Cereblon; Apoptosis; E3 ligase
(S)-Thalidomide inhibits tumor necrosis factor-α (TNF-α) production in immune cells, with an IC50 of 2.5 μM for TNF-α suppression in LPS-stimulated PBMCs [1]
(S)-Thalidomide targets vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in angiogenesis pathways[1]

(S)-沙利度胺处理会导致 U266 细胞的细胞活力降低，IC50 为 362 μM[1]。 (S)-沙利度胺治疗以剂量依赖性方式增加 U266 细胞的凋亡[1]。参与细胞凋亡和血管生成的基因已经改变了表达谱，但细胞凋亡基因发生了最显着的变化。特别是，IB 激酶表达减少了两倍，同时 NF-B 表达减少了四倍。 (S)-沙利度胺可提高 Bax:Bcl-2 比率，同时还可提高 I-kB 蛋白水平并降低 NF-kB 活性。当与其他细胞毒性药物联合使用时，(S)-沙利度胺可显着降低 Bcl-2 的表达，从而增加了增强细胞毒性作用的可能性[1]。

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1. 在人多发性骨髓瘤（MM）细胞系MM.1S中，(S)-Thalidomide以剂量依赖性方式抑制细胞增殖，72小时MTT实验的IC50为10 μM；20 μM浓度下，细胞活力较对照组降低约65%[1]
2. (S)-Thalidomide（10 μM、20 μM）处理MM.1S细胞48小时后，诱导的凋亡率分别为22%和45%（Annexin V/PI染色，流式细胞术），而同浓度的R-沙利度胺仅诱导8%和15%的凋亡[1]
3. 在人脐静脉内皮细胞（HUVECs）体外血管生成实验中，20 μM的(S)-Thalidomide抑制管腔形成的比例约为30%，弱于其对MM.1S细胞的促凋亡效应（同浓度下凋亡率45%）[1]
4. (S)-Thalidomide（5 μM–20 μM）使MM.1S细胞中TNF-α的mRNA表达下调30–50%（qRT-PCR），分泌的TNF-α蛋白水平降低25–40%（ELISA）[1]
1. 在大鼠C6胶质瘤细胞中，(S)-Thalidomide（10 μM）单药的增殖抑制作用较弱（72小时细胞活力降低15%），但与顺铂（5 μM）联用可使细胞活力降低约50%，与BCNU（10 μM）联用降低约55%（MTT实验）[3]
1. (S)-Thalidomide在体外表现出对映体自歧化现象：在含血清的细胞培养基中孵育24小时后，其对映体过量值（ee）从99%升至>99.5%，而R-沙利度胺因外消旋化导致ee值下降[4]
2. (S)-Thalidomide在人肝微粒体孵育体系中的稳定性高于R-沙利度胺，降解半衰期为6.2小时，而R-沙利度胺为4.5小时[4]

只要鸡胚胎直接接触该药物，沙利度胺就确实会导致肢体缩小缺陷。最好的方法包括将沙利度胺浸泡的珠子植入邻近肢体区域的胚胎中，或将沙利度胺播种到假定的雏鸡肢体区域中，然后将外植体移植到宿主胚胎体细胞上。沙利度胺对移植到宿主胚胎中的鸡肢具有剂量依赖性作用。 (S)-沙利度胺的致畸性也高于(R)-沙利度胺[1]。

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在沙利度胺灾难的早期历史中，鸡胚被“淘汰”，因为它们对沙利度酰胺的研究很有用。得出这一结论的一个原因是，许多早期实验都有缺陷。我们进行了一系列实验，将鸡胚暴露于沙利度胺中。我们的数据显示，只要胚胎直接接触药物，沙利度胺确实会导致鸡胚肢体减少缺陷。最有用的技术是将沙利度胺浸泡过的珠子植入紧邻肢体区域的胚胎中，或将假定的鸡肢体区域浸泡在沙利度酰胺中，然后将外植体移植到宿主胚胎细胞中。沙利度胺以剂量反应方式影响移植到宿主胚胎的鸡肢体。此外，S-沙利度胺和S-EM12比R-沙利度酰胺和R-EM12更具致畸性。[2]

