In search of a potent small molecular PD-L1 inhibitor, we designed and synthesized a compound based on a 2-hydroxy-4-phenylthiophene-3-carbonitrile moiety. Ligand's performance was tested in vitro and compared side-by-side with a known PD-L1 antagonist with a proven bioactivity BMS1166. Subsequently, we modified both compounds to allow 18F labeling that could be used for PET imaging. Radiolabeling, which is used in drug development and diagnosis, was applied to investigate the properties of those ligands and test them against tissue sections with diverse expression levels of PD-L1. We confirmed biological activity toward hPD-L1 for this inhibitor, comparable with BMS1166, while holding enhanced pharmacological properties.

Centre for Medicines Discovery Nuffield Department of Medicine Alzheimer's Research UK Oxford Drug Discovery Institute NDM Research Building Roosevelt Drive OX3 7FZ Oxford U K

Department of Chemistry University of Crete Voutes 70013 Heraklion Greece

Department of Drug Design University of Groningen A Deusinglaan 1 9713 AV Groningen The Netherlands

Department of Nuclear Medicine and MolecularImaging University Medical Center Groningen University of Groningen Hanzeplein 1 9713 GZ Groningen The Netherlands

Department of Obstetrics and Gynecology University Medical Center Groningen University of Groningen Hanzeplein 1 9713 GZ Groningen The Netherlands

Department of Organic Chemistry Faculty of Chemistry Jagiellonian University Gronostajowa 2 30 387 Krakow Poland

Doctoral School of Exact and Natural Sciences Jagiellonian University Prof St Łojasiewicz St 11 30 348 Krakow Poland

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry and Czech Advanced Technology and Research Institute Palacky University in Olomouc Olomouc 77900 Czech Republic

See more in PubMed

Pardoll D. M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 2012, 12, 252–264. 10.1038/nrc3239. PubMed DOI PMC

Michot J. M.; Bigenwald C.; Champiat S.; Collins M.; Carbonnel F.; Postel-Vinay S.; Berdelou A.; Varga A.; Bahleda R.; Hollebecque A.; Massard C.; Fuerea A.; Ribrag V.; Gazzah A.; Armand J. P.; Amellal N.; Angevin E.; Noel N.; Boutros C.; Mateus C.; Lambotte O. Immune-related adverse events with immune checkpoint blockade: a comprehensive review. Eur. J. Cancer 2016, 54, 139–148. 10.1016/j.ejca.2015.11.016. PubMed DOI

European Medicines Agency . EMEA/H/C/003985—EPAR Product Information 2022. https://www.ema.europa.eu/en/documents/product-information/opdivo-epar-product-information_en.pdf (accessed Sept 01, 2022).

Opdivo (nivolumab) price, Farmacotherapeutisch Kompas. https://www.farmacotherapeutischkompas.nl/bladeren/preparaatteksten/n/nivolumab#kosten (accessed Sept 01, 2022).

Queva C.; Morrow M.; Hammond S.; Alimzhanov M.; Babcock J.; Foltz I.; Kang J. S.; Sekirov L.; Boyle M.; Chodorge M.. Targeted binding agents against b7-h1. WO 2011066389 A1, 2011.

Nastri H. G.; Iffland C.; Leger O.; An Q.; Cartwright M.; Mckenna S. D.; Sood V. D.; Hao G.. Anti-pd-l1 antibodies and uses thereof. WO 2013079174 A1, 2013.

Irving B.; Chiu H.; Maecker H.; Mariathasan S.; Lehar S. M.; Wu Y.; Cheung J.. Anti-PD-L1 antibodies, compositions and articles of manufacture. U.S. Patent 8,217,149 B2, 2012.

