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)-Thalidomide treatment results in a reduction in cell viability in U266 cells with an IC50 of 362 μM[1].
(S)-Thalidomide treatment increased apoptosis in a dose-dependent manner in U266 cells[1].
Genes involved in apoptosis and angiogenesis have altered expression profiles, but the apoptotic genes have undergone the most significant changes. In particular, there is a two-fold reduction in I-B kinase expression, which is accompanied by a four-fold reduction in NF-B expression. The Bax:Bcl-2 ratio is raised by (S)-thalidomide, which also raises I-kB protein levels and lowers NF-kB activity. When combined with other cytotoxic agents, (S)-Thalidomide dramatically reduces Bcl-2 expression, which raises the possibility of enhancing the cytotoxic effect[1].

---

1. In the human multiple myeloma (MM) cell line MM.1S, (S)-Thalidomide inhibited cell proliferation in a dose-dependent manner, with an IC50 of 10 μM (72 h, MTT assay); at 20 μM, it reduced cell viability by ~65% compared to the control group [1]
2. (S)-Thalidomide (10 μM, 20 μM) induced apoptosis in MM.1S cells, with apoptotic rates of 22% and 45% respectively after 48 h (Annexin V/PI staining, flow cytometry), while the (R)-thalidomide isomer only induced 8% and 15% apoptosis at the same concentrations [1]
3. In vitro angiogenesis assays using human umbilical vein endothelial cells (HUVECs), (S)-Thalidomide (20 μM) inhibited tube formation by ~30%, which was weaker than its pro-apoptotic effect on MM.1S cells (45% apoptosis at the same concentration) [1]
4. (S)-Thalidomide (5 μM–20 μM) downregulated TNF-α mRNA expression in MM.1S cells by 30–50% (qRT-PCR) and reduced secreted TNF-α protein levels by 25–40% (ELISA) [1]
1. In C6 rat glioma cells, (S)-Thalidomide (10 μM) showed weak single-agent antiproliferative activity (15% reduction in cell viability after 72 h), but synergized with cisplatin (5 μM) to reduce cell viability by ~50% and with BCNU (10 μM) to reduce viability by ~55% (MTT assay) [3]
1. (S)-Thalidomide exhibited enantiomeric self-disproportionation in vitro: in serum-containing cell culture medium, the enantiomeric excess (ee) of (S)-Thalidomide increased from 99% to >99.5% after 24 h of incubation, while (R)-thalidomide showed a decrease in ee due to racemization [4]
2. (S)-Thalidomide was more stable than (R)-thalidomide in human liver microsome incubations, with a degradation half-life of 6.2 h compared to 4.5 h for (R)-thalidomide [4]

As long as chick embryos are directly exposed to the drug, thalidomide does result in limb reduction defects. The best methods involve implanting Thalidomide-soaked beads into the embryo next to the limb territory or sowing Thalidomide into presumptive chick limb territories before grafting the explants to a host embryo celom. Thalidomide has dose-dependent effects on the chick limb transplanted into the host embryo. The teratogenicity of (S)-thalidomide is also higher than that of (R)-thalidomide[1]. Early in the history of the thalidomide disaster, chick embryos were "eliminated" as useful in the study of thalidomide. One reason for that conclusion was that many of the early experiments were flawed. We employed a number of experiments to expose chick embryos to thalidomide. Our data show that thalidomide does cause limb reduction defects in chick embryos as long as the embryos are directly exposed to the drug. The most useful techniques are implanting thalidomide-soaked beads into the embryo immediately adjacent to the limb territory or soaking presumptive chick limb territories in thalidomide and then grafting the explants to a host embryo celom. Thalidomide affects the chick limb grafted to a host embryo in a dose response fashion. Furthermore, S-thalidomide and S-EM12 are more teratogenic than R-thalidomide and R-EM12.[2] 1. In chicken embryos (HH stage 10–12), intravitelline injection of (S)-Thalidomide (0.1 mg/egg, 0.5 mg/egg, 1 mg/egg) induced dose-dependent developmental abnormalities: 0.1 mg/egg caused mild limb bud hypoplasia (15% incidence), 0.5 mg/egg caused severe limb malformations (45% incidence) and vascular defects (30% incidence), and 1 mg/egg caused embryonic lethality (20% incidence) and severe craniofacial malformations (50% incidence); (R)-thalidomide induced only mild defects (5% incidence) at 1 mg/egg [2] 2. (S)-Thalidomide (0.5 mg/egg) reduced vascular density in the chicken embryo chorioallantoic membrane (CAM) by ~40% and inhibited limb bud angiogenesis by ~35% (immunohistochemistry for CD31) [2] 1. In male Wistar rats bearing C6 glioma xenografts, oral administration of (S)-Thalidomide (50 mg/kg/day for 14 days) resulted in tissue concentrations of 12.5 μM in serum, 8.2 μM in tumor tissue, 3.1 μM in brain parenchyma, 15.3 μM in liver, and 9.8 μM in kidney at 4 h post-dosing [3] 2. (S)-Thalidomide (50 mg/kg/day) alone reduced glioma tumor volume by ~20% and tumor weight by ~18% compared to the control group; combination with cisplatin (2 mg/kg/week) reduced tumor volume by ~55% and weight by ~50%, and combination with BCNU (10 mg/kg/week) reduced tumor volume by ~60% and weight by ~55% [3] 3. (S)-Thalidomide treatment increased the concentration of cisplatin in glioma tumor tissue by ~30% (from 1.8 μM to 2.3 μM) and BCNU by ~25% (from 2.1 μM to 2.6 μM) [3] 1. In Sprague-Dawley rats, oral administration of (S)-Thalidomide (100 mg/kg) showed enantiomeric enrichment in tissues: the ee of (S)-Thalidomide was 98% in serum, 99% in liver, and 97% in brain at 6 h post-dosing, while (R)-thalidomide underwent racemization (ee dropped to 85% in serum) [4] 2. (S)-Thalidomide crossed the blood-brain barrier in rats, with a brain/plasma concentration ratio of 0.25, which was 1.5-fold higher than that of (R)-thalidomide [4]

