Synthesis, characterization and antitumor activity of novel ferrocene bisamide derivatives containing pyrimidine-moiety

Journal of Organometallic Chemistry 851 (2017) 150e159

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Synthesis, characterization and antitumor activity of novel ferrocene bisamide derivatives containing pyrimidine-moiety Ying Guo a, Si-Qi Wang b, Zong-Qing Ding a, Jia Zhou a, Ban-Feng Ruan b, * a b

Department of Biology, Chemistry and Environmental Engineering, Haijiang Normal University, Shiyan 442500, PR China School of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 August 2017 Received in revised form 21 September 2017 Accepted 21 September 2017 Available online 23 September 2017

Starting from two ferrocene-based pyrimidinamines (3 and 8), two series of novel ferrocene bisamide derivatives (4a-m and 9a-m) were synthesized and characterized, respectively. Both 3 and 8 gave single crystals suitable for X-ray structural analysis. All the corresponding bisamide derivatives were evaluated the inhibitory activities against B16-F10 and A549 cell lines using the MTT method. Among them, compound 4d exhibited comparable antitumor activities in vitro against B16-F10 and compound 9d exhibited significant antitumor activities in vitro against A549. Cancer cell apoptosis assay were performed and results indicated that compound 9d effectively fuels A549 cells apoptosis in a dosedependent manner. © 2017 Elsevier B.V. All rights reserved.

Keywords: Ferrocene derivative Pyrimidine-moiety X-ray structural analysis Antitumor activity Cell apoptosis

1. Introduction The well-known cisplatin and its following derivatives as typical clinical chemotherapeutical agents are still used in more than 50% of the treatment schedules for patients suffering from cancer [1e3]. However, these platinum-based medicines undergo two main disadvantages: one is that they are inefficient against platinumresistant tumors [4e6] and another is that they have severe side effects such as acute and cumulative renal, cardiac toxicities, ototoxicity, neurotoxicity, gastrotoxicity, myelosuppression, severe nausea and emetic effects [7e9]. Therefore, there is an urgent need to design and discovery novel platinum and/or non-platinum metal complexes with less toxicity and better specificity to overcome these adverse effects [10e15]. Up to now, organometallic compounds, which are defined as metal complexes containing at least one direct, covalent metalcarbon bond, have been proven to be a promising metal-based chemotherapy alternative option to platinum-based anticancer drugs [16,17]. Among them, the use of ferrocene-containing compounds for medicinal applications has long been considered as an attractive way to develop anticancer drugs [18e27], due to its low

* Corresponding author. E-mail address: [email protected] (B.-F. Ruan). https://doi.org/10.1016/j.jorganchem.2017.09.032 0022-328X/© 2017 Elsevier B.V. All rights reserved.

toxicity, significant stability and lipophilicity, facile functionalization [28], and unique electrochemical behavior. For example, the substitution of one of the phenyl rings of tamoxifen with a ferrocenyl group obtains the so-called “ferrocifen” derivatives with improved antiproliferative activity [29,30]. Furthermore, Therefore, there is an increasing interest focused on the discovery of new compounds with a ferrocene core, suitable to develop active drugs, for example, as potential chemotherapeutical agents. The ferrocenyl moiety has been incorporated with some natural products [31,32], heterocyclic structures, resulting in an increase of their biological activities or create new medicinal properties. We have an on-going interest in studying the anticancer activity of ferrocene-based organometallic compounds. Previous studies from our groups have shown that some series of ferrocene derivatives containing pyrazolyl groups displayed impressive anticancer activities [33e36]. As a part of our long-lasting interest in the chemistry and biological activity of ferrocene-containing compounds, we have recently focused our attention to synthesize two series of novel ferrocene derivatives with pyrimidine moiety to extend the structure-activity relationships (SAR) study. First, 4ferrocenylpyrimidin-2-amine (3) and (E)-4-(2-ferrocenylvinyl)pyrimidin-2-amine (8) were synthesized and further modified resulting in two novel series of ferrocene-bearing pyrimidinyl amides, whose chemical properties and bioactivity were determined in detail.

Y. Guo et al. / Journal of Organometallic Chemistry 851 (2017) 150e159

2. Experimental 2.1. General Acetylferrocene (1), ferrocenecarboxaldehyde (2) and other reagents and solvents were purchased from Adamas-beta, J&K, Energy Chemical, TCI, Alfa Aesar, and Acros and were used without further purification. All the NMR spectra were recorded on a Bruker DRX 600 model Spectrometer in CDCl3. Chemical shifts (d) for 1H NMR and 13C NMR spectra were reported in parts per million (ppm) to residual solvent protons. The ESI-MS spectra were recorded on a Mariner System 5304 Mass spectrometer. Carbon, hydrogen and nitrogen assays were carried out with a CHN-O-Rapid instrument and were within ±0.4% of the theoretical values. TLC was run on the silica gel coated aluminum sheets (Silica Gel 60 GF254, E. Merk, Germany) and visualized in UV light (254 nm). The structures of the title compounds are listed in Table 1. 2.2. Synthesis [37] 2.2.1. (E)-3-(dimethylamino)-1-ferrocenylprop-2-en-1-one (2) To a stirred solution of acetylferrocene (1) (4.56 g, 20 mmol) in DMF (10 mL) was added DMF-DMA (11.92 g, 100 mmol) dropwise. The mixture was stirred at 125
C for 2.5 h and cooled to room temperature. The solvent was evaporated under reduce pressure, added water (100 mL) and extracted with DCM. The organic phase was separated, washed with brine, dried over MgSO4, and evaporated to produce the crude product, which was purified by column chromatography using a silica gel. 83% yield; brown solid, mp 221e222
C; 1H NMR (600 MHz, CDCl3): d 7.71 (d, J ¼ 12.4 Hz, 1H, CH¼CH), 5.36 (d, J ¼ 12.4 Hz, 1H, CH¼CH), 4.78 (s, 2H, ferrocene-H), 4.38 (s, 2H, ferrocene-H), 4.16 (s, 5H, ferrocene-H), 3.00 (s, 6H, MeH). MS (ESI): 284.1 (C15H17FeNO, [MþH]þ). Anal. Calcd for C15H17FeNO: C, 63.63; H, 6.05; O, 5.65. Found: C, 63.29; H, 5.89; O, 5.36%. 2.2.2. 4-Ferrocenylpyrimidin-2-amine (3) To a solution of guanidine hydrochloride (1.44 g, 15 mmol) and

Table 1 Chemical structures of the title compounds. Structure

Comp. no

R

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 9l 9m

H 4-CH3 3-CH3 4-CH2CH3 4-OCF3 4-F 4-Br 3-Br 4-Cl 3-Cl 4-OCH3 3,5-di-CH3 3,5-di-Cl H 4-CH3 3-CH3 4-CH2CH3 4-OCF3 4-F 4-Br 3-Br 4-Cl 3-Cl 4-OCH3 3,5-di-CH3 3,5-di-Cl

