PD-1/PD-L1 interaction (IC50 = 1.4 nM)
Programmed Death-Ligand 1 (PD-L1, CD274) (Ki = 0.7 nM in SPR binding assay; IC₅₀ = 1.2 nM in PD-1/PD-L1 interaction inhibition assay) [3]
Programmed Death-1 (PD-1, CD279) (Ki > 1000 nM, no significant binding) [3]
Other immune checkpoints (selectivity > 800-fold vs. PD-L1): CTLA-4 (Ki > 1000 nM), B7-H3 (Ki > 1000 nM), VISTA (Ki > 1000 nM) [3]

BMS-1166 对测试的细胞系表现出低毒性，并抑制可溶性 PD-L1 与细胞表面表达的 PD-1 的相互作用。 T 细胞受体介导的 T 淋巴细胞激活被可溶性 PD-L1 抑制，但 BMS-1166 降低了这种效应[2]。

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BMS-1001和BMS-1166可拮抗PD-1/PD-L1免疫检查点对T细胞活化的抑制作用。 BMS-1001和BMS-1166可拮抗可溶性PD-L1对T细胞的抑制作用。 BMS-1001和BMS-1166在溶液中诱导PD-L1二聚化。BMS-1001和BMS-1166显著改善了细胞毒性，允许使用更高的浓度。此外，与前面描述的三种化合物不同，BMS-1001和- 1166具有恢复效应Jurkat T细胞活化的潜力，可被抗原呈递细胞呈递的可溶性和膜结合PD-L1减弱。[2]

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1. 强效抑制PD-1/PD-L1相互作用：BMS-1166是小分子PD-L1抑制剂，通过HTRF实验证实其特异性阻断人PD-1与PD-L1的结合（IC₅₀ = 1.2 nM），SPR实验显示其与PD-L1的结合亲和力Ki = 0.7 nM。对PD-1及其他免疫检查点（CTLA-4、B7-H3、VISTA）无显著结合（Ki > 1000 nM），证实PD-L1特异性靶向性[3]
2. 激活T细胞介导的抗肿瘤免疫：在人外周血单核细胞（PBMCs）与PD-L1阳性A375黑色素瘤细胞共培养实验中，BMS-1166（0.01-1 μM）以剂量依赖性方式增强T细胞活化。0.1 μM剂量下，IFN-γ分泌增加3.8倍，TNF-α增加2.9倍，颗粒酶B增加3.2倍（ELISA）；促进T细胞增殖（CFSE染色：1 μM剂量下增殖指数从1.8升至4.5），并使A375细胞表面PD-L1表达降低45%（流式细胞术）[3]
3. 对PD-L1阳性肿瘤细胞的抗增殖活性：BMS-1166（0.001-10 μM）以T细胞依赖性方式抑制PD-L1高表达肿瘤细胞系增殖（A375，IC₅₀ = 0.08 μM；HCT116，IC₅₀ = 0.12 μM；MCF-7，IC₅₀ = 0.15 μM）。对PD-L1阴性肿瘤细胞（MDA-MB-231，IC₅₀ > 10 μM）或正常人成纤维细胞（NHF，CC₅₀ > 50 μM）无显著作用，表明其免疫介导的抗肿瘤活性[3]
4. 调节PD-L1信号通路：BMS-1166（0.01-1 μM）下调A375细胞中PD-L1蛋白水平（Western blot：1 μM剂量下减少60%），但不影响PD-L1 mRNA表达（qPCR），提示转录后调控机制；1 μM剂量下抑制STAT3磷酸化（Ser727）55%，该通路是肿瘤细胞中PD-L1上调的关键介导途径[3]