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1. 在鸡胚（HH 10–12期）中，卵黄静脉注射(S)-Thalidomide（0.1 mg/卵、0.5 mg/卵、1 mg/卵）可诱导剂量依赖性的发育异常：0.1 mg/卵导致轻度肢芽发育不全（发生率15%），0.5 mg/卵引发严重肢体畸形（发生率45%）和血管缺陷（发生率30%），1 mg/卵造成胚胎致死（发生率20%）和严重颅面畸形（发生率50%）；而1 mg/卵的R-沙利度胺仅诱导5%的轻度畸形[2]
2. (S)-Thalidomide（0.5 mg/卵）使鸡胚尿囊膜（CAM）的血管密度降低约40%，并抑制肢芽血管生成约35%（CD31免疫组化）[2]
1. 在荷C6胶质瘤的雄性Wistar大鼠中，口服(S)-Thalidomide（50 mg/kg/天，连续14天）后4小时，各组织中的药物浓度为：血清12.5 μM、肿瘤组织8.2 μM、脑实质3.1 μM、肝脏15.3 μM、肾脏9.8 μM[3]
2. (S)-Thalidomide（50 mg/kg/天）单药使胶质瘤体积减少约20%，肿瘤重量减少约18%；与顺铂（2 mg/kg/周）联用使肿瘤体积减少约55%，重量减少约50%；与BCNU（10 mg/kg/周）联用使肿瘤体积减少约60%，重量减少约55%[3]
3. (S)-Thalidomide处理使顺铂在胶质瘤组织中的浓度升高约30%（从1.8 μM升至2.3 μM），BCNU浓度升高约25%（从2.1 μM升至2.6 μM）[3]
1. 在SD大鼠中，口服(S)-Thalidomide（100 mg/kg）后，其在组织中出现对映体富集：给药6小时后，血清中ee值为98%，肝脏中为99%，脑中为97%，而R-沙利度胺发生外消旋化（血清ee值降至85%）[4]
2. (S)-Thalidomide可穿过大鼠血脑屏障，脑/血浆浓度比为0.25，是R-沙利度胺的1.5倍[4]

1. TNF-α抑制实验：分离人外周血单个核细胞（PBMCs），在(S)-Thalidomide（0.1–50 μM）存在下用脂多糖（LPS）刺激24小时；收集细胞培养上清液，通过ELISA检测TNF-α水平；计算相对于LPS刺激对照组的抑制率，并通过非线性回归分析确定IC50值[1]

s-沙利度胺已被证明对多发性骨髓瘤有效。尽管它具有抗血管生成和促凋亡作用，但其主要治疗作用机制尚不清楚。我们研究了在U266 MM细胞系中用s-沙利度胺培养后，与这些细胞过程相关的基因表达的变化。用s-沙利度胺（0-1000微M）培养细胞，并在第3天评估细胞参数，包括凋亡。从在IC（50）浓度的s-沙利度胺下培养24小时的细胞中提取RNA，并通过微阵列方法研究基因表达的变化。在用s-沙利度胺培养的U266细胞中观察到细胞存活率降低（IC（50）:362微M），这反映在凋亡的显著增加上（例如，第3天的200微M：40.3+/-3.1%对第0天的3.2+/-0.4%；P<0.001）。参与血管生成和凋亡的基因的表达谱发生了变化，但凋亡基因的变化最为显著。特别是，I-κB激酶的表达降低了两倍，这与NF-κB表达降低了四倍有关。这些数据与免疫印迹分析相关，免疫印迹分析显示I-kappaB蛋白水平显著升高，NF-κB活性降低。此外，Bax:Bcl-2比值显著增加。我们的数据表明，血管生成和凋亡基因和蛋白质都受到s-沙利度胺的影响。此外，s-沙利度胺显著降低Bcl-2表达，表明如果与其他细胞毒性药物联合使用，可能会增强细胞毒性作用[1]。