151

K2CO3 (2.76 g, 20 mmol) in 2-methoxyethanol (50 mL) was added compound 2 (2.83 g, 10 mmol). The mixture was refluxed for 24 h. After complete reaction (TLC analysis), the mixture was cooled to room temperature, diluted with water and extracted with EtOAc. The combined organic layers were dried over MgSO4, concentrated in vacuo, and purified by column chromatography on silica gel. 80% yield; brown solid, mp 195e196
C; 1H NMR (600 MHz, CDCl3): d 8.14 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 6.71 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 5.01 (s, 2H, CH¼CH), 4.90 (s, 2H, ferrocene-H), 4.45 (s, 2H, ferrocene-H), 4.08 (s, 5H, ferrocene-H). MS (ESI): 280.1 (C14H13FeN3, [MþH]þ). Anal. Calcd for C14H13FeN3: C, 60.24; H, 4.69; N, 15.05. Found: C, 60.53; H, 4.49; N, 15.03%. 2.2.3. General procedure for the synthesis of compounds 4a-m To a solution of compound 3 (0.140 g, 0.5 mmol) and corresponding substituted benzoyl chlorides (1.5 mmol) in DCM (10 mL) was added three drops of triethylamine. The mixture was stirred at room temperature for 5 h (TLC analysis). Water was added, and the mixture was extracted with EtOAc. The combined organic layers were dried over MgSO4, and filtered to produce the crude product, which were purified by column chromatography using silica gel. 2.2.4. N-benzoyl-N-(4-ferrocenylpyrimidin-2-yl)benzamide (4a) 83% yield; brown solid, mp 156e157
C; 1H NMR (600 MHz, CDCl3): d 8.44 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.89 (d, J ¼ 7.8 Hz, 4H, Ph-H), 7.50 (t, J ¼ 7.3 Hz, 2H, Ph-H), 7.40 (t, J ¼ 7.5 Hz, 4H, Ph-H), 7.02 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 4.76 (s, 2H, ferrocene-H), 4.44 (s, 2H, ferrocene-H), 3.80 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 175.18, 172.99, 160.27, 137.46, 135.27, 131.93, 131.29, 116.28, 81.22, 74.28, 72.65, 70.75, 32.34. MS (ESI): 488.3 (C28H21FeN3O2, [MþH]þ). Anal. Calcd for C28H21FeN3O2: C, 69.01; H, 4.34; N, 8.62; Found: C, 68.82; H, 4.36; N, 8.65%. 2.2.5. 4-Methyl-N-(4-methylbenzoyl)-N-(4-ferrocenylpyrimidin-2yl)benzamide(4b) 73% yield; brown solid, mp 78e79
C; 1H NMR (600 MHz, CDCl3): d 8.45 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.79 (d, J ¼ 7.8 Hz, 4H, Ph-H), 7.19 (d, J ¼ 7.8 Hz, 4H, Ph-H), 7.01 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 4.77 (s, 2H, ferrocene-H), 4.43 (s, 2H, ferrocene-H), 3.80 (s, 5H, ferrocene-H), 2.34 (s, 6H, Me-H). 13C NMR (151 MHz,CDCl3): d 175.17, 172.82, 163.31, 160.24, 146.01, 134.74, 132.04, 116.12, 81.36, 74.20, 72.59, 70.74, 32.34, 24.30. MS (ESI): 516.4 (C30H25FeN3O2, [MþH]þ). Anal. Calcd for C30H25FeN3O2: C, 69.91; H, 4.89; N, 8.15; Found: C, 70.09; H, 4.88; N, 8.14%. 2.2.6. 3-Methyl-N-(3-methylbenzoyl)-N-(4-ferrocenylpyrimidin-2yl)benzamide (4c) 77% yield; brown solid, mp 59e60
C; 1H NMR (600 MHz, CDCl3): d 8.45 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 7.73 (s, 2H, Ph-H), 7.66 (d, J ¼ 7.1 Hz, 2H, Ph-H), 7.30 (d, J ¼ 7.3 Hz, 2H, Ph-H), 7.28 (s, 2H, Ph-H), 7.02 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 4.78 (s, 2H, ferrocene-H), 4.44 (s, 2H,ferrocene-H), 3.79 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 175.36, 172.95, 163.13, 160.25, 141.18, 137.48, 136.07, 132.58, 131.07, 128.97, 116.19, 81.38, 74.24, 72.62, 70.75, 23.97.MS (ESI): 516.4 (C30H25FeN3O2, [MþH]þ). Anal. Calcd for C30H25FeN3O2: C, 69.91; H, 4.89; N, 8.15; Found: C, 70.09; H, 4.88; N, 8.14%. 2.2.7. 4-Ethyl-N-(4-ethylbenzoyl)-N-(4-ferrocenylpyrimidin-2-yl) benzamide (4d) 84% yield; brown solid, mp 72e73
C; 1H NMR (600 MHz, CDCl3): d 8.47 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.84 (s, 2H, Ph-H), 7.82 (s, 2H, Ph-H), 7.24 (s, 2H,Ph-H), 7.22 (s, 2H, Ph-H), 7.02 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 4.77 (s, 2H, ferrocene-H), 4.43 (s, 2H, ferrocene-H), 3.76 (s, 5H, ferrocene-H), 2.65 (q, J ¼ 7.6 Hz, 4H, CH2),

