1,3,5-triazines inhibit osteosarcoma and avert lung metastasis in a patient-derived orthotopic xenograft mouse model with favorable pharmacokinetics

Document Type : Original Article


1 Department of Orthopedic Oncology, Yantai Shan Hospital, Yantai, 264003, China

2 Department of Orthopedics, The Second Affiliated Hospital Zhejiang University School of Medicine, Hangzhou, 310006, China

3 Departments of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, China

4 Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, China


Objective(s): Osteosarcoma is a major solid malignant tumor of bone, possessing significant burden on healthcare due to non-availability of specific anticancer agents. The current study was conducted to identify novel 1,3,5-triazine derivatives against osteosarcoma. 
Materials and Methods: The compounds were synthesized in a straight-forward two-step reaction and subsequently tested against PI3K and mTOR kinase and anticancer activity against osteosarcoma cells (MG-63, U2-OS, and Saos-2). The effect of the most potent compound was evaluated on apoptosis and cell phase of Saos-2 cells. The pharmacological activity was further established in the patient-derived orthotopic xenograft (PDOX) mouse model. 
Results: The developed compounds 8 (a-f) showed significant inhibitory activities against PI3K, mTOR, and OS cells. Among the tested series, compound 8a showed highly potent PI3K/mTOR inhibitory activity with significant anticancer activity against Saos-2 cells compared with Imatinib as standard. It also induces apoptosis and causes G2/M arrest in Saos-2 cells. Compound 8a significantly improved body weight, reduced tumor volume, and inhibited lung metastasis in athymic nude mice in a PDOX mouse model. It also showed optimal pharmacokinetic parameters in SD rats. 
Conclusion: In summary, 1,3,5-triazine analogs were identified as new PI3K/mTOR inhibitors against osteosarcoma.


