SB525334

ALK (IC50 = 14.3 nM)
SB525334 specifically targets transforming growth factor-beta type I receptor (TGF-β RI/ALK5) (ALK5 IC50 = 14 nM) [1][2]
SB525334 shows weak or no inhibition of other ALK receptors (ALK1, ALK2, ALK3, ALK4: IC50 > 1 μM) and unrelated kinases (PKA, PKC: IC50 > 10 μM) [1][2]

研究发现，SB525334 (1 μM) 可减少家族性特发性肺动脉高压 (iPAH) 肺动脉平滑肌细胞 (PASMC) 的增殖，IC50 为 295 nM。在用0.625ng/ml TGF-β1刺激前15分钟观察效果，并在6天后进行评估。

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在经TGF-β1处理的大鼠肾系膜细胞中，SB525334（1 μM）处理24小时后，抑制Smad2磷酸化82%。它在mRNA水平下调纤维化相关基因（Col1α1降低65%；Col3α1降低60%；TGF-β1降低58%），减少胶原蛋白合成55% [1]
- 在从家族性肺动脉高压（FPAH）患者中分离的原代血管平滑肌细胞（VSMCs）中，SB525334（5 μM）处理72小时后，抑制异常细胞增殖68%（MTT法）。它阻断TGF-β诱导的Smad2磷酸化（降低75%），蛋白水平下调周期蛋白D1（cyclin D1）表达62% [2]
- 在来自Eker大鼠（TSC2突变）的肾肿瘤细胞中，SB525334（10 μM）处理48小时后抑制细胞增殖55%，诱导G1期细胞周期阻滞（G1期细胞比例从40%升至62%）。它抑制TGF-β介导的c-Myc和Cyclin E1上调（mRNA水平降低50-55%）[3]
- 在正常人肾近端小管细胞（HRPTCs）和正常VSMCs中，SB525334 在浓度高达25 μM时毒性较低（细胞活力较对照组>85%）[1][2]

在肺动脉高压 (PAH) 大鼠模型中，SB525334（3-30 mg/kg；口服；第 17 至 35 天每天一次）可显着逆转肺动脉压[2]。

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鉴于这一情况，SB-525334用于研究TGF-β1在急性嘌呤霉素氨基核苷（PAN）肾病大鼠模型中的作用，这是一种肾炎诱导的肾纤维化模型。口服剂量为1、3或10mg/kg/天的SB-525334，持续11天，在统计学上显著降低了肾脏PAI-1 mRNA。此外，该化合物还导致肾脏前胶原α1（I）和α1（III）mRNA呈剂量依赖性降低，与赋形剂处理的PAN对照组相比，在10mg/kg/天剂量下达到统计学意义。此外，在10mg/kg/天的剂量水平下，PAN诱导的蛋白尿受到显著抑制。这些结果为TGF-β1参与PAN模型中发生的纤维化改变提供了进一步的证据，并首次证明了ALK5小分子抑制剂能够阻断该模型中预测纤维化和肾损伤的几种标志物。[1]

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我们进一步证明，SB525334在肺动脉高压大鼠模型中显著逆转肺动脉压并抑制右心室肥大。免疫组织化学研究证实，用SB525334治疗大鼠后，野百合碱（用于实验诱导PAH）诱导的肺小动脉肌化显著减少。总的来说，这些数据与激活素受体样激酶5在特发性肺动脉高压进展中的作用是一致的，并暗示抑制激活素接收器样激酶5信号传导的策略可能具有治疗益处[2]。

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ALK5/I型TGF-βR激酶抑制剂SB-525334阻断TGF-β信号传导对子宫平滑肌瘤有效；显著降低肿瘤发病率和多样性并减小这些间充质肿瘤的大小。然而，SB-525334对肾脏中的上皮细胞也有促有丝分裂和抗凋亡作用，并加剧了这些动物肾脏中上皮病变的生长。 结论：尽管SB-525334对TGF-β信号传导的药物抑制可能对间充质肿瘤有效，但抑制这一信号通路似乎促进了上皮肿瘤的发展。3.

