English

• 1.   Introduction

  Cancer is still a leading life-threatening disease worldwide. The incidence and mortality rates of cancer in China account to 23.7% and 30%, respectively, across the glo- be.^[1,2] This figure will further rise because of aging, intensification of industrialization and urbanization, lifestyle modifications, etc. Thus, the burden of cancer cannot be ignored. Although chemotherapy has emerged as one of the main strategies for the treatment of tumors, numerous drugs have produced multi-drug resistance and fatal side effects in clinical.^[3] As a consequence, it is of great significance to develop effective and side-effect anticancer drugs in the face of global cancer problems. Pyrimidine skeleton and heterocyclic annulated pyrimidines, elemental structural motifs of several synthetic and natural occurring products, attract great attention due to their remarkable biological activities such as antitumor, ^[4,5] anti-inflamma- tory, ^[6] anti-hepatitis, ^[7] anti-diabetic^[8] and antimicrobial^[9] etc. For example, trifluorothymidine (1) can be used as a potential chemotherapeutic agent for the treatment of brain tumors, ^[10] compound 2 is a potent and orally available agent of hedgehog signaling pathway inhibitor with improved physicochemical and pharmacokinetic proper- ties, ^[11] and compound 3 has promising activity against Hela cell line (IC₅₀＝2.22 μmol/L) (Figure 1).^[12]

  Figure 1

  

  Figure 1.  Representative anti-tumor agents and design of 2, 4, 6-substituted pyrimidines

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  Besides, hydrazine derivatives have recently received considerable attention on account of their diverse biological activities including antifungal, ^[13] anti-bacterial, ^[14] anti-inflammatory, anti-ulcerogenic, ^[15] antiviral, ^[16,17] antioxidant, ^[18] antitumor^[19] and so on.^[20] Therefore, hydrazine as an active building block has important value and research significance in the design and synthesis of antitumor drugs. With the aim of identifying new antitumor agents, a series of novel 2, 4, 6-substituted pyrimidine derivatives based on molecular hybridization strategy were synthesized and evaluated for their antitumor activities.

  2.   Results and discussion

  2.1   Chemistry

  The synthetic strategy for the preparation of the target compound was depicted in Scheme 1. Firstly, compound 8 was prepared via cyclization reaction of ethyl 4, 4, 4-trifluo- roacetoacetate with thiourea in the presence of potassium hydroxide under reflux in ethanol for 3 h. Next, compound 8 and bromopropyne were reacted in N, N-dimethylforma- mide (DMF) at 90 ℃ for 4 h to obtain compound 9. Then compound 9 was added to phosphorus oxychloride and then heated to 90 ℃ for 3 h to yield compound 10. Compound 10 was reacted with hydrazine hydrate in anhydrous ethanol at 80 ℃ for 2 h to obtain compound 11. Finally, compound 11 with appropriate aromatic aldehyde reacted in anhydrous ethanol at 80 ℃ for 3~6 h to obtain the target compounds 12a~12o. The structures of compounds 12a~12o were fully characterized by ¹H NMR, ¹³C NMR and HRMS.

  Scheme 1

  

  Scheme 1.  Synthesis of hydrazone-substituted pyrimidine derivatives

  Reagents and conditions: (a) CH₃CH₂OH, KOH, 80 ℃, 3 h; (b) N, N-dimethylformamide, 90 ℃, 4 h; (c) POCl₃, 90 ℃, 3 h; (d) CH₃CH₂OH, hydrazine hydrate, 80 ℃, 2 h; (e) CH₃CH₂OH, substituted benzaldehyde, 80 ℃, 3~6 h

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  2.2   Cytotoxic activity

  The anti-proliferative activities of the target compounds were evaluated against human cancer cell lines of different origins including MCF-7 (human breast cancer cell), MGC-803 (human gastric cancer cell), PC-3 (human prostate cancer cell), Hela (human cervical cancer cell) and A549 (human lung cancer cell) via the methyl thiazolyl tetrazolium (MTT) assay. 5-Fluorouracil (5-Fu) was employed as the positive drug. The results of preliminary biological evaluation were summarized in Table 1.

