Cytotoxic and Antimicrobial Activity of Dehydrozingerone based Cyclopropyl Derivatives

A small series of 1‐acetyl‐2‐(4‐alkoxy‐3‐methoxyphenyl)cyclopropanes was prepared, starting from dehydrozingerone (4‐(4‐hydroxy‐3‐methoxyphenyl)‐3‐buten‐2‐one) and its O‐alkyl derivatives. Their microbiological activities toward some strains of bacteria and fungi were tested, as well as their in vitro cytotoxic activity against some cancer cell lines (HeLa, LS174 and A549). All synthesized compounds showed significant antimicrobial activity and expressed cytotoxic activity against tested carcinoma cell lines, but they showed no significant influence on normal cell line (MRC5). Butyl derivative is the most active on HeLa cells (IC50 = 8.63 μm), while benzyl one is active against LS174 and A549 cell lines (IC50 = 10.17 and 12.15 μm, respectively).

A pungent constituent of ginger rhizome dehydrozingerone (1; 4-(4-hydroxy-3-methoxyphenyl)-3buten-2-one), as a half analogue of curcumin, is present in different bioactive compounds, showing broad spectrum of biological activities, such as anti-inflammatory, antidepressant, antibacterial, antiviral, anticancer [6 -12] and many others. Although conjugate enone system is presented in this phenolic compound, its structure differs from chalcones in possessing the methyl group connected to carbonyl instead of the aryl one. This unique chalcone-like structure, planar enone system and aromatic ring offer bifunctional site for various transformations. As cyclopropane ring is present in a huge number of molecules isolated from nature, [13] such as terpenes, fatty acids, alkaloids and steroids so it is no surprise that many of them show pronounced biological activities, from enzyme inhibition of herpes roteases [14] [15] to antibiotic, herbicidal, antitumor, and antiviral properties. [16 -18] Also, well-known are chrysanthemic acid, pyrethrin, and pyrethroid derivatives, as compounds related to natural and synthetic insecticides, with good insecticidal activities. [19] [20] This motif is attached to main frame of the molecules at different ways. Cyclopropyl group is connected to C-atom in heterocyclic fragment [21] or nitrogen atom in ciprofloxacin derivatives, [22] to carbon chain of C 29 sterols, [23] as 1,1-dichloro derivatives of diaryl cyclopropanes. [24] [25] It is also present in combretastatin derivatives as cyclopropyl-vinyl or a cyclopropyl-amide bridge, [26] [27] in cyclopropyl indolequinones [28] or cinnamic acid derivatives. [29] Based on data from different sources, nearly 50% of all modern scientific drugs on the market are natural products or natural-based materials and they play an important role in a drug design in pharmaceutical industries. [30] Although many drugs have been introduced into the market, their response to therapy is still poor. For this reasons more efficient drugs should be developed.
Radiotherapy and chemotherapy, as the most frequently used procedures in cancer treatment, are not specific to cancer cells and are known to cause severe and often adverse side effects, such as gastrointestinal reactions, immune suppression, bone marrow suppression, hair loss, nerve injury, and even development of secondary malignancies. Therefore, we decided to modify dehydrozingerone, as a natural product easily accessible by simple synthetic procedure, into a new group of compounds. In this way attractive motif, cyclopropyl ring was introduced into the molecule. Microbiological and cytotoxic properties of synthesized compounds had been tested.

Results and Discussion
Chemistry Due to our research interest for the synthesis of molecules exhibiting some biological activity, [12] [31 -34] herein we described the synthesis and characterization, starting from dehydrozingerone (4-(4-hydroxy-3methoxyphenyl)-3-buten-2-one; 1), of a series of compounds 2a -2j. In these compounds, the transolefinic bond is replaced by a conformationally restricted and inherent ring strain cyclopropyl moiety, affording a series of novel cyclopropyl derivatives 3a -3j.
Dehydrozingerone was synthesized following previously described procedure. [35] Alkylation of their free phenolic group was performed in the boiling acetone solution in the presence of corresponding alkyl halide and anh. K 2 CO 3 , yielding alkyl derivatives, 4-(4-alkoxy-3-methoxyphenyl)-3-buten-2-ones 2a -2j. Isolated alkyl derivatives were used as substrates in Corey-Chaykovsky reaction. Knowing that this reaction is stereospecific, the series of trans-cyclopropyl products were synthesized (Scheme 1). The prepared compounds, 1-acetyl-2-(4-alkoxy-3-methoxyphenyl)cyclopropanes 3, were characterized by their spectral data (IR, MS, 1 H-and 13 C-NMR). All four protons from the cyclopropane ring have a different environment, and each of them is coupling with others and four groups of signals are presented. As result of it, each proton has three different coupling constants. The crystal structure of 1-acetyl-2-(4-isopropoxy-3-methoxyphenyl)cyclopropane (3c) was also reported.

