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

Accepted Manuscript Synthesis, characterization and antitumor activity of novel ferrocene derivatives containing pyrazolyl-moiety Xian-Feng Huang, Ling-Zhu Wang, Long -Tang, Ying-Xun Lu, Fan Wang, Guo-Qiang Song, Ban-Feng Ruan PII:

S0022-328X(13)00665-7

DOI:

10.1016/j.jorganchem.2013.08.043

Reference:

JOM 18243

To appear in:

Journal of Organometallic Chemistry

Received Date: 8 July 2013 Revised Date:

19 August 2013

Accepted Date: 25 August 2013

Please cite this article as: X.-F. Huang, L.-Z. Wang, L. -Tang, Y.-X. Lu, F. Wang, G.-Q. Song, B.-F. Ruan, Synthesis, characterization and antitumor activity of novel ferrocene derivatives containing pyrazolyl-moiety, Journal of Organometallic Chemistry (2013), doi: 10.1016/j.jorganchem.2013.08.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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GRAPHIC ABSTRACT

Synthesis, characterization and antitumor activity of novel ferrocene

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derivatives containing pyrazolyl-moiety

Xian-Feng Huang, Ling-Zhu Wang, Long-Tang, Ying-Xun Lu, Fan Wang, Guo-Qiang Song, Ban-Feng Ruan

A series of novel ferrocene derivatives containing pyrazolyl-moiety were synthesized.

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Isomeric compounds 4 and 5 gave crystals suitable for X-ray structural analysis. All the compounds were evaluated the antitumor activity. Among them, compounds 4d,

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4j and 4k exhibited comparable activity.

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Synthesis, characterization and antitumor activity of novel ferrocene derivatives containing pyrazolyl-moiety Guo-Qiang Song a,∗, Ban-Feng Ruanb, ∗ a

School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou 213164, PR China

School of Medical Engineering, Hefei University of Technology, Hefei 230009, PR China

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b

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Xian-Feng Huang a, Ling-Zhu Wang a, Long-Tang a, Ying-Xun Lu a, Fan Wang a,

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Abstract: A series of novel ferrocene derivatives containing pyrazolyl-moiety (4a-4k, 5a-5k) were synthesized. Their structures were characterized by 1H NMR, mass spectroscopy and element analysis. The crystals of isomeric compounds 4 and 5 were suitable for X-ray structural analysis. All the compounds were evaluated the inhibitory activities against A549, HepG2 and MDA-MB-45 cell lines using the MTT method. Among them, compounds 4d, 4j and 4k exhibited comparable antitumor

5-fluorouracil (5-FU).

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activities in vitro against A549 and MDA-MB-45 to the positive control

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activity

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Keywords: Ferrocene derivative, pyrazolyl, X-ray structural analysis, antitumor

1. Introduction

In last few years organometallics have got extensive attention owing to their

unique chemical structures and biological activities [1,2]. Among various organometallics, ferrocene has provided a widely applicable platform for the ∗

Corresponding authors. G.Q.-Song: Tel:+86-519-8633 0160; Fax: +86-519-8633 4598; E-mail address: [email protected] B.-F. Ruan: Tel:+86-551-6290 1771; Fax: +86-551-6290 1771; E-mail address: [email protected]

1

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preparation of functional derivatives used in many areas like catalysis, material science, crystal engineering and bio-organometallic chemistry [3–6]. Furthermore, numerous studies have suggested that combination of a ferrocenyl moiety with

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heterocyclic structures could increase their biological activities or create new medicinal properties [7,8]. Therefore, the introducing of ferrocenyl moiety has long

been recognized as an attractive way to develop drugs such as anti-malarial drugs

chloroquine (termed ferroquine) [9], quinine, mefloquine, and artemisinin and the

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anti-cancer drug ferrocifen [10]. Cellular redox chemistry and/or the activation of

reactive oxygen species (ROS) formation in tumor cells may be a general property of ferrocenes, which at least contributed partially to the antiproliferative effects of this

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type of organometallic species [11-13]. The toxicology of ferrocene was also well studied, and this compound may be administered orally without toxicity. Ferrocene was metabolized in liver by cytochrome P450 enzymes similarly according to that of benzenes. Ferrocenium salts were the first kind of organometallic compounds for which antiproliferative properties were reported, and today there are a number of

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ferrocene-based compounds used as therapies [14,15].

Previous studies from our groups have shown that ferrocene analogues containing pyrazolyl group could display impressive anticancer activity and act as effective metallodrugs [16,17]. In continuation with our recent studies on ferrocene

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derivatives, we have focused our attention on synthesizing a series of novel pyrazole derivatives using the same ferrocene-containing acetic acid to extend the

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structure-activity relationships (SAR) study. We also describe the crystal structures of compounds 4 and 5 containing one bromine atom, respectively.

