Enitociclib (VIP152; BAY-1251152) is a highly potent and selective inhibitor of cyclin-dependent kinase 9 (CDK9) in complex with cyclin T1 (CycT1), a core component of positive transcription elongation factor b (P-TEFb). - Ki for human CDK9/CycT1: 0.3 nM (surface plasmon resonance, SPR) [3]
- IC₅₀ for human CDK9/CycT1 kinase activity: 0.8 nM (homogeneous time-resolved fluorescence, HTRF assay) [3]
- High selectivity over other CDKs/kinases: IC₅₀ > 1000 nM for CDK1/CycB, CDK2/CycE, CDK4/CycD1, CDK6/CycD3, CDK7/CycH; inhibition rate < 10% for 290+ other kinases at 100 nM [3]
- In cellular assays, it inhibits P-TEFb-mediated phosphorylation of RNA polymerase II (RNAPII) at Ser2 with IC₅₀ = 1.2 nM [3]

1. 跨癌种抗增殖活性： - 血液系统恶性肿瘤（文献3）：IC₅₀（72小时CellTiter-Glo实验）——MV-4-11（急性髓系白血病AML，0.9 nM）、Raji（伯基特淋巴瘤，1.2 nM）、Jurkat（T细胞白血病，1.5 nM）、U266（多发性骨髓瘤MM，2.3 nM）[3]
- MYC⁺淋巴瘤细胞（文献5）：IC₅₀——SU-DHL-4（0.8 nM）、OCI-Ly10（1.1 nM）、Farage（1.3 nM）；维奈托克耐药SU-DHL-4细胞（IC₅₀=0.9 nM，与亲本细胞相当）[5]
- 多发性骨髓瘤（MM）细胞（文献6）：IC₅₀——RPMI-8226（1.1 nM）、U266（1.5 nM）、MM.1S（1.8 nM）；患者来源CD138⁺原代MM细胞（IC₅₀范围：1.2–3.5 nM）[6]
- 实体瘤（文献3）：IC₅₀——HCT116（结直肠癌，3.5 nM）、A549（肺癌，4.8 nM）、MDA-MB-231（乳腺癌，5.2 nM）[3]

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2. 转录调控与癌蛋白下调： - MYC⁺淋巴瘤细胞（SU-DHL-4，文献5）：1 nM 埃尼托昔利布处理4小时，p-RNAPII（Ser2）降低70%，MYC蛋白降低65%（蛋白质印迹法）；5 nM处理8小时，MCL1降低70%，BCL2降低60%[5]
- MM细胞（RPMI-8226，文献6）：2 nM处理6小时，p-RNAPII（Ser2）降低65%，MCL1降低60%，BCL2降低55%；qPCR显示短半衰期转录本（MYC、MCL1）降低40–50%，对管家基因（GAPDH、ACTB）无影响[6]
- AML细胞（MV-4-11，文献3）：5 nM处理8小时，MYC降低70%，MCL1降低65%[3]

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3. 凋亡诱导： - SU-DHL-4细胞（文献5）：1 nM处理24小时，Annexin V⁺细胞比例达35%；5 nM处理达80%（流式细胞术）；5 nM浓度下caspase-3/7活性升高4.2倍[5]
- RPMI-8226细胞（文献6）：2 nM处理24小时，Annexin V⁺细胞达55%；5 nM处理48小时，TUNEL⁺细胞增加70%（免疫荧光）[6]
- MV-4-11细胞（文献3）：10 nM处理24小时，Annexin V⁺细胞达85%[3]

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4. 早期数据（文献1、2、4）： - WO2014076091A1：埃尼托昔利布（当时称“化合物X”）抑制CDK9/CycT1（IC₅₀<5 nM）及Raji细胞增殖（IC₅₀<5 nM）[1]
- Cancer Res 2017/AACR 2017：对AML细胞具有强效P-TEFb抑制活性（IC₅₀<1 nM）及MYC下调作用[2][4]

1. MYC⁺淋巴瘤异种移植模型（文献5）： - 模型：SU-DHL-4细胞（1×10⁶个）皮下接种于NSG小鼠；肿瘤体积达100–150 mm³时开始给药。 - 药效：静脉注射（IV）埃尼托昔利布（10、20 mg/kg，每周1次，连续3周，溶解于10% DMSO/40% PEG400/50%生理盐水）： - 10 mg/kg：肿瘤生长抑制率（TGI）=85%，无完全消退（CR）； - 20 mg/kg：TGI=98%，6只小鼠中5只实现CR（肿瘤<50 mm³持续>28天）； - 机制：20 mg/kg组肿瘤组织中p-RNAPII（Ser2）降低75%，MYC降低80%（vs溶媒组）[5]

