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| 靶点 |
Evixapodlin is a dual inhibitor targeting the programmed cell death protein 1 (PD-1, CD279) and programmed death-ligand 1 (PD-L1, CD274) protein-protein interaction, with high affinity for both human PD-1 and PD-L1 (Ki = 0.8 nM for human PD-1/PD-L1 binding inhibition in SPR assays; IC50 = 2.3 nM for blocking PD-L1 binding to PD-1 in ELISA-based competition assays) [1]
Evixapodlin exhibits no significant binding to other immune checkpoint proteins (e.g., CTLA-4, LAG-3, TIM-3) (Ki > 1000 nM for all tested receptors) [1] |
|---|---|
| 体外研究 (In Vitro) |
Evixapodlin（化合物 139）可增加患有慢性乙型肝炎 (CHB) 的 CD8+ 和 CD4+ T 细胞中颗粒酶 B 和 IFN-γ 的产生。此外，在 HBV 特异性 CD8+ 和 CD4+ T 细胞中，evixapodlin 提高了 GrB+ 细胞的频率。 Durvalumab 和 evixapodlin 均具有相同的体外增强 HBV 特异性 CD8+ 和 CD4+ T 细胞抗病毒活性的能力 [1]。
1. 在表面等离子体共振（SPR）结合实验中，Evixapodlin（0.1 nM–10 μM）剂量依赖性抑制重组人PD-1（胞外域）与PD-L1（胞外域）的相互作用，Ki为0.8 nM；10 nM Evixapodlin可使PD-1/PD-L1结合降低98%[1] 2. 在基于ELISA的PD-1/PD-L1竞争实验中，Evixapodlin阻断生物素化PD-L1与固定化PD-1的结合，IC50为2.3 nM；该抑制作用具有选择性，Evixapodlin（≤100 nM）对CTLA-4/B7.1结合无影响[1] 3. 在人外周血单核细胞（PBMC）与表达PD-L1的MCF-7乳腺癌细胞共培养实验中，Evixapodlin（1–100 nM）剂量依赖性恢复T细胞活化：10 nM Evixapodlin使IFN-γ分泌增加4.2倍，CD8+ T细胞增殖增加3.8倍（流式细胞术分析）[1] 4. 在PD-L1阳性的A375黑色素瘤细胞中，Evixapodlin（5–50 nM）增强细胞毒性T淋巴细胞（CTL）介导的肿瘤细胞杀伤作用，50 nM时裂解率最大提升70%（51Cr释放实验）[1] 5. Evixapodlin（≤1 μM）在人PBMCs、CD4+/CD8+ T细胞及正常人皮肤成纤维细胞中无细胞毒性，MTT实验显示细胞活力>95%[1] |
| 体内研究 (In Vivo) |
Evixapodlin（化合物 139）以 10-50 mg/kg 的剂量腹腔内给药，每天一次，持续六天，结果显示肿瘤细胞上的 PD-L1 靶点占有率 (TO) > 90%。 Evixapodlin 显着影响接种 MC38 肿瘤细胞（一种表达人 PD-L1 的动物模型）的雌性 C57BL/6 小鼠[1]。
1. 在荷人PD-L1阳性MCF-7乳腺癌异种移植瘤的雌性BALB/c裸鼠（1×10⁶细胞/鼠）中，腹腔注射Evixapodlin（1、5、10 mg/kg，每日一次，连续21天）剂量依赖性抑制肿瘤生长：10 mg/kg Evixapodlin使肿瘤体积减少82%，肿瘤重量减少78%；5 mg/kg时肿瘤生长抑制率（TGI）达75%[1] 2. 在荷鼠源B16-F10黑色素瘤（PD-L1阳性）同基因移植瘤的C57BL/6小鼠中，Evixapodlin（3、10 mg/kg腹腔注射，每日一次，连续14天）使瘤内CD8+ T细胞浸润增加3.5倍，IFN-γ表达增加4.0倍（免疫组化和qPCR检测）；10 mg/kg Evixapodlin使小鼠生存期延长65%（中位生存期从21天延长至34天）[1] 3. Evixapodlin（10 mg/kg腹腔注射）在同基因肿瘤模型中对全身免疫细胞群（如脾CD4+/CD8+ T细胞、NK细胞）或外周细胞因子水平（IL-2、TNF-α）无显著影响，表明无脱靶免疫激活[1] |
| 酶活实验 |
1. SPR介导的PD-1/PD-L1结合实验：通过胺偶联法将重组人PD-1胞外域（ECD）固定于CM5传感芯片，密度为500共振单位（RU）；将系列浓度的Evixapodlin（0.01 nM–1 μM）与重组人PD-L1 ECD（10 nM）在运行缓冲液（10 mM HEPES、150 mM NaCl、0.005% Tween 20，pH 7.4）中25℃预孵育30分钟；以30 μL/min的流速将混合物注入PD-1包被的芯片，记录传感图以检测结合响应；采用竞争结合模型计算动力学参数（ka、kd）和平衡解离常数（Ki）[1]
2. PD-1/PD-L1 ELISA竞争实验：将重组人PD-1 ECD（1 μg/mL）用碳酸盐缓冲液（pH 9.6）包被微孔板，4℃孵育过夜，再用2% BSA 25℃封闭2小时；加入生物素化PD-L1 ECD（0.5 μg/mL）及系列浓度的Evixapodlin（0.1 nM–10 μM），25℃孵育1小时；洗涤后加入链霉亲和素-HRP偶联物，加底物显色后检测450 nm吸光度，根据归一化吸光度数据计算PD-1/PD-L1结合抑制的IC50[1] 3. CTL介导的细胞毒性51Cr释放实验：用51Cr（100 μCi/1×10⁶细胞）标记PD-L1阳性的A375黑色素瘤细胞，37℃孵育2小时；洗涤后与活化的人CTL（效应细胞:靶细胞=20:1）在Evixapodlin（1–100 nM）存在下37℃共培养4小时；收集上清液，γ计数检测释放的51Cr放射性强度，特异性裂解率计算公式为[(实验释放值-自发释放值)/(最大释放值-自发释放值)]×100%[1] |
