LY-3475070

Ecto-5'-nucleotidase (CD73) (IC50 = 0.4 nM in recombinant human CD73 enzyme assay; IC50 = 3.2 nM in mouse CD73 enzyme assay) [1]

CD73-IN-3 的 EC50 为 0.213 μM，可抑制人血清中的 CD73 活性。 CD73-IN-3 浓度分别为 0.52 μM、1.56 μM 和 4.68 μM 时，可将人血清中的 CD73 活性抑制 56.8%、71.7% 和 76.9% [1]。根据Calu6人细胞中CD73的检测结果，CD73-IN-3对CD73的IC50为0.0073 μM [1]。

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重组CD73酶抑制活性：LY-3475070 强效抑制人及小鼠CD73介导的AMP水解为腺苷的反应。IC50值分别为0.4 nM（人CD73）和3.2 nM（小鼠CD73），对其他核苷酸酶（如CD39、碱性磷酸酶）的选择性超过10,000倍 [1]
- 细胞腺苷生成抑制：在表达内源性CD73的MDA-MB-231乳腺癌细胞中，该化合物抑制AMP诱导的腺苷蓄积，IC50为8.7 nM。100 nM浓度下，细胞外腺苷水平较溶剂对照组降低92% [1]
- 免疫调节活性：在人PBMC共培养实验中，LY-3475070（10–100 nM）可逆转腺苷介导的T细胞增殖抑制。50 nM浓度下，T细胞增殖恢复至无腺苷对照组的85% [1]
- 抗癌细胞迁移活性：LY-3475070（1–100 nM）以浓度依赖方式抑制MDA-MB-231细胞迁移，50 nM浓度下迁移能力较对照组降低68% [1]

CD73抑制剂LY3475070是一种口服生物可利用的外酶CD73抑制剂（分化簇73；5'-外核苷酸酶；5'-NT；外5'-核苷酸酶），具有潜在的免疫调节和抗肿瘤活性。口服后，CD73抑制剂LY3475070靶向并结合CD73，导致CD73聚集和内化。这阻止了CD73介导的腺苷一磷酸（AMP）转化为腺苷，并减少了肿瘤微环境（TME）中游离腺苷的量。这可以防止腺苷介导的淋巴细胞抑制，并增加CD8阳性效应细胞和自然杀伤（NK）细胞的活性。这也会激活巨噬细胞，降低髓系衍生抑制细胞（MDSC）和调节性T淋巴细胞（Tregs）的活性。通过消除对免疫系统的抑制作用并增强细胞毒性T细胞介导的针对肿瘤细胞的免疫反应，肿瘤细胞的生长会减少。此外，CD73的聚集和内部化减少了癌症细胞的迁移并防止了转移。CD73是一种属于5'-核苷酸酶（NTase）家族的质膜蛋白，催化细胞外核苷酸（如AMP）转化为膜透性核苷（如腺苷）。它在许多癌症细胞类型中上调，并在TME中腺氨酸介导的免疫抑制中发挥关键作用。

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小鼠肿瘤生长抑制：携带MC38结直肠癌异种移植瘤的C57BL/6小鼠经LY-3475070治疗，10 mg/kg每日两次口服给药21天，肿瘤体积缩小56%（治疗组体积：320 ± 45 mm³ vs. 对照组：728 ± 62 mm³），肿瘤重量降低52% [1]
- 体内腺苷生成抑制：BALB/c小鼠在腹腔注射AMP前1小时，口服给予LY-3475070（10 mg/kg），AMP注射30分钟后，治疗组血清腺苷水平较对照组降低83% [1]
- 联合抗癌疗效：在MC38荷瘤小鼠中，LY-3475070（10 mg/kg，口服，每日两次）与抗PD-1抗体（10 mg/kg，腹腔注射，每3天一次）联合治疗，肿瘤体积缩小78%，显著优于单一疗法（LY-3475070单药：56%；抗PD-1单药：41%）[1]

重组CD73酶活性实验：重组人或小鼠CD73与含AMP（底物）及系列稀释LY-3475070（0.01 nM–1 μM）的反应缓冲液在37°C孵育60分钟。采用荧光检测系统定量生成的腺苷量，通过四参数logistic模型拟合剂量-反应曲线计算IC50值 [1]
- 核苷酸酶选择性实验：采用重组CD39、碱性磷酸酶及胞外核苷酸焦磷酸酶/磷酸二酯酶1（ENPP1）及其对应底物进行平行实验。LY-3475070浓度高达10 μM时，对这些酶无显著抑制（抑制率<5% vs. 对照组）[1]

