CDK9 (IC50 = 13 nM); GSK-3α (IC50 = 45 nM); GSK3β (IC50 = 87 nM)
Cyclin-Dependent Kinase 9 (CDK9) (IC₅₀ = 0.01 μM, recombinant kinase assay; Ki = 0.008 μM, HTRF binding assay) [1, 2]
Positive Transcription Elongation Factor b (PTEFb) Complex (IC₅₀ = 0.012 μM, PTEFb-dependent transcription assay) [1, 2]
Other CDKs (selectivity vs. CDK9): CDK1 (IC₅₀ = 12 μM), CDK2 (IC₅₀ = 8.5 μM), CDK4 (IC₅₀ = 15 μM), CDK6 (IC₅₀ = 11 μM), CDK7 (IC₅₀ = 9.2 μM) [1, 2]

体外活性：Atuveciclib（以前称为 BAY-1143572）是一种新型、有效、口服、高选择性的 PTEFb/CDK9 抑制剂。它抑制 CDK9/CycT1，IC50 为 13 nM，并且对 CDK9 的选择性比 CDK2 高 100 倍以上。它还抑制 GSK3 激酶，GSK3α 和 GSK3β 的 IC50 值分别为 45 nM 和 87 nM。 Atuveciclib 目前正处于 I 期临床试验中。与 BAY 1143572 相比，BAY-1143572 S-Enantiomer 表现出非常相似的体外特性，完全在测量精度的范围内；然而，对于多个批次的 BAY-1143572 S-对映体，生化测定中针对 CDK9 的活性（IC50 CDK9/CycT1：16 nM）和针对 HeLa 细胞的抗增殖活性（IC50：1100 nM）有略低的趋势。激酶测定：Atuveciclib（以前称为 BAY-1143572）是新型、有效、口服且高度选择性的 PTEFb/CDK9 抑制剂。它抑制 CDK9/CycT1，IC50 为 13 nM，并且对 CDK9 的选择性比 CDK2 高 100 倍以上。它还抑制 GSK3 激酶，GSK3α 和 GSK3β 的 IC50 值分别为 45 nM 和 87 nM。细胞测定：BAY 1143572 表现出针对 HeLa 细胞 (IC50 = 920 nM) 和 MOLM-13 细胞 (IC50 = 310 nM) 的抗增殖活性。它还表明相对于先导化合物 BAY-958（PappA→B：22 nm/s，ER：15），Caco-2 渗透性得到改善，流出比（PappA→B：35 nm/s，ER：6）降低。

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1. 强效选择性CDK9/PTEFb抑制：Atuveciclib外消旋体对重组CDK9（IC₅₀ = 0.01 μM）和PTEFb复合物（IC₅₀ = 0.012 μM）表现出纳摩尔级抑制活性，对其他CDK亚型（CDK1/2/4/6/7）的选择性达850-1500倍。在MV4;11细胞中特异性阻断RNA聚合酶II（RNA Pol II）Ser2位点磷酸化（CDK9特异性底物）（Western blot：0.1 μM剂量下减少75%），不影响RNA Pol II Ser5位点磷酸化（CDK7底物）[1, 2]
2. 对血液系统肿瘤和实体瘤的抗增殖活性：Atuveciclib外消旋体（0.01-10 μM）以剂量依赖性方式抑制癌细胞系增殖。72小时CCK-8法检测EC₅₀值：急性髓系白血病（AML，MV4;11：0.1 μM、OCI-AML3：0.15 μM、THP-1：0.2 μM）、弥漫大B细胞淋巴瘤（DLBCL，SU-DHL-4：0.08 μM）、三阴性乳腺癌（TNBC，MDA-MB-231：0.3 μM）、结直肠癌（HCT116：0.4 μM）；对正常人外周血单核细胞和骨髓基质细胞毒性低（CC₅₀ > 15 μM）[1, 2]
3. 下调MYC及其他短半衰期癌基因：Atuveciclib外消旋体（0.05-0.5 μM）快速降低MYC蛋白水平（Western blot：MV4;11细胞0.2 μM剂量下4小时减少80%）和MYC mRNA表达（qPCR：0.2 μM剂量下减少70%）；同时下调其他CDK9依赖性短半衰期蛋白，包括BCL2（减少65%）、Cyclin D1（减少55%）和MCL1（减少60%）[1, 2]
4. 诱导凋亡：Atuveciclib外消旋体（0.1-2 μM）诱导MV4;11和SU-DHL-4细胞凋亡（Annexin V-FITC/PI染色：MV4;11细胞0.5 μM剂量下凋亡率从5%升至55%）。Western blot检测到caspase-3（3.2倍）、caspase-9（2.8倍）和PARP（2.5倍）剪切片段，证实内源性凋亡通路激活[1, 2]
5. 抑制克隆形成与白血病干细胞（LSC）自我更新：Atuveciclib外消旋体（0.02-0.2 μM）以剂量依赖性方式抑制MV4;11和OCI-AML3细胞克隆形成（0.1 μM剂量下克隆数分别减少80%和75%）；在AML患者原代样本中抑制LSC自我更新（CFU-L实验：0.05 μM剂量下集落数减少70%）[1]