  Table 1

  

  Table 1.  Antitumor activities [IC₅₀/(μmol·L^－1)] of target compounds against five cancer cell lines

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  Compd.	R	MCF-7	MGC-803	PC-3	Hela	A549
  9	—	> 64	28.15±1.56	> 64	> 64	> 64
  10	—	> 64	26.56±1.53	> 64	> 64	> 64
  11	—	> 64	25.47±1.51	> 64	42.43±1.62	> 64
  12a	C6H5	8.23±1.27	11.38±1.40	3.52±0.90	27.87±1.44	33.09±1.62
  12b	2-ClC6H4	11.63±1.03	7.06±0.89	8.61±1.31	36.01±1.55	30.43±1.59
  12c	3-ClC6H4	12.80±1.45	10.62±1.16	3.82±0.87	26.79±1.42	46.55±1.77
  12d	4-ClC6H4	8.21±1.04	7.68±1.01	4.21±0.83	17.97±1.23	16.61±1.32
  12e	3, 4-F2C6H3	12.73±1.32	10.18±1.06	6.72±1.07	19.27±1.28	48.07±1.78
  12f	2-CH3C6H4	11.52±1. 37	11.47±1.46	9.71±1.26	33.68±1.52	27.45±1.54
  12g	4-CH3C6H4	12.76±1.05	10.61±1.38	9.47±1.32	42.04±1.62	28.78±1.56
  12h	2-CH3OC6H4	14. 21±1.63	31.43±1.62	19.27±1.07	> 64	> 64
  12i	3-CH3OC6H4	29.51±1.36	20.53±1.34	15.31±1.19	22.44±1.35	47.03±1.78
  12j	4-CH3OC6H4	43.81±1.56	55.10±1.87	30.46±1.48	> 64	> 64
  12k	2-Thienyl	8.32±1.13	9.11±1.03	6.83±0.97	16.40±1.25	24.05±1.44
  12l	2-Pyridinyl	9.18±1.32	3.63±0.82	1.37±0.31	23.89±1.37	7.85±1.00
  12m	3-Pyridinyl	11.33±1.35	5.78±1.04	9.66±1.42	28.45±1.45	49.15±1.80
  12n	4-Pyridinyl	13.14±1.54	5.38±0.92	7.27±1.17	21.06±1.32	53.37±1.83
  12o	4-[1, 1'-Biphenyl]	12.16±1.41	10.23±1.07	8.46±1.27	39.05±1.59	54.14±1.84
  5-Fub	—	13.52±0.93	12.82±0.71	9.83±0.58	12.19±1.26	10.38±1.25
  a Antitumor activity was assayed by exposure for 72 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). Dates are presented as the mean±SDs of three independent experiments. b Positive control.

  From the preliminary results in Table 1, the anti-proli- ferative activities of the intermediates 9, 10 and 11 possessed relatively weak anti-proliferative activity against the tested five cell lines. However, after a series of structural modifications, the anti-proliferative activities of the target compounds 12a~12o were obviously improved, especially for PC-3. With the anti-proliferative activities against the cancer cell lines in hand, next, the preliminary structure-activity relationships of the target compounds were explored. Firstly, compared with compounds 12b~12d, when the same electron-withdrawing group was introduced on the benzene ring, the anti-proliferative activities of the para-position against MCF-7, Hela and A549 were relatively better than the ortho and meta-positions. But the introduction of multiple electron-withdrawing groups such as compound 12e had little effect on the improvement of anti-proliferative activity. However, compared with compounds 12f~12j, when an electron-donating group was introduced on the benzene ring, different sites and the number of substituents had greater influence on the anti- proliferative activities, such as compounds 12h~12j contained electron-donating methoxy substituent, the anti- proliferative activity of the meta-position except MCF-7 was relatively better than the ortho and para-positions. When aromatic substituent containing hetero atom was introduced into the structure of the compound, its anti- proliferative activities against MCF-7, MGC-803 and PC-3 were significantly improved, compared with the positive control drug 5-fluorouracil. With the volume of aromatic substituent increasing, the anti-proliferative activity of the target compounds had been significantly decreased, such as compound 12o. The 18 compounds in this series had certain activities on several tested tumor cells, among which PC-3 had the best anti-proliferative activity. Among them, the anti-proliferative activities of compound 12l against MCF-7, MGC-803, PC-3, and A549 were 9.18, 3.63, 1.37, 7.85 μmol/L, respectively, significantly better than 5-fluorouracil.

