Synthesis and cytotoxic properties of novel (E)-3-benzylidene-7-methoxychroman-4-one derivatives

Background and the purpose of the study There has been increscent interest in the field of cancer chemotherapy by discovery and development of novel agents with high efficacy, low toxicity, and minimum side effects. In order to find new anticancer agents, we replaced the pyrazolone part of well-known cytotoxic agent SJ-172550 with 7-methoxychroman-4-one. Thus, a novel series of 3-benzylidene-4-chromanones were synthesized and tested in vitro against human cancer cell lines. Methods The title compounds were prepared by condensation of 7-methoxychroman-4-one with suitable aldehydes in appropriate alcohol in the presence of gaseous HCl. The antiproliferative activity of target compounds were evaluated against MDA-MB-231 (breast cancer), KB (nasopharyngeal epidermoid carcinoma) and SK-N-MC (human neuroblastoma) cell lines using MTT assay. Results Although the direct analog of SJ-172550 (compound 5d) did not show any cytotoxic activity against tested cell lines, but 2-(2-chloro-6-methoxyphenoxy)acetic acid methyl ester analog 5c showed some activity against MDA-MB-231 and SK-N-MC cells. Further modification of compound 5c resulted in the 3-chloro-4,5-dimethoxybenzylidene derivative 5b which demonstrated better cytotoxic profile against all tested cell lines (IC50 values = 7.56–25.04 μg/ml). Conclusion The results demonstrated that the cytotoxic activity of compound 5b against MDA-MB-231 and SK-N-MC cells is more than etoposide. Therefore, compound 5b prototype could be considered as novel cytotoxic agent for further developing new anticancer chemotherapeutics.


Introduction
Cancer has been known as one of the most impressive clinical problems in both developing and developed countries. In spite of improved diagnostic techniques and advances in prevention and chemotherapeutic management of cancer, the disease still afflicts millions of peoples in the world [1]. Cancer cells are defined by uncontrolled replications associated with self-sufficiency in growth signals, hyposensitivity to anti-growth signals, ongoing angiogenesis, metastasis, and evasion of apoptosis [2]. Anti-cancer agents cannot recognize cancer cells from normal cells, as a matter of fact, these agents usually act on metabolically active or rapidly proliferating cells [3]. Thus, there has been increscent interest in the field of cancer chemotherapy by discovery and development of novel agents with high efficacy, low toxicity, and minimum side effects.
During recent years, several researchers developed different chalcone-like compounds with anticancer activity through the introduction of heterocyclic scaffolds [4,5]. The chemical structure of chalcone is characterized by two aromatic rings connected by a three carbon, α,β-unsaturated carbonyl system (1,3-diphenyl-2-propen-1one) [6][7][8]. The highly significant advantage of chalcone derivatives as cytotoxic agents is the low propensity to interact with DNA; which omits the risk of mutagenesity as the common side effect of current chemotherapeutic agents [9].

Chemistry
All chemical reagents and solvents were provided from Merck AG (Darmstadt, Germany). The general procedures for the synthesis of 3-benzylidene-4-chromanones 5a-k, and aldehyde intermediates (compounds 7-9 and 11) are illustrated in Schemes 1 and 2, respectively. 7-Methoxychroman-4-one (4) was prepared as literature method [18,19]. Melting points of compounds were determined using Kofler hot stage apparatus and are uncorrected. The IR spectra were recorded on a Shimadzu 470 spectrometer by using potassium bromide disks. The NMR spectra were obtained using a Bruker 400 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA). Tetramethylsilane (TMS) was used as internal standard and chemical shifts (δ) are reported in ppm. Mass spectra were recorded on a Finnigan TSQ 70 spectrometer at 70 eV. Elemental analyses were carried out by using a HERAEUS CHN-O rapid elemental analyzer (Heraeus GmbH, Hanau, Germany) for C, H and N and the results are within ± 0.4% of the theoretical values.
Scheme 2 Synthesis of aldehyde intermediates 7-9 and 11. Reagents and conditions: (a) Cl 2 , CH 3 COOH; (b) R 2 OCOCH 2 Br, K 2 CO 3 , CH 3 COCH 2 CH 3 ; (c) methyl iodide, K 2 CO 3 , DMF. times with ethyl acetate (5 ml). The combined extracts was dried (Na 2 SO 4 ), and concentrated to give 8b as a white solid in 91% yield. 1  To a solution of compound 7b (500 mg, 2.5 mmol) in ethyl methyl ketone (10 ml), was added potassium carbonate (370 mg, 2.5 mmol) and ethyl bromoacetate (2.5mmol) successively. The mixture was stirred under reflux for 3 h and then the solvent was removed under reduced pressure. The residue was added to 10 ml of water and extracted three times with ethyl acetate (10 ml). The combined extracts was dried (Na 2 SO 4) and concentrated to give 8c as a white solid in 88% yield. 1

