Acylated mono-, bis- and tris- Cinchona-Based Amines Containing Ferrocene or Organic Residues: Synthesis, Structure and in Vitro Antitumor Activity on Selected Human Cancer Cell Lines

A series of novel functionalized mono-, bis- and tris-(S)-{[(2S,4R,8R)-8-ethyl-quinuclidin-2-yl](6-methoxyquinolin-4-yl)}methanamines including ferrocene-containing derivatives was obtained by the reaction of the precursor amine with a variety of acylation agents. Their in vitro antitumor activity was investigated against human leukemia (HL-60), human neuroblastoma (SH-SY5Y), human hepatoma (HepG2) and human breast cancer (MCF-7) cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay and the 50% inhibitory concentration (IC50) values were determined. Our data indicate that the precursor amine has no antitumor activity in vitro, but the bis-methanamines with ureido-, thioureido and amide-type linkers display attractive in vitro cytotoxicity and cytostatic effects on HL-60, HepG2, MCF-7 and SH-SY5Y cells. Besides 1H- and 13C-NMR methods the structures of the new model compounds were also studied by DFT calculations.


Introduction
Chemotherapy is one of the most important methods in fighting cancer and several members of modified natural alkaloids serve as deserving drugs against tumors. Well-known representatives of vinca alkaloids such as vinblastine, camptothecine, staurosporine and ellipticin [1][2][3][4] are typical examples. Cinchona alkaloids have been proved to be efficient antimalarial [5] and antibacterial drug candidates [6]. It is well-documented that the application of quinine derivatives in the field of cancer detection [7,8] and in chemotherapy [9][10][11][12][13][14] goes far back to the past. Since ferrocene-based molecules as anti-tumor agents are also promising matherials [15][16][17][18][19][20][21] with a wide range of biological activities [22] first we envisaged the synthesis of novel ferrocene-based mono-and bis-quinines containing amide, urea, thiourea and acylthiourea linkers providing hydrophilic character along with different hydrogen bond profile for the models subjected to in vitro assays. This choice of functional groups can also be reasoned by the following facts: (i) a number of aromatic urea derivatives play important role as anticancer agents [23]; (ii) similarly, urea-based prodrugs have been reported as candidates for melanocyte-directed enzyme therapy [24]; (iii) thiourea based molecules have been proved to be effective agents in the treatment of human promyelocytic leukemia [25]; (iv) the antiproliferative activity [26] and citotoxicity [27] of some acyl-thiourea derivatives are worth to be noted and a few patents have also been published in this field [28,29].
The pronounced efficiency of several drugs with C 2 -symmetry [30][31][32] and bis-quinolines [33][34][35] encouraged us to construct three ferrocene derivatives containing two quinine units with C 2 -symmetry and a reference benzene 1,3,5-tricarboxamide incorporating three quinine units with C 3 -symmetry. Two further purely organic models with one-and two quinine moieties, respectively, were also prepared as additional references.

Theoretical Calculations
Since the attempts to grow crystals suitable for X-ray analysis have failed so far, the preferred conformations of the new compounds with potential relevance for receptor binding properties were examined by routine DFT calculations [38]. The geometry optimization of the bis-and tris-cinchona derivatives (3-5, 7 and 8) was carried out using the appropriate symmetry constraint (C n n = 2,3). It was found that in each model compound the N-1 atom is situated in the proximity of the hydrogen atom of the NH group directly attached to the cinchona skeleton (calculated distances: 2.1-2.3 Å) to form a five membered chelate ring representing a hydrophilic segment of the molecule. As evidenced by its downfield-shifted 1 H-NMR signal, this NH group is also incorporated in an additional six-membered chelate ring in the acylthiourea derivatives 5 and 6 considerably decreasing the flexibility of these molecules. In the optimized structures of 25 the Cp-rings are in the eclipsed position relative to each other and the attached functional groups (amide, urea and acylthiourea) are practically coplanar with them (see Supporting Data for the atomic coordinates).

Structure Determination
The supposed structures of the new compounds investigated 2-8 are consistent with their spectral parameters, so only the following remarks are necessary: the C 2 -and C 3 -symmetric structures of 35, 7 and 8, respectively, are confirmed by the identical spectral data of the two or three chincona residues incorporated in these compounds. In acylthioureas 5 and 6 the presence of the chelate rings gains support from the significantly downfield-shifted 1 H-NMR signal of the NH group directly bonded to C-9 atom. The relative configuration of the cinchona unit in each compound investigated was evidenced by DNOE measurements revealing endo position of H-9 in the proximity of H-5  -and H-7  , respectively. Accordingly, NOE's were detected between H-6  and the proton of the NH group attached to C-9 atom. Their proximity is also reflected from the significant downfield shift of the H-6  signal relative to that of its germinal partner, H-6  (= 0.60.7 ppm). The relative configuration of C-8 centre gains support from the NOE interactions measured between H-2 endo -and H-8 atoms. On irradiation of the protons on the ethyl group significant enhancements of the intensity of H-7  and H-8 signals were observed indicating the relative configuration of the C-3 atom.