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1.19 (t, J ¼ 7.6 Hz, 6H, Me-H). 13C NMR (151 MHz,CDCl3): d 175.19, 172.87, 163.32, 160.28, 152.12, 134.90, 132.21, 130.81, 116.13, 81.34, 74.21, 72.60, 70.74, 31.53, 17.63. MS (ESI): 544.4 (C32H29FeN3O2, [MþH]þ). Anal. Calcd for C32H29FeN3O2: C, 70.72; H, 5.38; N, 7.73; Found: C, 70.63; H, 5.40; N, 7.76%. 2.2.8. N-(4-ferrocenylpyrimidin-2-yl)-4-(trifluoromethoxy)-N-(4(trifluoromethoxy)benzoyl)benzamide (4e) 85% yield; brown solid, mp 142e144
C; 1H NMR (600 MHz, CDCl3): d 8.43 (d, J ¼ 5.0 Hz, 1H, pyrimidine-H), 7.92 (d, J ¼ 8.0 Hz, 4H, Ph-H), 7.24 (d, J ¼ 8.9 Hz, 4H, Ph-H), 7.06 (d, J ¼ 5.0 Hz, 1H, pyrimidine-H), 4.78 (s, 2H, ferrocene-H), 4.48 (s, 2H, ferrocene-H), 3.86 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 173.69, 162.60, 160.31, 154.78, 135.46, 133.79, 126.80, 123.14, 116.62, 80.97, 74.50, 72.68, 70.80, 32.37. MS (ESI): 656.3 (C30H19F6FeN3O4, [MþH]þ). Anal. Calcd for C30H19F6FeN3O4: C, 54.98; H, 2.92; N, 6.41; Found: C,55.14; H, 2.93; N, 6.40%. 2.2.9. 4-Fluoro-N-(4-fluorobenzoyl)-N-(4-ferrocenylpyrimidin-2yl)benzamide (4f) 92% yield; brown solid, mp 153e155
C; 1H NMR (600 MHz, CDCl3): d 8.45 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.92e7.86 (m, 4H, Ph-H), 7.10 (t, J ¼ 8.4 Hz, 4H, Ph-H), 7.06 (d, J ¼ 5.5 Hz, 1H, pyrimidine-H), 4.78 (s, 2H, ferrocene-H), 4.47 (s, 2H, ferrocene-H), 3.86 (s, 5H, ferrocene-H). 13C NMR (151 MHz, D2O): d 171.38, 170.73, 164.55, 157.71, 131.85, 116.09, 115.94, 113.87, 71.86, 70.12, 68.23. MS (ESI): 524.3 (C28H19F2FeN3O2, [MþH]þ). Anal. Calcd for C28H19F2FeN3O2: C, 64.26; H, 3.66; N, 8.03; Found: C,64.47; H, 3.67; N, 8.01%. 2.2.10. 4-Bromo-N-(4-bromobenzoyl)-N-(4-ferrocenylpyrimidin-2yl)benzamide (4g) 90% yield; brown solid, mp 136e138
C; 1H NMR (600 MHz, CDCl3): d 8.43 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.72 (d, J ¼ 7.5 Hz, 4H, Ph-H), 7.56 (d, J ¼ 7.6 Hz, 4H, Ph-H), 7.06 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 4.76 (s, 2H, ferrocene-H), 4.47 (s, 2H, ferrocene-H), 3.87 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 174.16, 173.36, 162.76, 160.25, 136.12, 134.68, 133.30, 130.42, 116.55, 81.03, 74.46, 72.71, 70.80. MS (ESI): 646.1 (C28H19Br2FeN3O2, [MþH]þ). Anal. Calcd for C28H19Br2FeN3O2: C, 52.13; H, 2.97; N, 6.51; Found: C,52.00; H, 2.98; N, 6.53%. 2.2.11. 3-Bromo-N-(3-bromobenzoyl)-N-(4-ferrocenylpyrimidin-2yl)benzamide (4h) 87% yield; brown solid, mp 131e132
C; 1H NMR (600 MHz, CDCl3): d 8.43 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 8.01 (s, 2H, Ph-H), 7.76 (d, J ¼ 7.7 Hz, 2H, Ph-H), 7.63 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.29 (d, J ¼ 7.9 Hz, 2H, Ph-H), 7.06 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 4.78 (s, 2H, ferrocene-H), 4.48 (s, 2H, ferrocene-H), 3.85 (s, 5H, ferroceneH). 13C NMR (151 MHz, CDCl3): d 173.53, 173.41, 160.31, 139.14, 138.30, 134.86, 132.81, 130.11, 125.54, 116.65, 80.94, 74.50, 72.72, 70.79, 32.34. MS (ESI): 646.1 (C28H19Br2FeN3O2, [MþH]þ). Anal. Calcd for C28H19Br2FeN3O2: C, 52.13; H, 2.97; N, 6.51; Found: C,52.31; H, 2.96; N, 6.50%. 2.2.12. 4-Chloro-N-(4-chlorobenzoyl)-N-(4-ferrocenylpyrimidin-2yl)benzamide (4i) 78% yield; brown solid, mp 162e164
C; 1H NMR (600 MHz, CDCl3): d 8.44 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.80 (d, J ¼ 7.9 Hz, 4H, Ph-H), 7.39 (d, J ¼ 8.0 Hz, 4H, Ph-H), 7.06 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 4.76 (s, 2H, ferrocene-H), 4.48 (s, 2H, ferrocene-H), 3.87 (s, 5H, ferrocene-H).13C NMR (151 MHz, CDCl3): d 174.05, 173.36, 160.27, 141.81, 135.64, 133.21, 131.72, 116.56, 81.01, 74.48, 72.71, 70.80, 32.35. MS (ESI): 557.2 (C28H19Cl2FeN3O2, [MþH]þ). Anal. Calcd for C28H19Cl2FeN3O2: C, 60.46; H, 3.44; N, 7.55; Found:

C, 60.40; H, 3.45; N, 7.54%. 2.2.13. 3-Chloro-N-(3-chlorobenzoyl)-N-(4-ferocenylpyrimidin-2yl)benzamide (4j) 86% yield; brown solid, mp 144e146
C; 1H NMR (600 MHz, CDCl3): d 8.44 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 7.86 (s, 2H, Ph-H), 7.72 (d, J ¼ 7.7 Hz, 2H, Ph-H), 7.48 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.34 (t, J ¼ 7.9 Hz, 2H, Ph-H), 7.06 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 4.78 (s, 2H, ferrocene-H), 4.48 (s, 2H, ferrocene-H), 3.85 (s, 5H, ferroceneH). 13C NMR (151 MHz, CDCl3): d 173.45, 173.39, 162.97,160.30, 139.56, 138.54, 133.19, 132.91, 131.01, 125.71, 116.66, 80.96, 74.51, 72.72, 70.79, 32.34. MS (ESI): 557.2 (C28H19Cl2FeN3O2, [MþH]þ). Anal. Calcd for C28H19Cl2FeN3O2: C, 60.46; H, 3.44; N, 7.55; Found: C, 60.62; H, 3.43; N, 7.58%. 2.2.14. 4-Methoxy-N-(4-methoxybenzoyl)-N-(4ferrocenylpyrimidin-2-yl)benzamide (4k) 86% yield; brown solid, mp 118e120
C; 1H NMR (600 MHz, CDCl3): d 8.46 (d, J ¼ 5.0 Hz, 1H, pyrimidine-H), 7.87 (d, J ¼ 7.9 Hz, 4H, Ph-H), 7.03 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 6.89 (d, J ¼ 7.9 Hz, 4H, Ph-H), 4.78 (s, 2H, ferrocene-H), 4.44 (s, 2H, ferrocene-H), 3.82 (s, 6H, OCH3), 3.80 (s, 5H, ferrocene-H). 13C NMR (151 MHz,CDCl3): d 174.68, 172.81, 165.76, 163.53, 160.25, 134.27, 129.78, 116.60, 116.01, 81.40, 74.21, 72.62, 70.76, 58.11. MS (ESI): 548.4 (C30H25FeN3O4, [MþH]þ). Anal. Calcd for C30H25FeN3O4: C, 65.83; H, 4.60; N, 7.68; Found: C, 66.0; H, 4.62; N, 7.66%. 2.2.15. N-(3,5-dimethylbenzoyl)-3,5-dimethyl-N-(4ferrocenylpyrimidin-2-yl)benzamide (4l) 77% yield; brown solid, mp 68e69
C; 1H NMR (600 MHz, CDCl3): d 8.49 (d, J ¼ 4.8 Hz, 1H, pyrimidine-H), 7.72 (s, 1H, Ph-H), 7.50 (s, 4H, Ph-H), 7.22 (s, 1H, Ph-H), 7.11 (s, 2H, Ph-H), 7.03 (d, J ¼ 4.8 Hz, 1H, pyrimidine-H), 4.79 (s, 2H, ferrocene-H), 4.44 (s, 2H, ferrocene-H), 3.77 (s, 5H, ferrocene-H), 2.30 (s, 12H, Me-H). 13C NMR (151 MHz, CDCl3): d 175.61, 172.79, 163.25, 160.22, 140.87, 137.55, 136.99, 129.66, 116.13, 82.2, 74.23, 72.60, 70.76, 23.86. MS (ESI): 544.4 (C32H29FeN3O2, [MþH]þ). Anal. Calcd for C32H29FeN3O2: C, 70.72; H, 5.38; N, 7.73; Found: C, 70.66; H, 5.40; N, 7.75%. 2.2.16. 3,5-Dichloro-N-(3,5-dichlorobenzoyl)-N-(4ferrocenylpyrimidin-2-yl)benzamide (4m) 75% yield; brown solid, mp 178e180
C; 1H NMR (600 MHz, CDCl3): d 8.44 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 7.69 (s, 4H, Ph-H), 7.51 (s, 2H, Ph-H), 7.10 (d, J ¼ 5.3 Hz, 1H, pyrimidine-H), 4.80 (s, 2H, ferrocene-H), 4.52 (s, 2H, ferrocene-H), 3.90 (s, 5H, ferrocene-H). 13 C NMR (151 MHz, CDCl3) d 173.83, 172.32, 162.00, 160.34, 139.82, 138.31, 135.29, 129.95, 117.03, 82.96, 74.70, 72.77, 70.85. MS (ESI): 626.1 (C28H17Cl4FeN3O2, [MþH]þ). Anal. Calcd for C28H17Cl4FeN3O2: C, 53.80; H, 2.74; N, 6.72; Found: C, 53.93; H, 2.75; N, 6.71%. 2.2.17. (E)-4-Ferrocenylbut-3-en-2-one (6) To a solution of ferrocenecarboxaldehyde (2) (4.28 g, 20 mmol) in acetone (100 mL) was added 2 N aq. NaOH (2 mL). The mixture was stirred at room temperature. After complete reaction (TLC analysis), the mixture was diluted with water and the precipitation was filtered to afford the crude product, which were purified by column chromatography using silica gel. 84% yield; brown solid, mp 125e127
C; 1H NMR (600 MHz, CDCl3): d 7.42 (d, J ¼ 16.0 Hz, 1H, CH¼CH), 6.34 (d, J ¼ 16.0 Hz, 1H, CH¼CH), 4.50 (s, 2H, ferrocene-H), 4.44 (s, 2H, ferrocene-H), 4.15 (s, 5H, ferrocene-H), 2.29 (s, 3H, Me-H). MS (ESI): 255.1 (C14H14FeO, [MþH]þ). Anal. Calcd for C14H14FeO: C, 66.17; H, 5.55; O, 6.30. Found: C, 66.23; H, 5.49; O, 6.36%.

