1,1-Bis(4-hydroxyphenyl)-2-ferrocenylbutane

Abstract

Ferrociphenols are anticancer organometallic molecules bearing a ferrocene group linked, at least, to one para-phenol moiety via a double bond. Up to the present, their biological activity has been thought to be linked to their oxidation within cells to form a reactive quinone-methide metabolite with the participation of this central double bond. To prove this assertion, the alkenyl entity of ferrociphenol 1a (1,1-bis-(4-hydroxyphenyl)-2-ferrocenylbut-1-ene) was reduced by triethylsilane in an acidic medium to obtain the alkyl counterpart 1,1-bis(4-hydrophenyl)-2-ferrocenylbutane. 1,1-bis(4-hydrophenyl)-2-ferrocenylbutane was fully characterized by ¹H NMR (including COSY), ¹³C NMR, HRMS, IR, elemental analysis and X-ray diffraction (XRD). Although missing the central double bond, this compound remains biologically active, opening the way to a new family of anticancer ferrocene-containing molecules.

1. Introduction

In the fight against cancer, the interest in organometallic complexes, particularly ferrocene-containing molecules, has increased significantly over the last two decades [1,2,3,4,5,6,7,8,9,10,11,12]. Within this family of molecules, ferrociphenols show high cytotoxic activity against various cancer cell lines. Initially, one of the three phenyl groups of Tamoxifen, a selective estrogen receptor modulator (SERM) used in the adjuvant treatment of hormone-dependent breast cancer, was replaced by a ferrocenyl group to form a new compound called Ferrocifen, by analogy to Tamoxifen. Ferrociphenol 1a (Figure 1) is a precursor in the synthesis of 4-Hydroxyferrocifen 1b, i.e., the ferrocenyl version of 4-hydroxytamoxifen, the active metabolite of Tamoxifen. Compound 1b showed antiproliferative activity against the hormone-dependent human breast cancer MCF-7 cell line as well as against the triple-negative MDA-MB-231 cell line. By serendipity, we found that precursor 1a was also very active against both the MDA-MB-231 and MCF-7 cell lines [13]. The cytotoxic activity of 1a and 1b is associated with the [ferrocene-alkene-p-phenol] moiety that participates in metabolite formation by oxidation (see below) [14]. In the twenty years following these discoveries, we synthesized many ferrociphenols, sometimes with great variation in structure—for instance, the cyclic version of 1a (ferrocenophane 2) or the replacement of a phenyl group by an alkyl group (ferrocenyl derivatives of diethylstilbestrol 3a–d)—all sharing the central double bond linking the ferrocenyl and aryl groups and being active to very active. For example, the IC₅₀s of the acyclic compounds 1a [13], 1b [14], 1c [15], 1d [16], 3a, 3b, 3c and 3d acting on MDA-MB-231 cells are 0.64, 0.5, 0.11, 0.035, 1.14, 0.14, 0.28 and 0.36 µM, respectively. Ferrocenophane 2 has an IC₅₀ of 0.089 µM. This systematic activity was attributed to oxidation within cells, yielding a quinone methide able to covalently bind to proteins [17]. The formation of quinone methide proceeds via a multistep mechanism involving two one-electron oxidation steps and two deprotonation steps [6].

We wondered whether a disconnection between the ferrocenyl group and the phenol group would have any effect on the biological activity of ferrociphenol 1a. Thus, we decided to reduce its double bond by chemical hydrogenation using triethylsilane and trifluoroacetic acid (TFA). As a great simplification, TFA brings H⁺ and triethylsilane brings H⁻. We have already used this method to reduce carbocations generated by TFA from an alcohol, from an alkene or even directly from a ketone. The reduced compound 4 was obtained from 1a in moderate yield (60%) after three days of reaction at room temperature in dichloromethane in the presence of TFA and HSiEt₃. Surprisingly, compound 4 was still cytotoxic to MDA-MB-231 cells, with an IC₅₀ that only doubled (IC₅₀ = 0.94 ± 0.11 μM) with respect to 1a (IC₅₀ = 0.64 ± 0.06 μM). This opens up possibilities for the widening of the diversity of active ferrocenyl compounds, starting with reduced versions of our most active compounds, i.e., 1b–d and 2. As the first and leading molecule of this new family, 4 was fully characterized by ¹H NMR, ¹³C NMR, COSY, IR, elemental analysis and X-ray diffraction (XRD).

