---
title: "D(+)-Biotin–Conjugated Resorcinol Dibenzyl Ethers: Tumor-Targeted **PD-L1** Inhibitor SW-1 for Pre"
id: "frontiers-in-immunology-10-rational-design-of-d-biotin-conjugated-resorcinol-dibenzyl-ethers-as-tumor"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-10-rational-design-of-d-biotin-conjugated-resorcinol-dibenzyl-ethers-as-tumor"
content_type: "clinical_feed_article"
specialty: "General"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1879839"
published_at: "2026-09-03T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# D(+)-Biotin–Conjugated Resorcinol Dibenzyl Ethers: Tumor-Targeted **PD-L1** Inhibitor SW-1 for Pre
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-10-rational-design-of-d-biotin-conjugated-resorcinol-dibenzyl-ethers-as-tumor
- **Specialty:** [General](https://medichelpline.com/clinical-feed/general.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1879839)
- **Published At:** 2026-09-03T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- This study reports a rational hybridization strategy to create **D(+)-biotin**–conjugated resorcinol dibenzyl ether compounds aimed at tumor-selective inhibition of the **PD-1/PD-L1** immune checkpoint. - The lead compound, **SW-1**, demonstrated potent and selective disruption of PD-1/PD-L1 binding with an IC50 of 5.6 nM and high binding affinity to both human and murine PD-L1 as measured by in vitro assays and SPR. - Molecular docking and 100 ns molecular dynamics simulations supported stable binding of SW-1 at the **PD-L1** dimer interface with key intermolecular interactions, and MM/PBSA was used to estimate binding free energy. - All synthesized compounds reported had ≥95% purity by HPLC; full spectroscopic characterization (1H/13C NMR, HRMS) is provided for SW-1 through SW-7. - Pharmacokinetic studies in rats indicated favorable systemic exposure and a suitable half-life for in vivo application (primary PK parameters are reported in the source's Table 2). - In a B16-F10 melanoma syngeneic mouse model, SW-1 produced greater tumor growth inhibition (TGI = 64.9%) than a PD-L1 antibody comparator (TGI = 44.2%), increased intratumoral CD8+ T-cell infiltration, and did not cause obvious body weight loss. - Safety assessments included serum cardiac biomarkers and in vivo tolerability reporting; the authors note favorable in vivo safety without obvious weight loss (serum biomarker data are summarized in the source Table 3). - The design rationale leverages tumor-overexpression of the sodium-dependent multivitamin transporter (**SMVT**) to promote receptor-mediated uptake of the **D(+)-biotin**–tagged inhibitors, aiming to restrict systemic PD-L1 engagement and reduce immune-related adverse events associated with systemic checkpoint blockade. - The work positions SW-1 as a promising lead candidate combining target potency, tumor-directed delivery, favorable PK, and preclinical antitumor efficacy for further development in precision tumor-targeted immunotherapy.
## Clinical Analysis & Structured Key Points
Frontiers | Rational design of D(+)-biotin-conjugated resorcinol dibenzyl ethers as tumor-targeted PD-L1 inhibitors for precision cancer immunotherapy ORIGINAL RESEARCH article Front. Immunol. , 03 September 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1879839 Published in Frontiers in Immunology Cancer Immunity and Immunotherapy 7 impact factor 11.3 citescore Part of a Research Topic Remodeling the tumor microenvironment and rational combinations in early-stage cancer immunotherapy Submission open 8623 views 7 articles Editor & Reviewers Edited by H J Hongfei Jiang Reviewed by J W Jie Wang Z Z Zhengjun Zhou N F Natalie Fuchs Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Scheme 1 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Table 