In Vitro Activity of the Compounds on Human Tumor Cell Cultures
We have determined the cytotoxic and cytostatic activity of the compounds in vitro on four human tumor cell lines: HL-60 leukemia, HepG2 hepatoma, MCF-7 breast adenocarcinoma and SH-SY5Y neuroblastoma cell cultures and expressed them as IC 50 values. Therefore cells were treated with the compounds at 10 −4 to 10 2 M concentration range and the viability of the cells was determined by MTT-assay.
The data summarized in Table 1 show that the precursor amine 1 has no antitumor activity in vitro on the tested human cancer cell cultures. Among the investigated ferrocene derivatives 25 the diamide 3 proved to be the most active on each type of tumor cells (the IC 50 values of its cytotoxicand cytostatic effects fall into the ranges between 0.72-1.70 µM and 0.40-1.00 µM, respectively). It is worth to emphasize that the presence of an additional quinine amide moiety in 3 induces a dramatic enhancement in the in vitro antitumor activity compared to that of the analogue mono-amide 2. The outstanding efficiency of 3 can probably be attributed to a cooperation of the two functionalities adopting optimal conformation by practically unrestricted rotation of the two Cp rings.
Significant differences are discernible between the activities of ferrocene-based bis-urea 4 and bis-acylthiourea 5. While 4 shows considerable activities on each investigated cell line, compound 5 has only selective cytotoxic and cytostatic effect on the HL-60 cells. The spectacularly decreased activity of 5 may be associated with the intramolecular hydrogen bonds and the increased rigidity of the two acylthiourea units stabilized by their chelate structure.
Among the purely organic models thiourea 7 of C 2 -symmetry exhibited significant cytotoxic and cytostatic effects against each tested cancer cell line, especially on HL-60 and SH-SY5Y cultures (cytotoxic effect: IC 50 = 1.80 and 0.84 µM, cytostatic activity: 10.20 and 4.20 µM, respectively). Acylthiourea 6 and tris-amide 8 with C 3 -symmetry also displayed remarkable activities (with higher IC 50 values: Between 17.60 and 32.20 µM; 2.30 and 8.90 µM, respectively) without in vitro cytotoxic effect on MCF-7 cells. On the other hand, these molecules were slightly cytostatic on the same cell lines after overnight incubation (Table 1).
The IR spectra were run in KBr disks on a Bruker IFS-55 FT-spectrometer controlled by Opus 3.0 software. Optical rotations were measured with a Zeiss Polamat A polarimeter. The 1 H-and 13 C-NMR spectra were recorded in CDCl 3 or DMSO-d 6 solution in 5 mm tubes at RT, on a Bruker DRX-500 spectrometer at 500.13 ( 1 H) and 125.76 ( 13 C) MHz, with the deuterium signal of the solvent as the lock and TMS as internal standard. DEPT spectra were run in a standard manner, using only a Θ = 135° pulse to separate the CH/CH 3 and CH 2 lines phased "up" and "down", respectively. The 2D-COSY, HMQC and HMBC spectra were obtained by using the standard Bruker pulse programs. The exact mass measurements were performed using a Q-TOF Premier mass spectrometer (Waters Corporation, 34 Maple St, Milford, MA, USA) in positive electrospray mode.