  3.   Conclusion

  In summary, a series of novel hydrazone-substituted pyrimidine derivatives were designed, synthesized and evaluated for their anti-proliferative activity against MCF-7, MGC-803, PC-3, Hela and A549 human cancer cell lines. Most of the synthesized compounds showed moderate to potent activity against five selected cancer cell lines. Among them, compound 12l displayed the most potent anti-proliferative activity against PC-3 cell line. Taken together, these results suggested that compound 12l might be a valuable lead compound for anti-tumor agents.

  4.   Experimental section

  4.1   Materials

  Silicagel: Qingdao Kangyexin Medicinal Silicone Desiccant Company Limited. Column chromatography silica gel: Yantai Jiangyou Silicone Development Company Limited. Potassium hydroxide: Tianjin Yongda Chemical Reagent Company Limited; Anhydrous Ethanol: Tianjin Yongda Chemical Reagent Company Limited. N, N-Di- methylformamide: Tianjin Yongda Chemical Reagent Company Limited. All reagents and solvents were purchased from commercial sources and were used without further purification. Melting points were determined on an X-5 micro melting apparatus and are uncorrected. ¹H NMR and ¹³C NMR spectra were measured using a DPX-DPX- 400 superconducting nuclear magnetic resonance instrument. Chemical shifts (δ) are given relative to TMS. High-resolution mass spectra were measured using a Waters-Micromass Q-TofMicro High Resolution Determination of tetragonal-flight time tandem mass spectrometer.

  4.2   MTT assay

  Cells in the logarithmic growth phase were seeded in 96-well plates at 3000~5000 cells per well. After the cells were cultured for 24 h, different concentrations of compounds were treated for 72 h, respectively. MTT (methyl thiazolyl tetrazolium, Solarbio) was added to each well at a final concentration of 0.5 mg/mL. After 4 h in a 37 ℃ incubator, the medium was aspirated. 150 mL of dimethyl sulfoxide (DMSO) was then added to each well to dissolve the formazan, and the plate was shaken on a shaker for 10 min. The absorbance was measured by an enzyme-linked immunosorbent assay reader (BioTek, USA) at a wavelength of 490 nm, and the cell survival rate was measured. The concentration-response curve generated by SPSS 16.0 software was used to determine the concentration of compound (IC₅₀) required to inhibit 50% of cell growth. Results were Mean±SD of three independent experiments.

  4.3   Chemistry

  2-Mercapto-6-(trifluoromethyl)pyrimidin-4-ol (8), ^[21] 2- (prop-2-yn-1-ylthio)-6-(trifluoromethyl)pyrimidin-4-ol (9), ^[22] 4-chloro-2-(prop-2-yn-1-ylthio)-6-(trifluoromethyl)- pyrimidine (10)^[22] were synthesized according to the literatures.

  4.3.1   Synthesis of 4-hydrazinyl-2-(prop-2-yn-1-yl- thio)-6-(trifluromethyl)pyrimidine (11)

  Compound 10 (2.11 g, 8.42 mmol) was added to 20 mL of absolute ethanol at room temperature, heated to 80 ℃, and hydrazine hydrate (821 μL, 16.84 mmol) was slowly added. The reaction was completed by thin-layer chromatography (TLC). The reaction mixture was cooled to room temperature and filtered to obtain 4-hydrazinyl-2-(prop- 2-yn-1-ylthio)-6-(trifluoromethyl)pyrimidine (11), white solid, yield 81.7%. m.p. 123.1~124.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.30 (s, 1H), 6.93 (s, 1H), 4.68 (s, 2H), 3.94 (d, J＝2.5 Hz, 2H), 3.12 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.9,168.7,121.5,104.5, 74.2, 73.2, 33.5; HRMS (ESI) calcd for C₈H₈F₃N₄S [M+H]⁺ 249.0422, found 249.0424.

  4.3.2   General procedure for the synthesis of compounds 12a~12o

  Compound 11 (0.15 g, 0.609 mmol) was added in 6 mL of absolute ethanol at room temperature, heated to 80 ℃, and benzaldehyde (70.44 μL, 0.609 mmol) was added slowly. The reaction was completed by TLC, cooled to room temperature, and the mixture was stirred. Column chromatography [V(petroleum ether):V(ethyl acetate)＝6:1) gave compound 12a.

