ANTI-PROLIFERATIVE ACTIVITIES OF 4H-PYRAN DERIVATIVES SYNTHESIZED FROM BENZOYLACETONE

Benzoylacetone (1) underwent a series of multi-component reactions with aromatic aldehydes and malononitrile or ethyl cyanoacetate to produce the pyran derivatives 4a-f. The latter compounds reacted with malononitrile or ethyl cyanoacetate to yield the condensation products 5a-m. On the other hand, the reaction of 4af with either the diazonium salts 6a-c yielded the arylhydrazone derivatives 7a-i. The multi-component reaction of (1) with aromatic adehydes and cyclohexan-1,3-dione produced the pyran derivatives 9a-c. Compound 1 underwent the Gewald’s reactions with elemental sulfur and malononitrile or ethyl cyanacetate yielding the thiophene derivatives 10a,b. Evaluations of the synthesized products were carried out against some selected cancer cell lines and the most active compounds were further evaluated against the seventeen cancer cell lines classified according to the disease. Morphological changes of A549 cell line by the effect of compound 7k was studied using microenvironment of the lung tissue where an excellent results was obtained.


INTRODUCTION
Within the field of pharmaceutical and medicinal chemistry [1] the pyran derivatives were of the most important bioactive compounds. They attract the attention in recent years as a result of their wide range of biological activities among which their antitumor [2], COX-2 and HDAC inhibitory [3], anti-microbial [4], anti-mycobacterial [5], anti-inflammatory [6], antiviral [7], antifungal [8], anticonvulsant [9], antioxidant [10], analgesic [11,12] and antinociceptive [13]. According to WHO cancer can be defined as a generic term for a large group of diseases that can impair any part of the body beside it was known as malignant tumours and neoplasms. Metastasis is another term for defining cancer which involve the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. As the result of the large spread of cancer and the anticancer activities of 4H-pyran derivatives, many reports concerned with the synthesis and anticancer activities of such group of compounds [14,15]. Recently, we were focused through the production of a large number of 4Hpyran compounds via the multicomponent reactions of aromatic aldehyde, 1,3-dicarbonyl compounds and active methylene reagents followed by anti-proliferative evaluations [16,17]. The best way to get poly-substituted 4H-pyran derivatives is the use of multi-component reactions (MCRs) [18,19]. There are also many reports showed that compounds containing pyran nucleus could be a worthy choice for cyctotoxic activity against various human tumor cell lines together with their pharmacological activities [20][21][22][23][24][25]. In the light of the above findings, we demonstrated herein the synthesis of some new 4H-pyran and arylhydrazone derivatives that cannot be obtained via another way. In addition, the evaluation of the newly synthesized products towards human cancer cell lines and cancer cell lines classified according to the disease was done. The originality of this work appeared through the investigating of new heterocyclic compounds that can be used as anti-cancer agents.

RESULTS AND DISCUSSION
This work demonstrated the uses of benzoylacetone as starting compound for the synthesis of many heterocyclic derivatives and the progress of reactions were outlined through Schemes 1-4. The multi-component reaction of benzoylacetone (1) with each of malononitrile (2a) or ethyl cyanoacetate (2b) and benzaldehyde (3a), 4-methoxybenzaldehyde (3b) or 4-chlorobenzaldehyde (3c) using absolute ethanol together with a catalytic amount of triethylamine yielded the 1-(6amino-2-phenyl-4-aryl-4H-pyran-3-yl)ethanone derivatives 4a-f (Scheme 1). The analytical and spectral data were the tools that were used to confirm the structures of compounds 4a-f together with the studying of their reactions with some chemical reagents. Thus, the reaction of compounds 4a-f with malononitrile (2a) or ethyl cyanoacetate (2b) in an oil bath at 120 o C produced the Knoevenagel condensation products 5a-m (Scheme 2). The structures of compounds 5a-m were based on analytical and spectral data. Thus, the 1 H NMR spectrum of 5a (as an example) revealed the presence of a singlet at  2.92 for the CH3 group, a singlet at  4.87 ppm (D2O exchangeable) confirming the presence of the NH2 group, a singlet at  6.50 ppm due to the presence of the pyran H-4 and a multiplet at  7.24-7.56 ppm which was attributed to the two C6H5 groups. Beside, the 13 C NMR spectrum showed the presence of a signal at  38.7 for the CH3 group, two signals at  86.3, 89.4 confirming the C=C moiety, a signal at  90.2 for the pyran C-4, three signals at  116. 8,117.0, 117.2 confirming the presence of the three CN groups, signals at  120.3, 120.5, 121.4, 122.6, 123.2, 123.8, 124.3, 125.9 for the two C6H5 groups and four signals at  128.2, 130.8, 131.6, 133.2 due to the pyran C-2, C-3, C-5, C-6. Scheme 1. Synthesis of compounds 4a-f.
The azomethine -NHN=CH group, hydrazones and their derivatives represents an important class of compounds with many applications as anticancer activities against different cancer cell lines [26][27][28][29] for that reason many hydrazone derivatives were synthesized. To our knowledge azomethine were previously prepared from the reaction of carbaldehyde with hydrazine [30,31], however, in this work we succeeded to get such compounds through the reaction of diazonium salts with the acetyl group of compounds 4a-f. Moreover, we obtained a variety of arylhydrazone derivatives characterized with different substituents not only at the heterocyclic ring but also at the aryl moiety where this enabled study of structure activity relationship when tested against Scheme 2. Synthesis of compounds 5a-m.

