3,4'-吡喃并[2,3-c]吡唑螺吲哚酮衍生物的绿色合成及抗肺癌活性研究

doi:10.6023/cjoc202603039

摘要/Abstract

为寻找新型抗肺癌耐药活性化合物, 以5-苯基-2,4-二氢-3H-吡唑-3-酮、丙二腈和靛红为原料, 绿色合成了21个3,4'-吡喃并[2,3-c]吡唑螺吲哚酮衍生物. 体外抗肺癌活性研究表明, 该类化合物不仅对A549细胞具有良好的抑制活性, 同时对A549/DDP也具有良好的抑制活性, 特别是6'-氨基-7-氯-2-氧代-3'-苯基-2'H-螺[吲哚啉-3,4'-吡喃并[2,3-c]吡唑]-5'-甲腈(HDY-3)对耐药株A549/DDP的活性是阳性对照药物顺铂的92倍. 机制研究表明, 化合物HDY-3可以显著阻滞A549/DDP细胞G₀/G₁及G₂/M期, 诱导细胞凋亡, 抑制细胞迁移. 分子对接、分子动力学模拟及MM/GBSA法计算结合自由能的结果揭示, 化合物HDY-3与ERK1的复合物表现出较高的稳定性和较强的结合能力, 化合物HDY-3可能通过抑制ERK1来影响RAS-RAF-MEK1/2-ERK1/2信号通路, 从而发挥抗肿瘤活性. 以上结果表明化合物HDY-3可以作为先导化合物, 进一步开发成为高效的抗肺癌药物.

关键词: 3-螺吲哚酮, 绿色合成, 衍生物, 抗肿瘤活性

In an effort to discover novel compounds active against drug-resistant lung cancer, 21 3,4'-pyrano[2,3-c]pyrazole-spirooxindole derivatives were synthesized via an environmentally friendly procedure using 5-phenyl-2,4-dihydro-3H-pyrazol-3-one, malononitrile, and isatin as starting materials. In vitro anti-lung cancer activity evaluation revealed that these compounds exhibited potent inhibitory activity not only against parental A549 cells but also against cisplatin-resistant A549/DDP cells. In particular, 6'-amino-7-chloro-2-oxo-3'-phenyl-2'H-spiro[indoline-3,4'-pyrano[2,3-c]pyrazole]-5'-carboni-trile (HDY-3) exhibited exceptional potency against the A549/DDP cells, with activity 92-fold higher than that of the positive control drug cisplatin. Mechanistic studies indicated that HDY-3 significantly blocked the cell cycle of A549/DDP cells at both the G₀/G₁ and G₂/M phases, induced apoptosis, and suppressed cell migration. Furthermore, molecular docking, molecular dynamics simulations, and MM/GBSA binding free energy calculations revealed that the HDY-3-ERK1 complex exhibited high stability and strong binding affinity. The antitumor activity of HDY-3 is likely mediated through inhibition of ERK1, thereby interfering with the RAS-RAF-MEK1/2-ERK1/2 signaling pathway. Collectively, these results suggest that compound HDY-3 can serve as a lead compound for further development into highly effective anti-lung cancer drugs.

Key words: 3-spirooxindole, green synthesis, derivative, antitumor activity

引用此文

杨俊, 黄冬燕, 梁光平, 刘雄伟. 3,4'-吡喃并[2,3-c]吡唑螺吲哚酮衍生物的绿色合成及抗肺癌活性研究[J]. 有机化学, 2026, 46(10): 3970-3982.

Jun Yang, Dongyan Huang, Guangping Liang, Xiongwei Liu. Green Synthesis of 3,4'-Pyrano[2,3-c]pyrazole-spirooxindole Derivatives and Their Antiproliferation Activities against Lung Cancer[J]. Chinese Journal of Organic Chemistry, 2026, 46(10): 3970-3982.

导出引用 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

分享此文

图/表 15

图1. 含吡喃并[2,3-c]吡唑或3,4'-吡喃-螺吲哚酮骨架的活性化合物

Figure 1. Active compounds containing pyrano[2,3-c]pyrazole or 3,4'-pyrano spirooxindole scaffolds

图式1. 文献报道的相关工作及本工作采取的合成策略

Scheme 1. Literature-reported background and synthetic strategy in this work

Entry	Solvent	Temp./℃	Time/h	Yield/%
1	CH₃OH	60	12	83
2	EtOH	60	12	68
3	EtOAc	60	12	44
4	Toluene	60	12	0
5	DCM	60	12	76
6	H₂O	60	12	89
7	H₂O	40	18	70
8	H₂O	r.t.	24	0
9	H₂O	80	4	96