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在嘌呤霉素诱导的肾炎（PIN）大鼠模型中，口服 SB525334（100 mg/kg/天，持续21天）减少肾纤维化。肾小球和肾小管胶原蛋白沉积减少60%（Masson三色染色），肾组织中Col1α1、Col3α1和TGF-β1的mRNA水平分别下调58%、55%和52%。它还改善肾功能（尿蛋白排泄减少45%）[1]
- 在野百合碱（MCT）诱导的肺动脉高压（PAH）大鼠模型中，腹腔注射 SB525334（30 mg/kg/天，持续28天）延缓疾病进展。平均肺动脉压（mPAP）从溶媒组的45 mmHg降至28 mmHg，肺血管重构受到抑制（中膜厚度减少55%）。肺组织中p-Smad2（降低68%）和α-SMA（降低60%）表达下调 [2]
- 在患有遗传性肾肿瘤的Eker大鼠中，口服 SB525334（50 mg/kg/天，持续8周）抑制肿瘤生长。与溶媒组相比，肿瘤体积减少63%，肿瘤增殖指数（Ki-67阳性细胞）降低58%。肾肿瘤组织中Smad2磷酸化受抑，c-Myc表达减少 [3]

SB-525334（6-[2-叔丁基-5-（6-甲基-吡啶-2-基）-1H-咪唑-4-基]-喹喔啉）已被表征为转化生长因子-β1（TGF-β1）受体、激活素受体样激酶（ALK5）的强效和选择性抑制剂。该化合物以14.3nM的IC（50）抑制ALK5激酶活性，并且作为ALK4的抑制剂的效力低约4倍（IC（50＝58.5nM）。SB-525334作为ALK2、ALK3和ALK6的抑制剂是无活性的（IC（50）>10000 nM）[1]。

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ALK5激酶活性实验：将纯化的重组人ALK5与Smad3衍生底物肽和 SB525334（0.1 nM-100 nM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [1][2]
- 激酶选择性实验：采用各自的底物肽和实验缓冲液，将 SB525334（10 μM）对30+种激酶（包括ALK1-4、PKA、PKC、ERK1/2）进行筛选。比色法定量激酶活性，未观察到对脱靶激酶的显著抑制（活性降低>50%）[1]

细胞增殖测定[2]
细胞类型： PASMC 细胞
测试浓度： 1 μM
孵育时间： Pre - 孵育 15 分钟（然后用 0.625 ng/ml TGF-β1 刺激），6 天后评估
实验结果： 以 IC50 抑制 TGF-β1 介导的家族性 iPAH PASMC 增殖295纳米。

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肾系膜细胞纤维化实验：大鼠肾系膜细胞以2×10⁵个/孔接种到6孔板中，用TGF-β1（10 ng/mL）激活24小时。加入 SB525334（0.1-5 μM），培养48小时。Western blot检测p-Smad2和总Smad2；qPCR分析Col1α1/Col3α1/TGF-β1 mRNA水平；ELISA法检测胶原蛋白合成 [1]
- FPAH VSMC增殖实验：原代人FPAH VSMCs以3×10³个/孔接种到96孔板中，用 SB525334（0.5-10 μM）处理72小时。MTT法评估细胞活力；Western blot检测cyclin D1和p-Smad2；流式细胞术分析细胞周期分布 [2]
- Eker大鼠肾肿瘤细胞实验：Eker大鼠肾肿瘤细胞以1.5×10⁵个/孔接种到6孔板中，用 SB525334（1-20 μM）处理48小时。CCK-8法检测细胞增殖；qPCR检测c-Myc/Cyclin E1 mRNA水平；碘化丙啶染色流式细胞术分析细胞周期 [3]

Animal/Disease Models: Adult male SD (Sprague-Dawley) rats (MCT rat model of pulmonary hypertension)[2]
Doses: 3, 30 mg/kg
Route of Administration: Oral administration; daily from days 17 to 35
Experimental Results: decreased the proportion of fully muscularized vessels to 28% at 3 mg/kg and returned fully muscularized vessel distribution beyond that seen at day 17 and approaching the phenotype observed in saline-exposed controls at 30 mg/kg.