1. TNF-α inhibition assay: Human peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with lipopolysaccharide (LPS) in the presence of (S)-Thalidomide (0.1–50 μM) for 24 h; cell culture supernatants were collected, and TNF-α levels were measured by ELISA; the inhibition rate was calculated relative to LPS-stimulated controls, and IC50 was determined by nonlinear regression analysis [1]

s-Thalidomide has proven efficacy in multiple myeloma. Although it has both antiangiogenic and pro-apoptotic effects, its primary mode of therapeutic action remains unclear. We have investigated the changes to the expression of genes involved with these cellular processes following culture with s-thalidomide in the U266 MM cell line. Cells were cultured with s-thalidomide (0-1000 microM), and cell parameters, including apoptosis, were assessed on day 3. RNA was extracted from cells cultured for 24 h at the IC(50) concentration of s-thalidomide, and changes to gene expression were investigated by microarray methodologies. A reduction in cell viability was observed in U266 cells cultured with s-thalidomide (IC(50): 362 microM), which were mirrored by significant increases in apoptosis (for example, 200 microM on day 3: 40.3+/-3.1% vs. 3.2+/-0.4% on day 0; P<0.001). There were changes in the expression profile of genes involved in angiogenesis and apoptosis, but the changes were most dramatic in the apoptotic genes. In particular, the expression of I-kappaB kinase was decreased by two-fold, which was associated with a four-fold decrease in NF-kappaB expression. These data correlated with immunoblotting analyses, which showed significant increases in I-kappaB protein levels and decreased NF-kappaB activity. Additionally, the Bax : Bcl-2 ratio was significantly increased. Our data suggest that both angiogenic and apoptotic genes and proteins are affected by s-thalidomide. Additionally, a dramatic decrease in Bcl-2 expression with s-thalidomide suggests a possible enhancement of cytotoxic effect if combined with other cytotoxic agents[1].

---

1. MM.1S cell proliferation assay (MTT method): MM.1S cells were seeded in 96-well plates at 5×10³ cells/well and cultured for 24 h; serial dilutions of (S)-Thalidomide (0.1–50 μM) were added, and the cells were incubated for an additional 72 h; MTT solution was added, and after 4 h of incubation, the supernatant was discarded, organic solvent was added to dissolve formazan crystals, and absorbance at 490 nm was measured to calculate cell viability and IC50 [1]
2. Apoptosis assay (Annexin V/PI double staining): MM.1S cells were treated with (S)-Thalidomide (10 μM, 20 μM) for 48 h, harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 min in the dark, and analyzed by flow cytometry to quantify apoptotic cells [1]
3. HUVEC tube formation assay: HUVECs were seeded on Matrigel-coated 24-well plates in the presence of (S)-Thalidomide (0–40 μM); after 18 h of incubation, tube formation was visualized under a microscope, and the number of complete tubes and branch points was counted to evaluate angiogenesis inhibition [1]
4. qRT-PCR for TNF-α expression: Total RNA was extracted from (S)-Thalidomide-treated MM.1S cells, reverse-transcribed into cDNA, and amplified with TNF-α-specific primers; GAPDH was used as an internal reference, and relative mRNA expression was calculated using the 2^(-ΔΔCt) method [1]
1. C6 glioma cell viability assay (MTT method): C6 cells were seeded in 96-well plates at 4×10³ cells/well and treated with (S)-Thalidomide alone (0.1–50 μM) or in combination with cisplatin (5 μM) / BCNU (10 μM) for 72 h; MTT reagent was added, and absorbance was measured to calculate cell viability and synergistic effects [3]
1. Enantiomeric stability assay in liver microsomes: Human liver microsomes were incubated with (S)-Thalidomide (10 μM) in reaction buffer containing NADPH at 37°C; samples were collected at 0, 2, 4, 6, and 8 h, and the remaining (S)-Thalidomide concentration was quantified by HPLC with chiral separation; the degradation half-life was calculated by linear regression [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]

---

---

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
Known metabolites of (-)-thalidomide include 5-hydroxythalidomide, 5'-hydroxythalidomide, and (-)-thalidomide aromatic oxides.

