Farnesyl thiosalicylic acid amide   中文名称: 2-(3,7,11-trimethyldodeca-2,6,10-trienylsulfanyl)benzamide

RAS; PPMTase

法呢基硫代水杨酸酰胺（50 μM，24 小时）可降低 Panc-1 和 U87 细胞中总 Ras-GTP 和 K-Ras-GTP 的水平 [1]。
接下来，我们研究了FTS酰胺对panc-1和U87细胞的影响（图2C，D）。在6-100μM的浓度范围内，所有三种FTS-酰胺衍生物都导致两种细胞系的细胞数量呈剂量依赖性减少，表现出相似的效力（图2B，D）。在浓度高于50μM时观察到细胞死亡。两种细胞系中记录的IC50值在10-20μM范围内（表1）。有趣的是，与FTS相比，七种新化合物中有五种是Panc-1和U87细胞更有效的生长抑制剂，FTS的IC50值在Panc-1中为35μM，在U87细胞中为50μM。[1]

总之，这些结果表明，FTS羧基的某些修饰产生了能够在不同程度上抑制癌症细胞生长的活性化合物。此外，与FTS相比，一些修饰，即FTS加成转化为其甲酯、甲氧基甲酯、酰胺、甲基酰胺和二甲基酰胺衍生物，在抑制肿瘤细胞生长方面显示出更好的疗效。另一方面，与FTS本身相比，苄酯或异丙酯的生长抑制作用有所降低。[1]

这些结果共同表明，只有FTS酰胺衍生物模拟了母体化合物FTS的作用，而FTS酯的生长抑制活性可能包括Ras抑制以外的机制。也就是说，FTS的酰胺衍生物既表现出生长抑制活性，又影响了靶活性Ras蛋白；Panc-1细胞中的致癌K-Ras和U87细胞中的慢性活性N-Ras和K-Ras。 [1]

我们考虑了在所使用的培养条件下可能发生酯或酰胺水解，导致FTS本身形成的可能性。我们的结果不支持这种可能性。首先，与FTS相比，FTS-A和FTS-ME均表现出显著更高的生长抑制活性（表1）；如果它们被水解，那么这些化合物的效力将与FTS相似。鉴于之前的实验表明细胞以非常高的效率吸收法尼酰化肽，FTS衍生物的摄取也不太可能显著增加。其次，FTS-ME表现出与FTS不同的药理学（表2）；如果将其水解为FTS，我们会发现与FTS相比，FTS-ME对Ras-GTP的影响更强，但事实并非如此（表2）。第三，我们的下一组药代动力学实验如下所述，证实了FTS-A确实是稳定的。 总之，这些实验表明，FTS酰胺与FTS一样作为Ras抑制剂。与FTS相比，FTS-A似乎表现出相对较高的活性（约3-5倍），因此被用于随后的动物实验[1]。

法呢基硫代水杨酸酰胺（100 mg/kg，口服，一次）抑制Panc-1裸鼠模型中的肿瘤生长[1]。法呢基硫代水杨酸酰胺（100 mg/kg，口服，每日两次，14天）抑制U87裸鼠模型中的肿瘤生长[1]。药代动力学分析[1] 途径 剂量（mg/kg） 半衰期（分钟） Tmax（小时） Cmax（ng/mL） AUC0-inf（分钟*ng/mL） ig 100 314 110 371 108783

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FTS酰胺抑制裸鼠模型中人类肿瘤的生长[1]
接下来，我们使用裸小鼠模型研究了FTS-A对脑肿瘤生长的影响，该模型将人胶质母细胞瘤U87细胞植入纹状体区域。植入后四天，通过MRI扫描小鼠以确认肿瘤形成，并将其分为两组，每组七只对照动物和七只药物治疗动物。治疗在第一次MR成像后一天开始，随后在治疗开始后的第4、9和13天进行MR成像。每天口服两次FTS-A（100mg/kg）（见实验部分）。使用7-T BioSpec磁体在注射Gd-DTPA后采集T1和T2加权MR图像。T1加权图像用于测量肿瘤体积。T2加权图像用于获取有关炎症和心室扩大的信息。图5A显示了治疗前（基线图像）和治疗开始后第4天（第1次随访）和第9天（第2次随访）记录的代表性对照和FTS-A治疗小鼠的T1加权MR图像。对照小鼠的T1加权图像显示肿瘤体积随时间迅速增加。FTS-A小鼠随时间记录的肿瘤体积增加明显低于对照小鼠（图5A）。这些数据清楚地表明，与FTS-A治疗的小鼠相比，对照组小鼠体内积聚了更多的造影剂分子。重要的是，我们没有在对照组或药物治疗小鼠的大脑中观察到任何炎症反应（未显示）。