X-Ray Analysis
The crystal structure of molecule 3c and the selected geometrical parameters are given in Fig. 1, while the selected geometrical parameters are given in Table 1.
The C(3)-C(5) bond of the middle cyclopropane fragment (cp) is the longest bond in the structure. A slight elongation of this bond in comparison to the other two C-C bonds of the strained cp ring can be attributed to the presence of the acetyl and aryl substituents at the C(3) and C(5) atoms, respectively.
The angle opposite to the C(3)-C(5) bond also slightly expands at the expense of the remaining two angles ( Table 1). Similar asymmetry within the disubstituted cp ring has been observed in related crystal structures such as phenylcyclopropanecarboxylic acids. [36 -38] The angles outside the cp ring depend on the size of the attached substituents; thus, the angles between the C-C bonds of the cp ring and the corresponding bonds attaching the aryl and acetyl fragments have the average values of 121.3°and 117.6°, respectively.

Antimicrobial Activity
The antimicrobial activities of the investigated compounds against the test microorganisms are shown in Tables 2 and 3. The tested compounds demonstrated relatively strong antimicrobial activity inhibiting all tested microorganisms. The minimum inhibitory concentration (MIC) for these compounds relative to the tested bacteria ranged from 0.009 to 1.25 mg/ml ( Table 2). The strongest antibacterial activity was found in 3b compound inhibiting all the species of bacteria in low concentrations, especially Bacillus subtilis where measured MIC value was 0.009 mg/ml (stronger than streptomycin). Among the bacteria, Escherichia coli showed the highest resistance while B. subtilis was the most sensitive. The tested compound also inhibited the growth of the all tested fungi ( Table 3) but in slightly higher concentrations (MIC values were from 0.156 to 5 mg/ml). Among the fungi, Candida albicans appeared to be the most sensitive.
The antimicrobial activity was compared with the standard antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi). The experimental results showed that the tested compound had just slightly weaker effect than streptomycin (compound 3b even better than streptomycin), while ketoconazole had stronger activity than the tested samples as shown in Tables 2 and 3. In a negative control, DMSO had no inhibitory effect on the tested organisms.
In these experiments, antibacterial effect was observed against the both Gram-positive and Gramnegative bacteria but it should be noted that the Gram-negative bacteria were more resilient. It has been generally reported that the Gram-negative bacteria are more resistant than Gram-positive. [12][40] [41] This resistance is likely, due to the fact that Gramnegative bacteria have a wall which itself is surrounded by an outer complex membrane, slowing down the passage of hydrophobic compounds.  Bond length Angle Lacking outer membrane, Gram-positive bacteria are more susceptible to the antibiotic agents. [42] Compared to the bacteria, fungi were more resistant due to the more complex structure of the cell wall. [43] Cytotoxic Activity The cytotoxicity of cyclopropyl derivatives 3a -3j against the human carcinoma cell lines HeLa, A549, and LS174 was assessed by the MTT test, using cis-DDP as the control. The cytotoxic activity of tested compounds is given in  Table 5), indicates that all tested compounds exhibit superior selectivity comparing to cis-DDP (up to ten times). According to the supreme cytotoxicity and selectivity (low IC 50 and high selectivity index), compounds 3e, 3f, and 3h demonstrate the most promising cytotoxic activity against the selected carcinoma cell lines.

Conclusions
Dehydrozingerone, as an easily accessible natural product, was modified by the simple synthetic procedure. In the present study, the results clearly demonstrate  The good activity of synthesized compounds, fewer side effects, along with the lack of toxicity (very low cytotoxicity against MRC5 cells) and the feasible synthesis, underscore their value as promising novel scaffolds for the development of new antimicrobial and anticancer drugs.
Column chromatography: silica gel 60 (Merck, 230 -400 mesh ASTM). TLC: Silica gel 60 F254-precoated plates (Merck). IR Spectra: PerkinElmer Spectrum One FT-IR spectrometer with a KBr disc, m in cm À1 .   electro spray capillary was set at 4.3 kV and the cone at 40 V. The ion source temp. was set at 125°C and the nitrogen flow rates were 400 and 50 l/h, for desolvation and cone gas flow, resp. The collision energy was 40 eV. The melting point of products was determined by using MelTemp1000 apparatus.
General Procedure for the Synthesis of 4-(4-Alkoxy-3methoxyphenyl)-but-3-en-2-ones (2a -2j). A mixture of dehydrozingerone (1; 1.92 g, 10 mmol), appropriate alkyl halide (excess, 30 mmol) and K 2 CO 3 (7 g, anh.) in acetone (60 ml) was heated to reflux overnight under argon. Acetone and the excess of alkyl halide were evaporated under reduced pressure and the solid residue was dissolved in water. The mixture was distilled with steam to remove excess of alkyl halide and their side products. After cooling, the water mixture was extracted with CH 2 Cl 2 (3 9 50 ml). The combined extracts were washed with water and dried over anh. Na 2 SO 4 . After the removal of the main part of solvent, the residue was filtered over silica gel pad. Compounds 2e and 2j were isolated as oils, while others were isolated as white crystalline substances. General Procedure for the Synthesis of 1-Acetyl-2-(4alkoxy-3-methoxyphenyl)cyclopropanes (3a -3j). The solution of the ylide was prepared under nitrogen from sodium hydride (3 mmol, 0.15 g of 50% oil suspension), trimethyloxosulfonium iodide (0.726 g, 3.3 mmol) and DMSO (3 ml). Enone solution in DMSO (3 mmol in 5 ml) was added to this mixture while being stirred and cooled in a water bath. The mixture was stirred at r.t. for 2 h and then at 50°C for 1 h. The solvent mixture of toluene/AcOEt (95:5, 20 ml) was added to a reaction flask with stirring. The mixture was then poured into 50 ml of cold water and extracted with toluene. The extracts were washed twice with water, dried over anh. Na 2 SO 4 and evaporated, yielding pale yellow oil. The crude mixture was chromatographed on silica gel column, using hexane/AcOEt mixture (7:3).

