210细胞（约5]107个细胞，密度为106个细胞/ml）在37°C下单独或与10lM AG337一起孵育2小时。如前所述，通过中和的高氯酸提取物的阴离子交换HPLC测定核糖核苷酸谱。简言之，通过离心将细胞沉淀并重悬于0.2ml 0.7N高氯酸中。通过离心除去酸性沉淀物，并用固体KHCO3中和上清液。通过修改Garrett和Santi的程序，从中和的提取物中回收脱氧核糖核苷酸。用5 mM脱氧鸟苷（10 ul）和0.5 M NaIO4（40 ul）处理提取物的等分试样（175 ul），几分钟后用pH 7.5的4 M甲胺（25 ul）处理，并在37°C下孵育30分钟。通过在室温下用1 M鼠李糖（20 ul）孵育5分钟来减少过量的NaIO4。类似地处理含有ATP（2mM）和10mM dATP、dCTP、TTP和dGTP的标准品混合物以验证回收率。在Partisil-10 SAX柱上进行分离。用5mM NH4 H2 PO4（pH 2.5）至0.5M NH4 H2PO4（pH4.8）的线性梯度在40分钟内洗脱核糖核苷酸提取物（50ll），在254nm处进行检测。使用标准品在对照运行中验证了保留时间。使用5mM NH4 H2 PO4 pH 2.8∶0.5M NH4 H2PO4 pH 4.8（4:6）的流动相在同一柱上用等度洗脱分离脱氧核苷酸，并在284和270nm处进行检测。[1]

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细胞周期动力学[1]
通过流式细胞术分析，从碘化丙啶染色的细胞核的DNA直方图中确定细胞周期分布。将L1210细胞的指数生长培养物用0.7μM或3.5μM AG337的浓度和与未处理培养物相比的细胞周期动力学处理20小时。为了进行分析，将细胞固定在预冷至-20°C的95%乙醇中，在含有5%热灭活胎牛血清的磷酸盐缓冲盐水中洗涤，并用含有1 mg/ml RNase的20 ug/ml碘化丙啶处理。

Antitumor studies [1]
The thymidine kinase deficient L5178Y/TK~ tumor was maintained by passage in DBA/2 mice. For experiments, either 1]106 cells (i.p.) or 5]106 cells (i.m.) were injected into B6D2F1 hybrid mice. For i.m. studies, the tumor was implanted in the gastrocnemius muscle. The day of tumor inoculation was designated day 0, and treatment was initiated on day 1, 3 or 5 for 5—10 days. AG337 was dissolved in 5% dextrose and administered by i.p. or oral dosing. Antitumor effects were evaluated in animals bearing i.p. tumors by increase in life span compared to controls which received vehicle alone and for which the mean survival time was 26.3 days after tumor inoculation. Animals surviving to day 100 were designated cured. For the i.m. implanted tumor, the diameter of the leg at the site of inoculation was measured daily and animals were sacrificed at a fixed end point diameter of 14 mm, which represents an approximately 2.5-fold increase in size. Animals were considered cured if the leg diameter at day 35 was less than or equal to the leg diameter on day 0. Thymidine kinasereplete L1210 and P388 tumors were implanted i.p. at 1]106 cells per mouse (day 0) and treatment was initiated on day 3.

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Pharmacokinetics[1]
Studies were conducted in male Sprague-Dawley rats with jugular vein cannulated and exteriorized. After i.v. or oral administration of AG337, blood samples (200 ul) were withdrawn from the cannula and plasma separated by centrifugation. Plasma samples (100 ul) were extracted with 1 ml acetonitrile for 2 min, centrifuged at 2000 rpm for 5 min and the supernatant evaporated at 40°C under nitrogen. Dried samples were reconstituted with 300 ul mobile phase and AG337 levels quantitated by HPLC. Separation was effected using a C18 column (4.6 x 150 mm,5 lm) and isocratic elution with 0.05 M sodium phosphate buffer, pH 4.0 and acetonitrile (80:20); detection was at a wavelength of 230 nm. Plasma AG337 concentration-time data were fitted to multi-exponential equations using nonlinear regression analysis. Pharmacokinetic parameters, as follows, were calculated from the parameter estimates of the y-axis intercepts and rate constants: absorption, distribution, and elimination half lifes, volumes of distribution, and systemic clearance (Cl4 ). Absolute oral bioavailability was calculated as [AUCoral/AUCiv]]100 using model-independent values for AUC.

[1]. AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies. Cancer Chemother Pharmacol. 1996;37(6):509-17.

[2]. Characterization of the effect of AG337, a novel lipophilic thymidylate synthase inhibitor, on human head and neck and human leukemia cell lines. Int J Oncol. 1999 Dec;15(6):1245-50.

C14H14CL2N4OS

357.25

356.0265

C, 47.07; H, 3.95; Cl, 19.85; N, 15.68; O, 4.48; S, 8.97

152946-68-4

152946-68-4 (HCl salt); 147149-76-6;

Light yellow to yellow solid powder

4.54

109.96

CC1=C(C2=C(C=C1)N=C(NC2=O)N)SC3=CC=NC=C3.Cl.Cl

PJKVJJYMWOCLIJ-UHFFFAOYSA-N

InChI=1S/C14H12N4OS.2ClH/c1-8-2-3-10-11(13(19)18-14(15)17-10)12(8)20-9-4-6-16-7-5-9;;/h2-7H,1H3,(H3,15,17,18,19);2*1H

2-amino-6-methyl-5-pyridin-4-ylsulfanyl-3H-quinazolin-4-one;dihydrochloride

Nolatrexed HCl; Nolatrexed dihydrochloride; AG337; AG 337; AG-337; brand name: Thymitaq.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~50 mg/mL (~140.0 mM)
DMSO: ~41.7 mg/mL (~116.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (69.98 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。