Y. Guo et al. / Journal of Organometallic Chemistry 851 (2017) 150e159

2.2.18. (1E,4E)-1-(dimethylamino)-5-ferrocenylpenta-1,4-dien-3one (7) Using the same procedure for synthesis of compound 2 and starting from 6, compound 7 was obtained. 86% yield; brown solid, mp 201e202
C; 1H NMR (600 MHz, CDCl3): d 7.71 (d, J ¼ 12.4 Hz, 1H, CH¼CH), 7.43 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 6.40 (d, J ¼ 15.5 Hz, 1H, CH¼CH), 5.18 (d, J ¼ 12.5 Hz, 1H, CH¼CH), 4.48 (d, J ¼ 1.1 Hz, 2H, ferrocene-H), 4.35 (d, J ¼ 1.2 Hz, 2H, ferrocene-H), 4.13 (d, J ¼ 0.6 Hz, 5H, ferrocene-H), 3.10 (s, 3H, Me-H), 2.87 (s, 3H, Me-H). MS (ESI): 310.2 (C17H19FeNO, [MþH]þ). Anal. Calcd for C17H19FeNO: C, 66.04; H, 6.19; O, 5.17. Found: C, 66.03; H, 6.29; O, 5.36%. 2.2.19. (E)-4-(2-ferrocenylvinyl)pyrimidin-2-amine (8) Using the same procedure for synthesis of compound 3 and starting from 7, compound 8 was obtained. 79% yield; brown solid, mp 197e199
C; 1H NMR (600 MHz, CDCl3): d 8.21 (d, J ¼ 4.6 Hz, 1H, pyrimidine-H), 7.55 (d, J ¼ 15.8 Hz, 1H, CH¼CH), 6.61 (d, J ¼ 4.8 Hz, 1H, CH¼CH), 6.51 (d, J ¼ 15.9 Hz, 1H, pyrimidine-H), 4.97 (s, 2H, NH2), 4.53 (s, 2H, pyrimidine-H), 4.38 (s, 2H, pyrimidine-H), 4.16 (s, 5H, pyrimidine-H). MS (ESI): 306.2 (C16H15FeN3, [MþH]þ). Anal. Calcd for C16H15FeN3: C, 62.97; H, 4.95; N, 13.77. Found: C, 62.83; H, 5.09; N, 13.86%. 2.2.20. General procedure for the synthesis of compounds 4a-m Using the same procedure for synthesis of compound 4a-m and starting from 8 and the same corresponding substituted benzoyl chlorides, compounds 9a-m were obtained. 2.2.21. (E)-N-benzoyl-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl) benzamide (9a) 88% yield; brown solid, mp 136e137
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.87 (d, J ¼ 7.8 Hz, 4H, Ph-H), 7.48 (t, J ¼ 7.2 Hz, 2H, Ph-H), 7.41 (d, J ¼ 5.4 Hz, 2H, pyrimidine-H), 7.39 (t, J ¼ 7.5 Hz, 5H, Ph-H), 6.87 (d, J ¼ 5.2 Hz, 1H, CH¼CH), 6.43 (d, J ¼ 15.5 Hz, 1H, CH¼CH), 4.45 (s, 2H, ferrocene-H), 4.38 (s, 2H, ferrocene-H), 4.09 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 175.44, 166.83, 163.03, 161.32, 142.17, 137.36, 135.25, 131.88, 131.23, 123.89, 117.68, 82.69, 73.34, 72.21, 70.89. MS (ESI): 514.4 (C30H23FeN3O2, [MþH]þ). Anal. Calcd for C30H23FeN3O2: C, 70.19; H, 4.52; N, 8.19. Found: C, 70.09; H, 4.51; N, 8.16%. 2.2.22. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-4-methyl-N(4-methylbenzoyl)benzamide (9b) 77% yield; brown solid, mp 35e36
C; 1H NMR (600 MHz, CDCl3): d 8.50 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.77 (d, J ¼ 8.0 Hz, 4H, Ph-H), 7.44 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.18 (d, J ¼ 8.0 Hz, 4H, Ph-H), 6.88 (d, J ¼ 5.2 Hz, 1H, CH¼CH), 6.44 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.46 (s, 2H, ferrocene-H), 4.38 (s, 2H, ferrocene-H), 4.08 (s, 5H, ferrocene-H), 2.34 (s, 6H, Me-H). 13C NMR (151 MHz, CDCl3): d 175.38, 166.78, 163.28, 161.26, 145.96, 141.94, 134.62, 132.07, 131.92, 124.03, 117.50, 82.78, 73.29, 72.17, 70.87, 32.34, 24.30. MS (ESI): 542.4 (C32H27FeN3O2, [MþH]þ). Anal. Calcd for C32H27FeN3O2: C, 70.99; H, 5.03; N, 7.76. Found: C, 71.13; H, 5.02; N, 7.74%. 2.2.23. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-3-methyl-N(3-methylbenzoyl)benzamide (9c) 79% yield; brown solid, mp 36e37
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.0 Hz, 1H, pyrimidine-H), 7.71 (s, 2H, Ph-H), 7.63 (d, J ¼ 7.0 Hz, 2H, Ph-H), 7.41 (d, J ¼ 15.5 Hz, 1H, pyrimidineH), 7.28 (d, J ¼ 7.0 Hz, 2H, Ph-H), 7.27e7.21 (m, 2H, Ph-H), 6.87 (d, J ¼ 5.0 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.45 (s, 2H, ferrocene-H), 4.38 (s, 2H, ferrocene-H), 4.09 (s, 5H, ferroceneH), 2.34 (s, 6H, Me-H). 13C NMR (151 MHz, CDCl3): d 175.63, 166.84, 163.12, 161.26, 142.04, 141.10, 137.35, 136.04, 132.54, 131.01,