  4-(2-Benzylidenehydrazinyl)-2-(prop-2-yn-1-ylthio)-6- (trifluoromethyl)pyrimidine (12a): White solid, yield 52.5%. m.p. 156.1~156.7 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.19 (s, 1H), 8.23 (s, 1H), 7.97 (d, J＝7.3 Hz, 1H), 7.80~7.74 (m, 2H), 7.46 (d, J＝6.4 Hz, 2H), 7.27 (s, 1H), 4.00 (d, J＝2.4 Hz, 2H), 3.19 (t, J＝2.4 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.9,167.3,162.0,145.8,133.8,132.8,130.1,129.2,128.8, 95.9, 79.9, 73.4, 18.6; HRMS (ESI) calcd for C₁₅H₁₂F₃N₄S [M+H]⁺ 337.0735, found 337.0734.

  4-(2-(2-Chlorobenzylidene)hydrazinyl)-2-(prop-2-yn-1- ylthio)-6-(trifluoromethyl)pyrimidine (12b): White solid powder, yield 62.7%. m.p. 143.7~144.8 ℃; ¹H NMR (400MHz, DMSO-d₆) δ: 12.33 (s, 1H), 8.62 (s, 1H), 8.13~8.05 (m, 1H), 7.52~7.46 (m, 1H), 7.46~7.39 (m, 2H), 7.27 (s, 1H), 4.00 (d, J＝2.5 Hz, 2H), 3.19 (t, J＝2.5 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.0,162.0,141.6,132.9,131.2,131.1,129.8,127.4,126.9,121.9,119.2, 96.0, 79.7, 73.4, 18.6. HRMS (ESI) calcd for C₁₅H₁₁ClF₃N₄S [M+H]⁺ 371.0345, found 371.0344.

  4-(2-(3-Chlorobenzylidene)hydrazinyl)-2-(prop-2-yn-1- ylthio)-6-(trifluoromethyl)pyrimidine (12c): White solid, yield 59.8%. m.p. 149.5~150.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.28 (s, 1H), 8.19 (s, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.48 (d, J＝4.7 Hz, 2H), 7.34 (s, 1H), 3.99 (d, J＝2.5 Hz, 2H), 3.18 (t, J＝2.5 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.9,162.1,144.2,136.1,133.7,130.7,129.7,126.4,125.7,121.9,119.2, 96.2, 79.9, 73.4, 18.6; HRMS (ESI) calcd for C₁₅H₁₁ClF₃N₄S [M+H]⁺ 371.0345, found 371.0344.

  4-(2-(4-Chlorobenzylidene)hydrazinyl)-2-(prop-2-yn-1- ylthio)-6-(trifluoromethyl)pyrimidine (12d): White solid, yield 60.3%. m.p. 143.5~143.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.11 (s, 1H), 8.51 (s, 1H), 7.86 (d, J＝7.6 Hz, 1H), 7.31~7.25 (m, 3H), 7.20 (s, 1H), 3.99 (d, J＝2.5 Hz, 2H), 3.19 (t, J＝2.5 Hz, 1H); ¹³C NMR (101MHz, DMSO-d₆) δ: 169.9,161.9,144.9,136.6,130.9,129.7,126.2,121.9,119.2, 79.9, 73.4, 18.6; HRMS (ESI) calcd for C₁₅H₁₁ClF₃N₄S [M+H]⁺ 371.0345, found 371.0344.

  4-(2-(3, 4-Difluorobenzylidene)hydrazinyl)-2-(prop-2- yn-1-ylthio)-6-(trifluoromethyl)pyrimidine (12e): White solid, yield 33.1%. m.p. 153.1~153.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.26 (s, 1H), 8.16 (s, 1H), 7.94 (d, J＝8.3 Hz, 1H), 7.64~7.57 (m, 1H), 7.51 (dd, J＝10.3, 8.4 Hz, 1H), 7.36 (s, 1H), 3.98 (d, J＝2.6 Hz, 2H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.9,161.9,159.8,151.0,149.2,143.0,124.4,119.1,118.0,116.5,115.4, 96.2, 80.1, 73.7, 18.5; HRMS (ESI) calcd for C₁₅H₁₀F₅N₄S [M+H]⁺ 373.0546, found 373.0547.