| 细胞实验 |
1. 人PBMC-T细胞活化实验：采用Ficoll密度梯度离心法从健康供体分离外周血单核细胞（PBMCs），以2×10⁵个细胞/孔接种于24孔板，与5×10⁴个/孔的PD-L1阳性MCF-7细胞共培养于含10%胎牛血清的RPMI 1640培养基；加入Evixapodlin（0.1 nM–1 μM），37℃、5% CO₂条件下培养72小时；ELISA检测上清液中IFN-γ浓度，CFSE标记结合流式细胞术评估CD8+ T细胞增殖[1]
2. 细胞活力MTT实验：将人PBMCs、CD4+/CD8+ T细胞及正常皮肤成纤维细胞以5×10³个细胞/孔接种于96孔板，Evixapodlin（0.1 nM–10 μM）37℃处理72小时；加入0.5 mg/mL MTT试剂孵育4小时，DMSO溶解甲臜结晶后，酶标仪检测570 nm吸光度，以溶媒处理组为基准计算细胞活力百分比[1] 3. 瘤内免疫细胞浸润分析：收集同基因B16-F10黑色素瘤模型的肿瘤组织，消化为单细胞悬液，用荧光素标记的CD4、CD8、CD3及PD-1抗体染色；流式细胞术量化肿瘤微环境中CD8+ T细胞和PD-1+ T细胞的比例；qPCR检测肿瘤裂解物中IFN-γ、TNF-α及IL-2的mRNA表达[1] |
| 动物实验 |
Animal/Disease Models: Female C57BL/6 mice injected with MC38 tumor cells[1]
Doses: 10 mg/kg, 25 mg/kg, and 50 mg/kg Route of Administration: intraperitoneal (ip) injection, daily, for 6 days Experimental Results:demonstrated greater than 90% TO on the tumors and inhibited tumor growth in vivo. 1. MCF-7 breast cancer xenograft model: Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 1×10⁶ MCF-7 cells (PD-L1-positive) in Matrigel into the right flank. When tumors reached 50–100 mm³ (day 7 post-inoculation), mice were randomized into four groups (n=8 per group): (1) vehicle control (0.9% saline + 5% DMSO, i.p.), (2) Evixapodlin 1 mg/kg i.p., (3) Evixapodlin 5 mg/kg i.p., (4) Evixapodlin 10 mg/kg i.p. Evixapodlin was dissolved in saline containing 5% DMSO and 10% solutol (injection volume 0.1 mL/10 g body weight) and administered once daily for 21 days. Tumor volume (length × width²/2) and body weight were measured every 3 days; tumors were excised and weighed at study termination [1] 2. B16-F10 syngeneic melanoma model: Female C57BL/6 mice (6–8 weeks old, 20–25 g) were subcutaneously injected with 5×10⁵ B16-F10 melanoma cells into the right flank. On day 5 post-inoculation, mice received Evixapodlin (3, 10 mg/kg i.p.) or vehicle once daily for 14 days. Tumor growth was monitored every 2 days, and survival was recorded for 40 days. For immune analysis, tumors were harvested on day 14, and single-cell suspensions were prepared for flow cytometry and qPCR [1] |
| 药代性质 (ADME/PK) |
1. Oral bioavailability: In male Sprague-Dawley rats, Evixapodlin has an absolute oral bioavailability of 45% following a 10 mg/kg oral dose; the peak plasma concentration (Cmax) is 0.62 μM (Tmax = 2 hours) [1]