2. Results and Discussion

2.1. Synthesis of 4

Compound 4 was synthesized from compound 1a by reduction with triethylsilane and trifluoroacetic acid (TFA) in a dichloromethane solution at room temperature for three days, with a yield of 60% (Scheme 1). Recrystallization from dichloromethane provided crystals suitable for X-ray diffraction analysis. Compared to that of 1a, the ¹H NMR spectrum of 4 (Supplementary Figures S1 and S2) shows the appearance of signals assigned to the two new protons in positions 1 and 2 of the butane skeleton, namely, a doublet for H1 partially hidden by the signal of the ferrocene protons (Figure S1) at 3.99 ppm and a multiplet for H2 at 3.15 ppm. Both protons were coupled together, as shown by the COSY experiment (Figure S2). Anisotropy due to the generation of a stereogenic center also made the protons of the methylene (H3) and ferrocenyl (C₅H₄) groups inequivalent. These two methylene protons, H3, were coupled with H2 and not H1 (Figure S2), confirming this attribute. In the ¹³C NMR spectrum, two quaternary carbon atoms disappeared and were replaced by two CH groups (C1 and C2) at 55.1 and 46.0 ppm, respectively (Figures S3 and S4).

2.2. X-Ray Crystal Structure Determination

The crystal structure of 4 was determined by X-ray diffraction (XRD) (Figure 2). Orange crystals were obtained and grown by the slow evaporation of a CH₂Cl₂ solution under ambient conditions. Under these conditions, compound 4 crystallized in the P$\overline{1}$ space group (triclinic system). The asymmetric unit contains one molecule of 4 and a half-molecule of CH₂Cl₂. Crystallographic and refinement structure data are available in

Table 1. Crystallographic and refinement structure data for 4.

Parameter	Value	Parameter	Value
CCDC deposit number	2393186	Crystal size (mm²)	0.23 × 0.16 × 0.14
Empirical formula a	C26.5H27ClFeO2	Wavelength λ (Å)	0.71073
Moiety Formula	C26H26FeO2, 0.5 (CH2Cl2)	2ϴ range (°)	3.646–59.998
Formula weight (g/mol)	468.78	Miller indices ranges	−11 ≤ h ≤ 11, −16 ≤ k ≤ 16, −17 ≤ l ≤ 15
Temperature (K)	200
Crystal system	Triclinic
Space group	P$\overline{1}$	Measured reflections	20976
a (Å)	8.4426 (9)	Unique reflections	6734
b (Å)	11.6967 (14)	Rint/Rsigma	0.0275/0.0312
c (Å)	12.4258 (10)	Reflections [I ≥ 2σ(I)]	5273
α (°)	78.249 (7)	Restraints	77
β (°)	83.969 (7)	Parameters	336
γ (°)	76.411 (11)	Goodness of fit F²	1.032
Volume (Å3)	1165.6 (2)	Final R indices bc [all data]	R1 = 0.0606, wR2 = 0.1093
Z	2
ρcalc (g/cm3)	1.336	Final R indices bc [I ≥ 2σ(I)]	R1 = 0.0411, wR2 = 0.0973
Absorption coefficient μ (mm−1)	0.782 (MoKα)
F(000)	490	Largest diff. peak/hole (e/Å3)	0.54/−0.63

^a Including solvent molecules (if present); ^b $R1=\sum \left|\left|F_o\right|-\left|F_c\right|\right|/\sum \left|F_o\right|$; ^c $wR2=\sqrt{\sum \left(w\left(F_o^2-F_c^2\right)\right)/\sum \left(w{\left(F_o^2\right)}^2\right)}$.

