Multi-component Reactions of Cyclohexan-1,3-diketones to Produce Fused Pyran Derivatives with Antiproliferative Activities and Tyrosine Kinases and Pim-1 Kinase Inhibitions

In this work the multi-component reactions of either of the arylhydrazocyclohexan-1,3-dione derivatives 3a–c with either of benzaldehyde (4a), 4-chlorobenzaldehyde (4b) or 4-methoxybenzaldehyde (4c) and either malononitrile (5a) or ethyl cyanoacetate (5b) giving the 5,6,7,8-tetrahydro-4H-chromene derivatives 6a–r, respectively, are presented. The reaction of two equivalents of cyclohexan-1,3-dione with benzaldehyde gave the hexahydro-1H-xanthene-1,8(2H)-dione derivative 7. On the other hand, the multi-component reactions of compound 1 with dimedone and benzaldehyde gave 13. Both of 7 and 13 underwent heterocyclization reactions to produce fused thiophene, pyran and thiazole derivatives. Selected compounds among the synthesized compounds were tested against six cancer cell lines where most of them gave high inhibitions; especially compounds 3b, 3c, 6b, 6c, 6d, 6f, 6i, 6m, 6n, 8b, 14a, 15 and 16 being the most cytotoxic compounds. Further tests against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR and Pim-1 kinase showed that compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 were the most potent of the tested compounds toward the five tyrosine kinases and compounds 3c, 6c, 6d, 6n and 15 displayed the highest inhibitions toward Pim-1 kinase.


Introduction
Pyran derivatives are known as important class of compounds that exist in nature and have many applications 1 especially fused pyrans are important core units comprising many natural products. Due to their various kinds of biological activities pyrans and their fused derivatives attracted the attention within the last few years. It was reported that benzo[b]pyran derivatives were excellent anticancer compounds that give good results at very low concentrations. 2 Many 2-amino-4H-pyran derivatives have various applications within industry like their uses as photoactive materials, 3 pigments, 4 and potentially biodegradable agrochemicals. 5 In addition, naphthopyrans have many application with optical studies due to their ability to generate a yellow color on being irradiated with UV light (van). In addition, pyranochalcones have many applications like antimutagenic, antimicrobial, antiulcer, and antitumor activities. [6][7][8] Pyrans and their fused derivatives showed different kinds of biological activities. The attachments of heterocyclic ring to the pyran ring improve many of the biological effects of the resulting molecules. Especially the 4H-pyran derivatives exhibited wide range of biological activities with great interests such as antimicrobial, 9 antiviral, 10,11 mutagenicity, 12 antiproliferative, 13 sex pheromone, 14 antitumor, 15 cancer therapy, 16 and central nervous system activity. 17 Some of these compounds were applied in industrial chemistry as they can be used in many cosmetic manufacturing and through the field of agrochemicals. 18 Such high importance of pyrans and their derivatives together with the ease of their synthesis with high yields direct many works through their synthesis. Mohareb  This encouraged our research group to be attracted toward the synthesis of pyran derivatives through the uses of β-diketones. The produced compounds showed high antiproliferative activities against cancer cell lines together with high inhibitions toward tyrosine kinases. [19][20][21][22][23][24][25] Through our present work we adopted multi-component reactions of either arylhydrazonocyclohexan-1,3-dione, aromatic aldehydes and cyanometylene derivatives together with using the produced molecule as a suitable starting material for subsequent heterocyclization to obtain a variety of fused derivatives. The anti-proliferative activities of the synthesized compounds and their inhibitions toward tyrosine kinases were determined.

Experimental
For newly synthesized compounds melting points were determined and are given as uncorrected values. For all compounds the IR spectra (KBr discs) were measured using a FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer. The 1 H NMR spectra were measured using Varian Gemini-300 (300 MHz) and Jeol AS 500 MHz instruments. Measurements were performed in DMSO-d 6 as the solvent using TMS as the internal standard and chemical shifts are expressed as δ ppm. The MS spectra (EI) were measured using Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. The microanalytical CHN data were obtained from the Micro-analytical Data Unit at Cairo University and were performed on Vario EL III Elemental analyzer. Screening of compounds against the cancer cell lines and tyrosine kinases were performed through The National Cancer Institute at Cairo University.