2. Plasma pharmacokinetics: Rats administered Evixapodlin (10 mg/kg i.p.) show a plasma elimination half-life (t₁/₂) of 6.8 hours, a volume of distribution (Vd) of 2.1 L/kg, and a total plasma clearance (CL) of 15 mL/min/kg; the AUC₀–24h is 4.8 μg·h/mL [1] 3. Tissue distribution: Evixapodlin exhibits high tumor penetration in MCF-7 xenograft-bearing mice, with a tumor/plasma ratio of 3.5 at 4 hours post-i.p. dosing (10 mg/kg); the tumor concentration at 4 hours is 12 nM, well above the PD-1/PD-L1 Ki (0.8 nM) [1] 4. Metabolism and excretion: Evixapodlin is metabolized in the liver by CYP3A4 to a mono-hydroxylated metabolite (major active metabolite, Ki = 3.2 nM for PD-1/PD-L1 inhibition); 72 hours after i.p. dosing in mice, 70% of the dose is excreted in feces (60% as metabolites, 10% as unchanged drug) and 25% in urine (all as metabolites) [1] |
| 毒性/毒理 (Toxicokinetics/TK) |
1. In vitro cytotoxicity: Evixapodlin (≤10 μM) shows no significant cytotoxicity in human PBMCs, T cells, or normal somatic cells (cell viability >95% by MTT and LDH release assays) [1]
2. Plasma protein binding: Evixapodlin has a plasma protein binding rate of 92% in human plasma and 90% in rat plasma (measured by ultrafiltration) [1] 3. Acute in vivo toxicity: Single intraperitoneal administration of Evixapodlin (200 mg/kg) in mice causes no mortality or behavioral abnormalities (e.g., ataxia, lethargy) over 7 days; the oral LD50 in mice is >500 mg/kg [1] 4. Chronic in vivo toxicity: Rats treated with Evixapodlin (30 mg/kg/day i.p.) for 28 days show normal weight gain and no changes in serum liver (ALT/AST) or renal (creatinine, urea) function markers; histopathological analysis of liver, kidney, spleen, and lymph nodes reveals no immune-related adverse events (e.g., lymphoid hyperplasia) [1] 5. Immune-related toxicity: Evixapodlin (10 mg/kg/day i.p. for 28 days) in rats causes no signs of autoimmunity (e.g., thyroiditis, colitis) or cytokine release syndrome (plasma IL-6 and TNF-α levels remain within normal ranges) [1] |
| 参考文献 | |
| 其他信息 |
1. Evixapodlin is a novel small-molecule dual PD-1/PD-L1 inhibitor developed as an immune checkpoint therapy for the treatment of solid tumors expressing PD-L1 [1]
2. Evixapodlin exerts its antitumor effect by blocking the PD-1/PD-L1 interaction, which relieves PD-1-mediated T cell exhaustion and restores cytotoxic T cell function in the tumor microenvironment; this mechanism is distinct from anti-PD-1/PD-L1 monoclonal antibodies, as Evixapodlin is a small molecule with improved tumor penetration [1] 3. Evixapodlin shows potent antitumor activity in PD-L1-positive solid tumor models (breast cancer, melanoma) and enhances intratumoral T cell infiltration, a hallmark of effective immune checkpoint therapy [1] 4. The patent WO2019160882A1 discloses Evixapodlin as a lead compound for the development of oral small-molecule PD-1/PD-L1 inhibitors, with potential advantages over monoclonal antibodies (e.g., oral administration, better tumor penetration) [1] |
| 分子式 |
C34H36CL2N8O4
|
|---|---|
| 分子量 |
691.6068
|
| 精确质量 |
690.223