1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68:394–424. 
2. Czarnecka AM, Synoradzki K, Firlej W, Bartnik E, Sobczuk P, Fiedorowicz M, et al. Molecular biology of osteosarcoma. Cancers 2020; 12:1–27. 
3. Durfee RA, Mohammed M, Luu HH. Review of osteosarcoma and current management. Rheumatol Ther 2016; 3:221–243. 
4. Bielack SS, Hecker-Nolting S, Blattmann C, Kager L. Advances in the management of osteosarcoma. F1000Res 2016; 5:2767-2777. 
5. Savage DG, Antman KH. Imatinib mesylate — a new oral targeted therapy. N Engl J Med 2002; 346:683–693. 
6. O’Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003; 348:994–1004. 
7. Verweij J, Van Oosterom A, Blay JY, Judson I, Rodenhuis S, Van Der Graaf W, et al. Imatinib mesylate (STI-571 Glivec®, GleevecTM) is an active agent for gastrointestinal stromal tumours, but does not yield responses in other soft-tissue sarcomas that are unselected for a molecular target: results from an EORTC soft tissue and bone sarcom. Eur J Cancer 2003; 39:2006–2011. 
8. El Hajj Dib I, Gallet M, Mentaverri R, Sévenet N, Brazier M, Kamel S. Imatinib mesylate (Gleevec®) enhances mature osteoclast apoptosis and suppresses osteoclast bone resorbing activity. Eur J Pharmacol 2006; 551:27–33. 
9. O’Sullivan S, Naot D, Callon K, Porteous F, Horne A, Wattie D, et al. Imatinib promotes osteoblast differentiation by inhibiting PDGFR signaling and inhibits osteoclastogenesis by both direct and stromal cell-dependent mechanisms. J Bone Miner Res 2007; 22:1679–1689. 
10. Fierro F, Illmer T, Jing D, Schleyer E, Ehninger G, Boxberger S, et al. Inhibition of platelet-derived growth factor receptorβ by imatinib mesylate suppresses proliferation and alters differentiation of human mesenchymal stem cells in vitro. Cell Prolif 2007; 40:355–366. 
11. Gobin B, Moriceau G, Ory B, Charrier C, Brion R, Blanchard F, et al. Imatinib mesylate exerts anti-proliferative effects on osteosarcoma cells and inhibits the tumour growth in immunocompetent murine models. PLoS One 2014; 9:1-12. 
12. Bishop MW, Janeway KA. Emerging concepts for PI3K/mTOR inhibition as a potential treatment for osteosarcoma. F1000Res 2016; 5:1-6. 
13. Penel-Page M, Ray-Coquard I, Larcade J, Girodet M, Bouclier L, Rogasik M, et al. Off-label use of targeted therapies in osteosarcomas: data from the french registry OUTC’S (observatoire de i’utilisation des thérapies ciblées dans les sarcomes). BMC Cancer 2015; 15:854-862. 
14. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 2009; 9:28–39. 
15. Srivastava JK, Dubey P, Singh S, Bhat HR, Kumawat MK, Singh UP. Discovery of novel 1,3,5-triazine-thiazolidine-2,4-diones as dipeptidyl peptidase-4 inhibitors with antibacterial activity targeting the S1 pocket for the treatment of type 2 diabetes. RSC Adv 2015; 5:14095–14102. 
16. Solankee A, Kapadia K, Ana Ćirić, Soković M, Doytchinova I, Geronikaki A. Synthesis of some new S-triazine based chalcones and their derivatives as potent antimicrobial agents. Eur J Med Chem 2010; 45:510–518. 
17. Singh UP, Singh RK, Bhat HR, Subhashchandra YP, Kumar V, Kumawat MK, et al. Synthesis and antibacterial evaluation of series of novel tri-substituted-s-triazine derivatives. Med Chem Res 2011; 20:1603–1610. 
18. Kumar Ghosh S, Saha A, Hazarika B, Pratap Singh U, Raj Bhat H, Gahtori P. Design, facile synthesis, antibacterial activity and structure-activity relationship of novel di- and tri-substituted 1,3,5-triazines. Lett Drug Des Discov 2012; 9:329–335. 
19. Bhat HR, Pandey PK, Ghosh SK, Singh UP. Development of 4-aminoquinoline-1,3,5-triazine conjugates as potent antibacterial agent through facile synthetic route. Med Chem Res 2013; 22:5056–5065. 
20. Singh B, Bhat HR, Kumawat MK, Singh UP. Structure-guided discovery of 1,3,5-triazine-pyrazole conjugates as antibacterial and antibiofilm agent against pathogens causing human diseases with favorable metabolic fate. Bioorganic Med Chem Lett 2014; 24:3321–3325. 
21. Singh UP, Bhat HR, Gahtori P. Antifungal activity, SAR and physicochemical correlation of some thiazole-1,3,5-triazine derivatives. J Mycol Med 2012; 22:134–141. 
22. Singh UP, Bhat HR, Gahtori P, Singh RK. Hybrid phenylthiazole and 1,3,5-triazine target cytosolic leucyl-tRNA synthetase for antifungal action as revealed by molecular docking studies. Silico Pharmacol 2013; 1:3-11. 
23. Milata V, Reinprecht L, Kizlink J. Synthesis and antifungal efficacy of 1,3,5-triazines. Acta Chim Slovaca 2012; 5:95–99. 
24. Bhat HR, Singh UP, Yadav PS, Kumar V, Gahtori P, Das A, et al. Synthesis, characterization and antimalarial activity of hybrid 4-aminoquinoline-1,3,5-triazine derivatives. Arab J Chem 2016; 9:625–631. 
25. Bhat HR, Singh UP, Thakur A, Kumar Ghosh S, Gogoi K, Prakash A, et al. Synthesis, antimalarial activity and molecular docking of hybrid 4-aminoquinoline-1,3,5-triazine derivatives. Exp Parasitol 2015; 157:59–67. 