X-Ray Diffraction Experiment
The diffraction data for molecule 3c were collected at r.t. on Agilent Gemini S diffractometer equipped with CuK a radiation (k = 1.54184 A). Data reduction and empirical absorption correction were performed with CrysAlisPro. [49] The crystal structure was solved by direct methods, using Sir2002 [50] and refined using SHELXL [51] by full-matrix least-squares on F 2 . All H-atoms were placed geometrically [C-H = 0.93 -0.98 A] and refined using the riding model with isotropic displacement parameters set to 1.2 or 1.5 times the U eq values of the parent C-atoms. Crystallographic data and refinement parameters are listed in Table S1. The software used for the preparation of the materials for publication: WinGX, [52] Mercury, [53] PLATON, [54] PARST. [55] Antimicrobial Activity Antimicrobial activities of tested compounds were evaluated against ten microorganisms, including five The bacteria isolates were picked from overnight cultures in Mueller-Hinton agar and the suspensions were prepared in sterile distilled water by adjusting the turbidity to match 0.5 McFarland standards to approximately 10 8 CFU/ml. The fungal suspensions were prepared from 3-to 7-day-old cultures that grown on a potato dextrose agar except for C. albicans that was maintained on Sabourad dextrose (SD) agar. The spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530 nm, and then further diluted to approximately 10 6 CFU/ml, following the procedure recommended by NCCLS. [56] The 96-well microtiter assay using resazurin as the indicator of cell growth, [57] was employed for the determination of the MIC of the active components. The starting solutions of tested compounds were obtained by dissolving it in 5% DMSO. The twofold serial dilutions of tested compounds were made in a concentration range from 20 to 0.004 mg/ml in sterile 96-well plates containing Mueller-Hinton broth for bacterial cultures and a SD broth for fungal cultures. Then, fungal or bacterial suspensions were added to each well and finally, resazurin solution was added as an indicator to each well. The plates were prepared in triplicate, and placed in an incubator set at 37°C for 18 -24 h. The MIC was determined visually and defined as the lowest concentration of tested compounds preventing resazurin color change from blue to pink. Streptomycin and ketoconazole were used as positive controls while 5% DMSO was used as a negative control.

Cytotoxicity
Cells and Cell Culture. Human epithelial carcinoma HeLa cells, human lung carcinoma A549 cells and human colon carcinoma LS174 cells and nonmalignant human lung fibroblast cell line (MRC5) were obtained from American Type Culture Collection (Manassas, VA, USA). All cancer cell lines were cultured as a monolayer in the RPMI 1640 nutrient medium, with 10% (inactivated at 56°C) FBS, 3 mM of L-glutamine, and antibiotics, at 37°C in humidified air atmosphere with 5% CO 2 .
In vitro Cytotoxic Assay. In vitro assay for cytotoxic activity of tested compounds was performed when the cells reached 70 -80% of confluence. Stock solution (50 mg/ml) of compounds was dissolved in corresponding medium to the required working concentrations. Neoplastic HeLa cells (5000 cells per well), A549 cells (5000 cells per well), and LS174 cells (5000 cells per well) as well as non-cancerous MRC5 (5000 cells per well) were seeded into 96-well microtiter plates, and 24 h later, after cell adherence, five different, double diluted concentrations of investigated compounds were added to the wells. Final compounds concentrations were 200, 100, 50, 25, and 12.5 lg/ml except for the control wells, where only nutrient medium was added. The cultures were incubated for the next 72 h. The effect on cancer cell survival was determined 72 h after the addition of tested compounds, by the MTT test. [58] Briefly, 20 ll of MTT solution (5 mg/ml PBS) was added to each well and incubated for a further 4 h at 37°C in 5% CO 2 and humidified air. Subsequently, 100 ll of 10% SDS was added to solubilize the formazan crystals formed from MTT after the conversion by mitochondrial dehydrogenases of viable cells. Absorbencies proportional to the number of viable cells were measured using a microplate reader (Multiskan EX, Thermo Scientific, Finland) at 570 nm. Each experiment was performed in triplicate and independently repeated at least four times.

Supplementary Material
Supporting information for this article is available on the WWW under https://doi.org/10.1002/cbdv.201700077.