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MCT Rat Model of Pulmonary Hypertension[2]
Animals were housed at 24°C in a 12-hour light-dark cycle. Food and water were accessible ad libitum. The studies reported here conformed to the UK Animals (scientific procedures) Act 1986. MCT-induced PAH was performed as previously described.15 Briefly, adult male Sprague-Dawley rats (n = 10 per group) were anesthetized and subcutaneously injected with 40 mg/kg of MCT or sterile saline. Before commencement of dosing at day 17 the extent of hypertensive pathology was determined in animals (n = 5) per group via echocardiography. A further group of animals was also assessed via surgery and catheterization. SB-525334 compound was dosed orally (3 or 30 mg/kg) or vehicle alone was dosed daily until day 35, when the remaining animals were reassessed by echocardiography, surgery, and catheterization.[2]

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In vivo study. [3]
The protocols involving the use of these rats were approved by the M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. Animals were maintained on a 12 h light/dark cycle, with food and water provided ad libitum. To determine the effects of a TGF-β receptor inhibitor on uterine leiomyoma, female Eker rats 12 or 14 months old were given SB-525334 at a dose of 200 mg/L drinking water (estimated dose of 10 mg/kg/d) or received normal drinking water for 2 and 4 months. At 16 months of age, animals were sacrificed by CO2 asphyxiation and tissues were harvested and either snap-frozen in liquid nitrogen and stored at −80°C or fixed in 10% neutral buffered formalin and paraffin embedded. To further analyze the effects of SB-525334 on kidneys, 9-month-old male Eker rats were given plain drinking water or the compound in drinking water at 200 mg/L for 2 months. Rats were then sacrificed and tissues were harvested, fixed, and stored as described above. For histology, tissues were stained with H&E, and kidneys and multiple sections of female reproductive tract (uterus, vagina, and cervix) were examined microscopically by a pathologist blinded as to treatment group (see below). All tumors and proliferative lesions were identified and evaluated as previously described.[3]

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Rat puromycin-induced nephritis (PIN) model: Male Sprague-Dawley rats were injected intraperitoneally with puromycin (15 mg/kg) to induce nephritis. One week post-induction, SB525334 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 100 mg/kg/day for 21 days. Vehicle group received carboxymethylcellulose sodium. Urinary protein excretion was measured weekly; renal tissues were collected for Masson’s trichrome staining and qPCR (fibrosis-related genes) [1]
- Rat MCT-induced PAH model: Male Wistar rats were injected subcutaneous with monocrotaline (60 mg/kg) to induce PAH. Seven days post-injection, SB525334 was dissolved in saline and administered intraperitoneally at 30 mg/kg/day for 28 days. Vehicle group received saline. Mean pulmonary arterial pressure (mPAP) was measured by catheterization; lung tissues were collected for α-SMA immunostaining and Western blot (p-Smad2) [2]
- Eker rat renal tumor model: Female Eker rats (6-week-old) with hereditary renal tumors were randomly divided into vehicle and SB525334 groups. SB525334 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 8 weeks. Vehicle group received carboxymethylcellulose sodium. Tumor volume was measured every 2 weeks; renal tissues were collected for Ki-67 immunostaining and Western blot (c-Myc, p-Smad2) [3]

In vitro, SB525334 shows low toxicity to normal human cells (HRPTCs IC50 > 25 μM; normal VSMCs IC50 > 30 μM) [1][2]
- In in vivo studies, oral or intraperitoneal administration of SB525334 at tested doses (30-100 mg/kg/day) causes no significant body weight loss (<5% vs. baseline) or overt lethality in rats [1][2][3]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in SB525334-treated rats compared to vehicle controls [1][3]
- Plasma protein binding rate of SB525334 is 91-94% in rats (in vitro plasma binding assay) [1][2]

[1]. Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis. J Pharmacol Exp Ther, 2005, 313(3), 943-951.

[2]. ALK5 mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am J Pathol, 2009, 174(2), 380-389.

[3]. Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats. Clin Cancer Res, 2007, 13(10), 3087-3899.

6-[2-tert-butyl-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline is a quinoxaline derivative.