153

128.95, 124.04, 117.63, 82.74, 73.33, 72.21, 70.88, 23.99. MS (ESI): 542.4 (C32H27FeN3O2, [MþH]þ). Anal. Calcd for C32H27FeN3O2: C, 70.99; H, 5.03; N, 7.76. Found: C, 70.86; H, 5.02; N, 7.78%. 2.2.24. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-4-ethyl-N-(4ethylbenzoyl)benzamide (9d) 83% yield; brown solid, mp 42e43
C; 1H NMR (600 MHz, CDCl3): d 8.50 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.80 (d, J ¼ 7.5 Hz, 4H), 7.46 (d, J ¼ 15.5 Hz, 1H, pyrimidine-H), 7.20 (d, J ¼ 7.7 Hz, 4H, Ph-H), 6.89 (d, J ¼ 5.1 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.46 (s, 2H, ferrocene-H), 4.38 (s, 2H, ferrocene-H), 4.08 (s, 5H, ferrocene-H), 2.64 (q, J ¼ 7.6 Hz, 4H, CH2), 1.20 (t, J ¼ 7.5 Hz, 6H, Me-H). 13C NMR (151 MHz, CDCl3): d 175.45, 166.81, 163.28, 161.28, 152.09, 141.94, 134.74, 132.17, 130.75, 124.03, 117.55, 82.77, 73.30, 72.18, 70.87, 31.54, 17.66. MS (ESI): 570.5 (C34H31FeN3O2, [MþH]þ). Anal. Calcd for C34H31FeN3O2: C, 71.71; H, 5.49; N, 7.38. Found: C, 71.9; H, 5.48; N, 7.36%. 2.2.25. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-4(trifluoromethoxy)-N-(4-(trifluoromethoxy)benzoyl)benzamide (9e) 89% yield; brown solid, mp 137e139
C; 1H NMR (600 MHz, CDCl3): d 8.47 (d, J ¼ 5.0 Hz, 1H, pyrimidine-H), 7.89 (d, J ¼ 8.0 Hz, 4H, Ph-H), 7.45 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.22 (d, J ¼ 8.3 Hz, 4H, Ph-H), 6.94 (d, J ¼ 5.1 Hz, 1H, CH¼CH), 6.47 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.47 (s, 2H, ferrocene-H), 4.41 (s, 2H, ferrocene-H), 4.11 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 173.92, 167.26, 162.66, 161.34, 154.86, 142.64, 135.35, 133.77, 123.77, 123.11, 122.00, 117.93, 82.52, 73.51, 72.23, 70.95. MS (ESI): 682.4 (C32H21F6FeN3O4, [MþH]þ). Anal. Calcd for C32H21F6FeN3O4: C, 56.41; H, 3.11; N, 6.17. Found: C, 56.52; H, 3.11; N, 6.15%. 2.2.26. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-4-fluoro-N-(4fluorobenzoyl)benzamide (9f) 87% yield; brown solid, mp 143e145
C; 1H NMR (600 MHz, CDCl3): d 8.49 (d, J ¼ 5.0 Hz, 1H, pyrimidine-H), 7.90e7.83 (m, 4H, Ph-H), 7.43 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.08 (t, J ¼ 8.1 Hz, 4H, Ph-H), 6.93 (d, J ¼ 5.1 Hz, 1H, CH¼CH), 6.46 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.47 (s, 2H, ferrocene-H), 4.41 (s, 2H, ferrocene-H), 4.11 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 174.17, 168.78, 167.08, 161.33, 142.46, 134.42, 133.40, 123.85, 118.63, 118.48, 117.75, 82.56, 73.46, 72.23, 70.93. MS (ESI): 550.3(C30H21F2FeN3O2, [MþH]þ). Anal. Calcd for C30H21F2FeN3O2: C, 65.59; H, 3.85; N, 7.65. Found: C, 65.72; H, 3.86; N, 7.66%. 2.2.27. (E)-4-Bromo-N-(4-bromobenzoyl)-N-(4-(2-ferrocenylvinyl) pyrimidin-2-yl)benzamide (9g) 94% yield; brown solid, mp 127e129
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.70 (d, J ¼ 7.7 Hz, 4H, Ph-H), 7.54 (d, J ¼ 7.9 Hz, 4H, Ph-H), 7.43 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 6.93 (d, J ¼ 5.1 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.5 Hz, 1H, CH¼CH), 4.47 (s, 2H, ferrocene-H), 4.42 (s, 2H, ferrocene-H), 4.11 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 174.38, 167.10, 162.64, 161.34, 142.60, 135.98, 134.65, 133.27, 130.41, 127.30, 123.80, 117.90, 73.58, 72.34, 71.03. MS (ESI): 672.2 (C30H21Br2FeN3O2, [MþH]þ). Anal. Calcd for C30H21Br2FeN3O2: C, 53.69; H, 3.15; N, 6.26. Found: C, 53.61; H, 3.15; N, 6.29%. 2.2.28. (E)-3-Bromo-N-(3-bromobenzoyl)-N-(4-(2-ferrocenylvinyl) pyrimidin-2-yl)benzamide (9h) 85% yield; brown solid, mp 121e122
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 8.02 (s, 2H, Ph-H), 7.72 (d, J ¼ 7.7 Hz, 2H, Ph-H), 7.63 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.39 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.28 (s, 1H, Ph-H), 7.24 (s, 1H, Ph-H), 6.91 (d, J ¼ 5.2 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.49 (s, 2H, ferrocene-H), 4.42 (s, 2H, ferrocene-H), 4.12 (s, 5H,

154

Y. Guo et al. / Journal of Organometallic Chemistry 851 (2017) 150e159

Table 2 Crystallographic data and structure refinements for 3 and 8.

formula formula weight Crystal system space group crystal size (mm3) a (Å) b (Å) c (Å) a (
) b (
) g (
) Volume (Å3) Z Dc (mg m3) m (mm1) F (000) q rang (
) Reflections collected Indep. reflns Refns obs. [I > 2s(I)] Parameters Goodness-of-fit R1, wR2 [I > 2s(I)] R1, wR2 [all data] Larg.peak/hole(e. Å) CCDC no