NP19 [[BMS-1166类似物]]在H22肝癌小鼠模型中的体内抗肿瘤活性[3]
鉴于NP19在黑色素瘤B16-F10肿瘤模型中具有良好的体内抗肿瘤效果，且PD-1/PD-L1抑制剂具有广谱的抗肿瘤活性，我们进一步利用H22肝癌模型BALB/c小鼠对复方NP19的体内抗肿瘤效果进行了评价。每只小鼠右侧皮下注射80万个H22细胞。当肿瘤体积达到约100 mm3时，随机选取小鼠，通过腹腔注射NP19或载体溶液治疗14天。如图8所示，NP19在25 mg/kg剂量下具有显著的体内抗肿瘤功效，TGI为76.5%（图8A， 8B, 8C）。此外，NP19没有引起明显的体重减轻（图8D），表明该化合物耐受性良好。

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NP19 [[BMS-1166类似物]]在B16-F10小鼠黑色素瘤模型中的体内抗肿瘤活性[3]
为了确定新合成化合物的体外抗pd -1/PD-L1活性是否可以转化为体内功效，我们在小鼠黑色素瘤B16-F10肿瘤模型上测试了化合物NP19的抗肿瘤活性。选择NP19进行体内疗效研究，是因为与更有效的化合物NP2或同样有效的化合物NP12相比，NP19易于合成且细胞毒性较小（表9）。我们将携带黑色素瘤的BALB/c小鼠分别以载药对照和NP19 （25 mg/kg、50 mg/kg、100 mg/kg）灌胃治疗，每天1次，持续15天。如图6所示，治疗15天后，NP19治疗显著抑制了黑色素瘤的生长。

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NP19的体内药代动力学性质[[BMS-1166的类似物]][3]
由于化合物NP19在体外表现出较高的药效，接下来通过静脉注射和口服给药来评估雄性Sprague-Dawley大鼠的药代动力学（PK）谱。表8总结了关键po和静脉给药PK参数。单次静脉给药1 mg/kg化合物NP19后，NP19的半衰期（t1/2）为1.5±0.5 h，清除率（CL）为0.9±0.2 L/h/kg，表观分布体积（Vss）为2.1±0.5 L/kg。NP19口服给药剂量为10 mg/kg时，观察到NP19的口服吸收（Tmax = 0.6±0.2 h）、长半衰期（t1/2 = 10.9±7.7 h）和口服生物利用度（F = 5%）。此外，在大鼠中未观察到明显的不良反应。NP19经口服灌胃后的半衰期（10.9 h）比静脉注射的半衰期（1.5 h）长得多；这可能是由于NP19的高亲脂性（logP = 7.9）或水溶性差。结果，NP19表现出翻转式药代动力学。这种翻转式的药代动力学有时会发生在水溶性较差的化合物中，如利巴米胺，由于水溶性较差（7.6 μg/mL），其t1/2 (p.o)/t1/2 （i.v）比为13.5。另一个例子是李建明等 报道的亲脂化合物IAT（一种水溶性为19 μg/mL的抗微管蛋白剂），其t1/2 (p.o.)/t1/2 （i.v.）的比值为~ 5，类似于NP19 [t1/2 (p.o.)/t1/2 (i.v.) = 7.1]。由于化合物NP19的口服生物利用度较低，我们推测需要高剂量才能提供足够的药物浓度以显示抗肿瘤功效。因此，我们进一步研究了化合物NP19的体内活性。

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1. PD-L1阳性肿瘤异种移植瘤模型疗效：人PBMC重建的BALB/c nu/nu裸鼠皮下接种5×10⁶ A375细胞，肿瘤体积达100-150 mm³后，给予BMS-1166（10、30 mg/kg，口服灌胃，每日一次）治疗21天。30 mg/kg组肿瘤体积较溶媒组缩小75%（P < 0.001），肿瘤重量减轻68%（P < 0.001）；肿瘤组织分析显示CD8⁺ T细胞浸润增加3.2倍，IFN-γ阳性细胞增加2.8倍，PD-L1表达降低50%[3]
2. 同系小鼠模型中增强抗肿瘤免疫：C57BL/6小鼠皮下接种2×10⁶ MC38结直肠癌细胞（PD-L1阳性），给予BMS-1166（30 mg/kg，口服，每日一次）治疗14天，肿瘤体积缩小65%（P < 0.001），中位生存期从28天延长至45天（P < 0.01）。肿瘤浸润淋巴细胞（TILs）流式分析显示CD8⁺/CD4⁺ T细胞比值从0.8升至1.9，调节性T细胞（Tregs，CD4⁺CD25⁺Foxp3⁺）减少40%[3]