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2. 多发性骨髓瘤异种移植模型（文献6）： - 皮下模型（RPMI-8226，NSG小鼠）：IV 埃尼托昔利布（15、30 mg/kg，每周1次，连续4周）： - 15 mg/kg：TGI=78%，中位生存期延长30%； - 30 mg/kg：TGI=92%，中位生存期延长50%（从35天延长至53天）； - 骨转移模型（RPMI-8226经尾静脉接种，NSG小鼠）：30 mg/kg IV每周1次，连续4周，溶骨性病变（μCT检测）减少65%，骨髓肿瘤负荷（流式细胞术）减少70%[6]

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3. 血液系统/实体瘤异种移植模型（文献3）： - MV-4-11（AML，SCID小鼠）：40 mg/kg IV每周1次，连续3周：TGI=95%，8只小鼠中3只CR； - Raji（淋巴瘤，裸鼠）：20 mg/kg IV每周1次，连续3周：TGI=80%，8只小鼠中1只CR； - HCT116（结直肠癌，裸鼠）：40 mg/kg IV每周1次，连续3周：TGI=78%[3]

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4. 药效学（PD）-药代动力学（PK）相关性（文献3、5）： - 给药后血浆浓度>5 nM（高于细胞IC₅₀）持续>24小时，与最大TGI相关；24小时时肿瘤组织药物浓度为血浆浓度的2.3–2.8倍[3][5]

1. CDK9/CycT1激酶活性实验（HTRF，文献3）： - 反应体系（25 μL）：50 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、1 mM DTT、200 μM ATP、0.5 μg/mL生物素化RNAPII Ser2肽底物、0.1 nM重组人CDK9/CycT1，及埃尼托昔利布（0.01–100 nM）。 - 孵育：37°C孵育60分钟；加入25 μL终止缓冲液（50 mM HEPES pH 7.5、0.2 M EDTA、链霉亲和素-XL665、Eu³⁺标记抗p-RNAPII Ser2抗体）终止反应。 - 检测：测定时间分辨荧光共振能量转移（TR-FRET）信号（615 nm为Eu³⁺发射光，665 nm为XL665发射光）；通过剂量-反应曲线计算IC₅₀[3]

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2. CDK9/CycT1结合实验（SPR，文献3）： - 重组人CDK9/CycT1（5 μg/mL）通过胺偶联固定于CM5传感芯片。 - 埃尼托昔利布（0.1–100 nM）在运行缓冲液（10 mM HEPES pH 7.4、150 mM NaCl、0.05% Tween-20）中以30 μL/min流速进样。 - 记录结合相（120秒）与解离相（300秒）信号；采用1:1结合模型计算Ki[3]

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3. 激酶选择性谱实验（文献3）： - 埃尼托昔利布（100 nM）通过放射性或发光实验检测对290余种激酶的抑制活性；除CDK9外，其他激酶抑制率均<10%[3]

1. 细胞活力实验（CellTiter-Glo，文献3、5、6）： - 细胞以1×10³个/孔接种于384孔板，培养过夜。 - 加入埃尼托昔利布（0.01–100 nM，10点稀释）；37°C、5% CO₂孵育72小时。 - 加入等体积CellTiter-Glo试剂；检测发光值。通过四参数逻辑回归计算IC₅₀[3][5][6]

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2. p-RNAPII/癌蛋白蛋白质印迹实验（文献3、5、6）： - 细胞（5×10⁵个/mL）经埃尼托昔利布（0.1–10 nM）处理4–8小时。 - RIPA缓冲液（含蛋白酶/磷酸酶抑制剂）裂解细胞；30 μg蛋白经SDS-PAGE分离后转移至PVDF膜。 - 膜与一抗（抗p-RNAPII Ser2、抗MYC、抗MCL1、抗β-肌动蛋白）及HRP标记二抗孵育；ECL法显影条带，光密度法量化[3][5][6]

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3. 凋亡实验（Annexin V/PI，文献3、5、6）： - 细胞经埃尼托昔利布（1–10 nM）处理24小时。 - PBS洗涤后，Annexin V-FITC/PI室温染色15分钟。 - 流式细胞术分析：量化Annexin V⁺/PI⁻（早期凋亡）与Annexin V⁺/PI⁺（晚期凋亡）细胞比例[3][5][6]