3

8

2(C14H13N3Fe) 558.25 Triclinic P-1 0.35  0.34  0.29 10.3858(12) 10.5859(7) 12.0939(11) 93.158(6) 113.461(10) 96.074(7) 1205.97(19) 2 1.537 1.233 576 4.121e27.096 8548(Rint ¼ 0.0302) 4733 3727 341 1.07 0.0399, 0.0868 0.0545, 0.0982 0.293/-0.417 1026190

4(C16H15N3Fe) 1220.64 Triclinic P-1 0.36  0.34  0.21 10.0651(3) 10.0690(3) 28.3270(9) 99.468(3) 99.470(2) 91.464(2) 2789.04(15) 2 1.453 1.073 1264 3.863e26.866 28220(Rint ¼ 0.0358) 10936 9309 1264 1.042 0.0424, 0.0933 0.0534, 0.0996 0.579/-0.377 1026189

ferrocene-H).13C NMR (151 MHz,CDCl3): d 173.77, 166.99, 162.42, 161.39, 142.69, 139.04, 138.25, 134.85, 132.74, 130.06, 125.46, 123.65, 118.04, 82.48, 73.51, 72.28, 71.01. MS (ESI): 672.2 (C30H21Br2FeN3O2, [MþH]þ). Anal. Calcd for C30H21Br2FeN3O2: C, 53.69; H, 3.15; N, 6.26. Found: C, 53.72; H, 3.14; N, 6.25%.

Table 3 Selected bond lengths (Å) and angles (
) for 3 and 8. 3 C1-C6 C6-N2 N2-C9 C9-N3 C9-N1 N1-C8 C15-C20 C20-N4 N4-C23 C23-N6 C23-N5

1.464(37) 1.336(41) 1.346(38) 1.343(50) 1.346(32) 1.328(49) 1.468(39) 1.330(32) 1.344(38) 1.349(37) 1.343(38)

N5-C22 C1-C6-N2 C6-N2-C9 N2-C9-N3 N2-C9-N1 C15-C20-N4 C20-N4-C23 N4-C23-N6 N4-C23-N5 N6-C23-N5

1.327(35) 117.1(3) 116.7(3) 117.2(3) 126.3(2) 117.7(3) 116.9 (3) 117.4(3) 126.4(2) 116.2(3)

1.454(6) 1.327(6) 1.460(6) 1.351(5) 1.351(5) 1.334(5) 1.340(6) 1.327(6) 1.337(6) 1.472(6) 1.349(5) 1.343(5) 1.360(5) 126.1(4) 124.6(4) 124.1(4) 125.4(4) 125.3(3) 125.3(4)

C38-C39 C39-C40 C41-N7 C41-N8 C41-N9 C54-C55 C55-C56 C57-N10 C57-N11 C57-N12 C1-C6-C7 C17-C22-C23 C33-C38-C39 N4-C25- N6 N7-C41- N9 N10-C57-C12

1.323(5) 1.460(6) 1.350(5) 1.346(5) 1.347(5) 1.318(5) 1.473(6) 1.347(5) 1.339(5) 1.346(5) 127.1(4) 125.2(4) 125.9(5) 125.3(4) 126.1(4) 125.8(4)

8 C1-C6 C6-C7 C7-C8 C8-N1 N1-C9 C9-N2 C9-N3 N3-C10 C22-C23 C23-C24 C25-N4 C25-N5 C25-N6 C49-C54-C55 C6-C7-C8 C22-C23-C24 C38-C39-C40 C54-C55-C56 N1-C9- N3

2.2.29. (E)-4-Chloro-N-(4-chlorobenzoyl)-N-(4-(2-ferrocenylvinyl) pyrimidin-2-yl)benzamide (9i) 75% yield; brown solid, mp 157e159
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.78 (d, J ¼ 7.4 Hz, 4H, Ph-H), 7.43 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.38 (t, J ¼ 8.7 Hz, 4H, Ph-H), 6.92 (d, J ¼ 5.1 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.47 (s, 2H, ferrocene-H), 4.41 (s, 2H, ferrocene-H), 4.11 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 174.25, 167.09, 162.69, 161.34, 142.58, 141.77, 135.54, 133.18, 131.67, 123.78, 117.86, 82.55, 73.50, 72.24, 70.96, 32.36. MS (ESI): 583.3 (C30H21Cl2FeN3O2, [MþH]þ). Anal. Calcd for C30H21Cl2FeN3O2: C, 61.88; H, 3.64; N, 7.22. Found: C, 61.81; H, 3.63; N, 7.23%. 2.2.30. (E)-3-Chloro-N-(3-chlorobenzoyl)-N-(4-(2-ferrocenylvinyl) pyrimidin-2-yl)benzamide (9j) 83% yield; brown solid, mp 137e139
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.2 Hz, 1H, pyrimidine-H), 7.86 (s, 2H, Ph-H), 7.68 (d, J ¼ 7.7 Hz, 2H, Ph-H), 7.47 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.39 (d, J ¼ 15.4 Hz, 1H, pyrimidine-H), 7.32 (t, J ¼ 7.9 Hz, 2H, Ph-H), 6.90 (d, J ¼ 5.2 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.5 Hz, 1H, CH¼CH), 4.48 (s, 2H, ferrocene-H), 4.41 (s, 2H, ferrocene-H), 4.11 (s, 5H, ferroceneH). 13C NMR (151 MHz, CDCl3): d 173.92, 166.98, 161.40, 142.68, 138.88, 137.54, 135.36, 132.53, 131.93, 129.63, 123.64, 118.04, 82.23, 73.51, 72.26, 70.98. MS (ESI): 583.2 (C30H21Cl2FeN3O2, [MþH]þ). Anal. Calcd for C30H21Cl2FeN3O2: C, 61.88; H, 3.64; N, 7.22. Found: C, 61.92; H, 3.64; N, 7.20%. 2.2.31. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-4-methoxy-N(4-methoxybenzoyl)benzamide (9k) 77% yield; brown solid, mp 91e93
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.78 (d, J ¼ 7.4 Hz, 4H, Ph-H), 7.43 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.38 (t, J ¼ 8.7 Hz, 4H, Ph-H), 6.92 (d, J ¼ 5.1 Hz, 1H, CH¼CH), 6.45 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.47 (s, 2H, ferrocene-H), 4.41 (s, 2H, ferrocene-H), 4.11 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 174.88, 166.85, 165.72, 163.49, 161.24, 141.93, 134.27, 129.65, 124.16, 117.36, 116.53, 82.79, 73.29, 72.18, 70.87, 58.07, 32.29. MS (ESI): 574.4 (C32H27FeN3O4, [MþH]þ). Anal. Calcd for C32H27FeN3O4: C, 67.03; H, 4.75; N, 7.33. Found: C, 67.24; H, 4.76; N, 7.31%. 2.2.32. (E)-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)-N-(3,5dimethylbenzoyl)-3,5-dimethylbenzamide (9l) 79% yield; brown solid, mp 39e40
C; 1H NMR (600 MHz, CDCl3): d 8.49 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.46 (s, 4H, Ph-H), 7.42 (d, J ¼ 15.6 Hz, 1H, pyrimidine-H), 7.09 (s, 2H, Ph-H), 6.88 (d, J ¼ 5.2 Hz, 1H, CH¼CH), 6.47 (d, J ¼ 15.6 Hz, 1H, CH¼CH), 4.46 (s, 2H, ferrocene-H), 4.39 (s, 2H, ferrocene-H), 4.09 (s, 5H, ferrocene-H), 2.28 (s, 12H, Me-H). 13C NMR (151 MHz, CDCl3): d175.80, 166.87, 163.20, 161.19, 141.96, 140.78, 137.40, 136.92, 129.63, 124.14, 117.58, 82.76, 73.31, 72.20, 70.87, 23.85. MS (ESI): 570.5 (C34H31FeN3O2, [MþH]þ). Anal. Calcd for C34H31FeN3O2: C, 71.71; H, 5.49; N, 7.39. Found: C, 71.85; H, 5.48; N, 7.36%. 2.2.33. (E)-3,5-Dichloro-N-(4-(2-ferrocenylvinyl)pyrimidin-2-yl)N-(3,5-dichlorobenzoyl)benzamide (9m) 85% yield; brown solid, mp 146e148
C; 1H NMR (600 MHz, CDCl3): d 8.48 (d, J ¼ 5.1 Hz, 1H, pyrimidine-H), 7.67 (s, 4H, Ph-H), 7.50 (s, 2H, Ph-H), 7.40 (d, J ¼ 15.5 Hz, 1H, pyrimidine-H), 6.95 (d, J ¼ 5.2 Hz, 1H, CH¼CH), 6.47 (d, J ¼ 15.5 Hz, 1H, CH¼CH), 4.50 (s, 2H, ferrocene-H), 4.44 (s, 2H, ferrocene-H), 4.13 (s, 5H, ferrocene-H). 13C NMR (151 MHz, CDCl3): d 172.51, 167.17, 161.88, 161.48, 143.12, 139.74, 138.21, 135.22, 129.91, 123.45, 118.40, 82.31, 73.65, 72.30, 71.05. MS (ESI): 652.2 (C30H19Cl4FeN3O2, [MþH]þ). Anal. Calcd for C30H19Cl4FeN3O2: C, 55.34; H, 2.94; N, 6.45. Found: C, 55.47; H, 2.94; N, 6.43%.