表1. 反应条件的优化

Table 1. Optimization of reaction conditions

Compd.	R¹	R²	R³	R⁴	R⁵	Yield/%
HDY-1	H	Cl	H	H	H	96
HDY-2	H	OCH₃	H	H	H	98
HDY-3	H	H	H	Cl	H	77
HDY-4	H	H	H	CH₃	H	82
HDY-5	H	Br	H	H	H	90
HDY-6	H	H	Cl	H	H	92
HDY-7	H	H	H	Br	H	76
HDY-8	Br	H	H	H	H	79
HDY-9	H	H	F	H	H	97
HDY-10	H	F	F	H	H	90
HDY-11	H	NO₂	H	H	H	93
HDY-12	H	H	H	NO₂	H	86
HDY-13	H	H	OCH₃	H	H	93
HDY-14	H	H	H	H	CH₃	77
HDY-15	H	F	H	H	H	87
HDY-16	Cl	H	Cl	H	H	90
HDY-17	H	Br	H	H	CH₃	82
HDY-18	H	H	H	F	CH₃	89
HDY-19	H	H	H	H	Ph	77
HDY-20	H	H	H	H	H	96
HDY-21	H	CH₃	H	H	H	91

表2. 反应底物的拓展

Table 2. Scope of the substrates

图2. 化合物HDY-2的X射线单晶衍射图

Figure 2. X-Ray crystallographic structures of HDY-2

图式2. 克级反应

Scheme 2. Gram-scale reaction

Compd.	IC₅₀^a/(μmol•L^-1)
Compd.	A549	A549/DDP	RF^b
HDY-1	0.044±0.001	0.711±0.018	16.03
HDY-2	0.297±0.006	0.453±0.006	1.53
HDY-3	0.115±0.002	0.027±0.001	0.23
HDY-4	0.111±0.004	0.885±0.030	7.97
HDY-5	1.201±0.021	0.024±0.001	0.02
HDY-6	0.105±0.003	0.073±0.003	0.70
HDY-7	1.502±0.007	0.471±0.007	0.31
HDY-8	2.899±0.086	0.228±0.004	0.08
HDY-9	0.089±0.003	0.420±0.002	4.72
HDY-10	0.463±0.010	0.759±0.015	1.64
HDY-11	0.422±0.017	0.030±0.001	0.07
HDY-12	3.851±0.156	0.800±0.007	0.21
HDY-13	0.013±0.001	0.085±0.004	6.79
HDY-14	0.259±0.011	0.238±0.003	0.92
HDY-15	0.181±0.004	0.332±0.008	1.83
HDY-16	0.205±0.005	0.418±0.007	2.04
HDY-17	0.107±0.003	1.321±0.027	12.37
HDY-18	0.189±0.006	0.411±0.008	2.18
HDY-19	0.255±0.005	3.477±0.038	13.66
HDY-20	0.112±0.002	0.304±0.004	2.71
HDY-21	0.037±0.001	0.065±0.001	1.73
Cisplatin	1.480±0.008	2.496±0.117	1.69

表3. 化合物HDY-1~HDY-21对A549和A549/DDP细胞的抗增殖活性

Table 3. Anti-proliferation activities of compounds HDY-1~HDY-21 against A549, A549/DDP cells

Compd.	IC₅₀^a/(μmol•L^-1)
Compd.	A549	A549/DDP	BEAS-2B	SI^b	SI^c
HDY-3	0.115±0.002	0.027±0.001	8.726±0.518	75.88	323.18
Cisplatin	1.480±0.008	2.496±0.117	2.539±0.244	1.71	1.02

表4. 化合物HDY-3对BEAS-2B细胞的抗增殖活性

Table 4. Anti-proliferation activities of compound HDY-3 against BEAS-2B cells

图3. AO/EB双荧光染色法结果

Figure 3. AO/EB dual fluorescence staining assay results

图4. (A) PI染色法和流式细胞术检测细胞周期分布及(B)细胞周期分布的量化分析(n=3, *P<0.001, 与空白组对比; **P>0.05, 与空白组对比)

Figure 4. (A) PI staining and flow cytometry assays performed to detect the cell cycle distribution and (B) quantitative representation of cell cycle distribution(n=3; *P<0.001, vs control; **P>0.05, vs control)

图5. (A) Annexin V/PI染色法和流式细胞术检测到的细胞凋亡结果及(B)细胞凋亡率的量化分析(n=3, *P<0.001, 与空白组对比)