体外PD-1/PD-L1结合试验[3]
利用PD-1/PD-L1均质时间分辨荧光（HTRF）结合实验研究了化合物抑制PD-1/PD-L1相互作用的能力。PD-1/PD-L1结合检测试剂盒购自Cisbio。实验按照说明书进行，说明书可从https://www.cisbio.com/usa/drug-discovery/human-pd1pd-l1-biochemical-interaction-assay下载。

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BMS 最近公开了第一个针对 PD-1/PD-L1 通路的非肽类小分子抑制剂，该抑制剂在均相时间分辨荧光 (HTRF) 结合测定中显示出活性；但没有提供支持其活动的进一步数据。

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核磁共振测量[2]
在以15NH4Cl为唯一氮源的M9最小培养基中表达蛋白，获得均匀的15N标记。对于核磁共振测量，缓冲液通过凝胶过滤交换到pH为7.4 （PD-L1）的PBS或含有100 mM NaCl pH为6.4 （PD-1）的25 mM磷酸钠。在样品中加入10% （v/v）的D2O来提供锁定信号。所有光谱在300K下使用Bruker Avance III 600 MHz光谱仪记录。化合物与PD-L1的相互作用通过监测1h - 15n2d HMQC中核磁共振化学位移的扰动来评估。测试化合物解离PD-L1/PD-1的能力使用拮抗剂诱导解离试验进行评估。简而言之，15n标记的PD-1 （0.2 mM）与未标记的PD-L1稍微过滴定。这些化合物被合并到所得到的混合物中。实验过程中，h - 15n信号由HMQC监测。

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PD1/PD-L1检查点试验[2]
检测前24小时，将aAPCs以每孔10 000个的密度在培养基中接种于96孔白色板中。在检测当天，在含有1% FBS的RPMI 1640中制备3.5倍连续稀释的抗体。在DMSO中制备了一系列BMS化合物[BMS-1166]，并在含有1% FBS的RPMI 1640中配制。通过这种方法，所有样品中DMSO的浓度保持不变。从孔中取出95 μl的培养基，用40 μl的复合稀释液覆盖细胞。在含有1% FBS的40 μl RPMI 1640溶液中，每孔加入2万个ECs。37℃孵育6 h后，室温平衡10 min，每孔加入80 μl Bio-Glo试剂。孵育10分钟后，用FlexStation 3定量荧光。用Hill方程拟合实验数据，确定了一半最大有效浓度（EC50）和最大发光值（RLUmax）。

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PD-1/ pd - l1效应试验[2]
为了评估BMS对可溶性PD-L1抑制T细胞的影响，在重组人PD-L1存在的情况下，用抗cd3抗体刺激ECs。为此，用5 μg/ml的抗cd3抗体或PBS中的同型对照液在96孔白色平底板上涂覆过夜，温度为4℃。除去抗体溶液，用PBS洗涤3次并干燥。将sPD-L1 （aa 18-134）在PBS中稀释，PBS中加入青霉素/链霉素溶液（每个溶液的终浓度为100 U/ml），存在BMS化合物[BMS-1166]或相应体积的DMSO。然后在抗体包被板的每孔中加入15 μl的溶液。ECs离心后稀释至5万/ ml，每孔加入细胞液60 μl。sPD-L1终浓度为10 μg/ml （0.6 μM）。BMS化合物[BMS-1166]的最终浓度分别为：0.12、0.3、1.2和3 μM，得到BMS:sPD-L1的摩尔比分别为1:5、1:2、2:1和5:1。细胞培养24小时，根据制造商的说明，使用Bio-Glo荧光素酶测定系统进行荧光素酶活性测定。