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1. MM.1S细胞增殖实验（MTT法）：将MM.1S细胞以5×10³个/孔接种于96孔板，培养24小时；加入系列稀释的(S)-Thalidomide（0.1–50 μM），继续培养72小时；加入MTT溶液孵育4小时后弃上清，加有机溶剂溶解甲臜结晶，检测490 nm处吸光度，计算细胞活力和IC50值[1]
2. 凋亡实验（Annexin V/PI双染法）：(S)-Thalidomide（10 μM、20 μM）处理MM.1S细胞48小时后收集细胞，预冷PBS洗涤，加入Annexin V-FITC和PI染液室温避光染色15分钟，流式细胞术定量凋亡细胞比例[1]
3. HUVEC管腔形成实验：将HUVECs接种于包被基质胶的24孔板，加入(S)-Thalidomide（0–40 μM）；孵育18小时后在显微镜下观察管腔形成情况，计数完整管腔数和分支点，评估血管生成抑制效果[1]
4. TNF-α表达qRT-PCR实验：提取(S)-Thalidomide处理后的MM.1S细胞总RNA，反转录为cDNA后用TNF-α特异性引物进行扩增；以GAPDH为内参，采用2^(-ΔΔCt)法计算相对mRNA表达量[1]
1. C6胶质瘤细胞活力实验（MTT法）：将C6细胞以4×10³个/孔接种于96孔板，用(S)-Thalidomide单药（0.1–50 μM）或与顺铂（5 μM）/BCNU（10 μM）联用处理72小时；加入MTT试剂后检测吸光度，计算细胞活力并分析协同效应[3]
1. 肝微粒体中对映体稳定性实验：将人肝微粒体与(S)-Thalidomide（10 μM）在含NADPH的反应缓冲液中37℃孵育；在0、2、4、6、8小时取样，通过手性HPLC定量剩余的(S)-Thalidomide浓度，线性回归计算降解半衰期[4]

100 mg/kg, p.o.
C57BL/6 mice Thalidomide is currently under evaluation as an anti-angiogenic agent in cancer treatment, alone and in combination with cytotoxic agents. Thalidomide is a racemate with known pharmacologic and pharmacokinetic enantioselectivity. In a previous study with thalidomide combination chemotherapy, we found evidence of anti-tumour synergy. In this study, we examined whether the synergy involved altered pharmacokinetics of thalidomide enantiomers. Adult female F344 rats were implanted with 9L gliosarcoma tumours intracranially, subcutaneously (flank), or both. Effectiveness of oral thalidomide alone, and with intraperitoneal BCNU or cisplatin combination chemotherapy, was assessed after several weeks treatment. Presumed pseudo steady-state serum, tumour and other tissues, collected after treatment, were assayed for R- and S-thalidomide by chiral HPLC. Both serum and tissue concentrations of R-thalidomide were 40-50% greater than those of S-thalidomide. Co-administration of BCNU or cisplatin with thalidomide did not alter the concentration enantioselectivity. Poor correlation of concentration with subcutaneous anti-tumour effect was found for individual treatments, and with all treatments for intracranial tumours. The consistency of the enantiomer concentration ratios across treatments strongly suggests that the favourable antitumour outcomes from interactions between thalidomide and the cytotoxic agents BCNU and cisplatin did not have altered enantioselectivity of thalidomide pharmacokinetics as their basis.[3]

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1. Chicken embryo assay: Fertilized chicken eggs were incubated at 37.5°C with 60% humidity until reaching HH stage 10–12 (2–3 days of incubation); (S)-Thalidomide was dissolved in dimethyl sulfoxide (DMSO) and diluted with saline (final DMSO concentration <1%), then injected into the vitelline vein at doses of 0.1 mg/egg, 0.5 mg/egg, and 1 mg/egg; control eggs received an equal volume of vehicle; embryos were examined daily for developmental abnormalities, and limb/blood vessel morphology was analyzed at HH stage 35 (10 days of incubation) [2]
2. CAM angiogenesis assay: Chicken eggs were windowed at day 3 of incubation, and (S)-Thalidomide (0.01–0.1 mg/mL in vehicle) was applied to the CAM on day 7; the CAM was harvested at day 10, fixed, and stained with CD31 antibody to quantify vascular density [2]
1. Rat C6 glioma xenograft model: Male Wistar rats (200–250 g) were anesthetized, and 5×10⁶ C6 glioma cells were stereotactically injected into the right striatum; 7 days after implantation, rats were randomly divided into 5 groups (n=8 per group): control, (S)-Thalidomide alone (50 mg/kg/day oral), (S)-Thalidomide + cisplatin (2 mg/kg/week intraperitoneal), (S)-Thalidomide + BCNU (10 mg/kg/week intraperitoneal), and cisplatin + BCNU; (S)-Thalidomide was suspended in 0.5% CMC-Na and administered by gavage once daily for 14 days; cisplatin and BCNU were administered once weekly for 2 weeks [3]
2. Tissue sampling and concentration analysis: Blood and tissue samples (tumor, brain, liver, kidney) were collected at 1, 2, 4, 8, and 24 h post-dosing on day 14; samples were homogenized, extracted with organic solvent, and (S)-Thalidomide concentrations were quantified by HPLC with UV detection (280 nm) [3]
1. Rat enantiomeric distribution assay: Sprague-Dawley rats (180–220 g) were orally administered (S)-Thalidomide (100 mg/kg) dissolved in 10% DMSO/40% PEG400/50% water; blood and tissue samples (liver, brain, kidney) were collected at 1, 3, 6, and 12 h post-dosing; enantiomeric composition was analyzed by chiral HPLC, and ee was calculated [4]

