CD73 generates immunosuppressive adenosine in the tumor microenvironment and is a promising target for cancer immunotherapy. We have designed and systematically studied diverse 2-substituted 7-deazapurine ribonucleoside 5'-O-bisphosphonates bearing a variety of (het)-aryl groups at position 6 and discovered their highly potent and selective CD73 inhibition activity. The most active compounds (with single-digit picomolar K i) contained bicyclic (het)-aryl groups at position 6 in combination with chlorine at position 2. Further optimization of pharmacokinetic properties identified inhibitors with low clearance, long half-life, high solubility, and excellent selectivity over CD39 and NTPDase3. They effectively suppressed adenosine formation in MDA-MB-231 cells, rescued CD8+ T cell activation, and were nontoxic to human fibroblasts. Overall, their profile compares favorably with AB680, a CD73 inhibitor currently in phase I/II clinical trials.

Department of Organic Chemistry Faculty of Science Charles University Prague Hlavova 8 Prague 2 CZ 12843 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 2 Prague 6 CZ 16610 Czech Republic

Zobrazit více v PubMed

Noringriis I. M., Donia M., Madsen K., Schmidt H., Haslund C. A., Bastholt L., Svane I. M., Ellebaek E.. Long-Term Clinical Outcome of Patients with Metastatic Melanoma and Initial Stable Disease during Anti-PD-1 Checkpoint Inhibitor Immunotherapy with Pembrolizumab. Br. J. Cancer. 2025;133:337–345. doi: 10.1038/s41416-025-03048-8. PubMed DOI PMC

Larkin J., Chiarion-Sileni V., Gonzalez R., Grob J.-J., Rutkowski P., Lao C. D., Cowey C. L., Schadendorf D., Wagstaff J., Dummer R., Ferrucci P. F., Smylie M., Hogg D., Hill A., Márquez-Rodas I., Haanen J., Guidoboni M., Maio M., Schöffski P., Carlino M. S., Lebbé C., McArthur G., Ascierto P. A., Daniels G. A., Long G. V., Bastholt L., Rizzo J. I., Balogh A., Moshyk A., Hodi F. S., Wolchok J. D.. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N. Engl. J. Med. 2019;381:1535–1546. doi: 10.1056/NEJMoa1910836. PubMed DOI

Boison D., Yegutkin G. G.. Adenosine Metabolism: Emerging Concepts for Cancer Therapy. Cancer Cell. 2019;36:582–596. doi: 10.1016/j.ccell.2019.10.007. PubMed DOI PMC

Knapp K., Zebisch M., Pippel J., El-Tayeb A., Müller C. E., Sträter N.. Crystal Structure of the Human Ecto-5′-Nucleotidase (CD73): Insights into the Regulation of Purinergic Signaling. Structure. 2012;20:2161–2173. doi: 10.1016/j.str.2012.10.001. PubMed DOI

Klemens M. R., Sherman W. R., Holmberg N. J., Ruedi J. M., Low M. G., Thompson L. F.. Characterization of Soluble vs Membrane-Bound Human Placental 5′-Nucleotidase. Biochem. Biophys. Res. Commun. 1990;172:1371–1377. doi: 10.1016/0006-291X(90)91601-N. PubMed DOI

Synnestvedt K., Furuta G. T., Comerford K. M., Louis N., Karhausen J., Eltzschig H. K., Hansen K. R., Thompson L. F., Colgan S. P.. Ecto-5′-Nucleotidase (CD73) Regulation by Hypoxia-Inducible Factor-1 Mediates Permeability Changes in Intestinal Epithelia. J. Clin. Invest. 2002;110:993–1002. doi: 10.1172/JCI15337. PubMed DOI PMC

Niemelä J., Ifergan I., Yegutkin G. G., Jalkanen S., Prat A., Airas L.. IFN-β Regulates CD73 and Adenosine Expression at the Blood–Brain Barrier. Eur. J. Immunol. 2008;38:2718–2726. doi: 10.1002/eji.200838437. PubMed DOI