1 In vitro PD-1/PD-L1 and antiproliferative activity of target compounds SW1-7 a . View in article Table 2 Primary pharmacokinetic properties of SW-1 in SD rats (n = 6, Mean ± SD). View in article Table 3 Serum cardiac biomarkers (n = 6, Mean ± SD). View in article ORIGINAL RESEARCH article Front. Immunol. , 03 September 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1879839 Rational design of D(+)-biotin-conjugated resorcinol dibenzyl ethers as tumor-targeted PD-L1 inhibitors for precision cancer immunotherapy J D Jing-Jing Du 1 R L Rongling Liu 1 L C Lei Chen 1 J Z Jian Zhang 2 * 1. Hubei Provincial Key Laboratory of Occurrence and Intervention of Kidney Diseases, Hubei Provincial Engineering Research Center of Immunotherapy Drugs for Renal Tumors, Hubei Polytechnic University, School of Medicine, Huangshi, Hubei, China 2. Huangshi Central Hospital, Huangshi, China Article metrics View details Abstract Objectives: This study aims to discover and characterize novel D(+)-biotin-conjugated PD-L1 inhibitors for precision tumor-targeted cancer immunotherapy. Methods: Integrated strategies, including computer-aided molecular simulation, antiproliferative activity (CCK8) assay, surface plasmon resonance (SPR), pharmacokinetic evaluation, and in vivo antitumor efficacy assessment, were performed. Results: Compound SW-1 showed potent and selective inhibitory activity against PD-1/PD-L1 interaction with an IC 50 of 5.6 nM, and high binding affinity to both human and murine PD-L1. Molecular docking and molecular dynamics simulations verified stable binding of SW-1 to the PD-L1 dimer interface with key intermolecular interactions. Pharmacokinetic studies in rats revealed favorable systemic exposure and a suitable half-life for in vivo application. In the B16-F10 melanoma mouse model, SW-1 achieved a more potent tumor growth inhibition (TGI = 64.9%) than the PD-L1 antibody (TGI = 44.2%), effectively promoted CD8 + T cell infiltration into the tumor microenvironment, and maintained favorable in vivo safety without obvious body weight loss. Conclusions: SW-1, as a highly potent biotin-conjugated PD-L1 small-molecule inhibitor, exhibits outstanding target activity, favorable pharmacokinetic properties, potent antitumor efficacy, and high safety. These results highlight SW-1 as a promising lead candidate for further development in precision tumor-targeted immunotherapy. 1 Introduction Targeting the PD-1/PD-L1 immune checkpoint axis has become one of the most pivotal and rapidly evolving frontiers in modern anticancer drug discovery and cancer immunotherapy ( 1 – 3 ). Monoclonal antibody (mAb)-based PD-1/PD-L1 inhibitors have achieved significant clinical milestones across various solid tumors; however, their extensive therapeutic applicability remains constrained by several inherent pharmacological and safety limitations, including excessively prolonged systemic exposure (half-life often exceeding 2–3 weeks), modest objective response rates (typically 15–25% in unselected populations), and a notable occurrence of immune-related adverse events (irAEs) ( 4 – 6 ). Clinically important irAEs encompass immune-mediated colitis and, more critically, checkpoint inhibitor-associated myocarditis—a rare yet frequently fatal complication that considerably affects patient safety, treatment continuity, and long-term survival prospects ( 7 , 8 ). In light of these limitations, small-molecule PD-L1 inhibitors have emerged as a compelling and urgently needed alternative, offering distinct pharmacological advantages: oral bioavailability, tunable pharmacokinetics (including shorter half-lives enabling rapid dose adjustment or discontinuation), and reduced risk of Fc-mediated effector functions—thereby mitigating off-target immune activation and improving