Zak K. M.; Grudnik P.; Guzik K.; Zieba B. J.; Musielak B.; Dömling A.; Dubin G.; Holak T. A. Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1). Oncotarget 2016, 7, 30323–30335. 10.18632/oncotarget.8730. PubMed DOI PMC

Zhang F.; Wei H.; Wang X.; Bai Y.; Wang P.; Wu J.; Jiang X.; Wang Y.; Cai H.; Xu T.; Zhou A. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade. Cell Discovery 2017, 3, 17004.10.1038/celldisc.2017.4. PubMed DOI PMC

Magiera-Mularz K.; Skalniak L.; Zak K. M.; Musielak B.; Rudzinska-Szostak E.; Berlicki Ł.; Kocik J.; Grudnik P.; Sala D.; Zarganes-Tzitzikas T.; Shaabani S.; Dömling A.; Dubin G.; Holak T. A. Bioactive Macrocyclic Inhibitors of the PD-1/PD-L1 Immune Checkpoint. Angew. Chem., Int. Ed. 2017, 56, 13732–13735. 10.1002/anie.201707707. PubMed DOI PMC

Chupak L. S.; Ding M.; Martin S. W.; Zheng X.; Hewawasam P.; Connolly T. P.; Xu N.; Yeung K.-S.; Zhu J.; Langley D. R.; Scola P. M.. Compounds useful as immunomodulators. WO 2015034820 A1, 2015.

Wang Y.; Gu T.; Tian X.; Li W.; Zhao R.; Yang W.; Gao Q.; Li T.; Shim J. H.; Zhang C.; Liu K.; Lee M. H. A small molecule antagonist of PD-1/PD-L1 interactions acts as an immune checkpoint inhibitor for NSCLC and melanoma immunotherapy. Front. Immunol. 2021, 12, 654463.10.3389/fimmu.2021.654463. PubMed DOI PMC

Maxinovel Pty. Ltd. . MAX-10181 Given Orally to Patients With Advanced Solid Tumor (NCT 04122339). https://clinicaltrials.gov/ct2/show/NCT04122339 (accessed Sept 01, 2022).

Gilead Sciences . Study to Evaluate Safety, Tolerability, Pharmacokinetics, and Efficacy of GS-4224 in Participants With Advanced Solid Tumors (NCT04049617). https://www.clinicaltrials.gov/ct2/show/NCT04049617 (accessed Sept 01, 2022).

Konstantinidou M.; Zarganes-Tzitzikas T.; Magiera-Mularz K.; Holak T. A.; Dömling A. Immune Checkpoint PD-1/PD-L1: Is There Life Beyond Antibodies. Angew. Chemie Int. Ed. 2018, 57, 4840–4848. 10.1002/anie.201710407. PubMed DOI PMC

Butera R.; Ważyńska M.; Magiera-Mularz K.; Plewka J.; Musielak B.; Surmiak E.; Sala D.; Kitel R.; de Bruyn M.; Nijman H. W.; Elsinga P. H.; Holak T. A.; Dömling A. Design, Synthesis, and Biological Evaluation of Imidazopyridines as PD-1/PD-L1 Antagonists. ACS Med. Chem. Lett. 2021, 12, 768–773. 10.1021/acsmedchemlett.1c00033. PubMed DOI PMC

Shaabani S.; Huizinga H. P. S.; Butera R.; Kouchi A.; Guzik K.; Magiera-Mularz K.; Holak T. A.; Dömling A. A patent review on PD-1/PD-L1 antagonists: small molecules, peptides, and macrocycles (2015-2018). Expert Opin. Ther. Pat. 2018, 28, 665–678. 10.1080/13543776.2018.1512706. PubMed DOI PMC

Guzik K.; Tomala M.; Muszak D.; Konieczny M.; Hec A.; Błaszkiewicz U.; Pustuła M.; Butera R.; Dömling A.; Holak T. A. Development of the Inhibitors that Target the PD-1/PD-L1 Interaction-A Brief Look at Progress on Small Molecules, Peptides and Macrocycles. Molecules 2019, 24, 2071.10.3390/molecules24112071. PubMed DOI PMC

