Original articles
Xiaowei Wu, Mengdi Dai, Rongrong Cui, Yulan Wang, Chunpu Li, Xia Peng, Jihui Zhao, Bao Wang, Yang Dai, Dan Feng, Tianbiao Yang, Hualiang Jiang, Meiyu Geng, Jing Ai, Mingyue Zheng, Hong Liu. Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyridazinone derivatives as covalent FGFR inhibitors[J]. Acta Pharmaceutica Sinica B, 2021, 11(3): 781-794

Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyridazinone derivatives as covalent FGFR inhibitors
Xiaowei Wua, Mengdi Daib,c, Rongrong Cuid, Yulan Wanga, Chunpu Lia, Xia Pengb, Jihui Zhaoa,c, Bao Wanga, Yang Daib, Dan Fenga, Tianbiao Yanga, Hualiang Jianga,d, Meiyu Gengb, Jing Aib,c, Mingyue Zhenga,c,d, Hong Liua
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China;
b Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China;
c University of Chinese Academy of Sciences, Beijing 100049, China;
d School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
Fibroblast growth factor receptors (FGFRs) have emerged as promising targets for anticancer therapy. In this study, we synthesized and evaluated the biological activity of 66 pyrazolo[3,4-d]pyridazinone derivatives. Kinase inhibition, cell proliferation, and whole blood stability assays were used to evaluate their activity on FGFR, allowing us to explore structure—activity relationships and thus to gain understanding of the structural requirements to modulate covalent inhibitors’ selectivity and reactivity. Among them, compound 10h exhibited potent enzymatic activity against FGFR and remarkably inhibited proliferation of various cancer cells associated with FGFR dysregulation, and suppressed FGFR signaling pathway in cancer cells by the immunoblot analysis. Moreover, 10h displayed highly potent antitumor efficacy (TGI = 91.6%, at a dose of 50 mg/kg) in the FGFR1-amplified NCI-H1581 xenograft model.
Key words:    Tyrosine kinase    Covalent FGFR inhibitors    Virtual screening    Pyrazolo[3,4-d] pyridazinone    Structure—activity relationships    Antitumor efficacy   
Received: 2020-06-02     Revised: 2020-07-23
DOI: 10.1016/j.apsb.2020.09.002
Funds: We gratefully acknowledge financial support from the National Natural Science Foundation of China (81620108027 and 21632008 to Hong Liu, 81773634 to Mingyue Zheng and 81773762 to Jing Ai), National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2018ZX09711002, China), the Major Project of Chinese National Programs for Fundamental Research and Development (2015CB910304 to Hong Liu), “Personalized Medicines—Molecular Signature-based Drug Discovery and Development”, and Strategic Priority Research Pro-gram of the Chinese Academy of Sciences (XDA12050201 to Mingyue Zheng, XDA12020000 to Meiyu Geng and XDA12020103 to Jing Ai). The Natural Science Foundation of China for Innovation Research Group (81821005 to Meiyu Geng, China). The Collaborative Innovation Cluster Project of Shanghai Municipal Commission of Health and Family Planning (2020CXJQ02 to Meiyu Geng, China).
Corresponding author: Jing Ai, Mingyue Zheng, Hong Liu     Email:jai@simm.ac.cn;myzheng@simm.ac.cn;hliu@simm.ac.cn
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Xiaowei Wu
Mengdi Dai
Rongrong Cui
Yulan Wang
Chunpu Li
Xia Peng
Jihui Zhao
Bao Wang
Yang Dai
Dan Feng
Tianbiao Yang
Hualiang Jiang
Meiyu Geng
Jing Ai
Mingyue Zheng
Hong Liu

1. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Canc 2009;9:28-39.
2. Wu P, Nielsen TE, Clausen MH. FDA-approved small-molecule kinase inhibitors. Trends Pharmacol Sci 2015;36:422-39.
3. Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 2005;16: 139-49.
4. Turner N, Grose R. Fibroblast growth factor signaling: From development to cancer. Nat Rev Canc 2010;10:116-29.
5. Carter EP, Fearon AE, Grose RP. Careless talk costs lives: Fibroblast growth factor receptor signaling and the consequences of pathway malfunction. Trends Cell Biol 2015;25:221-33.
6. Heinzle C, Sutterlüty H, Grusch M, Grasl-Kraupp B, Berger W, Marian B. Targeting fibroblast-growth-factor-receptor-dependent signaling for cancer therapy. Expert Opin Ther Targets 2011;15: 829-46.