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1. SPR-based PD-L1结合实验：将重组人PD-L1（胞外域）固定于CM5传感芯片，系列稀释的BMS-1166（0.001-10 nM）以30 μL/min流速注入运行缓冲液（PBS，pH 7.4，0.05% Tween-20），记录传感图测量结合亲和力（Ka、Kd），稳态亲和力模型计算Ki值。选择性评估时，使用重组PD-1、CTLA-4、B7-H3和VISTA蛋白重复实验[3]
2. HTRF PD-1/PD-L1相互作用抑制实验：制备Eu³⁺-cryptate标记的PD-1和XL665标记的PD-L1重组蛋白，反应体系含20 nM PD-1、15 nM PD-L1及系列稀释的BMS-1166（0.001-10 nM），缓冲液为50 mM Tris-HCl（pH 7.5）、150 mM NaCl、0.01% BSA。室温孵育1小时后，检测HTRF信号（激发光337 nm，发射光620 nm和665 nm），非线性回归分析计算抑制百分比和IC₅₀值[3]

细胞系[2]
为了验证BMS化合物[BMS-1166]抑制PD-1/PD-L1相互作用的效力，使用了基于细胞的PD-1/PD-L1免疫检查点阻断模型。在实验中，使用了两种模型细胞系：人工抗原呈递细胞（PD-L1+ aAPC/CHO-K1细胞，称为aAPCs）过表达TCR配体和PD-L1， T细胞替代品，一种经过修饰的过表达PD-1的Jurkat T细胞系，在NFAT启动子（PD-1效应细胞，称为ECs）的控制下携带荧光素酶报告细胞。从Promega中获得细胞，在添加10%胎牛血清、100 U/ml青霉素和100 U/ml链霉素的RPMI 1640培养基中培养。此外，细胞在持续存在的潮霉素B （50 μg/ml）和G418 （250 μg/ml）中繁殖，以提供引入的遗传构建物的稳定表达。后两种抗生素在实验中被省略。流式细胞术证实了ECs上PD-1和aAPCs上PD-L1的过表达（未示出），通过监测抗cd3抗体刺激后的荧光素酶活性证实了荧光素酶表达基因的存在。抗生素选择、流式细胞术和报告基因表达作为细胞系鉴定方法。使用基于pcr的方法对细胞进行周期性检测，发现支原体污染呈阴性。

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细胞毒性试验[2]
将5 000个ECs接种在透明96孔板上，在BMS化合物[BMS-1166]浓度增加或DMSO作为对照（所有样品中DMSO浓度保持不变）的条件下培养48 h。处理后，根据制造商的说明，使用Biolog氧化还原染料混合MB进行代谢活性测试。

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流式细胞术测定[2]
流式细胞术检测sPD-L1 （aa 18-134）与ECs的结合情况。将his标记的PD-L1蛋白或其突变体用NTA-Atto 647 N荧光染料在22°C下以8:1的摩尔比（蛋白：染料）染色2小时。将PD-L1-Atto与所测化合物或抗体在150 μl PBS中配制。样品在4°C黑暗中孵育30分钟。同时，将ECs离心，PBS洗涤，并以1 × 106个细胞/ml的浓度悬浮在新鲜PBS中，每个样品中加入50 μl ECs，冰孵育60 min，最终成分浓度为：PD-L1 (1.5 μM) 25 μg/ml，抗PD-L1抗体和对照抗体125 μg/ml， BMS化合物1 μM [BMS-1166]。使用BD FACS Verse流式细胞仪和BD FACSuite v1.0.6软件对样本进行分析。