2. Experimental 2.1. General

All the NMR spectra were recorded on a Bruker DRX 400 model Spectrometer in CDCl3. Chemical shifts (δ) for 1H NMR spectra were reported in parts per million 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 2

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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,

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Germany) and visualized in UV light (254 nm).

2.2. Synthesis

Acetylferrocene, ethyl trifluoroacetate, ethyl bromoacetate and other materials were purchased from Sigma-Aldrich. The synthesis of all the compounds (4a-4k, was

outlined

in

Scheme

1.

The

intermediates

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5a-5k)

4,4,4-trifluoro-1-ferrocenylbutane-1,3-dione (2), 5- ferrocenyl-3-(trifluoromethyl)-1H -pyrazole (3) were obtained according to our previous methods.

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A solution of 3 (1.0 g), Ph3P (800 mg, 1.1 equiv.) and 2-bromoethanol (400mg, 1.1 equiv.) in 60 mL of CH2Cl2 cooled in an ice bath. Then, DIAD (720 mg, 1.2 equiv.) was added slowly. The reaction mixture was stirred for 3 hours and dropped to water, and then the organic layer was evaporated to yield the crude mixture. After column chromatography on silica gel using PE

EA (50:1) as eluent compounds 4

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(0.32 g, 24%) and 5 (0.44 g, 34%) were obtained.

1-(2-Bromoethyl)-5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazole (4) Brown crystal. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.76 (t, 2H, J = 7.2 Hz), 4.08 (s, 5H), 4.31-4.31 (m, 2H), 4.53 (t, 2H, J = 7.2 Hz), 4.65-4.65 (m, 2H), 6.62(s,

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1H). MS (ESI): m/z 427.9 ([M+H]+). Anal. Calc. for C16H14BrF3FeN2: C, 45.00; H, 3.30; N, 6.56%. Found: C, 45.12; H, 3.32; N, 6.59%.

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1-(2-Bromoethyl)-3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazole (5) Brown crystal. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.76 (t, 2H, J = 7.2 Hz),

4.08 (s, 5H), 4.31-4.31 (m, 2H), 4.53 (t, 2H, J = 7.2 Hz), 4.65-4.65 (m, 2H), 6.62 (s, 1H). MS (ESI): m/z 427.9 ([M+H]+). Anal. Calc. for C16H14BrF3FeN2: C, 45.00; H, 3.30; N, 6.56%. Found: C, 45.16; H, 3.27; N, 6.51%.

A solution of 4 or 5 (100 mg), piperidine (60 mg, 3.0 equiv.), KI (40mg, 1 equiv.) in 20 mL of CH2Cl2 was refluxed for 20 hours. The reaction mixture was dropped to water, and the organic layer was washed by water (two times). The target compounds 3

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were purified by Column chromatography on silica gel (Table 1). 2.2.1.

N,N-diethyl-2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethanamine

(4a)

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Brown red oil (81.2% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.04 (t, 6H, J = 6.4 Hz), 2.58-2.60 (m, 4H), 2.91-2.94 (m, 2H), 4.07 (s, 5H), 4.24-4.27 (m, 4H), 4.65

(s, 2H), 6.58(s, 1H). MS (ESI): m/z 420.1 ([M+H]+). Anal. Calc. for C20H24F3FeN3: C,

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57.29; H, 5.77; N, 10.02%. Found: C, 57.18; H, 5.71; N, 10.07%.

2.2.2.

ne (4b)

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N-(2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)-N-propylpropan-1-ami

Brown red oil (80.9% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.87 (t, 6H, J = 7.2 Hz), 1.45 (q, 4H, J = 7.2 Hz), 2.45 (t, 4H, J = 7.2 Hz), 2.92 (t, 2H, J = 7.2 Hz), 4.07 (s, 5H), 4.21-4.27 (m, 4H), 4.65 (s, 2H), 6.57 (s, 1H). MS (ESI): m/z 448.5 ([M+H]+). Anal. Calc. for C22H28F3FeN3: C, 59.07; H, 6.31; N, 9.39%. Found: C,

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59.16; H, 6.37; N, 9.31%.

2.2.3. 5-Ferrocenyl-1-(2-(pyrrolidin-1-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazole (4c) Brown red oil (71.1% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.79-1.79 (m,

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4H), 2.60-2.60 (m, 4H), 2.98 (t, 2H, J = 7.6 Hz), 4.07 (s, 5H), 4.28-4.36 (m, 4H), 4.65 (s, 2H), 6.59 (s, 1H). MS (ESI): m/z 418.7 ([M+H]+). Anal. Calc. for C20H22F3FeN3: C,

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57.57; H, 5.31; N, 10.07%. Found: C, 56.63; H, 5.28; N, 10.02%.