therapeutic controllability ( 9 – 11 ). To date, numerous structurally diverse small-molecule PD-L1 inhibitors have advanced into preclinical development and early-phase clinical trials ( Figure 1 ) ( 12 – 14 ). Several non-antibody PD-L1 modulators are currently under clinical evaluation for solid tumors, including INCB086550 (NCT03762447, Incyte), CA-170 (NCT02812875, Aurigene Oncology), and GS-4224 (NCT04049617, Gilead Sciences). Despite this progress, a fundamental challenge persists: PD-L1 is constitutively expressed not only on tumor cells but also across diverse normal tissues—including lymphoid compartments (thymus, bone marrow, spleen, lymph nodes) and vital non-lymphoid organs (heart, lung, liver, kidney). Systemic, non-selective PD-L1 blockade inevitably disrupts peripheral immune homeostasis, leading to aberrant T-cell activation against self-antigens—a mechanism directly implicated in the onset, spectrum, and severity of irAEs ( 15 – 17 ). Thus, there remains a critical unmet need for PD-L1 inhibitors engineered for tumor-selective delivery, capable of preserving physiological PD-L1 function in healthy tissues while potently inhibiting its immunosuppressive activity within the tumor microenvironment ( 18 ). Figure 1 Small molecules with structural diversity targeting the PD-1/PD-L1 pathway. Reprinted (adapted) with permission from ref ( 18 ). Copyright {2023} American Chemical Society, license number 6277100092658. Accumulating evidence demonstrates that biotin receptors—particularly the sodium-dependent multivitamin transporter (SMVT)—are significantly overexpressed in multiple malignancies, including B16F10 melanoma, A549 lung adenocarcinoma, and MCF-7 breast cancer cells ( 19 – 21 ). This differential expression renders SMVT an attractive target for tumor-selective drug delivery. D(+)-Biotin, as a high-affinity endogenous ligand for SMVT, has been widely validated as a robust tumor-homing moiety. Conjugation of bioactive scaffolds to D(+)-biotin facilitates receptor-mediated uptake and intratumoral accumulation, markedly reducing off-target distribution and associated toxicity—aligning rigorously with the principles of precision oncology and spatially resolved immunotherapy ( Figure 2 ). Importantly, emerging mechanistic studies further reveal that biotin itself modulates dendritic cell maturation and enhances CD8 + T-cell infiltration and cytotoxic function—indicating intrinsic immunostimulatory properties that synergize with PD-L1 blockade rather than merely serving as a passive targeting handle ( 18 ). Figure 2 Small molecule agents that are modified with D(+)-biotin. Reprinted (adapted) with permission from ref ( 18 ). Copyright {2023} American Chemical Society, license number 6277100092658. Our group has dedicated extensive efforts to developing small-molecule PD-L1 inhibitors. Building on structure–activity relationship (SAR) studies, we previously identified resorcinol dibenzyl ether derivatives as a privileged scaffold exhibiting potent PD-L1 binding affinity (sub-micromolar IC 50 ), robust disruption of PD-1/PD-L1 protein–protein interaction, and significant antitumor efficacy in syngeneic mouse models. Beyond monovalent inhibition, we have also pioneered bifunctional modalities—including PD-L1-targeting PROTAC degraders and a combined strategy targeting both PD-L1 and DNA-PK—establishing versatile chemical platforms and translational proof of concept for next-generation immuno-oncology agents ( 22 – 24 ). Motivated by the dual functionality of D(+)-biotin—as both a validated tumor-selective delivery vector and an endogenous immunomodulator—we herein report a rational molecular hybridization strategy to design and synthesize a novel class of D(+)-biotin-conjugated resorcinol dibenzyl ether inhibitors. This design