Figure 5. (A) Apoptosis detected by Annexin V/PI staining and flow cytometry assays and (B) quantitative representation of apoptosis rate (n=3; *P<0.001, vs control)

图6. (A)划痕实验结果及(B)伤口愈合率的量化分析(n=3, *P<0.001, 与空白组对比)

Figure 6. (A) Wound healing assay results, and (B) quantitative representation of wound healing rate (n=3, *P<0.001, vs control)

图7. (A)化合物HDY-3的激酶谱预测及(B)化合物HDY-3与ERK1 (PDB: 2ZOQ)分子对接图

Figure 7. (A) Kinase profiling prediction for compound HDY-3, and (B) molecular docking compound HDY-3 with ERK1 (PDB: 2ZOQ)

图8. 化合物HDY-3与ERK1复合物的分子动力学模拟: (A)根均方偏差(RMSD); (B)复合物氨基酸残基的根均方波动(RMSF); (C)回旋半径(Rg); (D)溶剂可及表面积(SASA); (E)氢键数量; (F)复合物的自由能形貌图

Figure 8. Molecular dynamics simulation of the compound HDY-3-ERK1 complex: (A) Root mean square deviation (RMSD); (B) Root mean square fluctuation (RMSF) of the amino acid residues in the complex; (C) Radius of gyration (Rg); (D) Solvent accessible surface area (SASA); (E) Number of hydrogen bonds; (F) Free energy landscape of the complex

Complex	ΔE_vdW	ΔE_ele	ΔG_GB	ΔG_SA	ΔG_bind
HDY-3-ERK1	-151.7±10.1	-29.0±7.4	118.7±10.1	-17.7±1.0	-79.6±9.5

表5. MM/GBSA法计算化合物HDY-3与ERK1的结合自由能(kJ/mol)a

Table 5. Binding free energy (kJ/mol) of the compound HDY-3-ERK1 complex calculated by the MM/GBSA method