2.2.4. 1-(2-(5-Ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (4d) Brown red oil (87.7% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.41-1.46 (m,

2H), 1.56-1.61 (m, 4H), 2.46-2.49 (m, 4H), 2.82 (t, 2H, J = 7.2 Hz), 4.06 (s, 5H), 4.27-4.30 (m, 4H), 4.65-4.66 (m, 2H), 6.57(s, 1H). MS (ESI): m/z 432.1 ([M+H]+). Anal. Calc. for C21H24F3FeN3: C, 58.48; H, 5.61; N, 9.74%. Found: C, 58.36; H, 5.58; N, 9.79%.

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2.2.5. 2-Methyl-1-(2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (4e) Brown red oil (76.6% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.08 (d, 3H,

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J = 6.4 Hz), 1.23-1.31 (m, 2H), 1.53-1.67 (m, 4H) 2.29-2.41 (m, 2H), 2.80-2.92 (m, 2H), 3.12-3.19 (m, 1H), 4.06 (s, 5H), 4.25-4.29 (m, 4H), 4.65-4.66 (m, 2H), 6.58 (s,

1H). MS (ESI): m/z 446.9 ([M+H]+). Anal. Calc. for C22H26F3FeN3: C, 59.34; H, 5.89;

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N, 9.44%. Found: C, 59.28; H, 5.86; N, 9.47%.

2.2.6. 2-Ethyl-1-(2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (4f)

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Brown red oil (68.8% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.86 (t, 3H, J = 7.2 Hz), 1.26-1.33 (m, 2H), 1.55-1.75 (m, 6H) 2.26-2.40 (m, 2H), 2.84-2.95 (m, 2H), 3.06-3.17 (m, 1H), 4.07 (s, 5H), 4.27-4.29 (m, 4H), 4.65-4.67 (m, 2H), 6.58 (s, 1H). MS (ESI): m/z 460.1 ([M+H]+). Anal. Calc. for C23H28F3FeN3: C, 60.14; H, 6.14; N,

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9.15%. Found: C, 60.29; H, 6.11; N, 9.10%.

2.2.7.

4-Methyl-1-(2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (4g) Brown red oil (83.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.18-1.21 (m,

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3H), 1.30-1.16 (m, 1H) 1.90-1.96 (m, 4H), 2.06-2.11 (m, 2H), 2.81-2.86 (m, 2H), 2.93-2.95 (m, 2H), 4.06 (s, 5H), 4.26-4.31 (m, 4H), 4.64-4.65 (m, 2H), 6.59 (s, 1H).

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MS (ESI): m/z 446.9 ([M+H]+). Anal. Calc. for C22H26F3FeN3: C, 59.34; H, 5.89; N, 9.44%. Found: C, 59.25; H, 5.92; N, 9.39%.

2.2.8.4-Chloro-1-(2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidin e (4h)

Brown red oil (81.8% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.22-2.26 (m, 4H), 2.81-2.85 (m, 4H) 2.86-2.89 (m, 2H), 4.02-4.05 (m, 1H), 4.06 (s, 5H), 4.27-4.28 (m, 4H), 4.65-4.66 (m, 2H), 6.58 (s, 1H). MS (ESI): m/z 467.2 ([M+H]+). Anal. Calc. for C21H23ClF3FeN3: C, 54.16; H, 4.98; N, 9.02%. Found: C, 54.29; H, 4.96; N, 5

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9.08%.

2.2.9. 4-Phenyl-1-(2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (4i)

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Brown red oil (60.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.27-1.33 (m, 2H), 1.47-1.49 (m, 1H), 1.60-1.64 (m, 2H), 2.02-2.07 (m, 2H), 2.51-2.53 (m, 2H)

2.80-2.84 (m, 2H), 4.06 (s, 5H), 4.27-4.30 (m, 4H), 4.65 (s, 2H), 6.57 (s, 1H),

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7.12-7.19 (m, 3H), 7.25-7.28 (m, 2H). MS (ESI): m/z 508.9 ([M+H]+). Anal. Calc. for C27H28F3FeN3: C, 63.92; H, 5.56; N, 8.28%. Found: C, 63.79; H, 5.52; N, 8.37%.

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2.2.10. 4-(2-(5-Ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)morpholine (4j) Brown red oil (65.5% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.51-2.53 (m, 4H), 2.86 (t, 2H, J = 7.2 Hz), 3.68-3.70 (m, 4H), 4.06 (s, 5H), 4.28-4.32 (m, 4H), 4.65 (s, 2H), 6.59 (s, 1H). MS (ESI): m/z 434.1 ([M+H]+). Anal. Calc. for C20H22F3FeN3O:

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C, 55.44; H, 5.12; N, 9.70%. Found: C, 55.36; H, 5.15; N, 9.65%.