  4-(2-(2-Methylbenzylidene)hydrazinyl)-2-(prop-2-yn-1- ylthio)-6-(trifluoromethyl)pyrimidine (12f): White solid, yield 59.0%. m.p. 158.2~158.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.14 (s, 1H), 8.57 (s, 1H), 7.94 (dd, J＝7.7, 1.4 Hz, 1H), 7.46~7.39 (m, 1H), 7.25 (s, 1H), 7.10 (d, J＝8.2 Hz, 1H), 7.02 (t, J＝7.5 Hz, 1H), 3.98 (d, J＝2.6 Hz, 2H), 3.87 (s, 3H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.9,161.9,157.6,153.3,141.4,131.6,125.6,121.8,120.7,119.2,111.7, 95.9, 79.9, 73.4, 55.7, 18.6; HRMS (ESI) calcd for C₁₆H₁₅F₃N₄OS [M+OH]^－ 367.0840, found 367.0800.

  4-(2-(4-Methylbenzylidene)hydrazinyl)-2-(prop-2-yn-1- ylthio)-6-(trifluoromethyl)pyrimidine (12g): White solid, yield 48.0%. m.p. 152.7~153.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.12 (s, 1H), 8.18 (s, 1H), 7.65 (d, J＝8.1 Hz, 2H), 7.28 (d, J＝6.7 Hz, 1H), 7.25 (d, J＝4.0 Hz, 2H), 3.98 (d, J＝2.5 Hz, 2H), 3.35 (s, 3H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.9,161.9,153.6,146.0,140.0,131.1,129.4,127.0,122.0, 95.9, 79.9, 73.4, 21.0, 18.6; HRMS (ESI) calcd for C₁₆H₁₄F₃N₄S [M+H]⁺ 351.0891, found 351.0892.

  4-(2-(2-Methoxybenzylidene)hydrazinyl)-2-(prop-2-yn- 1-ylthio)-6-(trifluoromethyl)pyrimidine (12h): White solid, yield 47.3%. m.p.143.1~143.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.15 (s, 1H), 8.57 (s, 1H), 7.95 (dd, J＝7.7, 1.3 Hz, 1H), 7.45~7.39 (m, 1H), 7.25 (s, 1H), 7.11 (d, J＝8.2 Hz, 1H), 7.02 (t, J＝7.5 Hz, 1H), 3.98 (d, J＝2.6 Hz, 2H), 3.86 (s, 3H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.9,161.9,157.6,153.5,141.5,131.6,125.7,121.9,120.8,119.2,111.8, 95.9, 79.9, 73.4, 55.7, 18.6; HRMS (ESI) calcd for C₁₆H₁₄F₃N₄OS [M+H]⁺ 367.0840, found 367.0841.

  4-(2-(3-Methoxybenzylidene)hydrazinyl)-2-(prop-2-yn- 1-ylthio)-6-(trifluoromethyl)pyrimidine (12i): White solid, yield 47.5%. m.p. 147.8~148.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.18 (s, 1H), 8.18 (s, 1H), 7.37 (s, 1H), 7.35 (d, J＝2.5 Hz, 1H), 7.30 (s, 1H), 7.27 (s, 1H), 7.01 (ddd, J＝7.7, 2.5, 1.6 Hz, 1H), 3.99 (d, J＝2.5 Hz, 2H), 3.85~ 3.80 (m, 3H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.1,161.9,159.5,145.7,135.2,129.9,129.6,121.9,119.6,116.0 111.8, 96.0, 79.9 73.4, 55.1, 18.6; HRMS (ESI) calcd for C₁₆H₁₄F₃N₄OS [M+H]⁺ 367.0840, found 367.0842.