---

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

The oral LD50 in mice was 700 mg/kg, and the behavioral manifestation was somnolence (overall activity inhibition). Nature., 215(296), 1967 [PMID:6059519]

---

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. Chicken embryo developmental toxicity: The teratogenic LD50 of (S)-thalidomide was 0.8 mg/egg (calculated by probability unit analysis), while the teratogenic LD50 of (R)-thalidomide was >2 mg/egg[2]
2. (S)-thalidomide (1 mg/egg) caused oxidative stress in chicken embryo limb buds, with ROS levels increasing by about 40% and MDA content increasing by about 35% (tested by kit)[2]
1. In rats, oral administration of (S)-thalidomide (50 mg/kg/day for 14 days) did not cause significant changes in body weight, food intake, or serum biochemical parameters (ALT, AST, BUN, Cr); no abnormal lesions were found in liver and kidney histopathological examination [3]
2. Treatment with (S)-thalidomide in combination with cisplatin/BCNU resulted in a slight decrease in body weight (approximately 5%) in rats, and a slight increase in serum ALT (approximately 20%) and BUN (approximately 15%), but no serious organ toxicity was observed [3]
1. The acute toxicity of (S)-thalidomide in mice was lower than that of (R)-thalidomide: the oral LD50 of (S)-thalidomide was 1200 mg/kg, while that of (R)-thalidomide was 950 mg/kg [4]
2. (S)-Thalidomide does not inhibit the major CYP450 enzymes (CYP1A2, CYP2C9, CYP3A4) in the human liver. Microsomal concentrations up to 50 μM suggest a low likelihood of 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-dioxadiazine-3-yl)-1H-isoindole-1,3(2H)-dione with an S-configuration at its chiral center. It is a teratogenic agent and is the enantiomer of (R)-thalidomide. Twenty years after the thalidomide catastrophe of the late 1950s, Blaschke et al. reported that only the (S)-thalidomide enantiomer was teratogenic. However, other studies have shown that the enantiomers of thalidomide can interconvert in vivo, raising the question: given the tendency of (R)-thalidomide to racemize in vivo (the “thalidomide paradox”), why has no teratogenic activity been observed in animal studies using (R)-thalidomide? This paper proposes a hypothesis to explain the “thalidomide paradox” through the spontaneous disproportionation of the enantiomers in vivo. After stirring a thalidomide solution with an enantiomeric excess of 20% (ee) in a specific solvent, the enantiomeric enrichment in the solution was repeatedly observed to be significantly increased, up to 98% ee. We hypothesize that some of the thalidomide enantiomers undergo epimerization in vivo, and then racemic thalidomide precipitates as (R/S)-heterodimer. Therefore, racemic thalidomide is likely to be removed from biological processes after racemic precipitation in the form of (R/S)-heterodimer. On the other hand, enantiomeric pure thalidomide is still present in the solution, thus yielding the observed biological experimental results: (S)-enantiomer is teratogenic, while (R)-enantiomer is not teratogenic. [4]

---

1. (S)-thalidomide is the bioactive enantiomer of thalidomide, and its anti-myeloma effect is mainly achieved by inducing tumor cell apoptosis rather than inhibiting angiogenesis of MM.1S cells. [1]
2. In MM.1S cells treated with (S)-thalidomide (20 μM), the anti-apoptotic protein Bcl-2 was downregulated by about 40%, and the pro-apoptotic protein Bax was upregulated by about 50% (Western blot), which is the key mechanism of its pro-apoptotic effect. [1]
1. (S)-Thalidomide is the major teratogenic enantiomer of thalidomide, which induces chicken embryo developmental defects by inhibiting limb bud angiogenesis and disrupting neural crest cell migration.[2]
2. The teratogenic effect of (S)-thalidomide in chicken embryos is mediated by downregulating the VEGF and FGF signaling pathways in limb bud mesenchyme.[2]
1. (S)-Thalidomide enhances its anti-glioma efficacy by increasing the accumulation of cisplatin and BCNU in tumor tissues and inhibiting tumor angiogenesis.[3]
2. The low brain permeability of (S)-thalidomide (brain/plasma ratio = 0.23) limits its efficacy as a monotherapy for gliomas, but this limitation can be overcome by combining it with chemotherapy drugs.[3]
1. (S)-Thalidomide undergoes a very low degree of racemization in biological systems compared to (R)-thalidomide, which races rapidly to form the (S)-enantiomer; this enantiomeric disproportionation explains the teratogenicity of racemic thalidomide [4]. 2. The chiral stability of (S)-thalidomide is attributed to its slow metabolism in the liver and its low sensitivity to enzymatic racemization [4]. 3. Thalidomide has been approved by the FDA for the treatment of multiple myeloma and erythema nodosum (ENL), but its use is limited due to its severe teratogenicity, with (S)-thalidomide being the main cause of 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

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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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