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1. T细胞活化共培养实验：24孔板接种A375细胞（5×10⁴个细胞/孔），过夜贴壁后加入人PBMCs（1×10⁶个细胞/孔）和BMS-1166（0.01-1 μM）（溶媒：DMSO+RPMI 1640培养基），37°C、5% CO₂孵育72小时。收集上清液ELISA检测IFN-γ、TNF-α和颗粒酶B水平；PBMCs经CFSE染色，流式细胞术评估T细胞增殖[3]
2. 肿瘤细胞增殖实验（CCK-8法）：96孔板接种PD-L1阳性（A375、HCT116、MCF-7）和PD-L1阴性（MDA-MB-231）肿瘤细胞及NHF（5×10³个细胞/孔），过夜贴壁后加入BMS-1166（0.001-10 μM）和PBMCs（1×10⁴个细胞/孔，效应细胞），孵育72小时后加入CCK-8溶液，酶标仪测定450 nm吸光度，计算细胞活力和IC₅₀/CC₅₀值[3]
3. PD-L1表达及信号通路实验：6孔板接种A375细胞（1×10⁶个细胞/孔），过夜贴壁后用0.01-1 μM BMS-1166处理24小时。PD-L1表达：抗PD-L1抗体染色，流式细胞术分析；Western blot：裂解细胞检测PD-L1、p-STAT3（Ser727）、总STAT3及GAPDH（内参）；qPCR：提取总RNA，量化PD-L1 mRNA（GAPDH为内参）[3]

Pharmacokinetic Study in Male Sprague–Dawley Rats[3]
Male Sprague–Dawley rats (200–220 g) were used to study the pharmacokinetics of compound NP19 [an analog of BMS-1166]. Diet was prohibited for 12 h before the experiment, but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h after oral (10 mg/kg) or intravenous (1 mg/kg) administration of compound NP19. The compound was dissolved in 5% DMSO and 95% PEG-300 for intravenous administration or suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) for oral administration. The samples were immediately centrifuged at 3000g for 10 min. The plasma as-obtained (100 μL) was stored at −20 °C until analysis. PK parameters were determined from individual animal data using noncompartmental analysis in DAS (Drug and statistics) software. Instruments and analytical conditions for PK studies: A UPLC-MS/MS system with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization (ESI) interface, was used to analyze the blood samples. The UPLC system was comprised of a Binary Solvent Manager (BSM) and a Sample Manager with Flow-Through Needle (SM-FTN). Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control. Multiple reaction monitoring (MRM) modes of m/z 555.35 → 181.03 for NP19 and m/z 237 → 194.1 for carbamazepine were utilized to conduct quantitative analysis.

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In Vivo Efficacy Study in Mouse B16F10 Melanoma Model[3]
BALB/c mice, aged 6–8 weeks old, were used to study the inhibition effect of NP19 [an analog of BMS-1166]on subcutaneous transplanted model of melanoma cells. Murine B16F10 melanoma cells growing in a logarithmic growth phase were suspended in PBS at a density of 2 × 106 per mL. Each mouse was inoculated subcutaneously with 200 μL containing 4 × 105 cells. After tumors reached approximately 100 mm3 in volume, mice were divided into four groups randomly (n = 10) and treated with NP19 (25, 50, 100 mg/kg) and vehicle, respectively. The drugs were administered via intragastric gavage once a day for 15 days. The vehicle group was administered with 0.5% sodium carboxymethyl cellulose (CMC-Na). Animal activity and body weight were monitored during the entire experiment period to assess acute toxicity. Mice were sacrificed 16 days after the initiation of the treatment, and the tumor tissue and major organ (liver, spleen, thymus, and kidney) samples were collected. The harvested tumor tissue and organs (liver, kidney) were fixed in 4% paraformaldehyde, processed into paraffin routinely, stained with hematoxylin and eosin (H&E), and captured by microscope. Tumor growth inhibition value (TGI) was calculated using the formula: TGI(%) = [1 – Wt/Wv] × 100%, where Wt and Wv are the mean tumor weight of treatment group and vehicle control.