1. Synthesis of the Arylhydrazone Derivatives 3a-c
A solution of either the diazonium salts (0.01 mol) [prepared by the addition of a solution of sodium nitrite (0.70 g, 0.01 mol) in water (10 mL) to a cold solution of either aniline (0.93 g, 0.01 mol), 4-methylaniline (1.07 g, 0.01 mol) or 4-chloroaniline (1.27 g, 0.01 mol) dissolved in concentrated hydrochloric acid (10 mL, 18 mol) with continuous stirring] was added to a cold solution of any of the compounds 1 (1.12 g, 0.01 mol), in ethanol (50 mL) containing sodium acetate (3.0 g) with stirring. The whole reaction mixture was left at room temperature for 2 h and the formed solid product was collected by filtration.

6. 4. Inhibitions of Selected Compounds Against Pim-1 Kinase
Compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 were selected to examine their Pim-1 kinase inhibition activity (Table 3) as these compounds showed high inhibition against the tested cancer cell lines at a range of 10 concentrations and the IC 50 values were calculated. Compounds 3c, 6c, 6d, 6n and 15 most potently inhibited Pim-1 kinase with IC 50 values of 0.24, 0.27, 0.24, 0.28 and 0.32 µM, respectively. On the other hand, compounds 6f and 14a were less effective (IC 50 > 10 µM). These profiles in combination with cell growth inhibition data of compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 are listed in Table 3, indicating that Pim-1 is a potential target of these compounds where SGI-1776 was used as the positive control with IC 50 0.048 µM in the assay.

6. 5. Pan Assay Interference Compounds (PAINS)
Good antitumor drugs should give false positive results when evaluated within Pan Assay Interference Compounds (PAINS). 29,30 Compounds can be regarded as false positives due their binding interactions by forming aggregates 31-33 by being protein-reactive entities [34][35][36] or by directly interfering with assay signaling. Pan Assay Interference Compounds (PAINS) are chemical entities that are frequently false positive in HTS. PAINS have a tendency to non-specifically react with several biological targets moderately, then specifically disturbing one preferred target. 37 A number of disorderly functional groups are collected by numerous PAINS. 38 Unwanted compounds may negatively influence not only enzyme assays but also phenotypic  Table 4. Drug-like character of different compounds and standard drugs foretinib and SGI-1776 screens and show biological activity for the wrong reason. 39 PAINS violations of proposed compounds and reference drugs are given in Table 4. Almost all the compounds showed zero PAINS alert and can be used as good anticancer agents in the future without side effects.

Results and Discussion
Initially 2-arylhydrazonocyclohexan-1,3-dione was chosen as the model substrate for the synthesis of fused het-erocyclic compounds through studying its multi-component reactions with aromatic aldehydes and cyanomethylene reagents to give biologically active fused pyran derivatives. The arylhydrazone derivatives 3a-c were obtained through the coupling reaction between cyclohexane-1,3-dione (1) and either benzenediazonium chloride (2a), 4-methylbenzenediazonium chloride (2b) or 4-chlorobenzenediazonium chloride (2c) in ethanol solution containing the appropriate amount of sodium acetate. The multi-component reactions of either 3a, 3b or 3c with either of benzaldehyde (4a), 4-chlorobenzaldehyde (4b) or were in agreement with the proposed structure. Thus, the 1 H NMR spectrum showed the presence of two multiplets at δ 1.59-1.80 and 2.58-2.73 ppm equivalent to the six CH 2 groups, a singlet at δ 5.09 ppm for the pyran H-4 and a multiplet at δ 7.25-7.41 ppm corresponding to the C 6 H 5 group. In addition, the 13 C NMR spectrum showed signals at δ 26.3, 28.4, 32.6 equivalent to the six CH 2 groups, signal at 50.9 due to the pyran C-4, four signals at δ 120.6, 121.4, 123.6 and 125.8 for the phenyl carbons and a signal at δ 168.9 for the two symmetric C=O groups. Compound 7 showed interesting reactivity toward heterocyclization reactions through its reactions with some reagents. It was ready to undergo Gewald's thiophene [40][41][42] reaction to produce biologically active fused thiophene derivatives. Thus, the reaction of compound 7 with two folds of either malononitrile (5a) or ethyl cyanoacetate (5b) and elemental sulfur gave the dithieno[3,2-a:2' ,3'-j]xanthene derivatives 8a and 8b, respectively. On the other hand, compound 7