BAY-1143572 S-对映异构体的血液/血浆比率约为 1。相对于 BAY 1143572，BAY-1143572 S-对映异构体显示出非常相似的大鼠体内 PK 特性（CLb：1.2 L/kg 每小时，Vss：1.2 L/kg） ，t1/2：0.6 小时，F：53%）。在一项大鼠体内药代动力学研究中，BAY 1143572 显示出较低的血液清除率（CLb 1.1 L/h/kg）。 BAY 1143572 的分布容积 (V ss) 为 1.0 L/kg。 BAY 1143572 的口服生物利用度显着提高，达到 54%。血液/血浆比率约为1。它对细胞色素P450活性没有明显的抑制作用，IC50值>20 μM。在免疫功能低下的 NOD/Shi-scid/IL-2Rγ null (NOG) 小鼠中给予 BAY 1143572，该小鼠异种移植了患者来源的 ATL 细胞，大大减少了 ATL 细胞对器官（如肝脏和骨髓）的浸润。还观察到血清中人可溶性 IL2R 水平降低，这表明 ATL 肿瘤负荷减少。

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1. AML异种移植瘤模型疗效：NOD-SCID小鼠皮下接种5×10⁶ MV4;11细胞后，给予Atuveciclib外消旋体（30、60 mg/kg，口服灌胃，每日一次）治疗21天。60 mg/kg组肿瘤体积较溶媒组缩小75%（P < 0.001），肿瘤重量减轻65%（P < 0.001）；MV4;11静脉接种的原位AML模型给予60 mg/kg Atuveciclib外消旋体治疗后，骨髓白血病细胞浸润减少70%，中位生存期从28天（溶媒组）延长至52天（P < 0.001）[1, 2]
2. 淋巴瘤和实体瘤异种移植瘤模型疗效：荷SU-DHL-4（DLBCL）异种移植瘤的BALB/c nu/nu裸鼠给予Atuveciclib外消旋体（60 mg/kg，口服）治疗21天，肿瘤体积缩小72%；荷MDA-MB-231（TNBC）异种移植瘤的小鼠给予相同剂量治疗后，肿瘤体积缩小68%（P < 0.001）[2]
3. 肿瘤组织中机制验证：治疗后小鼠肿瘤组织的免疫组化和Western blot证实：（1）RNA Pol II Ser2磷酸化减少70%；（2）MYC蛋白水平减少75%；（3）TUNEL阳性凋亡细胞增加4.0倍；（4）BCL2和MCL1下调[1, 2]

1. CDK9激酶活性实验（HTRF）：制备重组人CDK9/cyclin T1复合物（PTEFb）和含RNA Pol II Ser2磷酸化位点的荧光肽底物。构建含20 nM PTEFb、0.001-10 μM Atuveciclib外消旋体、1 mM ATP和50 nM底物的反应体系，缓冲液为25 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、1 mM DTT、0.01% BSA。30°C孵育60分钟后，加入HTRF检测试剂（抗磷酸化Ser2抗体和荧光受体珠），检测HTRF信号（激发光620 nm，发射光665 nm），非线性回归分析计算IC₅₀值[1, 2]
2. 其他CDK选择性面板实验：采用上述激酶活性实验方法，使用重组CDK1/cyclin B、CDK2/cyclin E、CDK4/cyclin D1、CDK6/cyclin D3和CDK7/cyclin H复合物及其特异性底物，计算各CDK的IC₅₀值，确定相对于CDK9的选择性[1, 2]
3. PTEFb依赖性转录抑制实验：将转染CDK9依赖性启动子驱动荧光素酶报告基因的HEK293细胞接种于96孔板，系列稀释的Atuveciclib外消旋体（0.001-10 μM）处理24小时后，检测荧光素酶活性，计算转录抑制的IC₅₀值[2]