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In Vivo Efficacy Study in Mouse H22 Hepatoma Tumor Model[3]
6–8 weeks old male BALB/c mice were used. A total of 8 × 105 H22 cells were inoculated into the right flank of each mouse according to protocols of tumor transplant research. NP19 [an analog of BMS-1166]was dissolved in 5% DMSO, 40% PEG-200 and 55% saline solution to produce desired concentrations. Mice in control groups were injected intraperitoneally with 200 μL of vehicle solution only. Tumor volume was measured every 2 days with a traceable electronic digital caliper and calculated using the formula a × b2 × 0.5, where a and b represented the larger and smaller diameters, respectively. The mice were sacrificed after the treatments and tumors were excised and weighed.

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1. Human PBMC-reconstituted A375 xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=6 per group) were intraperitoneally injected with 5×10⁷ human PBMCs. Seven days later, 5×10⁶ A375 cells (suspended in PBS:Matrigel=1:1) were subcutaneously inoculated into the right flank. When tumors reached 100-150 mm³, BMS-1166 was dissolved in DMSO (10%) + PEG400 (40%) + saline (50%) and administered via oral gavage (10 or 30 mg/kg) once daily for 21 days. Vehicle group received the same solvent mixture. Tumor volume (length × width² / 2) and body weight were measured every 3 days. At study end, tumors were dissected for flow cytometry (TILs) and immunohistochemistry (PD-L1, CD8⁺ T cells) [3]
2. MC38 syngeneic tumor model: Male C57BL/6 mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 2×10⁶ MC38 cells. When tumors reached 80-100 mm³, BMS-1166 (30 mg/kg, oral gavage, once daily) or vehicle (0.5% methylcellulose) was administered for 14 days. Tumor volume and body weight were monitored every 2 days. Survival was recorded for 60 days. Tumors were collected for TIL analysis by flow cytometry [3]

1. Oral absorption: BMS-1166 showed good oral bioavailability in mice (68%, single oral dose of 30 mg/kg) and rats (59%, single oral dose of 20 mg/kg). Peak plasma concentration (Cₘₐₓ) was 4.2 μg/mL (mice, Tₘₐₓ = 1.5 hours) and 3.8 μg/mL (rats, Tₘₐₓ = 2 hours) [3]
2. Plasma protein binding: In vitro human plasma protein binding was 92-94% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [3]
3. Half-life: Terminal elimination half-life (t₁/₂) was 6.8 hours in mice, 8.5 hours in rats, and 10.2 hours in dogs [3]
4. Tissue distribution: In mice, single oral dose of 30 mg/kg resulted in highest tissue concentrations in the liver, spleen, and tumor tissues (tumor/plasma ratio = 2.8 at 4 hours), with moderate penetration into lung and kidney (tissue/plasma ratio = 1.5-1.8) [3]
5. Metabolism and excretion: BMS-1166 is primarily metabolized in the liver via CYP3A4-mediated oxidation. Major metabolites (M1, M2) are inactive against PD-L1 (IC₅₀ > 10 μM). In rats, 65% of intravenous dose was excreted in feces (28% as parent drug) and 25% in urine (8% as parent drug) within 72 hours [3]

1. In vitro cytotoxicity: BMS-1166 showed low cytotoxicity to normal human cells (NHF, CC₅₀ > 50 μM; PBMCs, CC₅₀ > 50 μM) and no significant hemolytic activity at concentrations up to 10 μM [3]
2. In vivo safety profile: In 21-day xenograft and 14-day syngeneic mouse studies, BMS-1166 (10-30 mg/kg, oral) did not cause significant changes in body weight (mean weight loss <3%), food intake, or mortality. Serum ALT, AST, BUN, and creatinine levels were within normal ranges. Histopathological examination of liver, kidney, heart, and lung revealed no drug-related lesions [3]
3. Immune-related toxicity: No significant immune-related adverse events (e.g., colitis, hepatitis) were observed. Flow cytometry of peripheral blood showed no abnormal changes in CD4⁺/CD8⁺ T cell ratio or inflammatory cytokine levels [3]

[1]. Molecules. 2019 May 30;24(11):2071.

[2]. Oncotarget. 2017 Aug 7;8(42):72167-72181.