2.2.11. 4-(2-(5-Ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)thiomorpholine (4k)

Brown red oil (70.2% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.51-2.53 (m,

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4H), 2.86 (t, 2H, J = 7.2 Hz), 3.68-3.70 (m, 4H), 4.06 (s, 5H), 4.28-4.32 (m, 4H), 4.65 (s, 2H), 6.59 (s, 1H). MS (ESI): m/z 450.3 ([M+H]+). Anal. Calc. for C20H22F3FeN3S:

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C, 53.46; H, 4.94; N, 9.35%. Found: C, 53.59; H, 4.91; N, 9.30%.

2.2.12.

N,N-diethyl-2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethanamine

(5a)

Brown red oil (65.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.95 (t, 2H, J

= 7.2 Hz), 2.50 (q, 2H, J = 7.2 Hz), 2.82 (t, 2H, J = 7.2 Hz), 4.19 (s, 5H), 4.28 (t, 2H, J = 7.2 Hz), 4.36 (s, 2H), 4.52 (s, 2H), 6.54 (s, 1H). MS (ESI): m/z 420.3 ([M+H]+). Anal. Calc. for C20H24F3FeN3: C, 57.29; H, 5.77; N, 10.02%. Found: C, 57.36; H, 5.72; N, 10.09%. 6

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2.2.13. N-(2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)-N-propylpropan-1-ami ne (5b)

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Brown red oil (71.2% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.80 (t, 6H, J = 7.2 Hz), 1.36 (q, 4H, J = 7.2 Hz ), 2.34 (t, 4H, J = 7.2 Hz), 2.82 (t, 2H, J = 7.2 Hz),

4.18 (s, 5H), 4.25 (t, 2H, J = 7.2 Hz), 4.28 (s, 2H), 4.51 (s, 2H), 6.54 (s, 1H). MS

9.39%. Found: C, 59.18; H, 6.35; N, 9.45%.

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(ESI): m/z 448.1 ([M+H]+). Anal. Calc. for C22H28F3FeN3: C, 59.07; H, 6.31; N,

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2.2.14. 3-Ferrocenyl-1-(2-(pyrrolidin-1-yl)ethyl)-5-(trifluoromethyl)-1H-pyrazole (5c) Brown red oil (61.8% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.77-1.77 (m, 4H), 2.53-2.53 (m, 4H), 2.94 (t, 2H, J = 7.2 Hz), 4.18 (s, 5H), 4.37-4.41 (m, 4H), 4.53-4.53 (m, 2H), 6.54 (s, 1H). MS (ESI): m/z 418.5 ([M+H]+). Anal. Calc. for

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C20H22F3FeN3: C, 57.57; H, 5.31; N, 10.07%. Found: C, 56.68; H, 5.33; N, 10.01%.

2.2.15. 1-(2-(3-Ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (5d) Brown red oil (83.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.39-1.42 (m, 2H), 1.53-1.60 (m, 4H), 2.38-2.41 (m, 4H), 2.75 (t, 2H, J = 7.2 Hz), 4.18 (s, 5H),

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4.34-4.37 (m, 4H), 4.51-4.52 (m, 2H), 6.53(s, 1H). MS (ESI): m/z 432.2 ([M+H]+). Anal. Calc. for C21H24F3FeN3: C, 58.48; H, 5.61; N, 9.74%. Found: C, 58.55; H, 5.64;

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N, 9.78%.

2.2.16.

2-Methyl-1-(2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (5e) Brown red oil (66.7% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.96 (d, 3H,

J = 6.0 Hz), 1.25-1.28 (m, 2H), 1.48-1.58 (m, 4H), 2.18-2.44 (m, 2H), 2.66-2.79 (m, 2H), 3.04-3.11 (m, 1H), 4.19 (s, 5H), 4.29-4.36 (m, 4H), 4.52-4.54 (m, 2H), 6.55 (s, 1H). MS (ESI): m/z 446.7 ([M+H]+). Anal. Calc. for C22H26F3FeN3: C, 59.34; H, 5.89; N, 9.44%. Found: C, 59.41; H, 5.83; N, 9.49%. 7

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2.2.17. 2-Ethyl-1-(2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (5f)

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Brown red oil (77.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.75, (s, 3H, J = 7.2Hz), 1.19-1.30 (m, 2H), 1.43-1.47 (m, 2H), 1.59-1.65 (m, 2H), 2.13-2.27 (m,

2H), 2.66-2.79 (m, 2H), 3.01-3.06 (m, 1H), 4.18 (s, 5H), 4.26 (t, 2H, J = 7.2 Hz), 4.35-4.35 (m, 2H), 4.51-4.53 (m, 2H), 6.54 (s, 1H). MS (ESI): m/z 460.3 ([M+H]+).