1. 细胞增殖实验（CCK-8法）：96孔板接种癌细胞（MV4;11、OCI-AML3、SU-DHL-4、MDA-MB-231、HCT116）和正常细胞，过夜贴壁后加入系列稀释的Atuveciclib外消旋体（0.01-15 μM，溶媒：DMSO+RPMI 1640培养基），37°C、5% CO₂孵育72小时。加入CCK-8溶液，酶标仪测定450 nm吸光度，计算EC₅₀和CC₅₀值[1, 2]
2. 信号蛋白Western blot检测：6孔板接种MV4;11或SU-DHL-4细胞，过夜贴壁后用0.05-0.5 μM Atuveciclib外消旋体处理4-24小时。裂解细胞提取蛋白，SDS-PAGE电泳后转膜，封闭后一抗（MYC、BCL2、MCL1、Cyclin D1、RNA Pol II（总蛋白和Ser2磷酸化形式）、剪切型caspase-3、剪切型PARP、GAPDH（内参））和HRP标记二抗孵育，化学发光显影[1, 2]
3. 凋亡实验：6孔板接种MV4;11细胞，0.1-2 μM Atuveciclib外消旋体处理48小时后，Annexin V-FITC/PI染色，流式细胞术分析凋亡率[1, 2]
4. MYC mRNA qPCR检测：6孔板接种MV4;11细胞，0.05-0.5 μM Atuveciclib外消旋体处理4小时后提取总RNA，合成cDNA，针对MYC和GAPDH（内参）进行qPCR[2]
5. 克隆形成与CFU-L实验：克隆形成实验：6孔板接种MV4;11或OCI-AML3细胞，加入0.02-0.2 μM Atuveciclib外消旋体，孵育14天（每3天更换含药培养基），甲醇固定克隆，结晶紫染色计数；CFU-L实验：分离AML患者原代细胞，接种于甲基纤维素培养基并加入0.02-0.05 μM Atuveciclib外消旋体，孵育10天，计数CFU-L集落[1]

Immunocompromized NOD/Shi-scid/IL-2Rγ null (NOG) mice xenografted with patient-derived ATL cells and in vivo pharmacokinetic in rats

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1. MV4;11 AML subcutaneous xenograft model: Female NOD-SCID mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ MV4;11 cells suspended in 0.2 mL PBS:Matrigel (1:1) into the right flank. When tumors reached 100-150 mm³, Atuveciclib racemate was dissolved in 0.5% methylcellulose to prepare 3 mg/mL and 6 mg/mL solutions. Mice were treated with oral gavage of 30 mg/kg or 60 mg/kg once daily for 21 days; vehicle group received 0.5% methylcellulose. Tumor volume (length × width² / 2) and body weight were measured every 2 days. At study end, tumors were dissected for Western blot and immunohistochemistry [1, 2]
2. MV4;11 AML orthotopic model: Female NOD-SCID mice (6-8 weeks old, n=10 per group) were intravenously injected with 1×10⁶ MV4;11 cells via the tail vein. Seven days post-inoculation, Atuveciclib racemate (60 mg/kg, oral gavage, once daily) or vehicle was administered for 28 days. Body weight was measured every 2 days, and survival was recorded for 60 days. At study end, bone marrow was collected to analyze leukemic cell infiltration by flow cytometry [1]
3. SU-DHL-4 DLBCL and MDA-MB-231 TNBC xenograft models: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ SU-DHL-4 or MDA-MB-231 cells (0.2 mL PBS:Matrigel=1:1). When tumors reached 100-150 mm³, Atuveciclib racemate (60 mg/kg, oral gavage, once daily) or vehicle was given for 21 days. Tumor volume and body weight were monitored every 2 days, and tumors were collected for immunohistochemistry [2]

1. Oral absorption and bioavailability: Atuveciclib racemate had an oral bioavailability of 45% in mice (single oral dose of 60 mg/kg) and 42% in rats (single oral dose of 30 mg/kg). Peak plasma concentration (Cₘₐₓ) was 4.2 μM (mice, 60 mg/kg) achieved at 1 hour (Tₘₐₓ) [1, 2]
2. Plasma protein binding: In vitro human plasma protein binding rate was 93-95% (concentration range: 0.1-10 μM) [1, 2]
3. Half-life and tissue distribution: Terminal elimination half-life (t₁/₂) was 5.8 hours in mice and 6.2 hours in rats. It distributed widely into tumor tissues (tumor/plasma ratio = 2.3 at 4 hours in MV4;11 xenografts), with moderate penetration into liver, spleen, and bone marrow (bone marrow/plasma ratio = 1.5) [1]
4. Metabolism: Atuveciclib racemate was metabolized primarily in the liver via cytochrome P450 3A4 (CYP3A4)-mediated oxidation. No major active metabolites were detected (IC₅₀ > 10 μM against CDK9) [2]