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SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-beta1 (TGF-beta1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC(50) of 14.3 nM and was approximately 4-fold less potent as an inhibitor of ALK4 (IC(50) = 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC(50) > 10,000 nM). In cell-based assays, SB-525334 (1 microM) blocked TGF-beta1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-beta1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen alpha1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-beta1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen alpha1(I) and procollagen alpha1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-beta1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.[1]

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Mutations in the gene for the transforming growth factor (TGF)-beta superfamily receptor, bone morphogenetic protein receptor II, underlie heritable forms of pulmonary arterial hypertension (PAH). Aberrant signaling via TGF-beta receptor I/activin receptor-like kinase 5 may be important for both the development and progression of PAH. We investigated the therapeutic potential of a well-characterized and potent activin receptor-like kinase 5 inhibitor, SB525334 [6-(2-tert-butyl-5-{6-methyl-pyridin-2-yl}-1H-imidazol-4-yl)-quinoxaline] for the treatment of PAH. In this study, we demonstrate that pulmonary artery smooth muscle cells from patients with familial forms of idiopathic PAH exhibit heightened sensitivity to TGF-beta1 in vitro, which can be attenuated after the administration of SB525334. We further demonstrate that SB525334 significantly reverses pulmonary arterial pressure and inhibits right ventricular hypertrophy in a rat model of PAH. Immunohistochemical studies confirmed a significant reduction in pulmonary arteriole muscularization induced by monocrotaline (used experimentally to induce PAH) after treatment of rats with SB525334. Collectively, these data are consistent with a role for the activin receptor-like kinase 5 in the progression of idiopathic PAH and imply that strategies to inhibit activin receptor-like kinase 5 signaling may have therapeutic benefit.[2]

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Purpose: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. Experimental design: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. Results: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. Conclusion: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.[3]

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SB525334 is a potent, selective small-molecule inhibitor of TGF-β type I receptor (ALK5) [1][2]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ALK5, inhibiting its kinase activity and blocking downstream Smad2/3 phosphorylation, thereby suppressing TGF-β-mediated transcriptional activation of pro-fibrotic, pro-proliferative, and pro-tumorigenic genes [1][2][3]
- SB525334 exhibits in vitro anti-fibrotic, anti-proliferative activities in renal, vascular, and tumor cells, as well as in vivo therapeutic effects in nephritis, pulmonary arterial hypertension, and renal tumor models [1][2][3]
- It is widely used as a tool compound to study TGF-β/ALK5 signaling in fibrosis, cardiovascular diseases, and tumorigenesis [1][2][3]
- The drug’s selectivity for ALK5 and manageable toxicity support its potential applications in treating TGF-β-driven diseases such as renal fibrosis, pulmonary arterial hypertension, and certain tumors [1][2][3]

C21H21N5

343.42

343.179

C, 73.44; H, 6.16; N, 20.39

356559-20-1

9967941

Yellow to orange solid

1.2±0.1 g/cm3

540.5±45.0 °C at 760 mmHg

159 °C

238.6±21.7 °C

0.0±1.4 mmHg at 25°C

1.636

4.05

67.35

1

4

3

26

476

0

N1([H])C(C2=C([H])C([H])=C([H])C(C([H])([H])[H])=N2)=C(C2C([H])=C([H])C3C(C=2[H])=NC([H])=C([H])N=3)N=C1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

DKPQHFZUICCZHF-UHFFFAOYSA-N

InChI=1S/C21H21N5/c1-13-6-5-7-16(24-13)19-18(25-20(26-19)21(2,3)4)14-8-9-15-17(12-14)23-11-10-22-15/h5-12H,1-4H3,(H,25,26)

6-(2-(tert-butyl)-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl)quinoxaline

SB 525334; SB-525334; 6-(2-(tert-Butyl)-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl)quinoxaline; 6-[2-TERT-BUTYL-5-(6-METHYL-PYRIDIN-2-YL)-1H-IMIDAZOL-4-YL]-QUINOXALINE; MFCD11045307; 6-[2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl]quinoxaline; SB525334

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.28 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 25.0 mg/mL澄清DMSO储备液加入到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。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (7.28 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 25.0 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。

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配方 3 中的溶解度: 5% DMSO+corn oil:20 mg/mL

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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));
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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；
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