1,1'-(Ferrocene-1,1'-diyl)-bis-{3-[(S)-((2S,4R,8R)-8-ethylquinuclidin-2-yl](6-methoxyquinolin-4-yl)methyl)}urea (4)
The amine (7.58 g, 23 mmol) and 1,1'-diisocyanatoferrocene [40] (2.6 g, 9.7 mmol) were stirred in dry THF at RT under argon overnight. After the evaporation of the reaction mixture the crude product was dissolved in DCM/MeOH (10:1) and the solution was passed through Celite and concentrated in vacuo. The residue was column chromatographed on silica with DCM/MeOH (10:1) and crystallized by EtOH (15 mL (5) 1,1'-bis-Isothiocyanatocarbonylferrocene was prepared from 1,1'-bis-chlorocarbonylferrocene (2.18 g, 7 mmol) according to the procedure employing potassium isothiocyanate as reagent and acetone as solvent [41]. This reactive intermediate was used without purification after acetone was removed by distillation and the residue was dissolved in THF. The amine (5.39 g, 17 mmol) and 1,1'-bis-(isothiocyanatocarbonyl)ferrocene dissolved in 100 mL of dry THF were stirred overnight at RT under argon. The reaction mixture was concentrated in vacuo. The residue was subjected to column chromatography on silica using DCM/MeOH (15:1) as eluent. The partially purified product was dissolved in EtOH and slowly precipitated by water. The precipitate was filtered off then the chromatography and the recrystallization were repeated in order to get rid of the traces of isothiocyanate reagent to afford the pure product as brick-red powder (120 mg, 2%). mp. 167-169 °C;  (6) The amine (3.00 g, 9.2 mmol) and benzoylisothiocyanate (1.51 g, 9.2 mmol) were dissolved in dry THF (100 mL). The reaction mixture was stirred overnight at RT and evaporated to dryness.  (7) The amine (7.58 g, 23 mmol) and thiocarbonyldiimidazole (TCDI; 2.08 g, 12 mmol) were stirred in dry THF (150 mL) under argon. After TCDI was slowly dissolved the solution was evaporated and the residue was subjected to flash column chromatography on silica using DCM/MeOH (15:1) as eluent. The resulted oily substance was crystallized by water-ethanol and thoroughly washed with boiling water to obtain the product as white powder (171 mg The amine (3.60 g 11 mmol), pyridine (0.89 mL 11 mmol) and DMAP (224 mg 1.8 mmol) were dissolved in dry DCM (100 mL). During vigorous stirring 1,3,5-tris-chlorocarbonylbenzene (0.66 mL, 3.7 mmol) was added to the solution in one portion. After stirring for 24 h the solution was poured onto ice. DCM was distilled off at atmospheric pressure. The resulted precipitate was filtered off and dried to yield the product as white powder (3.60 g 87%

In Vitro Cytostatic and Cytotoxic Activity of the Compounds
The cells were grown to confluency and were plated into 96-well plate with initial cell number of 5.0-7.5 × 10 3 per well. After 24 h incubation at 37 °C, cells were treated with the compounds in 200 μL final volume containing 1.0 v/v% DMSO. Cells were incubated with the compounds at 10 −4 -10 2 M concentration range for overnight. Control cells were treated with serum free medium (RPMI-1640 or DMEM) only or with DMSO (c = 1.0 v/v%) at 37 °C for overnight. After incubation the cells were washed twice with serum free (RPMI-1640 or DMEM) medium. To determine the in vitro cytostatic effect, cells were cultured for a further 72 h in serum containing medium. To measure the in vitro cytotoxicity of the compounds, MTT-assay was carried out immediately after the overnight treatment: The cell viability was determined by the following method using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) [42,43]. The solution of MTT (45 μL, 2 mg/mL) was added to each well which was reduced by the respiratory chain [42,43] and other electron transport systems [44] to form precipitated violet formazan crystals within the cell [45]. The amount of these crystals can be determined by spectrophotometry serving as an estimate for the number of mitochondria and hence the number of living cells in the well [46]. After 4 h of incubation the cells were centrifuged for 5 min (900 g) and supernatant was removed. The obtained formazan crystals were dissolved in 50 μL of DMSO and the optical density (OD) of the samples was measured at  = 540 and 620 nm, respectively, employing ELISA Reader instrument (iEMS Reader, Labsystems, Finland). OD 620 values were subtracted from OD 540 values and the percent of cytostasis or cytotoxicity was calculated using equation "Cytostatic effect/Cytotoxicity (%) = [1 − (OD treated /OD control )] × 100" (where OD treated and OD control correspond to the optical densities of the treated and the control cells, respectively). In each case two independent experiments were carried out with 4-8 parallel measurements. The 50% inhibitory concentration (IC 50 ) values were determined from the dose-response curves. The curves were defined using Microcal TM Origin1 (version 7.5) software: cytostasis (%) or cytotoxicty (%) was plotted as a function of concentration, fitted to a sigmoidal curve and, based on this curve, the half maximal inhibitory concentration (IC 50 ) value was determined representing the concentration of a compound required for 50% inhibition in vitro and expressed in micromoles.

Conclusions
Among the novel compounds reported in this contribution the bis-and tris-quinine derivatives exerted a dose-dependent in vitro antitumor activity at micromolar concentrations on the investigated tumor cell cultures. The ferrocene-based bis-amide 3 of pronounced activity can be considered as a promising lead structure for the development of a novel class of therapeutical agents. The highly promising results also obtained for a metallocene model containing two ureido functional groups (compound 4) suggest that 1,1'-disubstituted ferrocene unit with easily rotating Cp-rings seems to be highly beneficial to the desired activity allowing the molecule to adopt a conformation in which the two cooperating groups are situated in optimal distance from each other. On the other hand, the presence of rigid acylthiourea moiety stabilized by chelate structure dramatically decreases the activity on the investigated cell lines.