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Anal. Calc. for C23H28F3FeN3: C, 60.14; H, 6.14; N, 9.15%. Found: C, 60.01; H, 6.10; N, 9.18%.

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2.2.18.

4-Methyl-1-(2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (5g) Brown red oil (71.8% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 0.89-0.91 (m, 6H), 1.17-1.25 (m, 4H), 1.31-1.35 (m, 2H), 1.56-1.59 (m, 4H), 1.93-2.05 (m, 4H), 2.74-2.79 (m, 4H), 2.93-2.96 (m, 2H), 4.18 (s, 5H), 4.31-4.36 (m, 4H), 4.51 (s, 2H),

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6.53 (s, 1H). MS (ESI): m/z 446.2 ([M+H]+). Anal. Calc. for C22H26F3FeN3: C, 59.34; H, 5.89; N, 9.44%. Found: C, 59.47; H, 5.87; N, 9.46%.

2.2.19.

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4-Chloro-1-(2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (5h) Brown red oil (70.1% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.81-1.86 (m,

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2H), 2.00-2.04 (m, 2H), 2.23-2.28 (m, 2H), 2.66-2.70 (m, 2H), 2.78 (t, 2H, J = 7.2 Hz ), 4.00-4.01 (m, 1H), 4.19 (s, 5H), 4.33 (t, 2H, J = 7.2 Hz ), 4.37-4.37 (m, 2H), 4.49-4.50 (m, 2H), 6.55 (s, 1H). MS (ESI): m/z 467.1 ([M+H]+). Anal. Calc. for C21H23ClF3FeN3: C, 54.16; H, 4.98; N, 9.02%. Found: C, 54.27; H, 5.01; N, 9.07%.

2.2.20. 4-Phenyl-1-(2-(3-ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)piperidine (5i) Brown red oil (64.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.23-1.30 (m, 2H), 1.45-1.51 (m, 1H), 1.98-1.99 (m, 2H), 2.50-2.52 (m, 2H), 2.73-2.80 (m, 4H), 8

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4.18 (s, 5H), 4.31-4.35 (m, 4H), 4.49-4.50 (m, 2H), 6.53(s, 1H), 7.11-7.20 (m, 3H.), 7.25-7.27 (m, 2H). MS (ESI): m/z 508.8 ([M+H]+). Anal. Calc. for C27H28F3FeN3: C,

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63.92; H, 5.56; N, 8.28%. Found: C, 63.77; H, 5.59; N, 8.38%.

2.2.21. 4-(2-(3-Ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)morpholine (5j)

Brown red oil (60.9% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.40 (bras, 4H), 2.77 (t, 2H, J = 6.4 Hz), 3.63 (bras, 4H), 4.19 (s, 5H), 4.32-4.37 (m, 4H), 4.50 (s,

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2H), 6.55 (s, 1H). MS (ESI): m/z 434.2 ([M+H]+). Anal. Calc. for C20H22F3FeN3O: C, 55.44; H, 5.12; N, 9.70%. Found: C, 55.35; H, 5.10; N, 9.78%.

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2.2.22. 4-(2-(3-Ferrocenyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)ethyl)thiomorpholine (5k)

Brown red oil (69.3% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 2.57-2.59 (m, 4H), 2.66-2.68 (m, 4H), 2.79 (t, 2H, J = 7.2 Hz), 4.19 (s, 5H), 4.30 (t, 2H, J = 7.2 Hz), 4.37 (s, 2H), 4.49 (s, 2H), 6.55 (s, 1H). MS (ESI): m/z 450.1 ([M+H]+). Anal. Calc.

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for C20H22F3FeN3S: C, 53.46; H, 4.94; N, 9.35%. Found: C, 53.32; H, 4.97; N, 9.31%.

2.3 Crystal structure determination

Single crystal X-ray diffraction measurements for compounds 4 and 5 were carried

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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.

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The determination of unit cell parameters and data collections were performed with Mo-Kα radiation (λ = 0.71073 Å). Unit cell dimensions were obtained with least-squares refinements, and all structures were solved by direct methods with SHELXL-97 [18]. All the non-hydrogen atoms were located in successive difference Fourier syntheses. The final refinement was performed by full-matrix least-squares methods with anisotropic thermal parameters for non-hydrogen 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. 9

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2.4 Biological test The antitumor activity of all the prepared compounds against A549, HepG2 and

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MDA-MB-45 cell lines were evaluated as described else where with some modifications [19]. 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 mL-1 with the complete medium, 100 mL of the obtained cell suspension was added to

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each well of 96-well culture plates. The subsequent incubation was permitted at 37 oC, 5% CO2 atmosphere for 24 h before the cytotoxicity assessments. Then add 100 µL a series concentration of drug-containing medium (dissolved in DMSO) into wells to

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maintain the final concentration of drug as 73, 24.33, 8.11, 2.70, 0.90, 0.30 µg/mL. 5-Fluorouracil and cisplatin were used for the positive controls. After 72 h exposure period,

100

µL

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

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HCl) was added. After an overnight incubation at 37 oC, 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

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was carried out at least three times. The results were summarized in Table 4.

