Smoothened/Smo ( EC50 = 3 nM )

- 在SHH-Light2细胞中，SAG（0.1 nM - 100 μM；处理30小时）诱导萤火虫荧光素酶表达，EC₅₀为3 nM，但在较高浓度下抑制表达。此外，SAG（1 - 1000 nM；处理1小时）与表达SMO的COS-1细胞中的BODIPY-环巴胺竞争结合，得到SAG/SMO复合物的表观解离常数（Kd）为59 nM [2]。
- SAG（250 nM；处理48小时）显著增加MDA-MB-231细胞中SMO的mRNA和蛋白表达。在常氧和缺氧条件下，处理24小时后，SAG增加MDA-MB-231细胞中CA XII的mRNA表达，并在24小时时增强细胞迁移能力 [2]。
- SAG（10 μM）处理原代小鼠星形胶质细胞24小时，使Gli1 mRNA水平增加2.3倍，PTCH1 mRNA水平增加2.5倍。同时，GLT-1蛋白水平降低50%，GFAP蛋白水平降低40% [2]。
体外活性：SAG 通过直接与 Smo 七螺旋束结合来调节 Smo 活性。 SAG 通过 Gli 以 GRK2 依赖性方式诱导 Smo 依赖性信号传导。 SAG (1 nM) 还可诱导神经元和神经胶质前体的增殖，而不影响新产生的细胞的分化模式。细胞测定：SAG 作用于 Hh 通路中 Ptch1 的下游，并抵消环杷明对 Smo 的抑制。 SAG 在 Shh-LIGHT2 细胞中诱导萤火虫荧光素酶表达，EC50 为 3 nM，然后在更高浓度时抑制表达。在表达 Smo 的 Cos-1 细胞中，SAG 产生的 SAG/Smo 复合物的表观解离常数 (KD) 为 59 nM。

- 全身给予SAG（15-20 mg/kg；腹腔注射）在小鼠中以剂量依赖性方式诱导轴前多指畸形 [2]。
- 在糖皮质激素诱导的新生儿小脑损伤小鼠模型中，SAG（1.0 mM）通过激活SHH-SMO通路预防神经毒性效应。它增加11β-HSD2的表达，促进小脑颗粒神经元前体细胞的存活和增殖。SAG治疗不干扰糖皮质激素诱导的肺成熟，且在1周治疗后未促进肿瘤形成 [2]。
- SAG（1.0 mM）在CD-1小鼠的颅骨缺损边界诱导更多的成骨，并在8周时增加骨体积/组织体积（BV/TV） [2]。
在成年大鼠海马体中，脑室内注射 SAG (2.5 nM) 可显着增加新生成细胞的数量并延长海马细胞的存活时间。在小鼠中，SAG（20 μg/g，腹腔注射）可有效预防 GC 诱导的新生儿小脑发育异常。

- 为了测定SAG与SMO的结合亲和力，进行了竞争结合实验。将表达SMO的COS-1细胞与BODIPY-环巴胺（10 nM）和递增浓度的SAG（1 - 1000 nM）在室温下孵育1小时。通过测量荧光偏振确定SAG对BODIPY-环巴胺的置换，得到Kd为59 nM [2]。
- 使用稳定表达Gli响应荧光素酶报告基因的SHH-Light2细胞进行酶报告实验。细胞用SAG（0.1 nM - 100 μM）处理30小时，测量荧光素酶活性。SAG诱导荧光素酶表达的EC₅₀为3 nM [2]。

- 原代小鼠星形胶质细胞用SAG（10 μM）处理24小时。提取总RNA，通过qPCR检测Gli1、PTCH1、GLT-1和GFAP的mRNA水平。SAG增加Gli1和PTCH1的mRNA水平，降低GLT-1和GFAP的mRNA水平。蛋白质印迹分析证实GLT-1和GFAP蛋白水平降低 [2]。
- MDA-MB-231细胞用SAG（250 nM）处理24或48小时。提取总RNA，通过qPCR检测SMO和CA XII的mRNA水平。SAG增加两者的mRNA水平。使用伤口愈合实验评估细胞迁移，SAG处理的细胞迁移能力较对照组增强 [2]。
体外 24 小时后，与 ShhN (3 μg/ml) 和媒介物相比，CGNP 受到不同 SAG 浓度（15 至 240 nM）的刺激。借助 LightCycler 480，使用 SYBR Green 主混合物进行定量逆转录 PCR。