Western Blot分析[1]
细胞类型：U87
测试浓度：50 μM
孵育时间：24 小时
实验结果：降低了N-Ras-GTP、磷酸化ERK和磷酸化AKT的水平。

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生长抑制实验[1]
将U87和Panc-1细胞以每孔5000个细胞的密度铺在24孔板中并生长24小时。然后用含有所示浓度的每种化合物的培养基或含有0.1%DMSO的培养基（载体对照）代替培养基。将细胞培养5-7天，然后计数。数据以百分比表示：（药物处理培养物中的细胞数/对照中的细胞数量×100）。每个实验进行两次，共四次，并给出平均值±标准差。

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蛋白质免疫印迹[1]
U87或Panc-1细胞以1.5×106个细胞的密度铺在10cm的培养皿中，并在含有10%FCS的培养基中生长过夜。然后将培养基替换为含有50μM每种化合物或0.1%DMSO的培养基，并在药物存在下孵育细胞24小时。然后，我们用增溶缓冲液（50 mmol/L Tris-HCl（pH 7.6）、20 mmol/L MgCl2、200 mmol/L NaCl、0.5%NP40、1 mmol/L DTT和蛋白酶抑制剂）裂解细胞。赖氨酸盐用于通过谷胱甘肽S-转移酶（GST）-RBD（Raf的Ras结合结构域）下拉测定来确定Ras-GTP含量，然后用小鼠抗泛Ras抗体（1:2500）或Ras亚型特异性抗体（小鼠抗H-Ras、抗K-Ras和抗N-Ras（1:100）进行蛋白质免疫印迹，如别处所述。使用上述抗Ras抗体、兔抗磷酸ERK1/2抗体（1:10000）、兔抗磷酸盐Akt抗体（1:200）和兔抗β-微管蛋白（1:1000）通过免疫印迹法测定细胞裂解物中总Ras蛋白、磷酸细胞外信号调节激酶（ERK）、磷酸Akt和β-微管素的水平。免疫印迹分别暴露于过氧化物酶-山羊抗鼠IgG或过氧化物酶-羊抗兔IgG（1:2500）。通过增强化学发光观察蛋白质条带，并使用EZQuant-Gel计算机软件通过密度测定进行定量。

Pharmacokinetic Sampling and Analysis [1]
Four animals (male Sprague−Dawley rats, 275−285 g) were used to collect samples at each time point (10, 30, 50, 75, 105, 135, 165, 210, 270, and 330 min) taken after a single dose of 100 mg/kg FTS amide, po. Approximately 0.2 mL of blood was collected in sodium heparin containing tubes via the femoral artery while under anesthesia (urethane/chloralose, 33%/1% w/v) at each of the specified collection times. The blood samples were immediately placed on ice and centrifuged (6800 rpm for 5 min at 4 °C) within 5 min of collection. Plasma was then collected and immediately stored at −70 °C. Peak plasma concentration, time peak achieved, and half-life (Cmax, tmax, and t1/2) were calculated. Area under the plasma concentration−time curves (AUC) was also determined. These analyses were conducted using Win-Nonlin.