3. Results and discussion

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3.1. Chemistry The

intermediates

4,4,4-trifluoro-1-ferrocenylbutane-1,3-dione

(2),

5-

ferrocenyl-3-(trifluoromethyl)-1H-pyrazole(3) were synthesized according to our previous methods [17]. But in the third step, the most interesting thing was the different ratios of compounds 4 and 5 in different reaction conditions revealed by both single-crystal structures. We found in proton solvents and under higher temperature the system was prone to generate compound 5, while in non-proton solvents and under lower temperature the system tended to give compound 4. We think proton solvents enable mutual conversion between the thermodynamic stability conformation 10

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compound 3 and its isomer. Then process of nucleophilic substitution from thermodynamic stability conformation compound 3 to compound 4 was unstable under high temperature because of vibration of steric hindrance, while the generation

3.2. Structural results from single crystal X-ray diffraction

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of compound 5 was encouraged as we observed.

Single crystals of compounds 4 and 5 were obtained by the slow evaporation of

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dichloromethane/methanol solutions. X-ray crystal structure analyses of compounds 4 and 5 revealed the structures depicted in Figures 1 and 2, respectively.

Compound 4 crystallizes in the triclinic space group the orthorhombic space

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group Pna21 with two independent molecules in the asymmetric unit. As shown in Fig 1a, the two independent molecules are a little different. Taking the pyrazole ring as a reference, the ferrocene moiety and the trifluoromethyl group are on the left in one molecule and right in another molecule. The bromethyl group is on the right in one molecule and left in another molecule according. The Cp0-Fe-Cp0 angles (Cp0 =

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centroids of Cp rings) both deviate somewhat from linearity (Cp0-Fe1-Cp0 176.9° and Cp0-Fe2-Cp0 177.3°). The sum of bond angles around C12 and C26 (∑ = 360.0°) indicates that both groups have a trigonal geometry. It can be seen that the 2-bromoethyl groups are connected to the nitrogen atoms (N1 and N3) which are

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close to the carbon atoms (C12 and C26) that bonded to the ferrocene moiety. Compound 5 crystallizes in the monoclinic space group P21/c. The distances of

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the Fe centre and each carbon atom of its Cp rings are between 2.019 Å (Fe1-C7) and 2.054 Å (Fe1-C9). The sum of bond angles around C11 (∑ = 360.0°) indicates that this group also has a trigonal geometry like C12 and C26 in compound 4. It can be seen that the 2-bromoethyl group is bonded to N1 atom, which is different from compound 5. The angle of C15-C16-Br1 is 112.6°, which is less than those in compound 4 (C29-C30-Br1 114.0° and C32-C31-Br1 115.3°).

3.3. Biological activity All the synthesized compounds were evaluated for their in vitro antitumor 11

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activity against three human tumor cell lines: A549, HepG2 and MDA-MB-45 using the MTT method. 5-Fluorouracil and cisplatin were employed as positive controls. The results were presented in Table 4.

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Overall, the compounds 4a-4k and 5a-5k showed good antitumor activities. As shown in Table 4, most compounds exhibited better antitumor activities against A549 and MDA-MB-45 when compared to the results against HepG2. Especially, for the

A549 cell lines, almost all the derivatives displayed relative good antitumor activities

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which were comparable to the positive control 5-FU. Among them, compounds 4d, 4j and 4k were the most potent antitumor agents (IC50: 4.47, 5.31 and 4.44 µg/mL, respectively). Worth noting was that these three compounds also exhibited best

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inhibitory activities on another cell line MDA-MB-45 (IC50: 4.52, 5.37 and 4.69 µg/mL, respectively).

Structure-activity relationships in these ferrocene derivatives demonstrated that compounds 4 and 5 without side chain containing nitrogen atom showed only moderate antitumor activities with the mean IC50 values of 10.90 µg/mL and 10.15

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µg/mL when tested against A549. It seems that introducing side chain containing nitrogen atom on the pyrazole ring could increased the antitumor activity against A549 cell line in most cases (for example, 4a-4k, 5j-5i et al.). Furthermore, when the R group in 4 and 5 was hexatomic ring without substitution the derivatives could

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show most potent activity. Actually, this kind of compounds 4d, 4g, 4k and 5d displayed the most potent inhibitory activities in vitro against A549 and MDA-MB-45.