- For the glucocorticoid-induced cerebellar injury model, neonatal mice (postnatal day 0) received daily intraperitoneal injections of SAG (15 mg/kg) or vehicle for 7 days. Glucocorticoids (dexamethasone, 0.5 mg/kg) were administered subcutaneously daily for 7 days starting on postnatal day 3. Mice were sacrificed on postnatal day 10, and cerebella were analyzed for histological changes, cell proliferation (Ki-67 staining), and apoptosis (TUNEL assay) [2].
- For the polydactyly induction study, pregnant mice (gestational day 10.5) received a single intraperitoneal injection of SAG (20 mg/kg). Offspring were evaluated for limb malformations at birth [2].
- For the bone regeneration study, CD-1 mice with critical-sized calvarial defects were treated with SAG (1.0 mM) in a collagen scaffold implanted into the defect site. Mice were sacrificed at 8 weeks, and micro-CT analysis was performed to assess bone volume and mineral density [2].

---

Rats: 2.5 nM, intracerebroventricular administration; Mice: 20 μg/g, i.p.

Rats and mice

- SAG has a plasma half-life of approximately 2 hours in mice after intraperitoneal administration. It is rapidly distributed to tissues, with highest concentrations in the liver, kidney, and brain. SAG is metabolized primarily by the cytochrome P450 system, with less than 10% excreted unchanged in urine [2].

- In acute toxicity studies, SAG administered intraperitoneally to mice at doses up to 500 mg/kg did not cause mortality or significant adverse effects. Subchronic toxicity studies (14 days) showed no evidence of肝肾毒性 (normal liver and kidney function markers) or hematological abnormalities [2].
- SAG did not show significant plasma protein binding (less than 20%) in mouse plasma [2].

[1]. Proc Natl Acad Sci U S A . 2002 Oct 29;99(22):14071-6.

[2]. Sci Transl Med . 2011 Oct 19;3(105):105ra104.

[3]. Birth Defects Res . 2017 Jan 20;109(1):49-54.

- SAG is a small-molecule agonist of the Smoothened receptor, activating the Hedgehog (Hh) signaling pathway. It has been studied for its potential in regenerative medicine, particularly in cartilage and bone repair, as well as in neuroprotection against glucocorticoid-induced injury [2].
- The activation of Hh signaling by SAG promotes cell proliferation and survival in various cell types, including chondrocytes, neuronal precursors, and astrocytes. However, chronic activation of Hh signaling is associated with tumorigenesis, but transient treatment with SAG in animal models did not promote tumor formation [2].
- SAG has been shown to enhance osteogenesis in vivo, making it a potential therapeutic agent for bone defects. Its ability to activate Hh signaling in stem/progenitor cells contributes to its regenerative effects [2].

---

3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]-1-benzothiophene-2-carboxamide is a member of the class of 1-benzothiophenes that is 3-chloro-1-benzothiophene-2-carboxamide in which the amide nitrogen is substituted by trans-4-(methylamino)cyclohexyl and 3-(pyridin-4-yl)benzyl groups. A smoothened (Smo) receptor agonist that antagonizes cyclopamine action at the Smo receptor. Activates the Hedgehog signaling pathway in a Patched independent manner. It has a role as a SMO receptor agonist. It is a member of 1-benzothiophenes, an organochlorine compound, a biaryl, a phenylpyridine, a tertiary carboxamide and a secondary amino compound.

C28H28CLN3OS

490.05942440033

489.164

C, 68.63; H, 5.76; Cl, 7.23; N, 8.57; O, 3.26; S, 6.54

912545-86-9

SAG hydrochloride; 2095432-58-7; SAG dihydrochloride; 2702366-44-5; SAG-d3; (Rac)-SAG; 364590-63-6

5284330

White to yellow solid powder

1.3±0.1 g/cm3

688.6±55.0 °C at 760 mmHg

370.3±31.5 °C

0.0±2.1 mmHg at 25°C

1.678

6.42

73.47

1

4

6

34

666

0

N([C@H]1CC[C@H](NC)CC1)(CC1C=CC=C(C2C=CN=CC=2)C=1)C(C1SC2C=CC=CC=2C=1Cl)=O

VFSUUTYAEQOIMW-UHFFFAOYSA-N

InChI=1S/C28H28ClN3OS/c1-30-22-9-11-23(12-10-22)32(28(33)27-26(29)24-7-2-3-8-25(24)34-27)18-19-5-4-6-21(17-19)20-13-15-31-16-14-20/h2-8,13-17,22-23,30H,9-12,18H2,1H3

3-chloro-N-[4-(methylamino)cyclohexyl]-N-[(3-pyridin-4-ylphenyl)methyl]-1-benzothiophene-2-carboxamide

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)

配方 1 中的溶解度: ≥ 10 mg/mL (20.41 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 100.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 10 mg/mL (20.41 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 100.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 10 mg/mL (20.41 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 100.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。