7. Dieci MV, Arnedos M, Andre F, Soria JC. Fibroblast growth factor receptor inhibitors as a cancer treatment: From a biologic rationale to medical perspectives. Canc Discov 2013;3:264-79.
8. Wesche J, Haglund K, Haugsten EM. Fibroblast growth factors and their receptors in cancer. Biochem J 2011;437:199-213.
9. Dutt A, Salvesen HB, Chen TH, Ramos AH, Onofrio RC, Hatton C, et al. Drug-sensitive FGFR2 mutations in endometrial carcinoma. Proc Natl Acad Sci U S A 2008;105:8713-7.
10. Turner N, Pearson A, Sharpe R, Lambros M, Geyer F, LopezGarcia MA, et al. FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer. Cancer Res 2010;70:2085-94.
11. Helsten T, Elkin S, Arthur E, Tomson BN, Carter J, Kurzrock R. The FGFR landscape in cancer: Analysis of 4,853 tumors by nextgeneration sequencing. Clin Canc Res 2016;22:259-67.
12. Katoh M. Therapeutics targeting FGF signaling network in human diseases. Trends Pharmacol Sci 2016;37:1081-96.
13. Ronca R, Giacomini A, Rusnati M, Presta M. The potential of fibroblast growth factor/fibroblast growth factor receptor signaling as a therapeutic target in tumor angiogenesis. Expert Opin Ther Targets 2015;19:1361-77.
14. Katoh M. Fibroblast growth factor receptors as treatment targets in clinical oncology. Nat Rev Clin Oncol 2019;16:105-22.
15. Nakanishi Y, Akiyama N, Tsukaguchi T, Fujii T, Sakata K, Sase H, et al. The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor. Mol Canc Therapeut 2014;13:2547-58.
16. Gavine PR, Mooney L, Kilgour E, Thomas AP, Al-Kadhimi K, Beck S, et al. AZD4547: An orally bioavailable, potent, and selective inhibitor of the fibroblast growth factor receptor tyrosine kinase family. Cancer Res 2012;72:2045-56.
17. Guagnano V, Furet P, Spanka C, Bordas V, Le Douget M, Stamm C, et al. Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor receptor family of receptor tyrosine kinase. J Med Chem 2011;54:7066-83.
18. Collin MP, Lobell M, Hübsch W, Brohm D, Schirok H, Jautelat R, et al. Discovery of rogaratinib (BAY 1163877): A pan-FGFR inhibitor. ChemMedChem 2018;13:437-45.
19. Perera TPS, Jovcheva E, Mevellec L, Vialard J, de Lange D, Verhulst T, et al. Discovery and pharmacological characterization of JNJ-42756493 (erdafitinib), a functionally selective small-molecule FGFR family inhibitor. Mol Canc Therapeut 2017;16:1010-20.
20. Futami T, Okada H, Kihara R, Kawase T, Nakayama A, Suzuki T, et al. ASP5878, a novel inhibitor of FGFR1, 2, 3, and 4, inhibits the growth of FGF19-expressing hepatocellular carcinoma. Mol Canc Therapeut 2017;16:68-75.
21. Zhao G, Li WY, Chen D, Henry JR, Li HY, Chen Z, et al. A novel, selective inhibitor of fibroblast growth factor receptors that shows a potent broad spectrum of antitumor activity in several tumor xenograft models. Mol Canc Therapeut 2011;10:2200-10.
22. Verstovsek S, Vannucchi AM, Rambaldi A, Gotlib JR, Mead AJ, Hochhaus A, et al. Interim results from Fight-203, a phase 2, openlabel, multicenter study evaluating the efficacy and safety of pemigatinib (INCB054828) in patients with myeloid/lymphoid neoplasms with rearrangement of fibroblast growth factor receptor 1 (FGFR1). Blood 2018;132:690.
23. Zhou W, Hur W, McDermott U, Dutt A, Xian W, Ficarro SB, et al. A structure-guided approach to creating covalent FGFR inhibitors. Chem Biol 2010;17:285-95.
24. Tan L, Wang J, Tanizaki J, Huang Z, Aref AR, Rusan M, et al. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors. Proc Natl Acad Sci U S A 2014;111:E4869 77.
25. Hagel M, Miduturu C, Sheets M, Rubin N, Weng W, Stransky N, et al. First selective small molecule inhibitor of FGFR4 for the treatment of hepatocellular carcinomas with an activated FGFR4 signaling pathway. Canc Discov 2015;5:424-37.
26. Brown WS, Tan L, Smith A, Gray NS, Wendt MK. Covalent targeting of fibroblast growth factor receptor inhibits metastatic breast cancer. Mol Canc Therapeut 2016;15:2096-106.