Synthesis of 3-chloro-4,5-dimethoxybenzaldehyde (9a)
To a solution of 5-chlorovanillin (7a, 5 g, 26.8 mmol) in DMF (40 ml) was added potassium carbonate (3.7 g, 26.8 mmol) and iodomethane (4.56 g, 32.16 mmol) successively. The mixture was stirred at 80°C for 3 h, cooled to room temperature and poured to water (100 ml). The precipitated white solid was filtrated and washed with water to give 5.1 g of compound 9a in 94.8 yield. 1  General procedure for the preparation of compounds 11a-c A mixture of hydroxybenzaldehyde 10a-c (5 g, 40.95 mmol) and K 2 CO 3 (6 g, 40.95 mmol) in ethyl methyl ketone (100 ml) was stirred under reflux. After 1 h, methyl bromoacetate was added, and the mixture was allowed to stir under reflux for another 3 h. After the reaction was completed, ethyl methyl ketone was removed, and the residue was extracted with EtOAc (3 × 20 ml). The organic layer was dried (Na 2 SO 4 ) and evaporated to give methyl (formylphenoxy)acetate 11a-c [21].

Methyl (2-formylphenoxy)acetate (11a)
This compound was obtained using general procedure as a pale yellow oil without further purification in 85% yield. IR (KBr, cm

Cytotoxicity assay
The in-vitro cytotoxic activity of each synthesized compounds 5a-k was assessed using MTT colorimetric assay according to the literature method [22]. Each set of experiments was independently performed three times. For each compound, the concentration causing 50% cell growth inhibition (IC 50 ) compared with the control was calculated from concentration-response curves by regression analysis.
The corresponding aldehydes 7-9 and 11 were prepared as shown in Scheme 2. Chlorination of 3-alkoxy-4-hydroxybenzaldehyde 6a,b using acetic acid as a solvent gave 3-chloro-4-hydroxy-5-alkoxybenzaldehyde 7a,b which was reacted with suitable alkyl bromoacetate, in the presence of potassium carbonate to give compounds