Muszak D.; Surmiak E.; Plewka J.; Magiera-Mularz K.; Kocik-Krol J.; Musielak B.; Sala D.; Kitel R.; Stec M.; Weglarczyk K.; Siedlar M.; Dömling A.; Skalniak L.; Holak T. A. Terphenyl-Based Small-Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein-Protein Interaction. J. Med. Chem. 2021, 64, 11614–11636. 10.1021/acs.jmedchem.1c00957. PubMed DOI PMC

McRee D. E.Computational Techniques, Practical Protein Crystallography, 2nd ed.; Academic Press, 1999; p 91.

Gewald K.; Jablokoff H.; Hentschel M. Synthese und Reaktionen von 2-Hydroxy-3-cyan-thiophenen. J. Prakt. Chem. 1975, 317, 861–866. 10.1002/prac.19753170521. DOI

Huang Y.; Dömling A. The Gewald multicomponent reaction. Mol. Diversity 2011, 15, 3–33. 10.1007/s11030-010-9229-6. PubMed DOI

Wang K.; Kim D.; Dömling A. Cyanoacetamide MCR (III): three-component Gewald reactions revisited. J. Comb. Chem. 2010, 12, 111–118. 10.1021/cc9001586. PubMed DOI PMC

Huang Y.; Dömling A. 1,4-Thienodiazepine-2,5-diones via MCR (II): scaffold hopping by Gewald and Ugi-deprotection-cyclization strategy. Chem. Biol. Drug Des. 2010, 76, 130–141. 10.1111/j.1747-0285.2010.00990.x. PubMed DOI PMC

Huang Y.; Wolf S.; Bista M.; Meireles L.; Camacho C.; Holak T. A.; Dömling A. 1,4-Thienodiazepine-2,5-diones via MCR (I): synthesis, virtual space and p53-Mdm2 activity. Chem. Biol. Drug Des. 2010, 76, 116–129. 10.1111/j.1747-0285.2010.00989.x. PubMed DOI PMC

Skalniak L.; Zak K. M.; Guzik K.; Magiera K.; Musielak B.; Pachota M.; Szelazek B.; Kocik J.; Grudnik P.; Tomala M.; Krzanik S.; Pyrc K.; Dömling A.; Dubin G.; Holak T. A. Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells. Oncotarget 2017, 8, 72167–72181. 10.18632/oncotarget.20050. PubMed DOI PMC

Surmiak E.; Magiera-Mularz K.; Musielak B.; Muszak D.; Kocik-Krol J.; Kitel R.; Plewka J.; Holak T. A.; Skalniak L. PD-L1 Inhibitors: Different Classes, Activities, and Mechanisms of Action. Int. J. Mol. Sci. 2021, 22, 11797.10.3390/ijms222111797. PubMed DOI PMC

Mossine A. V.; Brooks A. F.; Makaravage K. J.; Miller J. M.; Ichiishi N.; Sanford M. S.; Scott P. J. Synthesis of [18F]Arenes via the Copper-Mediated [18F]Fluorination of Boronic Acids. Org. Lett. 2015, 17, 5780–5783. 10.1021/acs.orglett.5b02875. PubMed DOI PMC

Cheng T.; Zhao Y.; Li X.; Lin F.; Xu Y.; Zhang X.; Li Y.; Wang R.; Lai L. Computation of Octanol-Water Partition Coefficients by Guiding an Additive Model with Knowledge. J. Chem. Inf. Model. 2007, 47, 2140–2148. 10.1021/ci700257y. PubMed DOI

Brown J. A.; Dorfman D. M.; Ma F. R.; Sullivan E. L.; Munoz O.; Wood C. R.; Greenfield E. A.; Freeman G. J. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J. Immunol. 2003, 170, 1257–1266. 10.4049/jimmunol.170.3.1257. PubMed DOI