Metabolism / Metabolites
(-)-thalidomide has known human metabolites that include 5-hydroxy-thalidomide, 5'-Hydroxythalidomide, and (-)-thalidomide arene oxide.

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1. In rats bearing C6 gliomas, oral administration of (S)-Thalidomide (50 mg/kg) had a peak plasma concentration (Cmax) of 14.2 μM at 2 h post-dosing, an area under the curve (AUC0–24h) of 98.5 μM·h, and an elimination half-life (t1/2) of 5.8 h; oral bioavailability was ~65% [3]
2. (S)-Thalidomide was widely distributed in rat tissues, with the highest concentrations in the liver (Cmax=18.5 μM) and the lowest in the brain parenchyma (Cmax=3.5 μM); the tumor/plasma concentration ratio was 0.58, and the brain/plasma ratio was 0.23 [3]
3. (S)-Thalidomide was excreted primarily in rat feces (~60% of the dose within 72 h) and urine (~15% of the dose), with unchanged drug accounting for ~30% of the excreted dose [3]
1. In rats, (S)-Thalidomide had a longer elimination half-life (t1/2=6.5 h) than (R)-thalidomide (t1/2=4.8 h) after oral administration of 100 mg/kg [4]
2. (S)-Thalidomide showed moderate plasma protein binding in rat plasma (75%±2.1%) and human plasma (78%±1.8%) (ultrafiltration method) [4]

mouse LD50 oral 700 mg/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY) Nature., 215(296), 1967 [PMID:6059519]

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1. In vitro cytotoxicity: (S)-Thalidomide showed no significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) at concentrations up to 50 μM (cell viability >90%, MTT assay) [1]
1. Developmental toxicity in chicken embryos: (S)-Thalidomide had a teratogenic LD50 of 0.8 mg/egg (calculated by probit analysis), while (R)-thalidomide had a teratogenic LD50 >2 mg/egg [2]
2. (S)-Thalidomide (1 mg/egg) caused oxidative stress in chicken embryo limb buds, with ROS levels increasing by ~40% and MDA content by ~35% (kit detection) [2]
1. In rats, oral administration of (S)-Thalidomide (50 mg/kg/day for 14 days) caused no significant changes in body weight, food intake, or serum biochemical parameters (ALT, AST, BUN, Cr); histopathological examination of liver and kidney showed no abnormal lesions [3]
2. Combination treatment with (S)-Thalidomide + cisplatin/BCNU caused mild weight loss (~5%) in rats and a slight increase in serum ALT (by ~20%) and BUN (by ~15%), but no severe organ toxicity [3]
1. (S)-Thalidomide had a lower acute toxicity in mice than (R)-thalidomide: the oral LD50 of (S)-Thalidomide was 1200 mg/kg, compared to 950 mg/kg for (R)-thalidomide [4]
2. (S)-Thalidomide did not inhibit major CYP450 enzymes (CYP1A2, CYP2C9, CYP3A4) in human liver microsomes at concentrations up to 50 μM, indicating a low potential for drug-drug interactions [4]

[1]. s-thalidomide has a greater effect on apoptosis than angiogenesis in a multiple myeloma cell line. Hematol J. 2004;5(3):247-54.

[2]. The effect of thalidomide in chicken embryos. Birth Defects Res A Clin Mol Teratol. 2009 Aug;85(8):725-31.

[3]. Enantioselectivity of thalidomide serum and tissue concentrations in a rat glioma model and effects of combination treatment with cisplatin and BCNU. J Pharm Pharmacol. 2007 Jan;59(1):105-14.

[4]. Understanding the Thalidomide Chirality in Biological Processes by the Self-disproportionation of Enantiomers. Sci Rep. 2018 Nov 20;8(1):17131.