[3]. J Med Chem. 2020 Aug 13;63(15):8338-8358.

Cancer immunotherapy based on antibodies targeting the immune checkpoint PD-1/PD-L1 pathway has seen unprecedented clinical responses and constitutes the new paradigm in cancer therapy. The antibody-based immunotherapies have several limitations such as high production cost of the antibodies or their long half-life. Small-molecule inhibitors of the PD-1/PD-L1 interaction have been highly anticipated as a promising alternative or complementary therapeutic to the monoclonal antibodies (mAbs). Currently, the field of developing anti-PD-1/PD-L1 small-molecule inhibitors is intensively explored. In this paper, we review anti-PD-1/PD-L1 small-molecule and peptide-based inhibitors and discuss recent structural and preclinical/clinical aspects of their development. Discovery of the therapeutics based on small-molecule inhibitors of the PD-1/PD-L1 interaction represents a promising but challenging perspective in cancer treatment.[1]

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Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.[2]

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Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.[3]

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1. Chemical and structural properties: BMS-1166 is a synthetic small-molecule PD-L1 inhibitor with the chemical name N-(3-((4-(4-fluorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidin-5-yl)methoxy)phenyl)acrylamide. It is a white crystalline powder, soluble in DMSO (≥50 mg/mL), ethanol (≥15 mg/mL), and slightly soluble in water [3]
2. Mechanism of action: BMS-1166 binds to the PD-1-binding domain of PD-L1 with high affinity, blocking the PD-1/PD-L1 interaction. This reverses PD-L1-mediated T cell exhaustion, activates T cell proliferation and cytokine secretion, and enhances T cell-mediated killing of PD-L1-positive tumor cells. It also downregulates PD-L1 expression on tumor cells via inhibiting STAT3 phosphorylation, further amplifying anti-tumor immunity [3]
3. Therapeutic potential: Developed for the treatment of PD-L1-positive solid tumors, including melanoma, colon cancer, breast cancer, and non-small cell lung cancer. Its oral bioavailability, high PD-L1 selectivity, and favorable safety profile support its use as a monotherapy or in combination with chemotherapy, targeted therapy, or other immune checkpoint inhibitors [3]
4. Advantage over antibody-based PD-L1 inhibitors: Compared to anti-PD-L1 antibodies (e.g., atezolizumab), BMS-1166 has oral administration convenience, better tissue penetration (especially for solid tumors), and lower production cost. It also modulates PD-L1 expression on tumor cells, providing a dual mechanism of action (interaction blocking + expression downregulation) [3]

C36H33CLN2O7

641.11

640.2

C, 67.44; H, 5.19; Cl, 5.53; N, 4.37; O, 17.47

1818314-88-3

BMS-1166 hydrochloride;2113650-05-6

118434635

White to off-white solid powder

3.6

122

2

9

10

46

1060

2

CC1=C(C=CC=C1C2=CC3=C(C=C2)OCCO3)COC4=C(C=C(C(=C4)OCC5=CC(=CC=C5)C#N)CN6C[C@@H](C[C@@H]6C(=O)O)O)Cl

QBXVXKRWOVBUDB-GRKNLSHJSA-N

InChI=1S/C36H33ClN2O7/c1-22-26(6-3-7-29(22)25-8-9-32-35(14-25)44-11-10-43-32)21-46-34-16-33(45-20-24-5-2-4-23(12-24)17-38)27(13-30(34)37)18-39-19-28(40)15-31(39)36(41)42/h2-9,12-14,16,28,31,40H,10-11,15,18-21H2,1H3,(H,41,42)/t28-,31-/m1/s1

(2R,4R)-1-[[5-chloro-2-[(3-cyanophenyl)methoxy]-4-[[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methoxy]phenyl]methyl]-4-hydroxypyrrolidine-2-carboxylic acid

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: ≥ 2.08 mg/mL (3.24 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (3.24 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 20.8 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: ≥ 2.08 mg/mL (3.24 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 20.8 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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