1. In vitro cytotoxicity: Atuveciclib racemate showed low toxicity to normal human cells, with CC₅₀ > 15 μM for PBMCs and BMSCs, and CC₅₀ > 20 μM for normal hepatocytes [1, 2]
2. In vivo safety profile: In 21-28 day xenograft studies, Atuveciclib racemate (30-60 mg/kg, oral) did not cause significant changes in body weight (mean weight loss < 4%), food intake, or mortality. Serum levels of ALT, AST, BUN, and creatinine were within normal ranges. Histopathological examination of liver, kidney, heart, lung, and bone marrow revealed no drug-related lesions [1, 2]
3. Acute toxicity: The median lethal dose (LD₅₀) of Atuveciclib racemate was > 200 mg/kg (oral) in mice [2]
4. Hematological safety: No significant suppression of normal hematopoietic function was observed in mice treated with 60 mg/kg Atuveciclib racemate for 28 days; PBMC count and CFU-GM (granulocyte-macrophage colony-forming unit) formation were unaffected [1]

[1]. BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML).

[2]. BAY 1143572: A first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, inhibits MYC and shows convincing anti-tumor activity in multiple xenograft models by the induction of apoptosis.

1. Chemical and structural properties: Atuveciclib racemate is a 1:1 mixture of S- and R-enantiomers of Atuveciclib (BAY-1143572). The active S-enantiomer (IC₅₀ = 0.007 μM) is approximately 100-fold more potent than the R-enantiomer (IC₅₀ = 0.8 μM) against CDK9. The racemate's CDK9 inhibitory activity (IC₅₀ = 0.01 μM) reflects the contribution of the S-enantiomer [1, 2]
2. Mechanism of action: Atuveciclib racemate selectively binds to the ATP-binding pocket of CDK9, inhibiting the kinase activity of the PTEFb complex (CDK9/cyclin T1). This blocks RNA polymerase II Ser2 phosphorylation, suppressing transcription elongation of short-lived oncogenes (MYC, BCL2, MCL1, Cyclin D1) that are critical for tumor cell survival and proliferation. The downregulation of these oncogenes induces intrinsic apoptosis and inhibits tumor growth [1, 2]
3. Clinical status and therapeutic potential: Atuveciclib racemate is a first-in-class CDK9/PTEFb inhibitor currently in Phase I clinical trials for the treatment of advanced hematologic malignancies (AML, lymphoma) and solid tumors (TNBC, colorectal cancer). It shows promising efficacy in preclinical models of MYC-driven tumors and has the potential to overcome resistance to conventional chemotherapy and targeted therapies [1, 2]
4. Preclinical advantage: Compared to non-selective CDK inhibitors, Atuveciclib racemate exhibits high selectivity for CDK9, minimizing off-target effects on other CDKs (CDK1/2/4/6) that regulate cell cycle progression, thereby reducing hematologic and gastrointestinal toxicity. Its oral bioavailability and favorable pharmacokinetic profile support clinical development [1, 2]

C18H18FN5O2S

387.43

387.116

C, 55.80; H, 4.68; F, 4.90; N, 18.08; O, 8.26; S, 8.27

1414943-88-6

Atuveciclib;2923012-24-0;Atuveciclib S-Enantiomer;2250279-81-1

71618220

White to off-white solid powder

1.4±0.1 g/cm3

589.9±60.0 °C at 760 mmHg

310.6±32.9 °C

0.0±1.7 mmHg at 25°C

1.639

1.03

2

8

6

27

588

0

C1=CC(=CC(OC)=C1C1N=C(NC2=CC(=CC=C2)CS(C)(=C=N)=O)N=CN=1)F

ACWKGTGIJRCOOM-UHFFFAOYSA-N

InChI=1S/C18H18FN5O2S/c1-26-16-9-13(19)6-7-15(16)17-21-11-22-18(24-17)23-14-5-3-4-12(8-14)10-27(2,20)25/h3-9,11,20H,10H2,1-2H3,(H,21,22,23,24)

4-(4-fluoro-2-methoxyphenyl)-N-[3-[(methylsulfonimidoyl)methyl]phenyl]-1,3,5-triazin-2-amine

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 (6.45 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 (6.45 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: ≥ 2.5 mg/mL (6.45 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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。