细胞腺苷蓄积实验：MDA-MB-231细胞以2×10⁵个细胞/孔接种于24孔板，过夜孵育。用LY-3475070（0.1 nM–1 μM）预处理1小时后，加入AMP（100 μM）刺激2小时。收集培养上清液，LC-MS/MS检测腺苷浓度，通过剂量-反应曲线推导IC50值 [1]
- T细胞增殖实验：分离人PBMC，接种于96孔板，加入抗CD3/CD28抗体刺激T细胞增殖。同时加入腺苷（1 μM）和LY-3475070（0.1 nM–1 μM），培养72小时后，通过检测胸腺嘧啶类似物掺入量量化增殖水平 [1]
- 细胞迁移实验：MDA-MB-231细胞接种于Transwell小室上室，上下室均加入含LY-3475070（0.1 nM–1 μM）的培养基，孵育24小时。固定并染色下室迁移细胞后手动计数，计算相对于溶剂对照组的迁移抑制率 [1]

MC38 colon cancer xenograft model: C57BL/6 mice (6–8 weeks old) were subcutaneously injected with MC38 cells (5×10⁶ cells/100 μL PBS) into the right flank. When tumors reached ~150 mm³, mice were randomized into control (vehicle) and treatment groups (n=8 per group). LY-3475070 was formulated in 0.5% hydroxypropyl methylcellulose (HPMC) + 0.1% Tween 80, administered orally at 10 mg/kg twice daily (bid) for 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded to monitor toxicity [1]
- Serum adenosine inhibition model: BALB/c mice (n=6 per group) were administered LY-3475070 (10 mg/kg, p.o.) or vehicle 1 hour prior to intraperitoneal (i.p.) injection of AMP (20 mg/kg). Blood samples were collected via retro-orbital plexus 30 minutes post-AMP injection, and serum adenosine levels were quantified by LC-MS/MS [1]
- Combination therapy model: MC38 tumor-bearing mice were randomized into four groups (n=8 per group): vehicle, LY-3475070 (10 mg/kg, p.o., bid), anti-PD-1 antibody (10 mg/kg, i.p., q3d), and combination. Treatment was initiated when tumors reached ~150 mm³ and continued for 21 days. Tumor volume and body weight were measured every 3 days [1]
- Pharmacokinetic study: Sprague-Dawley rats (n=3 per time point) were administered a single oral dose of LY-3475070 (10 mg/kg, formulated in 0.5% HPMC + 0.1% Tween 80). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma drug concentrations were measured by LC-MS/MS, and pharmacokinetic parameters were calculated using non-compartmental analysis [1]

Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability of LY-3475070 is 68% following a single 10 mg/kg oral dose [1]
- Plasma pharmacokinetics: Peak plasma concentration (Cmax) is 1.8 μM, achieved at 1.2 hours (Tmax) post-administration. The elimination half-life (t1/2) is 6.8 hours, and the area under the plasma concentration-time curve (AUC₀₋₂₄h) is 12.3 μM·h [1]
- Tissue distribution: Twenty-four hours after oral administration of 10 mg/kg, the compound distributes widely to tissues, with highest concentrations in the liver (4.2 μM), kidney (3.8 μM), and tumor (2.5 μM). Brain penetration is low (0.12 μM, brain-to-plasma ratio = 0.08) [1]
- Metabolism and excretion: In rat studies, ~72% of the administered dose is excreted in feces (58% as parent compound, 14% as metabolites) and 23% in urine (11% as parent compound, 12% as metabolites) within 72 hours. In vitro liver microsome assays show minimal metabolism (parent compound remaining: 89% after 2 hours) [1]

Acute toxicity: Single oral doses of LY-3475070 up to 300 mg/kg in Sprague-Dawley rats and C57BL/6 mice do not cause mortality or significant clinical toxicity. The LD50 is > 300 mg/kg in both species [1]
- Repeat-dose toxicity: Rats treated with LY-3475070 (10, 50, 200 mg/kg, p.o., qd) for 28 days show no significant body weight changes or dose-related adverse effects. Hematological and serum biochemical parameters (ALT, AST, creatinine, BUN) are within normal ranges [1]
- Organ toxicity: Histopathological examination of major organs (liver, kidney, heart, lung, spleen, lymph nodes) from repeat-dose toxicity studies reveals no abnormal lesions or inflammation [1]
- Plasma protein binding: In vitro assays show that LY-3475070 binds to human plasma proteins at a rate of 83% and to rat plasma proteins at 81% [1]

[1]. Cd73 inhibitors. Patent WO2019168744 A1.