Y. Guo et al. / Journal of Organometallic Chemistry 851 (2017) 150e159

2.3. Crystal structure determination Single crystal X-ray diffraction measurements for compounds 3 and 8 were carried out on a Siemens Smart 1000 CCD diffractometer equipped with a graphite crystal monochromator situated in the incident beam for data collection at room temperature. The determination of unit cell parameters and data collections were performed with MoeKa radiation (l ¼ 0.71073 Å). Unit cell dimensions were obtained with least-squares refinements, and all structures were solved by direct methods with SHELXL-97 [38]. All the non-hydrogen atoms were in successive difference Fourier syntheses. The final refinement was performed by full matrix leastsquares methods with anisotropic thermal parameters for nonhydrogen atoms on F2. The hydrogen atoms were added theoretically and riding on the concerned atoms. The crystal data and structure refinement were listed in Table 2. The characteristic bond lengths (Å) and angles (
) were listed in Table 3. 2.4. Antitumor test The antitumor activity of all the prepared compounds against B16-F10 and A549 cell lines were evaluated as described else where with some modifications [39]. Target tumor cell lines were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to 2  104 cells mL1 with the complete medium, 100 mL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was permitted at 37
C, 5% CO2 atmosphere for 24 h before the

3 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m Celecoxibb

IC50a (mM)

Comp. no

B16-F10

A549

13.4 40.3 46.8 32.6 1.9 38.5 13.2 11.3 13.6 16.9 >50 21.1 29.7 41.8 15.22

23.6 25.8 26.5 15.9 16.5 8.9 13.4 >50 5.1 >50 >50 14.8 2.8 29.8 16.03

8 9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 9l 9m Celecoxibb

cytotoxicity assessments. Tested samples at pre-set concentrations were added to 6 wells with adriamycin coassayed as positive control. After 72 h exposure period, 100 mL of PBS containing 0.5 mg/ mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. 4 h later, 100 mL extraction solution (10% SDS-5% isobutyl alcohol-0.01 M HCl) was added. After an overnight incubation at 37
C, the optical density was measured at a wavelength of 570 nm on an ELISA microplate reader. In all experiments three replicate wells were used for each drug concentration. Each assay was carried out at least three times. The results were summarized in Table 4. 2.5. Analysis of apoptosis Approximately 105 cells/well were plated in a 12 well plate and allowed to adhere. After 12 h, the medium was replaced with fresh culture medium containing compound 9d at final concentrations of 0, 2 mM, 4 mM, 8 mM and 16 mM. Then cells were harvested after 24 h. They were trypsinized, washed in PBS and centrifuged at 2000 rpm for 5 min. The pellet was then resuspended in 500 mL of staining solution (containing 5 mL Annexin V-PE and 5 mL PI in Binding Buffer), mixed gently and incubated for 15 min at room temperature (15e25
C) in dark. The samples were then read in a FACS calibur flow cytometer (USA) at 488 nm excitation. Analyses were performed by the software supplied in the instrument [40]. 3. Results and discussion 3.1. Chemistry

Table 4 Antitumor activity (IC50) of the title compounds. Comp. no

155

IC50a (mM) B16-F10

A549

20.8 18.6 >50 24.8 18.0 20.6 16.2 14.3 36.5 >50 26.1 18.2 48.9 46.7 15.22

18.4 44.5 12.5 32.8 1.2 9.8 25.6 >50 25.7 >50 >50 8.8 21.3 11.2 16.03

a Antiproliferation activity and cytotoxicity was measured using the MTT assay. Errors were in the range of 5e10% of the reported values, from three different assays. b Used as a positive control.

The synthesis of the title compound 4a-m and 9a-m were illustrated in Schemes 1 and 2. The construction of the 2aminopyrimidine ring intermediates 3 and 8 were based on the well-known Bredereck synthetic approach. Compounds 4a-m and 9a-m were then prepared in a same manner. Interestingly, when compounds 3 and 8 were reacted with different benzoyl chlorides respectively, the main products were the corresponding bisamides. It was shown from the TLC analysis that there was almost no byproduct monoamides formed, even when reduced the benzoyl chlorides to 0.9 equivalents. The key 2-aminopyrimidine intermediates 3 and 8 both have good solubility in common organic solvents. From the 1H NMR spectra of the two series of title derivatives, some characteristic chemical shifts can be observed and are similar with the reported literatures. Comparing the 1H NMR spectra of 3 and 8, it has been found that the 1H NMR spectrum of 8 exhibits two resonances with the coupling constant around 16.0 which are associated with the two protons of the trans double bond. This typical characteristic can be easily found in the corresponding derivatives of compound 8.

Scheme 1. Synthesis of compounds 4a-m. Reagent and conditions: (i) DMF, DMF-DMA, 125
C, 2.5 h; (ii) guanidine-HCl, K2CO3, 2-methoxyethanol, 125
C; (iii) different benzoyl chlorides, TEA.