Xing Y., Ren Z., Jin R., Liu L., Pei J., Yu K.. Therapeutic Efficacy and Mechanism of CD73-TGFβ Dual-Blockade in a Mouse Model of Triple-Negative Breast Cancer. Acta Pharmacol. Sin. 2022;43:2410–2418. doi: 10.1038/s41401-021-00840-z. PubMed DOI PMC

Xia C., Yin S., To K. K. W., Fu L.. CD39/CD73/A2AR Pathway and Cancer Immunotherapy. Mol. Cancer. 2023;22:44. doi: 10.1186/s12943-023-01733-x. PubMed DOI PMC

Bhattarai S., Freundlieb M., Pippel J., Meyer A., Abdelrahman A., Fiene A., Lee S.-Y., Zimmermann H., Yegutkin G. G., Sträter N., El-Tayeb A., Müller C. E.. α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective Ecto −5′-Nucleotidase (CD73) Inhibitors. J. Med. Chem. 2015;58:6248–6263. doi: 10.1021/acs.jmedchem.5b00802. PubMed DOI

Bhattarai S., Pippel J., Meyer A., Freundlieb M., Schmies C., Abdelrahman A., Fiene A., Lee S., Zimmermann H., El-Tayeb A., Yegutkin G. G., Sträter N., Müller C. E.. X-Ray Co-Crystal Structure Guides the Way to Subnanomolar Competitive Ecto-5′-Nucleotidase (CD73) Inhibitors for Cancer Immunotherapy. Adv. Ther. 2019;2:1900075. doi: 10.1002/adtp.201900075. DOI

Bhattarai S., Pippel J., Scaletti E., Idris R., Freundlieb M., Rolshoven G., Renn C., Lee S.-Y., Abdelrahman A., Zimmermann H., El-Tayeb A., Müller C. E., Sträter N.. 2-Substituted α,β-Methylene-ADP Derivatives: Potent Competitive Ecto-5′-Nucleotidase (CD73) Inhibitors with Variable Binding Modes. J. Med. Chem. 2020;63:2941–2957. doi: 10.1021/acs.jmedchem.9b01611. PubMed DOI

Junker A., Renn C., Dobelmann C., Namasivayam V., Jain S., Losenkova K., Irjala H., Duca S., Balasubramanian R., Chakraborty S., Börgel F., Zimmermann H., Yegutkin G. G., Müller C. E., Jacobson K. A.. Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5′-Nucleotidase (CD73) Inhibitors. J. Med. Chem. 2019;62:3677–3695. doi: 10.1021/acs.jmedchem.9b00164. PubMed DOI PMC

Ge G.-H., Wang Q.-Y., Zhang Z.-H., Zhang X., Guo S., Zhang T.-J., Meng F.-H.. Small Molecular CD73 Inhibitors: Recent Progress and Future Perspectives. Eur. J. Med. Chem. 2024;264:116028. doi: 10.1016/j.ejmech.2023.116028. PubMed DOI

Sharif E. U., Kalisiak J., Lawson K. V., Miles D. H., Newcomb E., Lindsey E. A., Rosen B. R., Debien L. P. P., Chen A., Zhao X., Young S. W., Walker N. P., Sträter N., Scaletti E. R., Jin L., Xu G., Leleti M. R., Powers J. P.. Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors. J. Med. Chem. 2021;64:845–860. doi: 10.1021/acs.jmedchem.0c01835. PubMed DOI

Lawson K. V., Kalisiak J., Lindsey E. A., Newcomb E. T., Leleti M. R., Debien L., Rosen B. R., Miles D. H., Sharif E. U., Jeffrey J. L., Tan J. B. L., Chen A., Zhao S., Xu G., Fu L., Jin L., Park T. W., Berry W., Moschütz S., Scaletti E., Sträter N., Walker N. P., Young S. W., Walters M. J., Schindler U., Powers J. P.. Discovery of AB680: A Potent and Selective Inhibitor of CD73. J. Med. Chem. 2020;63:11448–11468. doi: 10.1021/acs.jmedchem.0c00525. PubMed DOI