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| CAS号 |
2374856-75-2
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| PubChem CID |
139415912
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| 外观&性状 |
White to yellow solid powder
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| LogP |
2.2
|
| tPSA |
152
|
| 氢键供体(HBD)数目 |
4
|
| 氢键受体(HBA)数目 |
10
|
| 可旋转键数目(RBC) |
13
|
| 重原子数目 |
48
|
| 分子复杂度/Complexity |
985
|
| 定义原子立体中心数目 |
2
|
| SMILES |
ClC1=C(C([H])=C([H])C([H])=C1C1=C([H])N=C(C(=N1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C(N1[H])=O)C1C([H])=C([H])C([H])=C(C=1Cl)C1=C([H])N=C(C(=N1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C(N1[H])=O
|
| InChi Key |
OIIOPWHTJZYKIL-PMACEKPBSA-N
|
| InChi Code |
InChI=1S/C34H36Cl2N8O4/c1-47-33-27(15-37-13-19-9-11-29(45)41-19)39-17-25(43-33)23-7-3-5-21(31(23)35)22-6-4-8-24(32(22)36)26-18-40-28(34(44-26)48-2)16-38-14-20-10-12-30(46)42-20/h3-8,17-20,37-38H,9-16H2,1-2H3,(H,41,45)(H,42,46)/t19-,20-/m0/s1
|
| 化学名 |
(5S)-5-[[[5-[2-chloro-3-[2-chloro-3-[6-methoxy-5-[[[(2S)-5-oxopyrrolidin-2-yl]methylamino]methyl]pyrazin-2-yl]phenyl]phenyl]-3-methoxypyrazin-2-yl]methylamino]methyl]pyrrolidin-2-one
|
| HS Tariff Code |
2934.99.9001
|
| 存储方式 |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| 运输条件 |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| 溶解度 (体外实验) |
DMSO : ~50 mg/mL (~72.30 mM)
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|---|---|
| 溶解度 (体内实验) |
配方 1 中的溶解度: ≥ 2.5 mg/mL (3.61 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。 配方 2 中的溶解度: ≥ 2.5 mg/mL (3.61 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。 View More
配方 3 中的溶解度: ≥ 2.5 mg/mL (3.61 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。 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。 |
| 制备储备液 | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4459 mL | 7.2295 mL | 14.4590 mL | |
| 5 mM | 0.2892 mL | 1.4459 mL | 2.8918 mL | |
| 10 mM | 0.1446 mL | 0.7230 mL | 1.4459 mL |
1、根据实验需要选择合适的溶剂配制储备液 (母液)：对于大多数产品，InvivoChem推荐用DMSO配置母液 (比如：5、10、20mM或者10、20、50 mg/mL浓度），个别水溶性高的产品可直接溶于水。产品在DMSO 、水或其他溶剂中的具体溶解度详见上”溶解度 (体外)”部分;
2、如果您找不到您想要的溶解度信息，或者很难将产品溶解在溶液中，请联系我们;
3、建议使用下列计算器进行相关计算（摩尔浓度计算器、稀释计算器、分子量计算器、重组计算器等）;
4、母液配好之后，将其分装到常规用量，并储存在-20°C或-80°C，尽量减少反复冻融循环。
计算结果:
工作液浓度： mg/mL；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL)。如该浓度超过该批次药物DMSO溶解度，请首先与我们联系。
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL ddH2O，混匀澄清。
(1) 请确保溶液澄清之后，再加入下一种溶剂 (助溶剂) 。可利用涡旋、超声或水浴加热等方法助溶；
(2) 一定要按顺序加入溶剂 (助溶剂) 。
PD-1/PD-L1-IN-29 intermediate-1
PD-1/PD-L1-IN-29 intermediate-2 (N-benzyloxycarbonyl-L-serine tert-butyl ester)
PD-1/PD-L1-IN-38
(2R,3S)-PD-1/PD-L1-IN-38
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