In vitro cytotoxic activity
The cytotoxic activity of synthesized compounds 5a-k was evaluated against three cell lines namely MDA-MB-231 (breast cancer), KB (nasopharyngeal epidermoid carcinoma) and SK-N-MC (human neuroblastoma) cells.
The results of cytotoxic assay were mentioned as IC 50 (μg/ml) of compounds in comparison with reference drug etoposide in Table 1.
In the case of MDA-MB-231 cell line, the IC 50 values of all compounds were ≤20 μg/ml with the exception of compounds 5d and 5e. Furthermore, compounds 5b and 5i exhibited the highest cytotoxic activity against this cell line (IC 50 < 10 μg/ml). Compound 5b was also the most potent derivative against KB cell line with IC 50 value of 25.04 μg/ml. Beside compound 5b, compound 5a exhibited good activity against KB cells, but remaining compounds 5c-k showed no activity against this cell line (IC 50 >100 μg/ml). Against SK-N-MC cells, compound 5b followed by compounds 5a and 5c showed significant inhibitory activity with IC 50 values of 9.64, 12.6 and 58.04 μg/ml, respectively.
Overall, it is clear that among the test compounds described in this study, the 3-chloro-4,5-dimethoxybenzylidene derivative 5b demonstrated better cytotoxic profile against all tested cell lines (IC 50 values = 7.56-25.04 μg/ml). Generally, the comparison of IC 50 values of compound 5b with those of etoposide demonstrated that the cytotoxic activity of compound 5b against MDA-MB-231 and SK-N-MC cells is more than etoposide.
In this work, as part of an ongoing program to find new cytotoxic agents, we have focused our attention on modification of the 3-benzylidene-4-chromanones and introducing new functionality on the benzylidene moiety.
Thus, we designed novel 3-benzylidene-4-chromanones that possessed a 2-(2-chloro-6-alkoxyphenoxy)acetic acid ester. These modifications were made on the basis of SJ-172550, a new cytotoxic agent possessing 2-(2-chloro-6ethoxyphenoxy)acetic acid methyl ester attached to the pyrazolone ring. Surprisingly, compound 5d, the chromanone analog of SJ-172550 showed no activity against tested cell lines. Also, the ethyl ester counterpart of 5d (compound 5e) was inactive against tumor cell lines. However, the 2-(2-chloro-6-methoxyphenoxy)acetic acid methyl ester analog 5c was active against MDA-MB-231 and SK-N-MC cells. We have briefly investigated the SAR of compounds by simplification of the functionality on the benzylidene part of the basic molecule.
As can be deduced from the cytotoxic data of compounds 5f-k which characterized by the lack of 2chloro-6-alkoxy functionality, the cytotoxic activity against MDA-MB-231 can be served by the simple phenoxyacetic acid ester derivatives. However, the lack of 2-chloro-6-alkoxy functionality results in the lack of activity against KB and SK-N-MC cells. Among the compounds 5f-k, butyl ester derivative 5i showed the highest activity against MDA-MB-231 being 3-fold more potent than standard drug etoposide.
To determine the effect of acetic acid ester substitution in compound 5c, we prepared both the 4-hydroxy derivative 5a and 4-methoxy analog 5b. When compared to 5c, both compounds had similar or better in vitro activities against tested cell lines.
The cytotoxic activities of regio-isomeric compounds 5f, 5j and 5 k against MDA-MB-231cells revealed that changing the position of oxyesteric group has led to non-significant changes in activities. As seen from data, in poly-substituted compounds changing of methoxy group on phenyl ring to ethoxy group (for example 5d versus 5c) dramatically decreased the cytotoxic potency in MDA-MB-231 and SK-N-MC cells.
In summary, in the pursuit for finding new cytotoxic agents, we replaced the pyrazolone part of well-known cytotoxic agent SJ-172550 with 7-methoxychroman-4one. Although the direct analog of SJ-172550 (compound 5d) did not show any cytotoxic activity against tested cell lines, but 2-(2-chloro-6-methoxyphenoxy) acetic acid methyl ester analog 5c showed some activity against MDA-MB-231 and SK-N-MC cells. Further modification of compound 5c resulted in the 3-chloro-4,5dimethoxybenzylidene derivative 5b which demonstrated better cytotoxic profile against all tested cell lines (IC 50 values = 7.56-25.04 μg/ml).
It is worthwhile to mention that, since we have originally designed the target compounds based on p53dependent cytotoxic agent SJ-172550, it was better using the latter compound as standard drug in our cytotoxic assay. However, our primary cytotoxic experiments on the closest compound to SJ-172550 (compound 5d) in a side-by-side comparison manner with etoposide revealed that compound 5d had no activity against cancer cell lines. On the other hand, simplified compounds 5a and 5b with more dissimilarity respect to the SJ-172550 showed better profile of cytotoxicity. Based on these results, it seems that a different mechanism is responsible for potential cytotoxic activity of compound 5b prototype.
We employed MTT cell viability assay as a standard and well-documented in vitro method for evaluation of the cytotoxic potential of designed compounds. Although these types of in vitro models are beneficial and promising as early screening tools for finding new lead compounds, but these models are associated with some limitations [23]. Thus, for efficacy and safety evaluation of lead compounds, conducting a method based on animal model is necessary in the next steps of study.
In conclusion, the results demonstrated that the cytotoxic activity of 3-(3-chloro-4,5-dimethoxybenzylidene)-7-methoxychroman-4-one (5b) against MDA-MB-231 and SK-N-MC cells is more than standard drug etoposide. Therefore, compound 5b prototype bearing 3-chloro-4,5dimethoxybenzylidene moiety could be considered as novel lead compound for further developing new anticancer chemotherapeutics. Although, compound 5b showed promising activity in vitro, but to identify a promising anticancer drug candidate that has good pharmacokinetic and toxicological profiles, the in vivo ADME-Tox studies of compound 5b prototype should be conducted.