Debien, L. P. P. ; Jaen, J. C. ; Kalisiak, J. ; Lawson, K. V. ; Leleti, M. R. ; Lindsey, E. A. ; Miles, D. H. ; Newcomb, E. ; Powers, J. P. ; Rosen, B. R. ; Sharif, E. U. . Modulators of 5 ’ -Nucleotidase, ecto and the use of thereof. U.S. Patent US10981944B2, 2021.

Šímová M., Ormsby T., Šinkevičiu̅tė U., Sirotová Veselovská L., Čermáková K., Hadzima M., Bartoň L., Staňurová J., Kramná A., Šácha P., Tichý M., Hocek M., Konvalinka J., Blazkova K.. Identification of 6-Aryl-7-Deazapurine Ribonucleoside Phosphonates as Inhibitors of Ecto-5′-Nucleotidase (CD73) ACS Pharmacol. Transl. Sci. 2025;8:2575–2585. doi: 10.1021/acsptsci.5c00180. PubMed DOI PMC

Malnuit V., Slavětínská L. P., Nauš P., Džubák P., Hajdúch M., Stolaříková J., Snášel J., Pichová I., Hocek M.. 2-Substituted 6-(Het)­Aryl-7-deazapurine Ribonucleosides: Synthesis, Inhibition of Adenosine Kinases, and Antimycobacterial Activity. ChemMedChem. 2015;10:1079–1093. doi: 10.1002/cmdc.201500081. PubMed DOI

Kim Y. A., Sharon A., Chu C. K., Rais R. H., Al Safarjalani O. N., Naguib F. N. M., El Kouni M. H.. Structure–Activity Relationships of 7-Deaza-6-Benzylthioinosine Analogues as Ligands of Toxoplasma Gondii Adenosine Kinase. J. Med. Chem. 2008;51:3934–3945. doi: 10.1021/jm800201s. PubMed DOI

Serafinowski P., Dorland E., Balzarini J., De Clercq E.. The Synthesis and Antiviral Activity of Some New S-Adenosyl-L-Homocysteine Derivatives and Their Nucleoside Precursors. Nucleosides, Nucleotides Nucleic Acids. 1995;14:545–547. doi: 10.1080/15257779508012423. DOI

Eldrup A. B., Prhavc M., Brooks J., Bhat B., Prakash T. P., Song Q., Bera S., Bhat N., Dande P., Cook P. D., Bennett C. F., Carroll S. S., Ball R. G., Bosserman M., Burlein C., Colwell L. F., Fay J. F., Flores O. A., Getty K., LaFemina R. L., Leone J., MacCoss M., McMasters D. R., Tomassini J. E., Von Langen D., Wolanski B., Olsen D. B.. Structure–Activity Relationship of Heterobase-Modified 2‘- C -Methyl Ribonucleosides as Inhibitors of Hepatitis C Virus RNA Replication. J. Med. Chem. 2004;47:5284–5297. doi: 10.1021/jm040068f. PubMed DOI

Seela F., Soulimane T., Mersmann K., Jürgens T.. 2,4-Disubstituted Pyrrolo­[2,3-d]­Pyrimidine α-d- and ß-d-Ribofuranosides Related to 7-Deazaguanosine. Helv. Chim. Acta. 1990;73:1879–1887. doi: 10.1002/hlca.19900730710. DOI

Nauš P., Perlíková P., Pohl R., Hocek M.. Sugar-Modified Derivatives of Cytostatic 6-(Het)­Aryl-7-Deazapurine Nucleosides: 2′-C-Methylribonucleosides, Arabinonucleosides and 2′-Deoxy-2′-Fluoroarabinonucleosides. Collect. Czech. Chem. Commun. 2011;76:957–988. doi: 10.1135/cccc2011082. DOI