.

Dichloromethane is disordered over two positions with an occupation rate of 42/8. The presence of a partial CH₂Cl₂ molecule in the asymmetric unit is confirmed by elemental analysis. The proportions of CH₂Cl₂ determined by XRD and elemental analysis are different (0.50 vs. 0.25 of a molecule of dichloromethane per molecule of 4, respectively). This difference is probably due to the vaporization of CH₂Cl₂ and the two analyses being performed at different temperatures and times. It is also interesting to note the presence of CH₂Cl₂ in the ¹H NMR results (Figure S1). Therefore, the presence of partial CH₂Cl₂ is only proved qualitatively.

Regarding 4, the C1-C2 bond length is 1.58 Å, corresponding to a simple bond. This result proves the reduction of the double bond of 1a. For comparison, the bond in 1a has a length of 1.38 Å (see CCDC 739363 [18]). The ethyl group bound to C1 is disordered between two very close positions with an occupancy rate of 60/40. Each phenol group is involved in hydrogen bonds. One is connected to the second phenol group of the neighboring molecule obtained by b-axis translation. The other is connected to the first phenol group of the neighboring molecule obtained using an inversion center, located under the two phenol groups of the first molecule. The network created by these H-bonds resembles an almost perfect square (O_i…O_j…O_i angles: 94.3 and 85.7°; distortion angle: 0°; Figure 3). H-bond lengths and angles are available in

Table 2. H-bond lengths and angles in the crystal structure of 4 at 200 K.

D-H…A	D-H (Å)	H…A (Å)	D…A (Å)	D-H…A (°)
O1-H1….O2 i	0.94(2)	1.85(2)	2.758(2)	161(2)
O2-H2…O1 ii	0.92(3)	1.88(3)	2.788(2)	170(2)

D—H-donor atom; H—hydrogen atom; A—H-acceptor atom. Symmetry codes: (i) x, −1 + y, z; (ii) 2 − x, 1 − y, 2 − z.

.

3. Materials and Methods

3.1. General Procedure

¹H and ¹³C-NMR spectra were acquired using Bruker 300 and 400 MHz spectrometers (Bruker France, 67166 Wissembourg, France, bs = broad singlet; d = doublet; t = triplet; m = multiplet). EI-MS was performed using a Nermag R 10-10C spectrometer; ESI-MS using a triple quadrupole mass spectrometer API 3000 LC–MS/MS system (Applied Biosystems, Sciex, Framingham, MA 01701, USA) in positive-ion mode; and high-resolution mass spectrometry (HRMS) using a Jeol MS 7000 instrument (JEOL Europe SAS, 78290 Croissy-sur-Seine, France). IR spectra were recorded on an FT-IR spectrometer (Tensor 27, Bruker France, 67166 Wissembourg, France) equipped with an ATR MIRacle (Pike Technologies inc., Madison WI 53719, USA) accessory. Elemental analyses were performed at the microanalysis laboratory of the CNRS (91190 Gif sur Yvette, France). HPLC purification was conducted on a reverse-phase semi-preparative Kromasil C18 column (402 58 Göteborg, Sweden). Thin-layer chromatography was performed on silica gel 60 GF254 (Merck KGaA, Darmstadt, 64293, Germany). Column chromatography was performed on silica gel 60 M (Merck KGaA, Darmstadt, 64293, Germany). Reagents were obtained from Sigma Aldrich (38297 Saint-Quentin-Fallavier Cedex) and used as received.