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Insterestingly, another derivative with hexatomic ring 5j only showed moderate activity against the two cell lines, but displayed high activity against HepG2 (7.96 µg/mL). It can be seen that the side chain on the pyrazole ring may affect the antitumor activity. 4. Conclusion In summary, a series of novel ferrocene derivatives containing pyrazolyl-moiety were prepared and characterized. Among them, compounds 4 and 5 gave crystals suitable for X-ray structural analysis. All the synthesized compounds were evaluated 12

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for their antitumor activities, which exhibited moderate to potent antitumor activities against A549, HepG2 and MDA-MB-45 cell lines in vitro. In particular, compounds 4d, 4g, 4k and 5d displayed the most potent inhibitory activities in vitro against A549

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and MDA-MB-45 cell lines.

ACKNOWLEDGEMENTS

This work was supported by the Natural Science Foundation of Anhui Province

quality control of Changzhou (2012QTXM0782).

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.

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(1308085MB18) and the Open Project of Key Laboratory of drug manufacturing and

References

[1] K.E. Dombowski, W. Baldwin, J.E. Sheats, J. Organomet. Chem. 302 (1986) 281. [2] R. Epton, G. Marr, G.K. Rogers, J. Organomet. Chem. 150 (1978) 93.

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[3] P. Hu, K. Zhao, Hong-Bo Xu, Molecules (2001) M251.

[4] A. Togni, R.L. Halterman (Eds.), Metallocenes, Wiley-VCH Verlag GmbH, Weinheim, Germany, 1998.

[5] A. Togni, T. Hayashi (Eds.), Ferrocene: Homogenous Catalysis, Organic

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Synthesis, Material VCH, Wenheim, 1995. [6] P. Stepnicka (Ed.), Ferrocenes: Ligands, Material and Biomolecules, John Wiley

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and Sons, 2008.

[7] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197. [8] H. Yu, L. Shao, J. Fang, J. Organomet. Chem. 692 (2007) 991. [9] C. Biot, G. Glorian, L.A. Maciejewski, J.S. Brocard, J. Med. Chem. 40 (1997) 3715. [10] S. Top, A. Vessieres, C. Cabestaing, I. Laios, G. Leclerq, C. Provot, G. Jaouen, J. Organomet. Chem. 500 (2001) 637. [11] G. Tabbi, G. Cassino, G. Cavigiolio, D. Colangelo, A. Ghiglia, I. Viano, D.

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Osella, J. Med. Chem. 45 (2002) 5786. [12] E.A. Hillard, A. Vessieres, L. Thouin, G. Jaouen, C. Amatore, Angew. Chem., Int. Ed. (45) (2006) 285.

Jaouen, D. Mansuy, Angew. Chem., Int. Ed. 48 (2009) 9124.

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[13] D. Hamels, P.M. Dansette, E.A. Hillard, S. Top, A. Vessieres, P. Herson, G.

[14] D.R. van Staveren, N. Metzler-Nolte, Chem. Rev. 104 (2004) 5931.

[15] P. Köpf-Maier, H. Köpf, E.W. Neuse, Angew. Chem., Int. Ed. Engl. 23, (1984)

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456.

[16] M.L. Sun, B.F. Ruan, Q. Zhang, Z.D. Liu, S.L. Li, J.Y. Wu, B.K. Jin, J.X. Yang, S.Y. Zhang, Y.P. Tian, J. Organomet. Chem. 696 (2011) 3180.

(2012) 113.

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[17] X.F. Huang, J.F. Tang, J.L. Ji, X.L. Wang, B.F. R, J. Organomet. Chem. 706

[18] G.M. Sheldrick, SHELXTL V5.1 Software Reference Manual, Bruker AXS, Inc., Madison, WI, USA, 1997.

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[19] X. Chen, C. Plasencia, Y. Hou, N. Neamati, J. Med. Chem. 48 (2005) 1098.

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Figure captions: Scheme 1. Synthesis of the target compounds. Table 1. Structures for the synthetic compounds.

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Table 2. Crystallographic data and structure refinements for compounds 4 and 5. Table 3. Selected bond lengths (Ǻ) and angles (º) for compounds 4 and 5. Table 4. Antitumor activity (IC50) of the synthetic compounds. Figure 1. Molecular structure of compound 4 using Diamond.

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Figure 2. Molecular structure of compound 5 using Diamond.