  4-(2-(4-Methoxybenzylidene)hydrazinyl)-2-(prop-2-yn- 1-ylthio)-6-(trifluoromethyl)pyrimidine (12j): White solid, yield 72.1%. m.p. 138.9~140.1 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.07 (s, 1H), 8.16 (s, 1H), 7.71 (d, J＝8.8 Hz, 2H), 7.23 (s, 1H), 7.01 (d, J＝8.8 Hz, 2H), 3.98 (d, J＝2.5 Hz, 2H), 3.81 (s, 3H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.8,161.8,160.9 145.8,128.7,126.4,122.0,119.2,114.3, 95.8, 79.9, 73.4, 55.3, 18.5; HRMS (ESI) calcd for C₁₆H₁₄F₃N₄OS [M+H]⁺ 367.0840, found 367.0839.

  2-(Prop-2-yn-1-ylthio)-4-(2-(thiophen-2-ylmethylene)- hydrazinyl)-6-(trifluoromethyl)pyrimidine (12k): White solid, yield 70.2%. m.p. 130.5~131.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.19 (s, 1H), 8.86 (s, 1H), 8.40 (s, 1H), 7.68 (d, J＝5.0 Hz, 1H), 7.49 (d, J＝2.9 Hz, 1H), 7.06 (s, 1H), 3.98 (d, J＝2.5 Hz, 2H), 3.18 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.0,161.6,155.7,140.9,138.5,133.8,131.0,130.8,129.0,128.0, 79.9, 73.5, 18.5; HRMS (ESI) calcd for C₁₃H₁₀F₃N₄S₂ [M+H]⁺ 343.0299, found 343.0300.

  2-(Prop-2-yn-1-ylthio)-4-(2-(pyridin-2-ylmethylene)- hydrazinyl)-6-(trifluoromethyl)pyrimidine (12l): White solid, yield 58.6%. m.p. 195.8~196.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.37 (s, 1H), 8.61 (d, J＝4.7 Hz, 1H), 8.25 (s, 1H), 8.11 (d, J＝7.9 Hz, 1H), 7.88 (dd, J＝10.9, 4.5 Hz, 1H), 7.46~7.39 (m, 1H), 7.35 (s, 1H), 4.00 (d, J＝2.5 Hz, 2H), 3.19 (t, J＝2.5 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.1,162.2,159.7,152.7,149.5,146.4,146.1,136.8,124.3,120.2, 96.2, 79.9, 73.5, 20.2, 18.6. HRMS (ESI) calcd for C₁₄H₁₁F₃N₅S [M+H]⁺ 338.0687, found 338.0686.

  2-(Prop-2-yn-1-ylthio)-4-(2-(pyridin-3-ylmethylene)- hydrazinyl)-6-(trifluoromethyl)pyrimidine (12m): White solid, yield 59.4%. m.p. 185.5~186.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.34 (s, 1H), 8.92 (s, 1H), 8.61 (d, J＝4.6 Hz, 1H), 8.24 (d, J＝8.3 Hz, 2H), 7.48 (dd, J＝7.8, 4.6 Hz, 1H), 7.36 (s, 1H), 3.99 (d, J＝2.4 Hz, 2H), 3.19 (d, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.0,162.2,150.6,148.7 142.9,136.9,133.5,129.8,123.9,121.9, 96.2, 79.9, 73.5, 18.6; HRMS (ESI) calcd for C₁₄H₁₁F₃N₅S [M+H]⁺ 338.0687, found 338.0685.

  2-(Prop-2-yn-1-ylthio)-4-(2-(pyridin-4-ylmethylene)- hydrazinyl)-6-(trifluoromethyl)pyrimidine (12n): White solid, yield 72.1%. m.p. 175.8~176.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.44 (s, 1H), 8.65 (d, J＝5.4 Hz, 2H), 8.19 (s, 1H), 7.74 (d, J＝5.9 Hz, 2H), 7.37 (s, 1H), 4.00 (d, J＝2.5 Hz, 2H), 3.19 (t, J＝2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.1,162.3,150.2,143.2,140.9,121.9,120.9,119.2, 96.3, 79.8, 73.5, 18.6; HRMS (ESI) calcd for C₁₄H₁₁F₃N₅S [M+H]⁺ 338.0687, found 338.0688.