Colclough N., Ruston L., Wood J. M., MacFaul P. A.. Species Differences in Drug Plasma Protein Binding. Med. Chem. Commun. 2014;5(7):963–967. doi: 10.1039/C4MD00148F. DOI

Gerber P. R., Müller K.. MAB, a Generally Applicable Molecular Force Field for Structure Modelling in Medicinal Chemistry. J. Comput.-Aided Mol. Des. 1995;9:251–268. doi: 10.1007/BF00124456. PubMed DOI

Nocentini A., Capasso C., Supuran C. T.. Small-Molecule CD73 Inhibitors for the Immunotherapy of Cancer: A Patent and Literature Review (2017–Present) Expert Opin. Ther. Pat. 2021;31:867–876. doi: 10.1080/13543776.2021.1923694. PubMed DOI

Pecina A., Fanfrlík J., Lepšík M., Řezáč J.. SQM2.20: Semiempirical Quantum-Mechanical Scoring Function Yields DFT-Quality Protein–Ligand Binding Affinity Predictions in Minutes. Nat. Commun. 2024;15:1127. doi: 10.1038/s41467-024-45431-8. PubMed DOI PMC

Lepsik, M. CD73_inhibitors_SQM; Mendeley Data: V2, 2025. 10.17632/j4rbk4gnr4.2. DOI

Fanfrlík J., Ruiz F. X., Kadlčíková A., Řezáč J., Cousido-Siah A., Mitschler A., Haldar S., Lepšík M., Kolář M. H., Majer P., Podjarny A. D., Hobza P.. The Effect of Halogen-to-Hydrogen Bond Substitution on Human Aldose Reductase Inhibition. ACS Chem. Biol. 2015;10:1637–1642. doi: 10.1021/acschembio.5b00151. PubMed DOI

Piovesan D., Tan J. B. L., Becker A., Banuelos J., Narasappa N., DiRenzo D., Zhang K., Chen A., Ginn E., Udyavar A. R., Yin F., Paprcka S. L., Purandare B., Park T. W., Kimura N., Kalisiak J., Young S. W., Powers J. P., Schindler U., Sivick K. E., Walters M. J.. Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity. Mol. Cancer Ther. 2022;21(6):948–959. doi: 10.1158/1535-7163.MCT-21-0802. PubMed DOI PMC

Allard B., Pommey S., Smyth M. J., Stagg J.. Targeting CD73 Enhances the Antitumor Activity of Anti-PD-1 and Anti-CTLA-4 mAbs. Clin. Cancer Res. 2013;19:5626–5635. doi: 10.1158/1078-0432.CCR-13-0545. PubMed DOI

Chen Q., Yin H., He J., Xie Y., Wang W., Xu H., Zhang L., Shi C., Yu J., Wu W., Liu L., Pu N., Lou W.. Tumor Microenvironment Responsive CD8+ T Cells and Myeloid-Derived Suppressor Cells to Trigger CD73 Inhibitor AB680-Based Synergistic Therapy for Pancreatic Cancer. Adv. Sci. 2023;10:2302498. doi: 10.1002/advs.202302498. PubMed DOI PMC

Tang T., Huang X., Lu M., Zhang G., Han X., Liang T.. Transcriptional Control of Pancreatic Cancer Immunosuppression by Metabolic Enzyme CD73 in a Tumor-Autonomous and -Autocrine Manner. Nat. Commun. 2023;14:3364. doi: 10.1038/s41467-023-38578-3. PubMed DOI PMC

RDKit: Open-source cheminformatics. https://www.rdkit.org.

Jakalian A., Jack D. B., Bayly C. I.. Fast, Efficient Generation of High-quality Atomic Charges. AM1-BCC Model: II. Parameterization and Validation. J. Comput. Chem. 2002;23:1623–1641. doi: 10.1002/jcc.10128. PubMed DOI