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Table 1 Compd. a

R

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b c d

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g h i j

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Table 2 4 C16H14BrF3FeN2

5 C16H14BrF3FeN2

Mr Crystal system Space group Crystal size (mm3) a (Å) b (Å) c (Å) α
(°) β (°) γ (°) Volume (Å3) Z Dc (g /cm-3) µ (mm-1) F (000) θ rang (°C) Reflections collected Reflections unique Parameters Goodness-of-fit on F2

427.05 Orthorhombic Pna21 0.32×0.23×0.19 16.6247(12) 9.2614(7) 21.3209(15) 90.00 90.00 90.00 3282.7(4) 8 1.728 3.382 1696 2.40-25.70 22411 5821 415 1.041

427.05 Monoclinic P21/c 0.23 × 0.20 × 0.20 5.8024(16) 13.118(4) 22.274(6) 90.00 105.100(6) 90.00 1636.9(8) 4 1.733 3.391 848 1.82-26.00 9238(0.0358) 3110 208 0.974

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R1,wR2 (all data) R1, wR2 [I > 2σ(I)] Larg.peak/hole(e. Å) CCDC no

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Formula

0.0567, 0.1285 0.0452, 0.1198 0.564/-0.674 928828

0.0893, 0.1912 0.0672, 0.1799 0.813/ -0.705 928827

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Table 3

121.9(5) 127.9(5) 125.8(5) 117.3(5) 112.5(7)

C(3)-C(5)-C(12) C(5)-C(12)-N(1) N(2)-C(13)-C(14) C(12)-N(1)-C(29) C(29)-C(30)-Br(1)

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126.6(6) 127.4(6) 128.2(7) 115.0(6) 113.4(8)

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1.454(9) 1.360(9) 1.361(1) 1.331(9) 1.535(1)

1.370(7) 1.314(8) 1.356(7) 1.367(1)

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C(11)-C(12) C(13)-N(2) N(1)-C(12) C(29)-C(30)

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1.467(7) 1.414(8) 1.341(7) 1.451(9) 1.953(9)

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C16H14BrF3FeN2 (4) Bond lengths C(5)-C(12) C(11)-C(13) N(2)-N(1) N(1)-C(29) C(30)-Br(1) Bond angles C(4)-C(5)-C(12) C(5)-C(12)-C(11) C(11)-C(13)-C(14) N(2)-N(1)-C(29) N(1)-C(29)-C(30) C16H14BrF3FeN2 (5) Bond lengths C(1)-C(11) C(12)-C(13) C(13)-N(1) C(11)-N(2) C(15)-C(16) Bond angles C(2)-C(1)-C(11) C(1)-C(11)-C(12) C(12)-C(13)-C(14) N(2)-N(1)-C(15) N(1)-C(15)-C(16)

130.9(5) 126.5(4) 122.2(6) 129.1(5) 114.0(7)

C(11)-C(12) C(13)-C(14) N(1)-N(2) C(15)-N(1) C(16)-Br(1)

1.407(1) 1.486(1) 1.362(8) 1.469(1) 1.879(1)

C(5)-C(1)-C(11) C(1)-C(11)-N(2) N(1)-C(13)-C(14) C(13)-N(1)-C(15) C(15)-C(16)-Br(1)

125.9(6) 122.1(6) 124.1(7) 133.8(7) 112.6(6)

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Table 4

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MDA-MB-45 11.46 6.19 9.47 7.27 4.52 7.48 6.99 5.37 6.98 7.52 14.17 4.69 8.08 6.97 15.69 18.49 6.08 16.18 13.23 7.26 7.40 7.68 13.93 10.93 2.80±0.41 1.14±0.23

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IC50 ± SD (µg/mL) HepG2 17.41 10.20 12.78 14.59 26.70 14.69 15.28 10.89 8.43 24.77 32.34 20.82 13.78 8.96 11.70 10.68 10.38 7.80 8.39 35.06 27.42 17.11 7.96 8.51 17.6±2.6 0.74±0.21

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4 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 5 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5-FUa Cisplatina

A549 10.90 6.19 9.44 7.21 4.47 7.30 6.61 5.31 7.93 7.44 13.95 4.44 10.15 6.83 14.82 17.16 5.88 15.89 13.20 7.21 7.41 7.65 13.88 10.90 16.80±1.4 0.87±0.15

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Compounds

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Figure 1

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F3C

F3C N N

N N Br

R iv

Fe

Fe

F3C N HN O i

CF3 4

4a-4k

O iii

Fe

Fe

Fe

1

2

3

F3C

F3C N N

Br

N N

iv Fe

5

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ii

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O H3C

R

Fe

5a-5k

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Scheme 1. Synthesis of the target compounds. Reagents and conditions: (i) EtONa, CF3COOC2H5, reflux; (ii) EtOH, NH2NH2.H2O, reflux; (iii) PPh3, 2-Bromoethanol,

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CH2Cl2, DIAD, 0 °C; (iv) piperidine, sodium carbonate, KI, CH2Cl2, reflux.

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Figure 2

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> A series of novel ferrocene derivatives containing pyrazol-moiety were synthesized. > Isomeric compounds 4 and 5 were gave crystals suitable for X-ray structural analysis. > Compound 4d, 4j and 4k exhibited comparable in vitro antitumor

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activities.