26. Bhat HR, Singh UP, Gahtori P, Ghosh SK, Gogoi K, Prakash A, et al. Antimalarial activity and docking studies of novel bi-functional hybrids derived from 4-aminoquinoline and 1,3,5-triazine against wild and mutant malaria parasites as pf-DHFR inhibitor. RSC Adv 2013; 3:2942–2952. 
27. Sunduru N, Sharma M, Srivastava K, Rajakumar S, Puri SK, Saxena JK, et al. Synthesis of oxalamide and triazine derivatives as a novel class of hybrid 4-aminoquinoline with potent antiplasmodial activity. Bioorganic Med Chem 2009; 17:6451–6462. 
28. Gahtori P, Ghosh SK, Parida P, Prakash A, Gogoi K, Bhat HR, et al. Antimalarial evaluation and docking studies of hybrid phenylthiazolyl-1,3,5-triazine derivatives: a novel and potential antifolate lead for Pf-DHFR-TS inhibition. Exp Parasitol 2012; 130:292–299. 
29. Bhat HR, Singh UP, Gahtori P, Ghosh SK, Gogoi K, Prakash A, et al. Synthesis, docking, in vitro and in vivo antimalarial activity of hybrid 4-aminoquinoline-1,3,5-triazine derivatives against wild and mutant malaria parasites. Chem Biol Drug Des 2015; 86:265–271. 
30. Lozano V, Aguado L, Hoorelbeke B, Renders M, Camarasa MJ, Schols D, et al. Targeting HIV entry through interaction with envelope glycoprotein 120 (gp120): synthesis and antiviral evaluation of 1,3,5-triazines with aromatic amino acids. J Med Chem 2011; 54:5335–5348. 
31. Srivastava JK, Awatade NT, Bhat HR, Kmit A, Mendes K, Ramos M, et al. Pharmacological evaluation of hybrid thiazolidin-4-one-1,3,5-triazines for NF-κB, biofilm and CFTR activity. RSC Adv 2015; 5:88710–88718. 
32. Cascioferro S, Parrino B, Spanò V, Carbone A, Montalbano A, Barraja P, et al. 1,3,5-Triazines: a promising scaffold for anticancer drugs development. Eur J Med Chem 2017; 142:523–549. 
33. Popowycz F, Fournet G, Schneider C, Bettayeb K, Ferandin Y, Lamigeon C, et al. Pyrazolo[1,5-a]-1,3,5-triazine as a purine bioisostere: access to potent cyclin-dependent kinase inhibitor (R)-roscovitine analogue. J Med Chem 2009; 52:655–663. 
34. Srivastava JK, Pillai GG, Bhat HR, Verma A, Singh UP. Design and discovery of novel monastrol-1,3,5-triazines as potent anti-breast cancer agent via attenuating epidermal growth factor receptor tyrosine kinase. Sci Rep 2017; 7:5851-5868. 
35. Yaguchi SI, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, et al. Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. J Natl Cancer Inst 2006; 98:545–556. 
36. Sciú ML, Sebastián-Pérez V, Martinez-Gonzalez L, Benitez R, Perez DI, Pérez C, et al. Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3. J Enzyme Inhib Med Chem 2019; 34:87–96. 
37. Singla P, Luxami V, Paul K. Synthesis and in vitro evaluation of novel triazine analogues as anticancer agents and their interaction studies with bovine serum albumin. Eur J Med Chem 2016; 117:59–69. 
38. Su Q, Xu B, Tian Z, Gong Z. Novel 1,3,5‐triazine‐nicotinohydrazide derivatives induce cell arrest and apoptosis in osteosarcoma cancer cells and inhibit osteosarcoma in a patient‐derived orthotopic xenograft mouse model. Chem Biol Drug Des 2022; 99:320–330. 
39. Huang X, Zhao J, Bai J, Shen H, Zhang B, Deng L, et al. Risk and clinicopathological features of osteosarcoma metastasis to the lung: a population-based study. J Bone Oncol 2019; 16:100230-100237. 
40. Guan B, Jiang C. Design and development of 1,3,5-triazine derivatives as protective agent against spinal cord injury in rat via inhibition of NF-ĸB. Bioorganic Med Chem Lett 2021; 41:127964-127971. 
41. Higuchi T, Miyake K, Oshiro H, Sugisawa N, Yamamoto N, Hayashi K, et al. Trabectedin and irinotecan combination regresses a cisplatinum-resistant osteosarcoma in a patient-derived orthotopic xenograft nude-mouse model. Biochem Biophys Res Commun 2019; 513:326–331. 
42. Li J, Yang Z, Li Y, Xia J, Li D, Li H, et al. Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment. Oncotarget 2016; 7:44763–44778. 
43. Jingwen B, Yaochen L, Guojun Z. Cell cycle regulation and anticancer drug discovery. Cancer Biol Med 2017; 14:348-362. 
44. Raghavendra NM, Pingili D, Kadasi S, Mettu A, Prasad SVUM. Dual or multi-targeting inhibitors: the next generation anticancer agents. Eur J Med Chem 2018; 143:1277–1300. 
45. Geromichalos GD, Alifieris CE, Geromichalou EG, Trafalis DT. Overview on the current status on virtual high-throughput screening and combinatorial chemistry approaches in multi-target anticancer drug discovery; part II. J BUON 2016; 21:1337–1358. 
46. Isakoff MS, Bielack SS, Meltzer P, Gorlick R. Osteosarcoma: current treatment and a collaborative pathway to success. J Clin Oncol 2015; 33:3029–3035. 
47. Kaste SC, Pratt CB, Cain AM, Jones-Wallace DJ, Rao BN. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer 1999; 86:1602–1608. 
48. Nataraj V, Rastogi S, Khan SA, Sharma MC, Agarwala S, Vishnubhatla S, et al. Prognosticating metastatic osteosarcoma treated with uniform chemotherapy protocol without high dose methotrexate and delayed metastasectomy: a single center experience of 102 patients. Clin Transl Oncol 2016; 18:937–944.