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4. 原代MM细胞实验（文献6）： - 磁珠分选法从患者骨髓中分离CD138⁺细胞。 - 以5×10⁴个/孔接种，埃尼托昔利布（0.1–10 nM）处理72小时。 - 台盼蓝排斥法检测活力；计算IC₅₀[6]

1. MYC⁺ lymphoma xenograft (SU-DHL-4, NSG mice): - 6–8-week-old female NSG mice (18–22 g) implanted subcutaneously with 1×10⁶ SU-DHL-4 cells (50% Matrigel/PBS). - Randomized into 3 groups (n=6/group): vehicle (10% DMSO/40% PEG400/50% saline), Enitociclib 10 mg/kg, 20 mg/kg. - IV injection via tail vein, once weekly for 3 weeks. - Tumor volume (V = 0.5×length×width²) measured twice weekly; mice euthanized if tumor > 1500 mm³ or weight loss > 20% [5]

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2. MM xenografts: - Subcutaneous model (RPMI-8226, NSG mice): 2×10⁶ cells (50% Matrigel/PBS) implanted subcutaneously. Dosed IV (15, 30 mg/kg, weekly × 4 weeks); tumor/weight monitored as above [6]
- Bone metastasis model (RPMI-8226, NSG mice): 5×10⁵ cells injected via tail vein. Dosed IV (30 mg/kg, weekly × 4 weeks); bone lesions assessed by μCT, bone marrow tumor burden by flow cytometry [6]

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3. AML/lymphoma/solid tumor xenografts: - MV-4-11 (SCID mice): 5×10⁶ cells (50% Matrigel/PBS) implanted subcutaneously; IV Enitociclib (10–40 mg/kg, weekly × 3 weeks) [3]
- Raji (nude mice): 1×10⁷ cells (PBS) implanted subcutaneously; IV 20 mg/kg weekly × 3 weeks [3]
- HCT116 (nude mice): 5×10⁶ cells (50% Matrigel/PBS) implanted subcutaneously; IV 40 mg/kg weekly × 3 weeks [3]

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4. PK study: - Female CD-1 mice (n=3/time point) administered Enitociclib 20 mg/kg IV. - Blood sampled at 0.083–24 h post-dose; plasma separated by centrifugation. - Drug concentration measured by LC-MS/MS; PK parameters: t₁/₂ = 6.8 h, CL = 12.3 mL/min/kg, Vss = 5.2 L/kg [3]

1. Preclinical PK in rodents/dogs: - Mice (IV 20 mg/kg): t₁/₂ = 6.8 h, CL = 12.3 mL/min/kg, Vss = 5.2 L/kg [3]
- Rats (IV 10 mg/kg): t₁/₂ = 8.2 h, CL = 9.5 mL/min/kg, Vss = 4.8 L/kg [3]
- Dogs (IV 5 mg/kg): t₁/₂ = 12.5 h, CL = 5.1 mL/min/kg, Vss = 3.9 L/kg [3]

2. Oral bioavailability: - Low oral absorption: F < 5% (mice), F < 3% (rats) due to poor solubility and first-pass metabolism; IV administration selected for clinical development [3]

3. Tissue distribution: - Mice (IV 20 mg/kg, 2 h post-dose): Drug concentrations (ng/g) – liver (1200), kidney (850), tumor (SU-DHL-4, 620) vs. plasma (270 ng/mL) [3][5]

4. Metabolism/excretion: - Metabolized primarily by human CYP3A4; major metabolites inactive (no CDK9 inhibition at 100 nM) [3]
- Rats (IV dose): 65% excreted in feces (30% unchanged), 20% in urine (5% unchanged) within 72 h [3]

1. Repeat-dose toxicity: - Rats (IV 2, 5, 10 mg/kg, daily × 14 days): NOAEL = 5 mg/kg; 10 mg/kg caused transient weight loss (≤10%), mild gastric mucosa hyperplasia, and platelet reduction (≤20%) [3]
- Dogs (IV 1, 3, 6 mg/kg, weekly × 4 weeks): NOAEL = 3 mg/kg; 6 mg/kg caused mild anemia (Hb ↓15%), lymphopenia (↓25%), and reversible ALT elevation (×2) [3]

2. Cardiac/genotoxicity: - No hERG inhibition (IC₅₀ > 10 μM, patch-clamp); no QT prolongation in dogs (≤6 mg/kg) [3]
- Negative in Ames test, in vitro micronucleus assay, and in vivo comet assay [3]

3. Plasma protein binding: - 98.5% (human plasma), 97.8% (mouse plasma), 98.2% (dog plasma) (equilibrium dialysis) [3]

4. Toxicity in efficacy studies: - MYC⁺ lymphoma model (20 mg/kg IV): No weight loss (>95% of initial weight) or organ histopathology (liver, kidney, heart) [5]
- MM model (30 mg/kg IV): No significant changes in ALT, AST, BUN, or creatinine vs. vehicle [6]

[1]. WO2014076091Al.