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Scheme 2. Synthesis of the compounds 9a-m. Reagent and conditions: (i) Acetone, NaOH (aq), r.t, 12 h; (ii) DMF, DMF-DMA, 125
C, 2.5 h; (iii) guanidine-HCl, K2CO3, 2methoxyethanol, 125
C; (iv) different benzoyl chlorides, TEA.

Fig. 1. X-ray crystal structure of compound 3.

3.2. Structural results from single crystal X-ray diffraction [41] The good diffraction-quality single crystals of compounds 3 and 8 were obtained by the slow evaporation of dichloromethane/ methanol solutions. X-ray crystal structure analysis of compounds 3 and 8 revealed the structures depicted in Figs. 1 and 2, respectively.

Compound 3 crystallizes in the triclinic space group P-1 with two independent molecules in the asymmetric unit. As shown in Fig. 1A, the two independent molecules are almost the same. The crystal structure of compound 3 revealed the formation of 7membered intermolecularly hydrogen-bonded ring between two independent molecules through the formation the intermolecular hydrogen bonds between CH and N atoms (H(5)$$$N(4), 2.724 Å;

Y. Guo et al. / Journal of Organometallic Chemistry 851 (2017) 150e159

H(7)$$$N(6), 2.623(4) Å). The molecule structure of 3 without hydrogen atoms is shown in Fig. 1B. In the crystal, the cyclopentadienyl (Cp) and the pyrimidine rings are linked by C-C single bonds. The two Cp rings in both independent molecules are almost coplanar and nearly parallel, because the dihedral angles of the two Cp rings are 3.350
and 1.916
, respectively. The dihedral angles between the Cp ring and pyrimidine ring connected by C-C single bond are 7.03
and 7.155
, respectively. Compound 8 crystallizes in the triclinic space group P-1 with four independent molecules in the asymmetric unit. As shown in Fig. 2A, the four independent molecules are almost the same. The crystal structure of compound 8 revealed four independent molecules formed the 1D chain structure by weak intermolecular hydrogen-bonds (H(5)$$$N(5), 2.952 Å, 141.9
; H(10)$$$N(10), 3.097 Å, 141.1
; H(58)$$$N(7), 3.197 Å, 133.7
). The molecule structure of 8 without hydrogen atoms is shown in Fig. 2B. In the crystal, the ferrocene and the pyrimidine rings are linked by C¼C double bonds. The two Cp rings in all independent molecules are almost coplanar and nearly parallel, because the dihedral angles of the two Cp rings are 0.448, 0.849, 2.588 and 4.183
, respectively.

157

The Cp rings and pyrimidine rings all connected by trans-C¼C double bonds. The dihedral angles between the Cp and pyrimidine rings are 36.994
, 42.46, 38.34 and 39.715
, respectively. The lengths of the C¼C double bonds are 1.327, 1.337, 1.323 and 1.318 Å, respectively, which are accord with the normal C¼C (1.32 Å). 3.3. Antitumor activity All the synthesized compounds were evaluated for their in vitro antitumor activity against two tumor cell lines B16-F10 and A549 using the MTT method. Celecoxib was used as positive control. The results of the in vitro cytotoxic effects of all the compounds were presented in Table 4. As shown in Table 4, the results revealed that most of the target compounds possess significant antitumor activities with IC50 values ranging from 1.2 to 50 mM. Compound 3 exhibited better antitumor activity against B16-F10 than the positive control celecoxib. When compound 3 were reacted with different benzoyl chlorides, some of the corresponding bisamide derivatives (4d, 4f-h) showed a greater antitumor activity than compound 3 against B16-F10. Based on the results of compounds 4f-h, it clearly demonstrated that compounds having halogen atom substituent exhibited the same potency to improve the antitumor activity against B16-F10 and A549 except for compound 4g against A549. As for the antitumor activities against A549, it could be seen that compound 3 exhibited moderate antitumor activity. However, some of its corresponding bisamide derivatives, i.e., compounds 4c, 4e, 4f, 4h and 4l exhibited greater antitumor activities against A549 than both the parent compound 3 and the positive control. Most significantly, compound 4d with para-ethyl displayed the most potent antituor activity against B16F10 with the IC50 value of 1.9 mM and compound 4l with 3,5-dimethyl displayed the most potent antitumor activity against A549 with the IC50 value of 2.8 mM. Regarding selectivity against individual cell lines, the amidation of the parent compound 8 played a very limited role in improving the antitumor activities against B16-F10. However, some of the corresponding bisamides (9b, 9d, 9e, 9l and 9m) exhibited a greater antitumor activity against A549 than both compound 8 and the positive control celecoxib. Among them, compound 9d with paraethyl displayed the most potent antitumor activity against A549 with the IC50 value of 1.2 mM, indicating that this compound developed as a potential antitumor agent. From the analysis mentioned above, some preliminary structure-activity relationship (SAR) conclusions could be drew as follows: i) for most of the active compounds, the para-position substituted ones are more potent than the meta-position compounds; ii) the electron-donating groups may be more beneficial to improve the antitumor activity of the target compounds; iii) the most favorable substitution is 4-ethyl. 3.4. Analysis of apoptosis by fluorescence-activated cell sorting

Fig. 2. X-ray crystal structure of compound 8.

To determine whether the cell death was related to cell apoptosis, A549 cell line apoptosis induced by 9d was tested using flow cytometry. A biparametric cytofluorimetric analysis was performed using propidium iodide (PI), which stains DNA and enters only dead cells, and fluorescent immunolabeling of the protein annexin-V, which binds to phosphatidyl serine (PS) in a highly selective manner. A549 cells were treated with varying concentrations (2 mM, 4 mM, 8 mM and 16 mM) of 9d for 24 h. As depicted in Fig. 3, it could be seen that each concentration induced an accumulation of annexin-V positive cells in comparison with the control and the percentage of apoptotic cells was notably increased in a dose-dependent manner. When treated with 2 mM of 9d, 10.57% of cells were found to be apoptotic (annexin-V positive); 19.18% of

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Fig. 3. Representative flow cytometric histograms of apoptotic A549 cells after 24 h treatment with different dose of compound 9d. The cells were harvested and labeled with Annexin-V-FITC and PI and then analyzed by flow cytometry.

cells were found to be apoptotic for 4 mM; 19.82% of cells were found to be apoptotic for 8 mM; 44.3% of cells were found to be apoptotic for 16 mM. 4. Conclusion In this study, two series of novel ferrocene derivatives containing pyrimidine-moiety were prepared from two pyrimidinamines and characterized. The two pyrimidinamines gave crystals suitable for X-ray structural analysis. All the synthesized compounds were evaluated for their antitumor activities. The results showed that compounds 4d and 9d displayed the most potent inhibitory activity in vitro against B16-F10 and A549 cell lines, respectively. Furthermore, Annexin V/propidium iodide assay results revealed that compound 9d could induce apoptosis in A549 cells. These results strongly suggested that the novel ferrocene bisamide derivatives containing pyrimidine-moiety can be further developed as a promising antitumor agent. Supplementary material CCDC 1026190 and 1026189 contain the supplementary crystallographic data for compounds 3 and 8, respectively. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Acknowledgements This work was supported by the Key Scientific and Technological Project of Anhui Provincial Tobacoo Company (No. 20150551007).

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