参考文献 22

[1]	(a) Siegel R. L.; Kratzer T. B.; Giaquinto A. N.; Sung H.; Jemal A. Ca-Cancer J. Clin. 2025, 75, 10. doi: 10.3322/caac.v75.1
	(b) Han B. F.; Zheng R. S.; Zeng H. M.; Wang S. M.; Sun K. X.; Chen R.; Li L.; Wei W. Q.; He J. J. Natl. Cancer Cent. 2024, 4, 47.
[2]	(a) Su P. L.; Furuya N.; Asrar A.; Rolfo C.; Li Z.; Carbone D. P.; He K. J. Hematol. Oncol. 2025, 18, 35. doi: 10.1186/s13045-025-01679-1
	(b) Zhai X.; Zhang Z.; Chen Y.; Wu Y.; Zhen C.; Liu Y.; Lin Y.; Chen C. J. Hematol. Oncol. 2025, 18, 37. doi: 10.1186/s13045-025-01690-6
[3]	Zheng X. H.; Jiang Z.; Li X. L.; Zhang C.; Li Z.; Wu Y.; Wang X.; Zhang C.; Luo H. B.; Xu J.; Wu D. Bioorg. Med. Chem. 2018, 26, 5934. doi: 10.1016/j.bmc.2018.10.044
[4]	Qvortrup K.; Jensen J. F.; Sørensen M. S.; Kouskoumvekaki I.; Petersen R. K.; Taboureau O.; Kristiansen K.; Nielsen T. E. PLoS One 2017, 12, 0162642.
[5]	Saundane A. R.; Vijaykumar K.; Vaijinath A. V. Bioorg. Med. Chem. Lett. 2013, 23, 1978. doi: 10.1016/j.bmcl.2013.02.036 pmid: 23454016
[6]	Patravale A. A.; Gore A. H.; Kolekar G. B.; Deshmukh M. B.; Choudhari P. B.; Bhatia M. S.; Prabhu S.; Jamdhade M. D.; Patole M. S.; Anbhule P. V. J. Taiwan Inst. Chem. Eng. 2016, 68, 105. doi: 10.1016/j.jtice.2016.09.034
[7]	(a) Reddy B. N.; Ramana C. V. Tetrahedron 2017, 73, 888. doi: 10.1016/j.tet.2016.12.074
	(b) Qing B.; Yang Z.; Wu Z. W.; Zhang Z. C.; Zhou Y. Q.; Yan X. L.; Liu Y. B.; Feng X. M. J. Am. Chem. Soc. 2025, 147, 7729. doi: 10.1021/jacs.4c17425
[8]	Zhang M. M.; Sha F.; Wu X. Y. Chin. J. Org. Chem. 2025, 45, 1369 (in Chinese). doi: 10.6023/cjoc202406026
	(张明美, 沙风, 伍新燕, 有机化学, 2025, 45, 1369.)
[9]	Xie J.; Xing X. Y.; Sha F.; Wu Z. Y.; Wu X. Y. Org. Biomol. Chem. 2016, 14, 8346. doi: 10.1039/C6OB01256F
[10]	Li Z. J.; Qu N.; Zhou J. Y.; Sun J. J.; Ren Q.; Meng J. Y.; Wang G. C.; Wang R. Y.; Liu J.; Chen Y. J.; Zhang S. L.; Zheng M. Y.; Li X. T. Nucleic Acids Res. 2024, 52, 489.
[11]	Kinoshita T.; Yoshida I.; Nakae S.; Okita K.; Gouda M.; Matsubara M.; Yokota K.; Ishiguro H.; Tada T. Biochem. Biophys. Res. Commun. 2008, 377, 1123. doi: 10.1016/j.bbrc.2008.10.127
[12]	(a) Degirmenci U.; Wang M.; Hu J. Cells 2020, 9, 198. doi: 10.3390/cells9010198
	(b) Xiao H.; Wang A. X.; Shuai W.; Qian Y. P.; Wu C. Y.; Wang X.; Yang P. P.; Sun Q.; Wang G.; Ouyang L.; Sun Q. Signal Transduction Targeted Ther. 2025, 10, 70. doi: 10.1038/s41392-025-02169-z
[13]	Yang S. H.; Sharrocks A. D.; Whitmarsh A. J. Gene 2013, 513, 1. doi: 10.1016/j.gene.2012.10.033
[14]	Voisin L.; Saba-El-Leil M. K.; Julien C.; Frémin C.; Meloche S. Mol. Cell. Biol. 2010, 30, 2918. doi: 10.1128/MCB.00131-10 pmid: 20368360
[15]	Yang J.; Mei M.; Wang X. H.; Liang G. P.; Zhuo J. R. CN 2026103585560, 2026.
[16]	Yang J.; Liang G. P.; Liu X. L. Synth. Commun. 2023, 53, 262. doi: 10.1080/00397911.2023.2164863
[17]	Yin Z. G.; Liu X. W.; Wang H. J.; Zhang M.; Liu X. L.; Zhou Y. New J. Chem. 2022, 46, 1295. doi: 10.1039/D1NJ05170A
[18]	Wang S. H.; Guang Y. F.; Liu X. J.; Yuan X. Y.; Yu G. X.; Li Y. R.; Zhang Y. B.; Song J.; Li W.; Zhang S. Y. Chin. J. Org. Chem. 2021, 41, 3617 (in Chinese). doi: 10.6023/cjoc202103059
	(王胜辉, 关永风, 刘秀娟, 原信颖, 蔚广曦, 李银茹, 张雁冰, 宋健, 李雯, 张赛扬, 有机化学, 2021, 41, 3617.)
[19]	Hu S. L.; Tong L. J.; Qin Q.; Wen J. X.; Li Y.; Feng F.; Wu K. Z.; Zhou Y.; Shang J. S.; Wang J. J.; Liu J. B.; Xie H.; Lu X. Y. J. Med. Chem. 2024, 67, 20531. doi: 10.1021/acs.jmedchem.4c01975
[20]	Wang B. C.; Shi T.; Jia S. L.; Wang E. Y.; Ruan X. Q.; Sheng C. Q.; Wu S. C.; Zhou Q. F. J. Med. Chem. 2025, 68, 1300. doi: 10.1021/acs.jmedchem.4c01859
[21]	(a) Zare F.; Ataollahi E.; Mardaneh P.; Sakhteman A.; Keshavarz V.; Solhjoo A.; Emami L. Sci. Rep. 2024, 14, 7749. doi: 10.1038/s41598-024-58485-x
	(b) Wang L.; Li Z. C.; Hu T. C.; Li Q. Y.; Zhang L. W.; Mei X. Y.; Qi X.; Liu S.; Kong W. J.; Luo J. S.; Wu A. G.; Huang F. H.; Li S. R.; Dai S.; Zhang C. X.; Li R.; Wu J. M. Adv. Sci. 2026, 13, e09807. doi: 10.1002/advs.v13.12
[22]	Yang J.; Huang D. Y.; Liang G. P.; Liu X. L. Chem. J. Chin. Univ. 2026, 47, 20260072 (in Chinese).
	(杨俊, 黄冬燕, 梁光平, 刘雄利, 高等学校化学学报, 2026, 47, 20260072.) doi: 10.7503/cjcu20260072