strategically merges the high-affinity PD-L1 inhibitory core with the SMVT-targeting biotin motif to achieve spatiotemporal precision: (i) retaining uncompromised PD-L1 blockade potency at the tumor site, and (ii) restricting systemic PD-L1 engagement through preferential accumulation in SMVT-overexpressing tumor tissue. Collectively, this work introduces a functionally integrated, tumor-directed PD-L1 inhibitor prototype—one that simultaneously addresses efficacy, selectivity, and safety bottlenecks—and offers a generalizable paradigm for developing safer, smarter, and more effective immunotherapeutics. 2 Materials and methods 2.1 Chemical general methods All reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise specified. BMS-202 ( InvivoChem , Cat. No. V0770) was obtained as a positive control. Reaction progress was monitored by thin-layer chromatography (TLC), with visualization under UV light (254 nm) and/or iodine vapor staining. Column chromatography was performed on silica gel (300–400 mesh, Qingdao Haiyang Chemical Co., Ltd.) using gradient elution. ¹H and ¹³C NMR spectra were acquired on a Bruker AV-400 spectrometer (400 MHz for ¹H; 101 MHz for ¹³C), with chemical shifts reported in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard. High-resolution mass spectra (HRMS) were recorded on a Waters mass spectrometer in electrospray ionization (ESI) positive. Compound purity was assessed by high-performance liquid chromatography (HPLC) equipped with a C 18 column (2.1 × 30 mm, 5 µm). Purity was determined from the UV chromatogram at 254 nm and confirmed to be ≥95% (area %) for all reported compounds. 2.2 Characterization and analytical test results of the target compounds Compound SW-1 ( N -(4’’-(((3-amino-2,2-dimethyl-3-oxopropyl)amino)methyl)-3’’-methoxy-2’-methyl-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 10.04 (s, 1H), 8.71 (s, 1H), 7.70 (s, 1H), 7.63 – 7.53 (m, 2H), 7.45 (d, J = 7.5 Hz, 1H), 7.35 (dd, J = 18.7, 8.3 Hz, 3H), 7.28 – 7.19 (m, 2H), 7.13 – 6.95 (m, 3H), 6.42 (d, J = 29.6 Hz, 2H), 4.31 (s, 1H), 4.12 (d, J = 26.4 Hz, 3H), 3.90 (s, 3H), 3.13 (s, 1H), 2.93 (s, 2H), 2.87 – 2.79 (m, 1H), 2.60 (s, 1H), 2.34 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 1.70 – 1.37 (m, 6H), 1.21 (s, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 179.54, 171.76, 163.14, 157.72, 142.80, 142.47, 142.40, 139.66, 132.49, 131.25, 129.22, 129.09, 128.91, 126.02, 124.16, 121.57, 120.16, 118.03, 112.40, 61.46, 59.61, 56.09, 55.81, 48.01, 36.65, 28.65, 28.51, 25.51, 24.16, 18.92. HRMS m/z: calcd for C 36 H 46 N 5 O 4 S [M+H] + 644.3271, found 644.3281. HPLC: t R 12.72 min, purity 98.36%. Compound SW-2 ( N -(4’’-(((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)methyl)-3’’-methoxy-2’-methyl-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 10.02 (s, 1H), 7.70 (s, 1H), 7.55 (s, 1H), 7.48 (s, 1H), 7.40 – 7.30 (m, 2H), 7.22 (d, J = 6.6 Hz, 2H), 7.03 (dd, J = 18.8, 8.7 Hz, 3H), 6.42 (d, J = 29.3 Hz, 2H), 5.19 (s, 2H), 4.44 – 4.13 (m, 4H), 3.87 (d, J = 7.1 Hz, 3H), 3.64 (s, 6H), 3.16 (d, J = 20.3 Hz, 2H), 2.83 (s, 1H), 2.64 – 2.55 (m, 1H), 2.34 (d, J = 6.4 Hz, 2H), 2.09 (d, J = 7.0 Hz, 3H), 1.91 (d, J = 7.3 Hz, 1H), 1.67 – 1.34 (m, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 171.75, 163.15, 157.60, 142.80, 142.48, 139.64, 132.49, 129.18, 129.07, 128.92, 126.02, 124.18, 120.15, 118.04, 112.35, 61.46, 59.61, 58.59, 56.14, 55.81, 40.90, 36.65, 28.65, 28.51, 25.51, 21.48, 18.92. HRMS m/z: calcd for C 35 H 45 N 4 O 6 S [M+H] + 649.3060, found 649.3032. HPLC: t R 13.31 min, purity 96.23%. Compound SW-3 ( N -(4’’-(((( S )-1-amino-3-hydroxy-1-oxopropan-2-yl)amino)methyl)-3’’-methoxy-2’-methyl-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 10.01 (s, 1H), 7.67 (s, 2H), 7.56 (d, J = 7.9 Hz, 1H), 7.46 – 