27. Brameld KA, Owens TD, Verner E, Venetsanakos E, Bradshaw JM, Phan VT, et al. Discovery of the irreversible covalent FGFR inhibitor 8-(3-(4-acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the treatment of solid tumors. J Med Chem 2017; 60:6516-27.
28. Joshi JJ, Coffey H, Corcoran E, Tsai J, Huang CL, Ichikawa K, et al. H3B-6527 is a potent and selective inhibitor of FGFR4 in FGF19-driven hepatocellular carcinoma. Cancer Res 2017;77:6999-7013.
29. Wang Y, Li L, Fan J, Dai Y, Jing A, Geng M, et al. Discovery of potent irreversible pan-fibroblast growth factor receptor (FGFR) inhibitors. J Med Chem 2018;61:9085-104.
30. Kalyukina M, Yosaatmadja Y, Middleditch MJ, Patterson AV, Smaill JB, Squire CJ. TAS-120 cancer target binding: Defining reactivity and revealing the first fibroblast growth factor receptor 1 (FGFR1) irreversible structure. ChemMedChem 2019;14:494-500.
31. Zhou Z, Chen X, Fu Y, Zhang Y, Dai S, Li J, et al. Characterization of FGF401 as a reversible covalent inhibitor of fibroblast growth factor receptor 4. Chem Commun 2019;55:5890-3.
32. Finlay MRV, Anderton M, Ashton S, Ballard P, Bethel PA, Box MR, et al. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J Med Chem 2014;57:8249-67.
33. Miller VA, Hirsh V, Cadranel J, Chen YM, Park K, Kim SW, et al. Afatinib versus placebo for patients with advanced, metastatic nonsmall-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): A phase 2b/3 randomised trial. Lancet Oncol 2012;13:528-38.
34. Davids MS, Brown JR. Ibrutinib: A first in class covalent inhibitor of bruton’s tyrosine kinase. Future Oncol 2014;10:957-67.
35. Wang Y, Dai Y, Wu X, Li F, Liu B, Li C, et al. The discovery and development of a series of pyrazolo[3,4-d]pyridazinone compounds as novel covalent FGFR inhibitors by rational drug design. J Med Chem 2019;62:7473-88.
36. Flanagan ME, Abramite JA, Anderson DP, Aulabaugh A, Dahal UP, Gilbert AM, et al. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J Med Chem 2014;57:10072-9.
37. Jackson PA, Widen JC, Harki DA, Brummond KM. Covalent modifiers: A chemical perspective on the reactivity of a,b-unsaturated carbonyls with thiols via hetero-Michael addition reactions. J Med Chem 2017;60:838-85.
38. Ascenzi P, Fanali G, Fasano M, Pallottini V, Trezza V. Clinical relevance of drug binding to plasma proteins. J Mol Struct 2014;1077: 4-13.
39. Wang J, Li-Chan XX, Atherton J, Deng L, Espina R, Yu L, et al. Characterization of HKI-272 covalent binding to human serum albumin. Drug Metab Dispos 2010;38:1083-93.
40. Dömötör O, Pelivan K, Borics A, Keppler BK, Kowol CR, Enyedy É A. Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187. J Pharmaceut Biomed Anal 2018;30:321-31.
41. Zhao Z, Bourne PE. Progress with covalent small-molecule kinase inhibitors. Drug Discov Today 2018;23:727-35.
42. Liu H, Ai J, Shen A, Chen Y, Wang X, Peng X, et al. c-Myc alteration determines the therapeutic response to FGFR inhibitors. Clin Canc Res 2017;23:974-84.
43. Nakanishi Y, Akiyama N, Tsukaguchi T, Fujii T, Satoh Y, Ishii N, Aoki M. Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1. Mol Canc Therapeut 2015;14: 704-12.
44. Harder E, Damm W, Maple J, Wu C, Reboul M, Xiang JY, et al. OPLS3: A force field providing broad coverage of drug-like small molecules and proteins. J Chem Theor Comput 2016;12: 281-96.
45. Frisch MJ, Schlegel HB, Scuseria GE, et al. Gaussian 09. Wallingford, CT, USA: Gaussian, Inc.; 2009.
46. Marenich AV, Cramer CJ, Truhlar DG. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J Phys Chem B 2009;113:6378-96.
47. Ai J, Chen Y, Peng X, Ji Y, Xi Y, Shen Y, et al. Preclinical evaluation of SCC244 (Glumetinib), a novel, potent, and highly selective inhibitor of c-Met in MET-dependent cancer models. Mol Canc Therapeut 2018;17:751-62.
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