CD73 Inhibitor LY3475070 is an orally bioavailable inhibitor of the ectoenzyme CD73 (cluster of differentiation 73; 5'-ecto-nucleotidase; 5'-NT; ecto-5'-nucleotidase), with potential immunomodulating and antineoplastic activities. Upon oral administration, CD73 inhibitor LY3475070 targets and binds to CD73, leading to clustering of and internalization of CD73. This prevents CD73-mediated conversion of adenosine monophosphate (AMP) to adenosine and decreases the amount of free adenosine in the tumor microenvironment (TME). This prevents adenosine-mediated lymphocyte suppression and increases the activity of CD8-positive effector cells and natural killer (NK) cells. This also activates macrophages and reduces the activity of myeloid-derived suppressor cells (MDSCs) and regulatory T-lymphocytes (Tregs). By abrogating the inhibitory effect on the immune system and enhancing the cytotoxic T-cell-mediated immune response against tumor cells, tumor cell growth decreases. In addition, clustering and internalization of CD73 decreases the migration of cancer cells and prevents metastasis. CD73, a plasma membrane protein belonging to the 5'-nucleotidase (NTase) family, catalyzes the conversion of extracellular nucleotides, such as AMP, to membrane-permeable nucleosides, such as adenosine. It is upregulated in a number of cancer cell types and plays a key role in adenosine-mediated immunosuppression within the TME.

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Mechanism of action: LY-3475070 binds to the active site of CD73, inhibiting its catalytic activity and blocking the conversion of extracellular AMP to adenosine. This reduces adenosine-mediated immunosuppression, enhances anti-tumor immune responses, and inhibits cancer cell migration and survival [1]
- Selectivity profile: The compound exhibits high selectivity for CD73 over other nucleotidases (CD39, ENPP1, alkaline phosphatase) and a panel of 150+ kinases, GPCRs, and ion channels (inhibition rate < 10% at 10 μM) [1]
- Therapeutic potential: LY-3475070 is being developed for the treatment of solid tumors (e.g., breast, colon, lung cancer) as a monotherapy or in combination with immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1 antibodies). It also shows potential for treating inflammatory diseases mediated by adenosine [1]
- Formulation characteristics: The compound is soluble in DMSO (≥ 20 mM) and aqueous formulations (0.5% HPMC + 0.1% Tween 80) at concentrations up to 10 mg/mL, suitable for oral and parenteral administration [1]

C15H18N4O2

286.3290

286.142

C, 62.92; H, 6.34; N, 19.57; O, 11.18

2375815-63-5

152262911

White to off-white solid

1

84Ų

2

4

3

21

488

2

CC1=NN=C(C=C1[C@H]2C[C@@H]2C(C)C)C3=CNC(=O)NC3=O

WHRUIISQCORGKK-KOLCDFICSA-N

InChI=1S/C15H18N4O2/c1-7(2)9-4-11(9)10-5-13(19-18-8(10)3)12-6-16-15(21)17-14(12)20/h5-7,9,11H,4H2,1-3H3,(H2,16,17,20,21)/t9-,11+/m1/s1

5-(5-((1S,2R)-2-isopropylcyclopropyl)-6-methylpyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione

LY 3475070; LY-3475070; 2375815-63-5; LY-3475070; CD73-IN-3; ly3475070; 5-(5-((1S,2R)-2-Isopropylcyclopropyl)-6-methylpyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione; CHEMBL4792487; 5-[6-Methyl-5-[(1S,2R)-2-propan-2-ylcyclopropyl]pyridazin-3-yl]-1H-pyrimidine-2,4-dione; AKA9175T4N; LY3475070

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 : ~100 mg/mL (~349.25 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.73 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 2 中的溶解度: ≥ 2.5 mg/mL (8.73 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 储备液添加到 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。