Ma J.; Li J.; Qian M.; Han W.; Tian M.; Li Z.; Wang Z.; He S.; Wu K. PD-L1 expression and the prognostic significance in gastric cancer: a retrospective comparison of three PD-L1 antibody clones (SP142, 28–8 and E1L3N). Diagn. Pathol. 2018, 13, 91.10.1186/s13000-018-0766-0. PubMed DOI PMC

Zhao Y. J.; Sun W. P.; Peng J. H.; Deng Y. X.; Fang Y. J.; Huang J.; Zhang H. Z.; Wan D. S.; Lin J. Z.; Pan Z. Z. Programmed death-ligand 1 expression correlates with diminished CD8+ T cell infiltration and predicts poor prognosis in anal squamous cell carcinoma patients. Cancer Manage. Res. 2017, 10, 1–11. 10.2147/CMAR.S153965. PubMed DOI PMC

Sasikumar P. G.; Sudarshan N. S.; Adurthi S.; Ramachandra R. K.; Samiulla D. S.; Lakshminarasimhan A.; Ramanathan A.; Chandrasekhar T.; Dhudashiya A. A.; Talapati S. R.; Gowda N.; Palakolanu S.; Mani J.; Srinivasrao B.; Joseph D.; Kumar N.; Nair R.; Atreya H. S.; Gowda N.; Ramachandra M. PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy. Commun. Biol. 2021, 4, 699.10.1038/s42003-021-02191-1. PubMed DOI PMC

Musielak B.; Kocik J.; Skalniak L.; Magiera-Mularz K.; Sala D.; Czub M.; Stec M.; Siedlar M.; Holak T. A.; Plewka J. CA-170 - A Potent Small-Molecule PD-L1 Inhibitor or Not?. Molecules 2019, 24, 2804.10.3390/molecules24152804. PubMed DOI PMC

Blevins D. J.; Hanley R.; Bolduc T.; Powell D. A.; Gignac M.; Walker K.; Carr M. D.; Hof F.; Wulff J. E. In Vitro Assessment of Putative PD-1/PD-L1 Inhibitors: Suggestions of an Alternative Mode of Action. ACS Med. Chem. Lett. 2019, 10, 1187–1192. 10.1021/acsmedchemlett.9b00221. PubMed DOI PMC

Ganesan A.; Ahmed M.; Okoye I.; Arutyunova E.; Babu D.; Turnbull W. L.; Kundu J. K.; Shields J.; Agopsowicz K. C.; Xu L.; et al. Comprehensive in vitro characterization of PD-L1 small molecule inhibitors. Sci. Rep. 2019, 9, 12392.10.1038/s41598-019-48826-6. PubMed DOI PMC

Sasikumar P. G.; Ramachandra M.; Prasad A.; Naremaddepalli S. S. S.. 3-substituted-1,2,4-oxadiazole and thiadiazole compounds as immunomodulators. WO 2016142886 A3, 2016.

Chen F. F.; Li Z.; Ma D.; Yu Q. Small-molecule PD-L1 inhibitor BMS1166 abrogates the function of PD-L1 by blocking its ER export. Oncoimmunology 2020, 9, 1831153.10.1080/2162402X.2020.1831153. PubMed DOI PMC

Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. 10.1016/0003-2697(76)90527-3. PubMed DOI

Liao Y.; Chen L.; Feng Y.; Shen J.; Gao Y.; Cote G.; Choy E.; Harmon D.; Mankin H.; Hornicek F.; Duan Z. Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells. Oncotarget 2017, 8, 30276–30287. 10.18632/oncotarget.16326. PubMed DOI PMC

Pettersen E. F.; Goddard T. D.; Huang C. C.; Couch G. S.; Greenblatt D. M.; Meng E. C.; Ferrin T. E. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612. 10.1002/jcc.20084. PubMed DOI

Trott O.; Olson A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2009, 31, 455–461. 10.1002/jcc.21334. PubMed DOI PMC