Biology Anticancer evaluations of the newly synthesized compounds
Using Foretinib as the positive control in such measurements, it was noticed that many of the synthesized compounds exhibited high potent anti-proliferative activity. Some of the synthesized compounds exhibited higher activity than the positive control Foretinib. The variations of substituents within the ring system beside the nature of the heterocyclic compound had a notable impact on the anti-proliferative activity. Through such measurements the IC50's of Foretinib demonstrated in Table 1 against the cancer cell lines were considered identical to the previously reported work [32][33][34].  Table 1 demonstrated that compounds 5a, 5c, 5d, 5e, 5f, 5g, 5h and 5i were the most cytotoxic compounds among these compounds. Table 1 showed that compounds 5e (X = Cl, R = R'= CN), 5f (X = Cl, R = COOEt, R' = CN) and 5g (X = Cl, R = CN, R' = COOEt) exhibited higher inhibitions than the reference  [32][33][34].

SAR's (structure activity relationship) of the synthesized compounds
Foretinib against U87MG and SMMC-7721 cell lines. On the other hand, compound 5h (X = Cl, R = R' = COOEt) showed higher inhibition than the reference only against U87MG cell line. It was surprisingly; within such series of compounds that compound 5i with the electron donating OCH3 group was the high cytotoxic derivative. It seemed that the presence of the two CN group had a great effect for its high inhibitions beside the conjugate effect of the OCH3 group with aryl moiety. Preferably among the arylhydrazone derivatives 7a-s, compounds 7c, 7e, 7f, 7g, 7h, 7i, 7k, 7l, 7m, 7o and 7p were the most cytotoxic compounds. Compounds 7c (X = H, R = CN, Y = Cl), 7g (X = Cl, R = CN, Y = H), 7h (X = Cl, R = COOEt, Y = H), 7i (X = Cl, R = CN, Y = Cl), 7k (X = Cl, R = COOEt, Y = Cl), 7l (X = Cl, R = CN, Y = CH3), 7m (X = Cl, R = COOEt, Y = CH3) and 7p (X = OCH3, R = CN, Y = Cl) exhibited good inhibitions against the six cancer cell lines. It is of interest to mention that compounds 7c, 7o and 7m exhibited higher inhibitions than the reference Foretinib against the three cancer cell lines MKN-45, U84MG and SMMC-7721. On the other hand, the presence of an electron donating methyl group (Y = CH3) in compounds 7e, 7f and 7i, and methoxy group (X = OCH3) in derivatives 7o and 7p resulted in high inhibitions for these compounds against A549, H460 and SMMC-7721 cell lines. Tabel 1 showed that compounds with the CH3 moiety like7e, 7f and 7i relatively exhibited lower inhibitions than compounds 7o and 7p with the OCH3 group. Our explanation for such finding was based on the conjugation effect of the OCH3 group with the double bonds of the phenyl group which might enhance the inhibitions. For the 4H-chromen-5(6H)-one derivatives 9a-c, compounds 9a (X = H) and 9c (X = Cl) were of high inhibitions while compound 9b (X = OCH3) exhibited low inhibitions this can be attributed to the steric effect of the OCH3 group which is larger H or Cl atom. Considering the thiophene derivatives 10a,b where compound 10a substituted with a relatively smaller CN exhibited higher inhibition than 10b substituted with a larger COOEt moiety.