3.2. Synthesis of 1,1-Bis(4-hydroxyphenyl)-2-ferrocenylbutane (4)

Ferrociphenol 1 (0.85 g; 2 mmol; M = 424.31; 1 equiv.) was dissolved in 50 mL of dichloromethane. Triethylsilane (0.96 mL; 6 mmol; M = 116.28; d = 0.728; 3 equiv.) and trifluoroacetic acid (2.07 mL; 26.9 mmol; M = 114.02; d = 1.535; 13.5 equiv.) were added to the solution. The mixture was stirred continuously for three days. Then, it was poured into a saturated bicarbonate solution and extracted twice with dichloromethane. The organic layers were combined, washed with water and dried over magnesium sulfate. After concentration under reduced pressure, the crude product was chromatographed on a silica gel column with cyclohexane/ethyl acetate (1:1) as an eluent. The second fraction, obtained as a red oil, corresponded to the desired product. It was further purified by reverse-phase semi-preparative HPLC using acetonitrile/water (4:1) as an eluent to fully remove excess triethylsilane. Compound 4 (0.51 g; 1.19 mmol; M = 426.33; yield = 60%) was obtained as an orange oil that was successfully recrystallized from dichloromethane for X-ray structure determination.

¹H NMR (300 MHz; acetone-d₆): δ = 1.02 (t, J = 7.4 Hz, 3H; CH₃: H4); 1.49–1.67 (m, 1H; CH₂: H3), 1.83–2.02 (m, 1H; CH₂: H3), 3.10–3.21 (m, 1H, CH-Et: H2), 3.52 (bs, 1H, CH; C₅H₄), 3.89 (bs, 1H, CH; C₅H₄), 3.99 (d, J = 7.6 Hz, 1H, CH-Ar₂: H1), 4.05 (bs, 2H; 2xCH C₅H₄), 4.06 (s, 5H; Cp), 6.64 (d, J = 8.7 Hz, 2H; C₆H₄), 6.73 (d, J = 8.7 Hz, 2H; C₆H₄), 6.93 (d, J = 8.7 Hz, 2H; C₆H₄), 7.09 (d, J = 8.7 Hz, 2H; C₆H₄), 8.04 (bs, 1H; OH), 8.09 (bs, 1H; OH). ¹³C NMR (75 MHz; acetone-d₆): δ = 12.6 (CH₃), 26.8 (CH₂), 46.0 (CH), 55.1 (CH), 67.2 (2CH; C₅H₄), 68.7 (CH; C₅H₄), 69.4 (5CH; Cp), 70.4 (CH; C₅H₄), 94.0 (C; C₅H₄), 115.3 (2xCH; C₆H₄), 115.6 (2xCH; C₆H₄), 130.4 (2xCH; C₆H₄), 131.2 (2xCH; C₆H₄), 134.8 (C), 136.7 (C), 156.2 (C-OH), 156.3 (C-OH). IR (ATR, ν cm⁻¹): 3472. MS (EI, 70 eV): m/z = 426 [M]^+., 227 [ferrocene-CH-Et]⁺, 199 [(HOC₆H₄)₂CH]⁺, 121 [FeCp]⁺. HRMS (EI, 70 eV, C₂₆H₂₆FeO₂, [M]^+.) calcd: 426.1283, found: 426.1284. Anal. Calcd for C₂₆H₂₆FeO₂(CH₂Cl₂)_1/4: C, 70.44; H, 5.96. Found: C, 70.24; H, 6.17.

3.3. X-Ray Diffraction Experiment

A single crystal of 4 was selected, mounted and transferred into a cold nitrogen gas stream. Intensity data were collected with a Nonius KappaCCD diffractometer using graphite-monochromated MoKα radiation. Data collection, unit-cell parameter determination, integration and data reduction were performed with the Nonius KappaCCD suite of programs [19,20,21]. The structure was solved with SHELXT [22], and non-hydrogen atoms were refined anisotropically by full-matrix least-squares methods with SHELXL [23] using Olex2 [24]. The structure was deposited at the Cambridge Crystallographic Data Centre with number CCDC 2393186 and can be obtained free of charge via www.ccdc.cam.ac.uk.