  4-(2-([1, 1'-Biphenyl]-4-ylmethylene)hydrazinyl)-2- (prop-2-yn-1-ylthio)-6-(trifluoromethyl)pyrimidine (12o): White solid, yield 72.3%. m.p. 150.2~150.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.23 (s, 1H), 8.26 (s, 1H), 7.86 (d, J＝8.3 Hz, 2H), 7.76 (d, J＝8.3 Hz, 2H), 7.72 (d, J＝7.5 Hz, 2H), 7.50 (t, J＝7.6 Hz, 2H), 7.40 (t, J＝7.3 Hz, 1H), 7.30 (s, 1H), 3.99 (d, J＝2.4 Hz, 2H), 3.18 (t, J＝2.5 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ: 192.6,169.8,161.9,145.3,141.4,139.1,132.6,130.3,128.9,127.5,126.9,126.5,119.3, 95.8, 79.8, 73.3, 18.4; HRMS (ESI) calcd for C₂₁H₁₆F₃N₄S [M+H]⁺ 413.1048, found 413.1049.

  Supporting Information   ¹H NMR and ¹³C NMR spectra of compounds 9, 10 and 11 as well as 12a~12o are available for free download from our website (http://sioc-journal.cn/).

  
  
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  Figure 1  Representative anti-tumor agents and design of 2, 4, 6-substituted pyrimidines

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  Scheme 1  Synthesis of hydrazone-substituted pyrimidine derivatives

  Reagents and conditions: (a) CH₃CH₂OH, KOH, 80 ℃, 3 h; (b) N, N-dimethylformamide, 90 ℃, 4 h; (c) POCl₃, 90 ℃, 3 h; (d) CH₃CH₂OH, hydrazine hydrate, 80 ℃, 2 h; (e) CH₃CH₂OH, substituted benzaldehyde, 80 ℃, 3~6 h

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  Table 1.  Antitumor activities [IC₅₀/(μmol·L^－1)] of target compounds against five cancer cell lines

  Compd.	R	MCF-7	MGC-803	PC-3	Hela	A549
  9	—	> 64	28.15±1.56	> 64	> 64	> 64
  10	—	> 64	26.56±1.53	> 64	> 64	> 64
  11	—	> 64	25.47±1.51	> 64	42.43±1.62	> 64
  12a	C6H5	8.23±1.27	11.38±1.40	3.52±0.90	27.87±1.44	33.09±1.62
  12b	2-ClC6H4	11.63±1.03	7.06±0.89	8.61±1.31	36.01±1.55	30.43±1.59
  12c	3-ClC6H4	12.80±1.45	10.62±1.16	3.82±0.87	26.79±1.42	46.55±1.77
  12d	4-ClC6H4	8.21±1.04	7.68±1.01	4.21±0.83	17.97±1.23	16.61±1.32
  12e	3, 4-F2C6H3	12.73±1.32	10.18±1.06	6.72±1.07	19.27±1.28	48.07±1.78
  12f	2-CH3C6H4	11.52±1. 37	11.47±1.46	9.71±1.26	33.68±1.52	27.45±1.54
  12g	4-CH3C6H4	12.76±1.05	10.61±1.38	9.47±1.32	42.04±1.62	28.78±1.56
  12h	2-CH3OC6H4	14. 21±1.63	31.43±1.62	19.27±1.07	> 64	> 64
  12i	3-CH3OC6H4	29.51±1.36	20.53±1.34	15.31±1.19	22.44±1.35	47.03±1.78
  12j	4-CH3OC6H4	43.81±1.56	55.10±1.87	30.46±1.48	> 64	> 64
  12k	2-Thienyl	8.32±1.13	9.11±1.03	6.83±0.97	16.40±1.25	24.05±1.44
  12l	2-Pyridinyl	9.18±1.32	3.63±0.82	1.37±0.31	23.89±1.37	7.85±1.00
  12m	3-Pyridinyl	11.33±1.35	5.78±1.04	9.66±1.42	28.45±1.45	49.15±1.80
  12n	4-Pyridinyl	13.14±1.54	5.38±0.92	7.27±1.17	21.06±1.32	53.37±1.83
  12o	4-[1, 1'-Biphenyl]	12.16±1.41	10.23±1.07	8.46±1.27	39.05±1.59	54.14±1.84
  5-Fub	—	13.52±0.93	12.82±0.71	9.83±0.58	12.19±1.26	10.38±1.25
  a Antitumor activity was assayed by exposure for 72 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). Dates are presented as the mean±SDs of three independent experiments. b Positive control.

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