In vitro anticancer activities of the synthesized compounds
Against a panel of approximately seventeen tumor cell lines at 10-fold dilutions of five concentrations (100, 10, 1.0, 0.1 and 0.01 µM) [35], the arylhydrazone derivatives 7c, 7k, 7l, 7m and 7o were selected for this assay. The tested compounds exhibited inhibitions (GI50 < 5 µM) against the cancer cell lines that are classified into groups according to the type of disease, the data were shown through Table 2. There are two factors in this work affecting inhibitions of compounds 7c. 7k, 7l, 7m and 7o, the substituent at C-3 of the pyran ring and the substituents at the 4-position of the two aryl group. Table 2

Determination of morphological changes of A549 cell line
The lung is a unique organ that should be protected against inhaled pathogens and toxins, without unbalance immune responses or compromise its vital function. For that matter microenvironment of the lung tissue is regulated through complex and refined cell interactions [36,37]. For that reason we studied the effect of compound 7k toward A549 cell line which was selected for studying the morphological changes. There are many reports concerned with morphological changes of other cell lines [38,39]. To understand the ability of compound 7k in apoptosis induction, various qualitative (morphological) and quantitative assays were performed on the A549 lung cancer cell line. The changes in the morphological features of A549 cells were observed after the treatment with compound 7k at different concentrations along with the untreated control cells. Further, images reported in Figure 1A were captured using phase-contrast microscopy after 72 h; reveal the characteristic apoptotic features like changes in morphology (shape, shrinkage) of the cell, reduction in the number of live cells. In the present study, A549 cells after treatment with compound 7k for 72 h exhibited the formation of apoptotic features such as the appearance of membrane blebs and inverse proportion in the number of cells with a concentration of compound tested as indicated in Figure 1B. Compound 7k was treated A549 cells after 72 h, on staining with DAPI visualized the chromatin condensation, pyknotic (inset of 1.25 μM), and condensed (bright colored: inset of 2.5 μM) nuclei formation as depicted in Figure  1C. Through figure 1the effect of compound 7k on A549 cells marked by the red cells indicating reduction of the size of the cancer cell line .

Chemistry
Melting points were recorded using an electrothermal digital melting point apparatus. Using a FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer the IR spectra were recorded. The 1 H NMR spectra were measured using Varian Gemini-300 (300 MHz) instrument. Moreover, the mass spectra were measured using Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. The CHNS microanalyses were using Vario EL III Elemental analyzer. The anti-proliferative evaluations were measured by the aid of the National Cancer Research Centre in Cairo.

Procedure of cell proliferation
Using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [40,41] the tested compounds were evaluated in vitro as anti-proliferative agents. The human lung carcinoma (A549), lung cancer (H460), human colorectal (HT29), gastric cancer cell (MKN-45), glioma cell line (U87MG) and cellosaurus cell line (SMMC-7721) were used in such evaluations ( Table 1). The procedure used for such measurements were carried out according to our previously reported work [42,43]. The mean values of three independent experiments, expressed as IC50 values, were presented in Table 1. The IC50's presented in this were considered as the mean values of three independent experiments.

CONCLUSION
In conclusion, a series of new pyran, chromen-5-one and thiophene derivatives were synthesized and identified. The products were evaluated as novel anti-cancer agents toward the six cancer cell lines namely the human lung carcinoma (A549), lung cancer (H460), human colorectal (HT29), gastric cancer cell (MKN-45), glioma cell line (U87MG) and cellosaurus cell line (SMMC-7721). The preliminary investigation showed that the pyran derivatives 4a, 4b, 4e, 5a, 5d, 5e, 5f, 5g, 5h, 5i; the arylhydrazone derivatives 7c, 7e, 7f, 7g, 7h, 7i, 7k, 7l, 7m, 7o, 7p, 7q; the chromen-5-one derivatives 9a, 9c and the thiophene derivative 10a exhibited from good to excellent potency against the six used cell lines. In most cases, the analysis of SARs indicated that compounds with the 4-chloro on the phenyl ring and the CN or COOEt on the pyran ring were more active than those with other substituents. Screening of compounds 7c, 7k, 7l, 7m and 7o against cancer cell lines classified according to the disease revealed that all of them expressed high inhibitions. Morphological changes of A549 cell line by the effect of compound 7k was studied using microenvironment of the lung tissue where an excellent result was obtained. In our laboratory, further studies on the structural optimization about these derivatives are still underway for future work.