7.31 (m, 4H), 7.21 (dd, J = 13.0, 7.5 Hz, 2H), 7.07 – 6.93 (m, 3H), 6.41 (d, J = 29.3 Hz, 2H), 5.21 (s, 1H), 4.32 (s, 1H), 4.15 (s, 1H), 3.92 (d, J = 5.4 Hz, 2H), 3.84 (s, 3H), 3.70 (s, 2H), 3.62 (s, 1H), 3.13 (s, 1H), 2.85 – 2.79 (m, 1H), 2.58 (d, J = 12.3 Hz, 1H), 2.33 (s, 2H), 2.08 (s, 3H), 1.53 (dd, J = 72.9, 27.2 Hz, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 171.82, 157.49, 142.73, 142.51, 139.57, 132.49, 130.43, 129.11, 128.94, 126.02, 124.22, 121.48, 120.18, 118.04, 112.19, 61.50, 59.65, 55.96, 55.80, 45.55, 36.65, 28.66, 28.51, 25.51, 18.95. HRMS m/z: calcd for C 34 H 42 N 5 O 5 S [M+H] + 632.2907, found 632.2916. HPLC: t R 13.02 min, purity 96.56%. Compound SW-4 ( N -(3’’-methoxy-2’-methyl-4’’-((((( S )-5-oxopyrrolidin-2-yl)methyl)amino)methyl)-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 9.99 (s, 1H), 7.67 (s, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.34 (dd, J = 12.4, 7.4 Hz, 2H), 7.23 (d, J = 10.2 Hz, 2H), 7.02 (dd, J = 17.9, 8.6 Hz, 3H), 6.40 (d, J = 28.5 Hz, 2H), 4.32 (s, 1H), 4.07 (s, 3H), 3.88 (s, 4H), 3.13 (s, 1H), 2.95 (s, 1H), 2.83 (dd, J = 12.3, 3.6 Hz, 1H), 2.58 (d, J = 12.4 Hz, 1H), 2.34 (d, J = 6.3 Hz, 2H), 2.17 (s, 3H), 2.08 (s, 3H), 1.81 – 1.37 (m, 9H). 13 C NMR (101 MHz, d 6-DMSO) δ 177.20, 171.80, 157.48, 144.12, 142.77, 142.49, 142.43, 139.59, 132.48, 130.87, 129.11, 128.95, 126.04, 124.23, 121.54, 120.17, 118.05, 112.40, 61.49, 59.64, 56.14, 55.80, 53.14, 51.45, 46.72, 36.65, 29.72, 28.66, 28.51, 25.51, 24.80, 18.93. HRMS m/z: calcd for C 36 H 44 N 5 O 4 S [M+H] + 642.3114, found 642.3126. HPLC: t R 12.82 min, purity 97.11%. Compound SW-5 ( N -(4’’-(((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)methyl)-3’’-methoxy-2,2’-dimethyl-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 9.37 (s, 1H), 7.42 – 7.35 (m, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.27 – 7.21 (m, 2H), 7.07 (d, J = 7.1 Hz, 1H), 6.98 (d, J = 6.9 Hz, 1H), 6.91 (d, J = 9.8 Hz, 2H), 6.45 (s, 1H), 6.37 (s, 1H), 4.31 (s, 1H), 4.15 (s, 1H), 3.81 (d, J = 10.3 Hz, 6H), 3.44 (s, 4H), 3.14 (s, 2H), 2.83 (d, J = 11.9 Hz, 1H), 2.58 (d, J = 12.5 Hz, 1H), 2.36 (s, 2H), 1.99 – 1.86 (m, 10H), 1.58 (d, J = 46.4 Hz, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 172.57, 171.56, 163.26, 161.02, 157.09, 142.71, 142.50, 133.12, 130.34, 129.10, 128.47, 126.58, 125.95, 125.75, 121.57, 114.50, 111.81, 61.47, 61.19, 60.92, 59.60, 55.85, 55.80, 36.08, 28.73, 28.52, 25.73, 21.62, 18.13, 15.39. HRMS m/z: calcd for C 36 H 47 N 4 O 6 S [M+H] + 663.3216, found 663.3231. HPLC: t R 13.09 min, purity 97.29%. Compound SW-6 ( N -(4’’-(((3-amino-2,2-dimethyl-3-oxopropyl)amino)methyl)-3’’-methoxy-2,2’-dimethyl-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 9.37 (s, 1H), 8.11 (s, 1H), 7.59 (s, 1H), 7.46 (d, J = 6.6 Hz, 1H), 7.39 – 7.33 (m, 3H), 7.28 – 7.20 (m, 3H), 7.11 – 7.07 (m, 2H), 7.02 – 6.96 (m, 2H), 6.44 (s, 1H), 6.37 (s, 1H), 4.32 (s, 1H), 4.14 (s, 3H), 3.91 (s, 3H), 3.14 (s, 1H), 2.98 (s, 2H), 2.84 (d, J = 11.7 Hz, 2H), 2.60 (s, 1H), 2.36 (s, 2H), 1.93 (s, 2H), 1.90 (s, 2H), 1.64 – 1.40 (m, 6H), 1.22 (s, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 179.60, 178.38, 171.61, 163.17, 157.89, 142.62, 142.49, 142.00, 137.18, 133.02, 131.55, 130.49, 129.01, 126.60, 126.08, 125.91, 125.13, 121.76, 112.44, 61.54, 59.66, 56.20, 55.92, 47.98, 46.92, 36.06, 28.74, 28.60, 25.82, 24.15, 23.67, 18.17, 15.52. HRMS m/z: calcd for C 37 H 48 N 5 O 4 S [M+H]+ 658.3427, found 658.3431. HPLC: t R 12.84 min, purity 97.11%. Compound SW-7 ( N -(4’’-(((( S )-1-amino-3-hydroxy-1-oxopropan-2-yl)amino)methyl)-3’’-methoxy-2,2’-dimethyl-[1,1’:3’,1’’-terphenyl]-3-yl)-5-((3a S ,4 S ,6a R )-2-oxohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamide) 1 H NMR (400 MHz, d 6-DMSO) δ 9.37 (s, 1H), 7.61 (s, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.39 – 7.32 (m, 2H), 7.30 – 7.19 (m, 2H), 7.08 (d, J = 7.4 Hz, 1H), 7.00 – 6.93 (m, 2H), 6.40 (d, J = 28.0 Hz, 2H), 4.32 (s, 1H), 4.16 (s, 1H), 3.86 (d, J = 20.2 Hz, 5H), 3.54 (s, 4H), 3.14 (s, 1H), 2.83 (d, J = 8.4 Hz, 1H), 2.58 (d, J = 12.3 Hz, 1H), 2.35 (s, 2H), 1.91 (d, J = 15.4 Hz, 6H), 1.64 (s, 2H), 1.46 (d, J = 45.0 Hz, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 175.06, 171.58, 167.42, 166.22, 163.45, 157.40, 146.41, 142.74, 142.36, 142.15, 132.85, 130.77, 130.54, 129.18, 128.78, 126.48, 126.09, 125.98, 124.99, 121.43, 112.13, 61.50, 59.63, 55.94, 55.85, 35.99, 28.68, 28.53, 25.76, 18.12, 15.44. HRMS m/z: calcd for C 35 H 44 N 5 O 5 S [M+H]+ 646.3063, found 646.3071. HPLC: t R 12.90 min, purity 96.96%. 2.3 Molecular dynamics simulations Structural files of target proteins were downloaded from the Protein Data Bank (PDB). We used AutoDock Vina to build protein-ligand complexes through molecular docking. All MD simulations were performed using GROMACS 2019 with the CHARMM36 force field. Each complex was embedded in a triclinic box, ensuring a 1.0 nm buffer zone between the solute and box walls. The system was solvated with TIP3P water, and counterions were added to neutralize charge and set ionic strength to 0.15 M, with periodic boundary conditions applied in all directions. The steepest-descent method was used to minimize energy, remove steric conflicts, and optimize initial structures. Two sequential equilibration processes were applied: a 100 ps NVT run at 310.15 K using the Berendsen thermostat, followed by a 100 ps NPT run at 1 bar controlled by the Berendsen barostat. The production simulation lasted 100 ns, with a 2 fs time step and the leap-frog integrator. Long-range electrostatic interactions were calculated with the PME method, van der Waals forces were characterized by the Lennard-Jones potential, and covalent bonds were restrained using LINCS. Trajectory data were analyzed by calculating RMSD, RMSF, Rg, SASA, hydrogen-bond number, and interaction energy, and by performing principal component analysis. The MM/PBSA method was finally utilized to estimate the protein-ligand binding free energy. 2.4 In vitro PD-1/PD-L1 binding assay The recombinant PD-L1 protein was engineered to include a His affinity tag at its C-terminus. This tag enables specific binding to a monoclonal antibody targeting the 6His sequence, which is coupled to XL665 fluorescent labels. Correspondingly, the recombinant PD-1 protein was produced with a GST fusion tag. The paired detection antibody against GST was conjugated to europium cryptate as the signal reporter. In 384-well plates, 2 μL serially diluted compounds, 4 μL His-PD-L1, and 4 μL GST-PD-1 were sequentially added per well (total 10 μL). Then, 10 µL of pre-mixed anti-Tag1-Eu 3+ and anti-Tag2-XL665 was added, the plate was sealed, and the plate was incubated for 2 hours at room temperature. Plates were incubated at room temperature in the dark for 120 min before dual-fluorescence detection at 665 nm (acceptor) and 620 nm (donor). 2.5 SPR analysis All recombinant proteins, unless otherwise noted, were obtained from Sino Biological Inc.: murine PD-L1 (Cat. No. 50010-M08H) and human PD-L1 (Cat. No. 10084-HNAH). SPR imaging assays were performed using a PlexArray HT system. Briefly, hPD-L1 and mPD-L1 were each diluted to 0.5 mg/mL in sterile ultra
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> MedicHelpline content is structured for research, educational, and professional discovery purposes. It does not constitute individual medical advice, clinical diagnosis, or treatment recommendations.
> Always verify dosing, contraindications, and regulatory alerts against official product labeling and primary regulatory sources before clinical decision-making.