[2]. Cancer Res (2017) 77 (13_Supplement): 984. https://aacrjournals.org/cancerres/article/77/13_Supplement/984/622158/Abstract-984-Identification-of-potent-and-highly

[3]. Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer. J Med Chem. 2021 Aug 12;64(15):11651-11674.

[4]. Identification of potent and highly selective PTEFb inhibitor BAY 1251152 for the treatment of cancer: from p.o. to i.v. application via scaffold hops [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meet. https://aacrjournals.org/cancerres/article/77/13_Supplement/984/622158/Abstract-984-Identification-of-potent-and-highly

[5]. Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription. Cancer Res Commun. 2023 Nov 9;3(11):2268-2279.

[6]. Enitociclib, a selective CDK9 inhibitor: in vitro and in vivo preclinical studies in multiple myeloma. Blood Neoplasia, 2025, 2(1): 100050.

1. Mechanism of action: - Enitociclib inhibits CDK9/CycT1 (P-TEFb), blocking RNAPII Ser2 phosphorylation and transcription elongation. This selectively downregulates short-half-life oncoproteins (MYC, MCL1, BCL2) critical for cancer survival, without affecting housekeeping genes [3][5][6]

2. Therapeutic focus: - Developed for hematological malignancies (AML, MYC⁺ lymphoma, MM) and solid tumors (colorectal, lung cancer); prioritizes MYC-driven or therapy-resistant (venetoclax-resistant) cancers [2][3][5][6]

3. Formulation rationale: - IV formulation (10% DMSO/40% PEG400/50% saline) selected due to low oral bioavailability. Weekly dosing is supported by PK/PD data (sustained concentrations above IC₅₀ and minimal cumulative toxicity) [3]

4. Patent and early development: - WO2014076091A1: Discloses Enitociclib’s chemical structure (pyrazolopyrimidine derivative) and initial CDK9 inhibitory activity [1]
- Cancer Res 2017/AACR 2017: Confirms efficacy in AML xenografts and P-TEFb inhibition [2][4]

5. Clinical relevance: - Efficacy in patient-derived MM cells and bone metastasis models supports potential for treating relapsed/refractory MM [6]
- CR in MYC⁺ lymphoma models addresses unmet need for MYC-targeted therapies [5]

C19H18F2N4O2S

404.433629512787

404.111

C, 56.43; H, 4.49; F, 9.40; N, 13.85; O, 7.91; S, 7.93

1610358-53-6

Enitociclib;1610358-56-9; 1610408-96-2 (R-isomer); 1610408-97-3 (S-isomer); 1610358-53-6; 1610358-56-9 (+); 1610358-59-2 (-); 1610368-59-6 (R-isomer HCl); 1610368-60-9 (S-isomer HCl); (-)-Enitociclib;1610358-59-2

74767009

White to light yellow solid powder

4.2

96.3

2

8

6

28

619

0

S(C)(CC1C=CN=C(C=1)NC1C=C(C(=CN=1)F)C1C=CC(=CC=1OC)F)(=N)=O

YZCUMZWULWOUMD-UHFFFAOYSA-N

InChI=1S/C19H18F2N4O2S/c1-27-17-8-13(20)3-4-14(17)15-9-19(24-10-16(15)21)25-18-7-12(5-6-23-18)11-28(2,22)26/h3-10,22H,11H2,1-2H3,(H,23,24,25)

5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-[4-[(methylsulfonimidoyl)methyl]pyridin-2-yl]pyridin-2-amine

(±)-BAY-1251152; (±)-BAY 1251152; (±)-BAY1251152; (-)-BAY-1251152; BAY-1251152; BAY 1251152; BAY1251152; (+)-BAY-1251152

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)

DMSO: ~50 mg/mL (~123.6 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.18 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 (6.18 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 (6.18 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;
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