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Tumor Implantation [1]
Nude CD1-Nu mice (25−30 g) were housed in barrier facilities on a 12 h light/dark cycle. Food and water were supplied ad libitum. On day zero, Panc-1 cells (5 × 106 cells in 0.1 mL) were implanted subcutaneously just above the right femoral joint. On day 10, the tumors were measured and the mice were divided into treated and control groups. The treated animals received daily po doses of 100 μL FTS amide (100 mg/kg), while the control animals received daily po doses of 100 μL vehicle solution. Approximately 18 days later, the tumors were extracted and weighed.
For intracranial tumor implantation, mice were anesthetized by intraperitoneal injection of ketamin (100 mg/kg) and xylazine hydrochloride (10 mg/kg) solution in 0.1 mL of PBS. Cell inoculation was performed on day zero by creating a mid-skull incision, and then a burr hole 1 mm lateral and 1 mm posterior to the bregma, 3 mm deep, into the striatum area. U-87 cells (0.5 × 106) were injected using a 1 mL of BASI gastight syringe connected to a pump, thus allowing slow release of the cells into the brain (1 μL/min, 5 min total). The mice were imaged on day 4 to verify tumor development as detailed in MRI procedures, and the mice were divided into two groups exhibiting similar tumor sizes. Drug treatments started on day 5: the treated group received po dosing of 50 μL of FTS amide (100 mg/kg) twice a day, while the control animals received po doses of 50 μL vehicle solutions twice a day. Followup MR images were taken on days 9, 14, and 18.

Pharmacokinetics of FTS-A[1]
First, we evaluated the pharmacokinetics of FTS-A after oral administration (100 mg/kg) in rats (see Experimental Section). Blood samples were collected at specified time points (10–330 min) after administration. The samples were then processed according to the method described in Experimental Section and analyzed by LC-MS/MS. The calibration curve of FTS-A showed a linear relationship from 1 to 2500 ng/mL. FTS-A was easily detected in rat plasma by LC-MS/MS with a detection limit of 1 ng/mL. The retention time of the test sample ion was 1.02 min compared to the expected retention time of the parent ion (FTS-A, 1.01–1.02 min), validating the effectiveness of the analytical method and confirming the stability of the compound in plasma (Figure 4A). Based on the non-compartmental model detailed in Experimental Section, the pharmacokinetic parameters of FTS-A were calculated using the plasma concentration curves of FTS-A at different time points (Figure 4B). The results, including terminal half-life (elimination half-life, t1/2), time to reach maximum plasma concentration (Cmax) (Tmax), Cmax value, and area under the plasma concentration-time curve (AUC), are summarized in Table 3 and compared with previously obtained FTS pharmacokinetic parameters. (27) As shown in Table 3, although the t1/2 value of FTS-A is more than twice that of FTS, its plasma bioavailability is lower than that of FTS; the Tmax value of FTS-A is almost twice that of FTS, but even when the dose of FTS-A is higher than that of FTS, its Cmax value and AUC value are significantly lower than those of FTS.

[1]. New derivatives of farnesylthiosalicylic acid (salirasib) for cancer treatment: farnesylthiosalicylamide inhibits tumor growth in nude mice models. J Med Chem. 2009 Jan 8;52(1):197-205.

Farnesylthiosalicylic acid amide is a sesquiterpene compound.

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Our results, along with previous studies using isoprene-modified small molecules, suggest that the structure of such molecules is a key determinant of their function. While FTS and its amide derivatives can act as Ras inhibitors, FTS esters exhibit low activity as Ras inhibitors and may interfere with the function of other isoprene-binding proteins, such as RhoGDI, thereby inhibiting cell growth. [1]

C22H31NOS

357.55

357.212

1092521-74-8

25187973

Typically exists as solid at room temperature

1.0±0.1 g/cm3

497.6±45.0 °C at 760 mmHg

254.7±28.7 °C

0.0±1.3 mmHg at 25°C

1.560

6.66

68.39

1

2

10

25

500

0

CC(C)=CCC/C(/C)=C/CC/C(/C)=C/CSC1=CC=CC=C1C(N)=O

GZTMFRUGZMZCRD-CFBAGHHKSA-N

InChI=1S/C22H31NOS/c1-17(2)9-7-10-18(3)11-8-12-19(4)15-16-25-21-14-6-5-13-20(21)22(23)24/h5-6,9,11,13-15H,7-8,10,12,16H2,1-4H3,(H2,23,24)/b18-11+,19-15+

2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]sulfanylbenzamide

FTS amide; Farnesyl Thiosalicylic Acid Amide; 1092521-74-8; 2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl]sulfanylbenzamide; 2-[[(2E,6E)-3,7,11-Trimethyl-2,6,10-dodecatrien-1-yl]thio]benzamide; FTS Amide;Salirasib Amide; Farnesyl Thiosalicylic Acid Amide (10 mg/mL in ethanol); Salirasib Amide; FTS Amide; Salirasib amide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。