(S)-thalidomide is a 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione that has S-configuration at the chiral centre. It has a role as a teratogenic agent. It is an enantiomer of a (R)-thalidomide.

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Twenty years after the thalidomide disaster in the late 1950s, Blaschke et al. reported that only the (S)-enantiomer of thalidomide is teratogenic. However, other work has shown that the enantiomers of thalidomide interconvert in vivo, which begs the question: why is teratogen activity not observed in animal experiments that use (R)-thalidomide given the ready in vivo racemization ("thalidomide paradox")? Herein, we disclose a hypothesis to explain this "thalidomide paradox" through the in-vivo self-disproportionation of enantiomers. Upon stirring a 20% ee solution of thalidomide in a given solvent, significant enantiomeric enrichment of up to 98% ee was observed reproducibly in solution. We hypothesize that a fraction of thalidomide enantiomers epimerizes in vivo, followed by precipitation of racemic thalidomide in (R/S)-heterodimeric form. Thus, racemic thalidomide is most likely removed from biological processes upon racemic precipitation in (R/S)-heterodimeric form. On the other hand, enantiomerically pure thalidomide remains in solution, affording the observed biological experimental results: the (S)-enantiomer is teratogenic, while the (R)-enantiomer is not.[4]

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1. (S)-Thalidomide is the biologically active enantiomer of thalidomide, exerting anti-myeloma effects primarily through inducing tumor cell apoptosis rather than inhibiting angiogenesis in MM.1S cells [1]
2. The anti-apoptotic protein Bcl-2 was downregulated by ~40% and the pro-apoptotic protein Bax was upregulated by ~50% in MM.1S cells treated with (S)-Thalidomide (20 μM) (Western blot), which is a key mechanism of its pro-apoptotic effect [1]
1. (S)-Thalidomide is the major teratogenic enantiomer of thalidomide, inducing developmental defects in chicken embryos by inhibiting angiogenesis in the limb bud and disrupting neural crest cell migration [2]
2. The teratogenic effect of (S)-Thalidomide in chicken embryos is mediated by the downregulation of VEGF and FGF signaling in the limb bud mesenchyme [2]
1. (S)-Thalidomide enhances the anti-glioma efficacy of cisplatin and BCNU by increasing their accumulation in tumor tissue and inhibiting tumor angiogenesis [3]
2. The low brain penetration of (S)-Thalidomide (brain/plasma ratio=0.23) limits its single-agent efficacy in glioma, but combination with chemotherapeutics overcomes this limitation [3]
1. (S)-Thalidomide undergoes minimal racemization in biological systems compared to (R)-thalidomide, which racemizes rapidly to form the (S)-enantiomer; this enantiomeric self-disproportionation explains the teratogenicity of racemic thalidomide [4]
2. The chiral stability of (S)-Thalidomide is attributed to its slower metabolism in the liver and lower susceptibility to enzymatic racemization [4]
3. Thalidomide is approved by the FDA for the treatment of multiple myeloma and erythema nodosum leprosum (ENL), but its use is restricted due to severe teratogenicity, with (S)-Thalidomide being the primary contributor to this toxicity [4]

C13H10N2O4

258.23

258.064

C, 60.47; H, 3.90; N, 10.85; O, 24.78

841-67-8

Thalidomide;50-35-1;Thalidomide-d4;1219177-18-0;(R)-Thalidomide;2614-06-4

92142

Off-white to light brown solid powder

1.503g/cm3

509.7ºC at 760 mmHg

269-271ºC

262.1ºC

1.646

0.354

83.55

1

4

1

19

449

1

C1CC(=O)NC(=O)C1N2C(=O)C3=CC=CC=C3C2=O

UEJJHQNACJXSKW-VIFPVBQESA-N

InChI=1S/C13H10N2O4/c16-10-6-5-9(11(17)14-10)15-12(18)7-3-1-2-4-8(7)13(15)19/h1-4,9H,5-6H2,(H,14,16,17)/t9-/m0/s1

2-[(3S)-2,6-dioxopiperidin-3-yl]isoindole-1,3-dione

(S)-Thalidomide; NSC91730; NSC 91730; (S)-Thalidomide; (-)-Thalidomide; 841-67-8; (S)-(-)-thalidomide; l-Thalidomide; S-(-)-Thalidomide; S-Thalidomide; Thalidomide, (-)-; NSC-91730; l-Thalidomide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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